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dissertation entitled

CHARACTERIZATION OF NOVEL GROUPS OF
CATECHOLAMINERGIC CELLS IN THE MALE PRAIRIE VOLE
EXTENDED AMYGDALA

presented by

KATHARINE VIRGINIA NORTHCU'IT

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CHARACTERIZATION OF NOVEL GROUPS OF CATECHOLAMINERGIC
CELLS IN THE MALE PRAIRIE VOLE EXTENDED AMYGDALA

By

Katharine Virginia Northcutt

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Neuroscience

2010

ABSTRACT

CHARACTERIZATION OF NOVEL GROUPS OF CATECHOLAMINERGIC CELLS
IN THE MALE PRAIRIE VOLE EXTENDED AMYGDALA

By
Katharine Virginia Northcutt

The neurobiology of copulatory and maternal behaviors has been well studied in
mammals, but much less is known about gregarious and monogamous social behaviors.
Prairie voles (Microtus ochrogaster) are one of few mammalian species that are highly
gregarious, monogamous, and biparental, and have been a useful rodent model for
studying the neural networks inﬂuencing these behaviors. Comparisons between the
prairie vole brain and closely related nonmonogamous rodents have revealed species
differences in neurotransmitter and receptor expression that contribute to differences in
social behavior. Recently, large populations of cells expressing tyrosine hydroxylase
(TH), the rate-limiting enzyme in catecholarnine synthesis, were discovered in the prairie
vole principal nucleus of the bed nucleus of the stria terminalis (pBST) and posterodorsal
medial amygdala (MeApd). Large numbers of TH-immunoreactive (TH-ir) cells have
never been described in these sites in other species. The pBST and MeApd are important
for the expression of sociosexual behaviors, and these cells may affect the social
behaviors typical of prairie voles. The experiments in this dissertation characterize the
distribution, hormone regulation, immediate-early gene (IEG) responses, and
neuroanatomical projections of these unique groups of cells in the male prairie vole brain.

The results of the ﬁrst experiment (Chapter 2) indicated that several
nonmonogamous species, laboratory rats, Syrian hamsters, and meadow voles, did not

have these large populations of TH—ir cells, further supporting the idea that these cells

may inﬂuence species-speciﬁc social behaviors. The results also indicated that male
prairie voles had 3-5 times more TH-ir cells in the pBST and the MeApd than did
females, and this sex difference was almost entirely due to differences in circulating
gonadal hormones during adulthood. The experiments in Chapter 3 examined
immediate-early gene expression in these TH-ir cells after males engaged in sociosexual
behaviors, and these experiments revealed that these groups of TH-ir neurons likely
inﬂuence gregariousness in male prairie voles, but may also be involved more
speciﬁcally in mating and its facilitation of pair bonding. Finally, neuroanatomical
tracing (Chapter 4) revealed that many TH-ir cells in the pBST and MeApd project to the
medial preoptic area (MPO), an area important for the expression of many sociosexual
behaviors.

These experiments demonstrate that TH expression in the pBST and MeApd
remains plastic in adulthood and is sensitive to circulating gonadal hormones, much like
other morphological and neurochemical features of the pBST and MeApd in rats, mice,
and hamsters. Furthermore, these cell groups are unique in that they may promote the
expression of all social behaviors, but also speciﬁc behaviors related to mating and pair
bonding. They also provide a species-speciﬁc catecholaminergic input to the MPO,
which has connections with other brain areas known to be important for pair bonding.
These experiments have provided information necessary for many future experiments that
can explore the precise function of these cells and their interactions with other
neurochemicals. This catecholaminergic network is likely essential for the afﬁliative and

monogamous behaviors typical of male prairie voles.

I dedicate this dissertation to Rascal. You were my best friend and partner in crime, and

thank you for always reminding me to embrace every moment.

iv

ACKNOWLEDGEMENTS

First and foremost, I thank my advisor, Dr. Joe Lonstein, for his wonderful
mentoring and support over the last ﬁve years. Joe allowed me to be independent, but
guided me whenever I needed it, and was always there to listen to my crazy ideas. I
cannot even begin to enumerate everything that I learned from Joe, and I would not be
ready to start my career if I had not had him as a mentor. I appreciate that he supported
my interests in teaching and outreach, even when it took time away from my research. I
also thank my other committee members, Drs. Cheryl Sisk, Marc Breedlove, and Keith
Lookingland for their guidance and helpful feedback as I planned and carried out these
projects.

I also thank current and former members of the Lonstein lab: Breyanna
Cavanaugh, Stephanie Miller, Carl Smith, and Ray Figueira. I had a great time working
with you, and appreciate your willingness to lend a hand whenever I needed it. I also
thank the students of the Neuroscience Program, who made this an extremely enjoyable
experience. I will miss our engaging science discussions and amazing adventures.

I thank my family, particularly my parents, for their support and encouragement
throughout my life. They believed in me when I did not believe in myself, and were
supportive when I said that I wanted to move across the country for graduate school. I
would not have ﬁnished this dissertation had it not been for their emotional support, and I
cannot thank them enough for always being available to listen. Finally, I thank all of the
members of the Buddies soﬁball teams for giving me a fun way to relieve stress, and for

becoming amazing friends.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................... ix
LIST OF FIGURES .................................................................................. x
LIST OF ABBREVIATIONS ...................................................................... xiv
CHAPTER 1: Introduction ............................................................................ 1
The prairie vole model ........................................................................... 3
The neurobiology of prairie vole sociosexual behaviors ..................................... 5
Dopamine and sociosexual behaviors ............................................................................ 8
The olfactory system and sociosexual behaviors ......................................................... 13
Hormone regulation of the pBST and MeApd ............................................................. 15
Hormonal inﬂuences on sociosexual behaviors in prairie voles .................................. 17
Overview of dissertation chapters ................................................................................ 18

CHAPTER 2: Sex and species differences in tyrosine hydroxylase-synthesizing cells of

the rodent extended amygdala ........................................................................................... 20
Abstract ........................................................................................................................ 21
Introduction .................................................................................................................. 23
Methods ....................................................................................................................... 25

Subjects .................................................................................................................. 25
Gonadectomy and hormone replacement ............................................................... 27
Perfusion, tissue collection, and immunocytochemistry ........................................ 28
Analyses ................................................................................................................. 30
Results .......................................................................................................................... 32
Species comparison ................................................................................................ 32
Sex difference and effects of gonadal hormones on TH immunoreactivity in
prairie voles ............................................................................................................ 39
Discussion .................................................................................................................... 46
Species differences in TH immunoreactivity ......................................................... 46
Sex differences and hormone sensitivity of TH-immunoreactive cells ................. 48
Functional considerations ...................................................................................... 51
Acknowledgements ...................................................................................................... 54

CHAPTER 3: Social contact elicits immediate-early gene expression in

catecholaminergic cells of the male prairie vole extended amygdala ................................ 55
Abstract ........................................................................................................................ 56
Introduction .................................................................................................................. 58
Methods ....................................................................................................................... 60

Subjects .................................................................................................................. 60
Experiment Ia: effects of 30-min social interactions on Fos expression ............... 61
F05 and TH dual immunocytochemistry .......................................................... 65

vi

Analysis of Fos and TH—ir cells ....................................................................... 66

Statistical analyses ........................................................................................... 67
Experiment Ib: effects of 30-min social interactions on Egr-l expression in TH-ir
cells ........................................................................................................................ 68

Egr-l and TH immunocytochemistry and analysis .......................................... 68

Statistical analyses ........................................................................................... 69
Experiment 11: effects of 24-h social interactions on F 03 expression .................... 70

Results .......................................................................................................................... 71
Experiment Ia: F os and TH expression after 30-min interactions ......................... 7]

Behavior ........................................................................................................... 71

TH expression .................................................................................................. 72

Fos expression ................................................................................................. 76

Fos expression within TH-ir cells .................................................................... 78

Correlations between the percentage of TH-ir cells expression F os and

behavior ............................................................................................................ 81
Experiment lb: Egr-l expression after 30-min social interactions ........................ 84

TH expression .................................................................................................. 84

Egr-l expression .............................................................................................. 85

Egr-l expression within TH-ir cells ................................................................. 87

Correlations between the percentage of TH-ir cells expressing Egr-l and

behavior ............................................................................................................ 87
Experiment II: Fos expression after 6- or 24-h social interactions ........................ 89

TH expression .................................................................................................. 89

F 05 expression .................................................................................................. 89

Fos expression within TH-ir cells .................................................................... 92

Discussion .................................................................................................................... 94
IEG expression after sociosexual interactions ....................................................... 94

IEG expression within TH— expressing cells after sociosexual interactions ........... 95

DA and prairie vole sociosexual behaviors ........................................................... 98
Acknowledgements .................................................................................................... l 00
CHAPTER 4: Unique catecholaminergic projections from the extended olfactory
amygdala to the medial preoptic area in male prairie voles ............................................. 101
Abstract ...................................................................................................................... 101
Introduction ................................................................................................................ 103
Experiment 1: Anterograde projections of the prairie vole pBST ............................. 106
Methods ............................................................................................................... 106

Subjects .......................................................................................................... 106

Stereotaxic surgery and tissue collection ....................................................... 106

Tissue collection and immunohistochemistry ................................................ 107

BDA microscope analysis .............................................................................. 108
Results .................................................................................................................. 108

Injection site ................................................................................................... 108

Projections to the lateral septum .................................................................... 109

Projections to the preoptic area and hypothalamus ........................................ 117

Projections to other regions of the BST ......................................................... l 17

vii

Projections to the amygdala ........................................................................... 119

Projections to mammillary bodies ................................................................. 119
Other projections ............................................................................................ 1 19
Overlap between BDA-labeled ﬁbers and TH-ir ﬁbers ................................. 120
Experiment 2: Retrograde analysis of TH-ir projections to the MPO, MeApd, and
pBST .......................................................................................................................... 122
Methods ................................................................................................................ 122
Subjects and stereotaxic surgery .................................................................... 122
Tissue collection and immunohistochemistry ................................................ 122
FG microscope analysis ................................................................................. 123
Results .................................................................................................................. 124
Injection site ................................................................................................... 124
F G injection into MP0 .................................................................................. 125
FG injection into pBST .................................................................................. 130
F G injection into MeApd ............................................................................... 132
Discussion .................................................................................................................. l 34
Methodological considerations ............................................................................ 134
Comparison of pBST projection in prairie voles and other rodents .................... 136
Implications of species-speciﬁc catecholaminergic network .............................. 138
Conclusions .......................................................................................................... 142
CHAPTER 5: Conclusions .............................................................................................. 143
Signiﬁcance of sex difference in TH-ir cells ............................................................. 143
Species comparison between TH-ir cells and others in pBST and MeApd .............. 144
Phenotype of TH-ir cells of the male prairie vole pBST and MeApd ....................... 148
Possible role of pBST and MeApd TH-ir cells in social motivation or ability .......... 150
Conclusions ................................................................................................................ l 54
REFERENCES ................................................................................................................ 155
viii

 

LIST OF TABLES

Table 3.1. The number of cells expressing TH and the number of dual-labeled cells
(Mean :I: SEM) in the pBST and MeApd after males interacted with one of several social
stimuli for 30 min. Groups with any of the same superscript letters do not signiﬁcantly
differ from each other ........................................................................................................ 73

Table 3.2. The number of cells expressing TH, Fos, both Fos and TH, and the percentage
of TH-ir cells also expressing Fos (Mean i SEM) after subjects interacted with one of
several social stimuli for 30 min. Groups with any of the same superscript letters do not
signiﬁcantly differ from each other. ................................................................................. 74

Table 3.3. Correlations between the percentage of TH-ir cells expressing Fos and the
expression of sexual behavior, paternal behavior, and all social behaviors during 30-min
interactions with a sexually receptive female, pups, or a familiar female sibling. See
Figure 3.4 for additional correlations associated with the duration of sexual behaviors...83

Table 3.4. The number of cells expressing TH, and the number of cells containing both
TH and Egr-l (Mean i SEM) in two sections through the pBST and three sections
through the MeApd after males interacted with one of several stimuli for 30 min. Groups
with any of the same superscript letters do not signiﬁcantly differ from each other ......... 84

Table 3.5. The number of cells expressing TH and the number of cells containing both
TH and Fos (Mean i SEM) in four sections through the pBST and seven sections through
the MeApd after males interacted with one of several stimuli for 6 or 24 h ..................... 90

Table 4.1. Estimated number of TH-ir neurons (i SEM) in regions throughout the

forebrain that project to the MPO, pBST, or MeApd in all subjects examined (also see
Figure 4.7). ...................................................................................................................... 126

ix

LIST OF FIGURES

Figure 1.1. Proposed neural network involved in pair bonding. Somatosensory input
from the penis and vagina likely projects to the periaqueductal gray (PAG) and nucleus of
the solitary tract (NTS), which in turn communicate with other areas of the forebrain. In
addition, vasopressin (AVP) release in the VP and LS, oxytocin (OT) release in the ACB
and medial amygdala (MeA), and dopamine (DA) in the ACB and prefrontal cortex
(PFC) are important for pair bonding. The location of cell bodies of these
neurotransmitters in prairie voles, as well as the connections with other brain sites, is
largely unknown. Figure adapted from Young et al., 2005 ................................................ 6

Figure 1.2. Depiction of dopamine receptor distribution in the nucleus accumbens in
naive and pair bonded male prairie voles. The upregulation of D1 receptors during pair
bond formation is thought to prevent the formation of additional pair bonds. Figure from
Edwards and Self, 2006. ..................................................................................................... 9

Figure 1.3. TH-ir cells in the pBST (left) and MeApd (right) of a male prairie vole. Scale
bar = 50 um ........................................................................................................................ 12

Figure 2.1. Photomicrographs of TH-ir cells in the pBST of representative prairie voles.
A: Male prairie vole at X 100 magniﬁcation. B: Male at X200 magniﬁcation. C: Female at
X200 magniﬁcation. LV, lateral ventricle; ac, anterior commissure. Scale bars = 100 um
inA;50p.minBandC ...................................................................................................... 34

Figure 2.2. Photomicrographs (X200 magniﬁcation) of TH-ir cells in the dorsal pBST of
representative male meadow voles, hamsters, and rats. Males of these species were
similar to females. Scale bars = 50 um .............................................................................. 35

Figure 2.3. Photomicrographs of TH-ir cells in the MeApd of representative prairie voles.
A: Male prairie vole at X 100 magniﬁcation. B: Male at X200 magniﬁcation. C: Female at
><200 magniﬁcation. opt, Optic tract. Scale bars = 100 pm in A; 50 pm in B,C ............... 37

Figure 2.4. Photomicrographs (X200 magniﬁcation) of TH-ir cells in the MeApd of
representative male meadow voles, hamsters, and rats. Males of these species were
similar to females. Scale bars = 50 um .............................................................................. 38

Figure 2.5. Number (Mean i SEM) of TH-ir cells in every other section of the dorsal
PBST of male and female prairie voles that were gonadally intact (Sham),
gonadectomized (GDX), or gonadectomized and implanted with a capsule ﬁlled with
testosterone (GDX + T). *Signiﬁcant difference between the sexes, collapsed across
treatment. or, GDX males signiﬁcantly different from other groups of males; [3, GDX + T
females signiﬁcantly different from other groups of females ............................................ 40

Figure 2.6. Photomicrographs (X200 magniﬁcation) of TH-ir cells in the dorsal pBST of
male (top) and female (bottom) prairie voles that were gonadally intact (Sham),

 

gonadectomized (GDX), or gonadectomized and implanted with a capsule ﬁlled with
testosterone (GDX + T). Scale bar = 50 pm ...................................................................... 41

Figure 2.7. Number (Mean :1: SEM) of TH-ir cells in every other section of the MeApd of
male and female prairie voles that were gonadally intact (Sham), gonadectomized
(GDX), or gonadectomized and implanted with a capsule ﬁlled with testosterone (GDX +
T). * Signiﬁcant difference between the sexes, collapsed across treatment. or, GDX males
signiﬁcantly different from other groups of males; B, GDX + T females signiﬁcantly
different from other groups of females .............................................................................. 43

Figure 2.8. Photomicrographs (X200 magniﬁcation) of TH-ir cells in the MeApd of male
(top) and female (bottom) prairie voles that were gonadally intact (Sham),
gonadectomized (GDX), or gonadectomized and implanted with a capsule ﬁlled with
testosterone (GDX + T). Scale bar = 50 um ...................................................................... 44

Figure 2.9. Number (mean i SEM) of TH-ir cells in every other section of the
dorsolateral thalamus of male and female prairie voles that were gonadally intact (Sham),
gonadectomized (GDX), or gonadectomized and implanted with a capsule ﬁlled with
testosterone (GDX + T) ..................................................................................................... 45

Figure 3.1. Number of Fos-ir cells (Mean i SEM) in four sections through the pBST and
seven sections through the MeApd after male subjects interacted with one of several
stimuli for 30 min. Bars with any of the same letters above them do not signiﬁcantly
differ from each other ........................................................................................................ 77

Figure 3.2. Percentage of TH-ir cells expressing Fos (Mean i SEM) in the pBST and
MeApd aﬁer male subjects interacted with one of several stimuli for 30 min. Bars with
any of the same letters above them do not signiﬁcantly differ from each other ................ 79

Figure 3.3. Photomicrographs of the pBST (top row) and MeApd (bottom row) from
representative males in the ALONE, NON EJAC, and EJAC groups in Experiment Ia.
Small arrows in top left panel indicate cells that are single-labeled with TH-
immunoreactivity (blue cytoplasmic label). Large arrows in top middle panel indicate
cells that are single-labeled with Fos-immunoreactivity (brown nuclear label). Dual-
labeled cells are highlighted at higher magniﬁcation in the two panels showing EJAC
males. Scale bar in bottom left panel = 50 um. Scale bar in bottom right panel inset = 10
um ...................................................................................................................................... 80

Figure 3.4. Correlations between the percentage of TH-ir cells in the pBST (left) or
MeApd (right) that also expressed Fos and the duration of time spent mounting and
thrusting by EJAC males (squares) and. NON EJAC males (circles) during a 30-min test
with a sexually receptive female. Time engaged in male sexual behavior (bottom row) is
the summed duration of mounting, thrusting, and ejaculating. See Table 3.3 for
correlations between other behaviors and the percentage of TH-ir cells that also expressed
Fos ...................................................................................................................................... 82

xi

Figure 3.5. Number of Egr-l-ir cells (Mean :1: SEM) in two sections of the pBST and
three sections through the MeApd after male subjects interacted with one of several
stimuli for 30 min. Bars with any of the same letters above them do not signiﬁcantly
differ from each other ........................................................................................................ 86

Figure 3.6. Percentage of TH-ir cells (Mean i SEM) in the pBST and MeApd that also
expressed Egr-l after male subjects interacted with one of several stimuli for 30 min.
Bars with any of the same letters above them do not signiﬁcantly differ from each other.

............................................................................................................................................ 88

Figure 3.7. Number of F os-ir cells (Mean :t SEM) in four sections through the pBST and
seven sections through the MeApd after male subjects mated, interacted with a familiar
female sibling, or remained alone for 6 or 24 h. Note that the Y-axis scales for both sites
are the same as that in Figure 3.]. Bars with any of the same letters above them do not
signiﬁcantly differ from each other ................................................................................... 91

Figure 3.8. Percentage of TH-ir cells also expressing Fos (Mean 1: SEM) in the pBST
and MeApd aﬁer male subjects mated, interacted with a familiar female sibling, or
remained alone for 6 or 24 h. Note that the Y-axis scales for both sites are the same as
that in Figure 3.2. Bars with any of the same letters above them do not signiﬁcantly
differ from each other ........................................................................................................ 93

Figure 4.1. Representation of BDA injection sites (gray shading) in the pBST for the
seven subjects (1-7) mapped onto atlas plate 21 from Swanson’s rat brain atlas (Swanson,
1998), and a photomicrograph of the BDA injection site (dark blue staining on right)
compared to TH-ir cell distribution (brown staining on left) of subject 1 (la) ............... 110

Figure 4.2. Relative distribution of BDA-labeled ﬁbers throughout the forebrain in one
representative subject receiving a BDA injection into the pBST, mapped onto plates 13-
37 (A-Y) of Swanson’s rat brain atlas (Swanson, 1998) ................................................. 111

Figure 4.3. Photomicrograph showing BDA-labeled ﬁbers (dark blue staining) and TH-ir
(brown staining) in the MPO (A) and MeApd (B). Both regions received dense
innervation from the BST that overlapped with the distribution of TH-ir ﬁbers ............. 118

Figure 4.4. Photomicrograph showing BDA-labeled ﬁbers (dark blue staining) and TH-ir
(brown staining) in the ACB (A) and LS (B). The ACB contained a dense plexus of TH-
ir ﬁbers, but few BDA-labeled ﬁbers. In the LS, BDA-labeled ﬁbers were primarily
found in the LSv and LSr subregions, and did not overlap with TH-ir ﬁbers in the LSc
(B) .................................................................................................................................... 121

Figure 4.5. Representation of the center of PG injection sites (gray shading) in the MPO
for the ﬁve subjects mapped onto plates 20 (1-4) and 21 (5) of Swanson’s rat brain atlas
(Swanson, 1998), and a photomicrograph of the PG injection site (brown staining on
right) in subject 1 (la). TH-ir cells and ﬁbers (blue staining) can also be seen on the left.

......................................................................................................................................... 127

xii

Figure 4.6. Photomicrographs of TH-ir (blue labeling) and FG-ir (brown labeling) cells
and ﬁbers in the pBST (panels A, B) and MeApd (panels C, D) in voles with F G injected
into the MPO (scale bars = 20 or 100 pm). Some double-labeled cells are indicated by
arrows. Photomicrographs of TH-ir cells in the pBST after FG injection in the MeApd
(panels E, F), and TH-ir cells in the MeApd after FG injection in pBST (panels G, H)
(scale bars = 20 or 100 um). Note the lack of double-labeled cells in panels E and
F ....................................................................................................................................... 128

Figure 4.7. Diagram of TH-ir cells throughout the forebrain and midbrain that project to
the MPO, pBST, and MeApd. Different arrow sizes represent the number of TH-ir cells
connecting the two regions (see Table 4.1) .................................................................... 129

Figure 4.8. Representation of PG injection sites in the pBST (gray shading) for the six
subjects (1-6) mapped onto plate 21 of Swanson’s rat brain atlas (Swanson, 1998) ....... 131

Figure 4.9. Photomicrograph of the F G injection site in the pBST of subject 4 (Figure 6)
showing FG-labeled cells (brown staining) and TH-ir cell and ﬁber distribution (blue
staining) ............................................................................................................................ 131

Figure 4.10. Representation of MeA and surrounding regions at plates 28 (A), 29 (B), and
30 (C) of Swanson’s brain atlas (Swanson, 1998), and F G injection sites in the MeApd
that were centered on corresponding plates (1, 2, 3, 4). 2a and 4a are photomicrographs
of the PG injection site (brown staining on right) in subjects 2 and 4. TH-ir cells and
ﬁbers (blue staining) can also be seen on the leﬁ ............................................................ 133

Figure 5.1. Hypothetical representation of neural activity in Newman’s “integrated social
behavior circuit” during male sexual behavior. Higher peaks indicate greater neuronal
activity in that brain site. Figure from Newman, 1999 ................................................... 152

Images in this dissertation are presented in color.

xiii

ACB

3C0

adBST

ADP

AHA

AHN

alBST

ARH

AV

avBST

AVP

AVPV

BDA

BMA

BST

ccBST

CEA

COA

DA

DAB

dBST

dlBST

LIST OF ABBREVIATIONS
nucleus accumbens
anterior commissure, olfactory limb
bed nuclei of the stria terminalis, anterior division, anterodorsal area
anterodorsal preoptic nucleus
anterior hypothalamic area
anterior hypothalamic nucleus
bed nuclei of the stria terminalis, anterior division, anterolateral area
arcuate nucleus hypothalamus
anteroventral nucleus thalamus
bed nuclei of the stria terminalis, anterior division, anteroventral area
anteroventral preoptic nucleus
anteroventral periventricular nucleus
biotinylated dextran amine
basomedial nucleus amygdala
bed nuclei of the stria terrninalis

bed nuclei of the stria terminalis, anterior division, anterodorsal area,
central core

central nucleus amygdala

cortical nucleus amygdala

dopamine

3,3’-diaminobenzidine

bed nuclei of the stn'a terminalis, posterior division, dorsal nucleus

bed nuclei of the stria terminalis, anterior division, dorsolateral nucleus

xiv

deST
DMH
ENT
FG

ﬁ

fuBST

IEG

ifBST

juBST
LH
LHA
LM
LPO
LS
LSc
LSc.d
LSc.v
LSr
LSr.d
LSr.v

LSv

bed nuclei of the stria terminalis, anterior division, dorsomedial nucleus
dorsomedial nucleus hypothalamus

entorhinal area

F luoroGold

ﬁmbria

bed nuclei of the stria terminalis, anterior division, fusiforrn nucleus
columns of the fomix

immediate-early gene

bed nuclei of the stria terminalis, posterior division, interfascicular nucleus
immunoreactive

bed nuclei of the stria terminalis, anterior division, juxtacapsular nucleus
lateral habenula

lateral hypothalamic area

lateral mammillary nucleus

lateral preoptic area

lateral septal nucleus

lateral septal nucleus, caudal part

lateral septal nucleus, caudal part, dorsal zone

lateral septal nucleus, caudal part, ventral zone

lateral septal nucleus, rostral part

lateral septal nucleus, rostral part, dorsal zone

lateral septal nucleus, rostral part, ventral zone

lateral septal nucleus, ventral part

XV

‘.. t-‘
l‘ '...
i

.5 n...

J.

MA
MD
MeA
MeAad
MeAav
MeApd
MeApv
MEPO
mgBST
MH
MM
MPN
MPO
NaPB
NDB
NGS
opt
ovBST
PA
PAG
pBST
PH

PM

magnocellular preoptic nucleus

mediodorsal nucleus thalamus

medial nucleus amygdala

medial nucleus amygdala, anterodorsal part

medial nucleus amygdala, anteroventral part

medial nucleus amygdala, posterodorsal part

medial nucleus amygdala, posteroventral part

median preoptic nucleus

bed nuclei of the stria terminalis, anterior division, magnocellular nucleus
medial habenula

medial mammillary nucleus

medial preoptic nucleus

medial preoptic area

sodium phosphate buffer

nucleus of the diagonal band

normal goat serum

optic tract

bed nuclei of the stria terminalis, anterior division, oval nucleus
posterior nucleus amygdala

periaqueductal gray

bed nuclei of the stria terminalis, posterior division, principal nucleus
posterior hypothalamic nucleus

premammillary nucleus

xvi

PS

PSCH

PV

PVH

PVT

RCH

thST

RT

SBPV

scBST

seBST

SF

SI

sm

SN

81

SUM

TBS

TEP

TH

TMV

trBST

TRS

parastrial nucleus

suprachiasmatic preoptic nucleus

periventricular nucleus hypothalamus

paraventricular nucleus hypothalamus

paraventricular nucleus thalamus

retrochiasmatic area

bed nuclei of the stria terrninalis, anterior division, rhomboid nucleus
reticular nucleus thalamus

subparaventricular zone hypothalamus

bed nuclei of the stria terminalis, anterior division, subcommissural zone
bed nuclei of the stria terminalis, posterior division, stria extension
septoﬁmbrial nucleus

substantia innominata

stria medullaris

substantia nigra

stria terrninalis

supramammillary nucleus

tris-buffered saline

temporal pole

tyrosine hydroxylase

tuberomammillary nucleus, ventral part

bed nuclei of the stria terminalis, posterior division, transverse nucleus

triangular nucleus septum

xvii

TU tuberal nucleus

V3 third ventricle

vBST bed nuclei of the stria terminalis, anterior division, ventral nucleus
VL lateral ventricle

VMH ventromedial nucleus hypothalamus

VP ventral pallidum

VTA ventral tegmental area

21 zona limita

xviii

 

1h

 

 

h 1

vlltc

CHAPTER 1:

Introduction

Monogamy is deﬁned as “a prolonged association and essentially exclusive
mating relationship between one male and one female,” and often occurs when breeding
opportunities are limited and offspring beneﬁt from biparental care (Wittenberger and
Tilson, 1980). Mating exclusivity is difﬁcult to determine, and individuals of “socially
monogamous” species do occasionally mate with conspeciﬁcs other than their partner
(Wittenberger and Tilson, 1980). In fact, in some monogamous species one litter can
contain pups from different fathers (Borkowska et al., 2009; Glenn et al., 2009; Goossens
et al., 1998; Solomon et al., 2004). Therefore, Kleiman (1977) has described four
characteristics that are often used to recognize monogamous species: 1) members of the
pair remain in close proximity during breeding and nonbreeding periods, 2) members of
the pair prefer to mate with their partner as opposed to an unfamiliar opposite-sex
conspeciﬁc, 3) unrelated conspeciﬁcs are rarely or never found in the pair’s territory, and
4) only one pair in a family group breeds. Most bird species are monogamous (Lack,
1968), but monogamy is rare in mammals, ﬁsh, reptiles, and amphibians (Bull, 2000;
Kleiman, 1977; Whiteman and Cote, 2004; Wittenberger and Tilson, 1980).

Monogamy can be further divided into two types, facultative (Type I) and
obligate (Type II) (Kleiman, 1977). Facultative monogamy occurs only at times when
the population density is so low that only one opposite-sex conspeciﬁc is available, and
in this type of monogamy males typically do not directly provide care to their offspring.

In contrast, obligately monogamous species require male participation in rearing young.

Obligate monogamy often co-occurs with cooperative breeding, a social structure in
which animals live in groups and some members take care of young that are not their own
(Bergmuller et al., 2007; Jennions and MacDonald, 1994). In obligately monogamous
family groups, adult and juvenile offspring of the breeding pair will typically help raise
younger litters, and often forego their own breeding opportunities (Carter and Roberts,
1997). Cooperatively breeding species usually live in environments with ecological
constraints that allow some members of the group to have greater reproductive success by
caring for other members of the group than breeding independently, such as low
availability of mates, high predator density that make dispersal risky, or poor quality of
breeding opportunities (Komdeur et al., 2008; Solomon and Getz, 1997).

The brains of obligately monogamous and cooperatively breeding species are
surely different than those of nonmonogamous species because their social behaviors
drastically differ. Neural networks must exist that allow breeding individuals to form
bonds with their partner, sires to care for their pups, nonbreeding individuals to display
alloparental behavior towards offspring that are not their own, and all individuals to show
gregarious behaviors necessary to live in a large social group. Prairie voles (Microtus
ochrogaster) have become a popular rodent model for investigating the neurobiology of
complex sociosexual behaviors more typical of humans and other primates because
prairie voles are obligately monogamous cooparative breeders that form lifelong pair
bonds after mating and engage in biparental care (Carter et al., 1995; Getz and Hoffman,
1986). Investigations of the prairie vole brain have revealed that it contains some neural
networks not present in non-monogamous rodents, and many Of these networks inﬂuence

social motivation.

 

Hp

The prairie vole model

Prairie voles are highly gregarious, both in the wild and in captivity, making them
a valuable model for laboratory and ﬁeld research (Carter et al., 1995; Getz and Hoffman,
1986). As cooperative breeders, they are often found in large social groups composed of
a reproductively active male and female, and several generations of their offspring (Getz
and Carter, 1980; Getz and Hofmann, 1986; Getz et al., 1993). In fact, as many as 70%
of males and females remain in the natal nest for life and forego opportunities for
reproduction (McGuire et al., 1993). Although many voles are in close contact with their
opposite-sex relatives throughout their lives, incest is prevented because females avoid
sniffing the anogenital region of their male relatives and the absence of exposure to these
male olfactory cues prevents the induction of estrus (Carter et al., 1980, 1987b, 1989;
Gavish et al., 1984). The prairie voles that do disperse from the nest become
reproductively active, and many of these animals form life-long pair bonds after mating,
as indicated by ﬁeld studies in which the same two voles were repeatedly captured in the
nest together until one or both members of the pair died (Getz and Hofmann, 1986; Getz
et al., 1993). Even after the death of a partner, only 10% of survivors form a new pair
bond (Getz et al., 1981). In the laboratory, pair bonding is inferred when a vole forms a
“partner preference” (e. g. Williams et al., 1992b). After a period of cohabitation with an
opposite-sex “partner,” the subject (either male or female) is placed in a three-chambered
apparatus with the partner tethered in one chamber and an unfamiliar opposite-sex
“stranger” tethered to another chamber. Subjects have access to all three chambers, and
prairie voles that have formed a partner preference spend signiﬁcantly more time in

contact with their partner than with the stranger. Mating with the partner prior to the test

facilitates the formation of partner preferences, particularly in males, but extended
cohabitation without mating is sometimes sufﬁcient for partner preference formation
(Insel et al., 1995; Williams et al., 1992a). Once the pair bond has formed, both males
and females are aggressive toward intruders, which may limit contact with unfamiliar
adults and help maintain the pair bond (Getz et al., 1981; Gobrogge et al., 2007, 2009).
Even in the laboratory, juvenile prairie voles are highly parental and often provide
care towards younger siblings (Lonstein and De Vries, 2001; Olazabal and Young, 2005;
Roberts et al., 1998; Wang and Novak, 1994). Interestingly, when males and females are
weaned from their natal cage before they are exposed to subsequent litters (and
presumably when they emigrate from the nest in the wild), adult virgin males are highly
paternal but most virgin females ignore or attack pups (Hayes and De Vries, 2007;
Lonstein and De Vries, 1999b, 2001; Olazabal and Young, 2005; Williams et al., 1992c).
However, after parturition, both sexes provide parental care, and males show the same
complement of parental behaviors as females, with the obvious exception of nursing
(Lonstein and De Vries, 1999a; McGuire and Novak, 1984; Oliveras and Novak, 1986).
Paternal care does inﬂuence survival of the offspring; female prairie voles are more likely
to maintain a pregnancy when their partners are present (Dewsbury, 1994; McGuire et al.,
1992), and litters develop more rapidly in the presence of the father (Wang and Novak,
1992). Thus, male prairie voles gain more reproductive success if they remain with their

mate, and this is likely one of the reasons that monogamy evolved in this species.

The neurobiology of prairie vole sociosexual behaviors

Studies examining the neurobiology of pair bonding and parental behavior in
prairie voles have highlighted the importance of several neurotransmitters, most notably
dopamine (DA), vasopressin (primarily in males), and oxytocin (primarily in females), in
the regulation of social behaviors (Wang and Aragona, 2004; Young et al., 2001, 2005,
2008). These neurotransmitters interact with one another to regulate pair bonding (Cho et
al., 1999; Lim et al., 2004c; Liu et al., 2001, 2003), and Young and colleagues (1998,
2001) have proposed that DA release during mating interacts with vasopressin or
oxytocin release from olfactory regions to allow animals to associate the rewarding
aspects of mating with the characteristics of that particular mate. In males, research has
focused on the release of DA in the nucleus accumbens (ACB) and vasopressin in the
ventral pallidum (VP) and lateral septum (LS) (Young et al., 2008), but little is known
about the neurons providing this input, or other areas of the brain that may also inﬂuence
social behaviors (see Figure 1.1). DA, vasopressin, and oxytocin are also important for
the expression of parental behaviors (Lim et al., 2004c; Lonstein, 2002; Olazabal and

Young, 2006; Wang et al., 1994a), but the neural networks involved are unclear.

 

Figure 1.1. Proposed neural network involved in pair bonding. Somatosensory input
from the penis and vagina likely projects to the periaqueductal gray (PAG) and nucleus of
the solitary tract (NTS), which in turn communicate with other areas of the forebrain. In
addition, vasopressin (AVP) release in the VP and LS, oxytocin (OT) release in the ACB
and medial amygdala (MeA), and dopamine (DA) in the ACB and prefrontal cortex
(PFC) are important for pair bonding. The location of cell bodies of these
neurotransmitters in prairie voles, as well as the connections with other brain sites, is
largely unknown. Figure adapted from Young et al., 2005.

The Microtus genus consists of a number of highly social species, such as the
prairie vole, but also many species that are neither monogamous nor biparental. This
diversity in social strategy has allowed a number of comparisons of neurotransmitter and
receptor expression among monogamous and nonmonogamous species to better
understand the neurobiology of social behaviors. These studies have revealed complex
species differences in the expression of vasopressin (Wang, 1995), vasopressin receptors
(Insel et al., 1994; Young et al., 1997), DA receptors (Aragona et al., 2006; Smeltzer et
al., 2006), oxytocin receptors (Insel and Shapiro, 1992; Olazabal and Young, 2006;
Smeltzer et al., 2006), corticotropin-releasing factor receptors (Lim et al., 2005), and

cocaine- and amphetamine-regulated transcript (Hunter et al., 2005) that may be related

to social structure. All of these species differences probably contribute to differences in

 

 

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or.“ [I

behavior, but interspecies variations in vasopressin receptor density have been the focus
of most research to date. Indeed, blocking vasopressin receptors in the ventral pallidum
(VP), a brain area with many more vasopressin receptors in social voles compared to
non-social voles, inhibits partner preference formation in male prairie voles (Lim and
Young, 2004). Interestingly, increasing the expression of vasopressin receptors in the VP
promotes social interactions in relatively non-social meadow voles, rats, and mice, and
can even facilitate partner preference formation in some animals (Landgraf et al., 2003;
Lim et al., 2004c, Young et al., 1999). These drastic changes in social interactions are
remarkable, but the complete repertoire of gregarious and monogamous behaviors typical
of prairie voles are not induced after these manipulations. In addition, although
differences in microsatellite length in the promoter region of the vasopressin Vla
receptor gene have been found to underlie species differences in receptor expression
(Hammock et al., 2004), Fink et al. (2006) reported that microsatellite length and Vla
receptor expression cannot completely explain differences in social behavior across the
Microtus genus. Furthermore, microsatellite length does not reﬂect the mating strategy
used by individual prairie voles (Ophir et al., 2008a, b). There are likely a number of
neural systems contributing to the expression of social behavior, and some of these have
yet to be discovered.

Species differences in DAergic systems have also been reported, and may
contribute to differences in social behavior. For example, male meadow voles have a
greater density of D1 DA receptors in the ACB and prefrontal cortex (PFC) than do male
prairie voles, while prairie voles have more D2 receptors in the PFC than do meadow

voles (Aragona et al., 2006; Smeltzer et al., 2006). Furthermore, while baseline

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extracellular DA levels are equivalent, male prairie voles have a greater increase in DA
release in the ACB after peripheral amphetamine adminstration than do meadow voles
(Curtis and Wang, 2007). This species difference in response to amphetamine could be
due to differences in numbers of DA transporters or DA autoreceptors on presynaptic
terminals in the ACB. The behavioral effects of these species differences are unknown,
but may contribute to variations in social structure given the role of DA in pair bonding

and parental behavior (see below).

Dopamine and sociosexual behaviors

DA, synthesized from tyrosine via the rate-limiting enzyme tyrosine hydroxylase
(TH) and L-amino acid decarboxylase, was one of the ﬁrst neurotransmitters proposed to
inﬂuence prairie vole social behaviors, and its release is necessary for the formation and
maintenance of pair bonds in both sexes of prairie voles. DA release in the ACB
increases during mating, and peripheral administration of the DA antagonist haloperidol
eliminates partner preference formation (Aragona et al., 2003). Furthermore, partner
preference formation can be elicited in the absence of mating if a D2 DA receptor agonist
is injected into the ACB (Aragona et al., 2006; Gingrich et al., 2000). However,
activation of D1 receptors in the ACB prevents partner preference formation, and an
upregulation of D1 receptors after pair bond formation is thought to ensure the stability of
the initial pair bond (see Figure 1.2, Aragona et al., 2006). Although DAergic projections
to the ACB necessary for DA’s facilitation of pair bonding are assumed to come from the

ventral tegmental area (VTA) (Curtis and Wang, 2005), it is likely that DA originating

from multiple brain sites might be important (Dominguez and Hull, 2005; Melis and

Argiolas, 1995; Miller and Lonstein, 2005).

a Bachelor vole D Bonded vole

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Nucleus accumbens Nucleus accumbens
neurons neurons

Figure 1.2. Depiction of DA receptor distribution in the nucleus accumbens in naive and
pair bonded male prairie voles. The upregulation of D1 receptors during pair bond
formation is thought to prevent the formation of additional pair bonds. Figure from
Edwards and Self, 2006.

DA neurotransmission is also necessary for the typical expression of parental
behavior in prairie voles. Systemically administered haloperidol increases the latency for
males to contact pups, decreases the amount of time that male and female prairie voles
spend licking their pups, and decreases the amount of time spent actively hovering over
pups without affecting motor systems (Lonstein, 2002). However, investigations of the
speciﬁc DAergic systems responsible for parental behaviors in prairie voles have not
been carried out. In rats, DA release (measured by microdialysis) increases in the ACB

after lactating females interact with pups (Champagne et al., 2004; Hansen et al., 1993),

and is also higher in the medial preoptic area (MPO) during lactation compared to the end

of pregnancy (Lonstein et al., 2003). D1 agonists injected into either the ACB or the
MPO decrease the time needed for females to show maternal behavior (Stolzenberg et al.,
2007). Conversely, selective lesions of DAergic terminals in the ACB or local injections
of DA antagonists into the ACB or MP0 disrupt maternal behaviors (Hansen et al.,
1991a, b, 1994; Keer and Stern, 1999; Miller and Lonstein, 2005; Numan et al., 2005).
Similar to the divergent role of D1 and D2 receptors in pair bonding, these receptors have
different functions in maternal behavior, at least in the MPO, as D1 receptor activation
stimulates retrieving and licking while D2 receptor activation inhibits quiescent nursing
behaviors (Miller and Lonstein, 2005). This relevant DA release likely arises from cell
bodies in the VTA and hypothalamus (Champagne et al., 2004; Keer and Stern, 1999;
Miller and Lonstein, 2005, 2009; Silva et al., 2003; Stolzenberg et al., 2007). Similar
mechanisms may be responsible for the expression of maternal and paternal behaviors in
prairie voles.

DA neurotransmission is also critical for male sexual behavior in at least some
rodents. In male rats, DA release in the MPO increases when males are exposed to
estrous females and during copulation (Dominguez et al., 2001; Hull et al., 1995), and
microinjections of DA agonists increase intromissions and ejaculations (Hull et al.,
1986), while microinjections of DA antagonists decrease ejaculations and penile reﬂexes
(Pehek et al., 1988; Warner et al., 1991). Interestingly, activity in the medial amygdala
(MeA) resulting from olfactory and other sensory inputs from females enhances DA
release in the MP0 to facilitate masculine copulatory behaviors (Dominguez and Hull,
2001). DAergic cells are not found in the rat MeA, but glutamatergic neurons in the

MeA synapse on terminals of DAergic cells coming from other regions, such as the

10

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periventricular hypothalamus or posterior hypothalamus (Miller and Lonstein, 2009;
Moore and Lookingland, 1995), to increase DA release (Dominguez and Hull, 2005).
DA release is also required in additional areas, including the dorsal striatum and ACB,
for the normal expression of copulatory behaviors in male rats (Melis and Argiolas,
1995). Interestingly, DA may be one of the mediators of hormonal effects on male
sexual behavior, as castration eliminates increases in DA release in the MPO (Hull et al.,
1995), and DA agonists can restore sexual behaviors in castrated rats or mice lacking the
estrogen receptor a gene (Malmnas, 1977; Scaletta and Hull, 1990; Wersinger and
Rissman, 2000).

DA’s role in sexual behavior in male prairie voles is less clear, as initial
investigations surprisingly found that DA may not be necessary. DA release is enhanced
in some brain areas, including the ACB, during mating in both male and female prairie
voles (Aragona et al., 2003; Curtis et al., 2003). However, Aragona and colleagues
(2003) reported no reduction in the number of mating bouts in males given systemic
haloperidol. The details of mating interactions resulting from this manipulation were not
reported, however, and it is possible that DA may be required for some aspects of their
mating, as it is for male and female rats.

Recently, during an investigation of DAergic systems that may be important for
the expression of parental behavior in prairie voles (Lansing and Lonstein, 2006), the
Lonstein laboratory discovered groups of neurons in the principal nucleus of the bed
nucleus of the stria terrninalis (pBST) and posterodorsal medial amygdala (MeApd) that
contained TH irmnunoreactivity (Figure 1.3; Northcutt et al., 2007). TH-immunoreactive

(TH-ir) cells were particularly numerous in males. Interestingly, large numbers of TH-ir

11

cells had never before been described in the pBST or MeApd of other vertebrates that
have been studied (Albanese et al., 1986; Hokfelt et al., 1984; Kalsbeek et al., 1992;
Smeets and Gonzalez, 2000), and may be part of neural networks inﬂuencing social
behaviors. Indeed, the BST and MeA process chemosensory and hormonal information,
have widespread projections throughout the forebrain (including extensive projections to
one another), and regulate sociosexual behaviors in mammals (Coolen and Wood, 1998;

Kling, 1972; Newman, 1999; Numan and Insel, 2003; Wood and Swarm, 2005).

 

 

 

 

Figure 1.3. TH-ir cells in the pBST (left) and MeApd (right) of a male prairie vole. Scale
bar = 50 pm.

TH-expressing neurons have never been described in the rat pBST or MeApd, but
similar groups may exist in male hamsters. Wommack et al. (2002) found TH-ir cells in
the pBST and MeApd of male hamsters, especially under conditions of social stress, but
in very low numbers (~10—1l cells per region). Many more TH-expressing cells are seen
in the pBST and MeApd in hamsters after axonal transport is inhibited by colchicine
(Asmus et al., 1992; Asmus and Newman, 1993), but the need for colchicine indicates

either that prairie voles have higher TH levels than hamsters under normal conditions or

that axonal transport of TH is different between the two species. Similar to previously-
discovered differences in neurotransmitter or receptor distribution, this species difference
in TH-immunoreactivity may also reﬂect differences in behavior, and the lack of large
numbers of TH-ir cells in rats and hamsters may partially explain why they do not engage
in high levels of afﬁliative behaviors, pair bonding, and paternal behavior. Furthermore,
these cell groups may contribute to DA release necessary for pair bonding and paternal
behavior, and may interact with other neurotransmitters known to be involved in

facilitating social interactions.

The olfactory system and sociosexual behaviors

The BST and MeA, as well as olfactory areas projecting to these regions, are
already implicated in regulating sociosexual behaviors in prairie voles and other rodents.
Lesions of the olfactory bulbs prevent partner preference formation in females and reduce
general afﬁliative behavior in male prairie voles (Curtis et al., 2001; Kirkpatrick et al.,
1994c). Furthermore, the expression of Fos, an immediate-early gene (IEG) product,
increases in the BST and MeA after male prairie voles cohabitate with a female, which
indicates that these areas may play a role in consolidating partner preferences (Cushing et
al., 2003; Lim and Young, 2004). The MeA may also be involved in pair bond formation
in primates, as the MeA of pair bonded titi monkey males shows greater glucose uptake
in PET scans than that of lone males (Bales et al., 2007). However, the speciﬁc role of
the BST and MeA in pair bond formation or maintenance in prairie voles or other

monogamous species is not known.

13

The BST and MeA are also involved in the expression of paternal behavior in
prairie voles. Lesions of the olfactory bulbs or the MeA decrease paternal behavior
(Kirkpatrick et al., 1994a, c), and Fos expression increases in the BST and MeA of male
prairie voles after exposure to pups (Kirkpatrick et al., 1994b). However, olfactory bulb
lesions have no effect on maternal behavior in females (Williams et al., 1992c),
suggesting that olfactory information is more important for promoting paternal behavior
than maternal behavior in prairie voles.

Although there is limited information about the function of the BST and MeA in
pair bonding and parental behavior, these regions clearly inﬂuence sexual behavior in
prairie voles and other rodents. Lesions of the main and accessory olfactory bulbs
prevent estrus induction in female prairie voles and eliminate mating in males (Curtis et
al., 2001; Kirkpatrick et al., 1994c; Lepri and Wysocki, 1987). Additionally, Fos
expression increases in the BST and MeA after male prairie voles cohabitate or mate with.
a female (Cushing et al., 2003; Lim and Young, 2004). The roles of subregions of the
prairie vole BST and MeA are unknown, but the pBST and MeApd are especially
important for chemosensory investigation of females and ejaculation in male rats
(Bressler and Baum, 1996; Coolen et al., 1997, 1998).

To date, research on BST and MeA neurochemistry important for prairie vole
sociosexual behaviors has focused on vasopressin projections originating in these
regions. Males have more vasopressinergic cells in the BST and MeA than do female
prairie voles, and more vasopressin-1r ﬁbers, presumably from BST and MeA cells, in the
lateral septum (LS) and lateral habenula (Barnshad et al., 1993; Lonstein and De Vries,

1999b; Wang, 1995). The ﬁrst idea that the extra-hypothalamic vasopressin system plays

14

a role in sociosexual behaviors in prairie voles came from evidence that microinjections
of vasopressin into the LS facilitate paternal behavior (Wang et al., 1994a). Furthermore,
injection of a Vla vasopressin receptor antagonist into the LS or VP blocks the formation
of partner preferences (Lim and Young, 2004; Liu et al., 2001), while injection into the
LS or MeA interferes with paternal behavior (Lim et al., 2004c; Wang et al., 1994a).
Finally, over-expression of Vla receptors in the VP facilitates partner preference
formation in male prairie vole and in the typically non-monogamous male meadow vole
(Lim et al., 2004c; Pitkow et al., 2001). Thus, the BST and MeA probably inﬂuence pair
bonding and paternal behaviors by regulating vasopressin production and release, but
there are likely other neurochemicals, such as DA, in these regions that regulate species-

speciﬁc social behaviors in prairie voles.

Hormone regulation of the pBS T and MeApd

The apparent sex difference in TH expression in the prairie vole pBST and
MeApd is not particularly surprising because pBST and MeApd morphology and
neurochemistry are sexually dimorphic in many rodents (Cooke et al., 1999; De Vries
and Miller, 1998; Hines et al., 1992; Malsbury and McKay, 1987, 1989; Micevych et al.,
1988). In rats and mice, males have a greater overall pBST and MeApd volume in both
regions, and larger neuronal soma sizes in the MeApd (Cooke et al., 1999; Durazzo et al.,
2007; Hines et al., 1992; Morris et al., 2005, 2008). Astrocyte number and complexity
are also sexually dimorphic in the MeApd (Johnson et al., 2008), as is the expression of
numerous neurotransmitters in both sites (De Vries et al., 1984, 1985; Malsbury and

McKay, 1994; Simerly and Swanson, 1987; Swann and Newman, 1992). Most sex

15

differences in anatomy, physiology, and behavior arise due to sex differences in gonadal
hormone levels in adulthood or during a critical period in development (usually the
perinatal period or puberty) (e. g. De Vries and Simerly, 2002), and the pBST and MeApd
seem exquisitely sensitive to circulating hormone levels in adulthood (Cooke et al., 1999,
2003; De Vries et al., 1984, 1985; Malsbury and McKay, 1994; Morris et al., 2008;
Simerly and Swanson, 1987; Swarm and Newman, 1992).

Sex differences in pBST and MeApd morphology and neurochemistry are thought
to regulate many aspects of sociosexual behaviors in rodents, including mating and odor
preferences. Positive correlations between neuronal soma size in the MeApd and
measures of sexual arousal in castrated and hormone-treated male rats suggest that
hormone-mediated changes in the morphology of MeApd cells may result in changes in
sexual behaviors (Cooke et al., 2003). Additionally, testosterone or estradiol implants in
the pBST or MeApd are able to reverse castration-induced declines in male sex behavior
(Huddleston et al., 2003; Rasia—Filho et al., 1991; Wood and Newman, 1995b, c), and
estradiol implants in the MeA induce receptive sexual behaviors in some female rats and
hamsters (Lisk and Barﬁeld, 1975; Sterner et al., 1992).

Adult gonadal steroids also alter the neurochemistry of the prairie vole BST and
MeA, which may change the expression of sociosexual behaviors. The number of
vasopressinergic cells in the BST and MeA and the number of vasopressinergic ﬁbers in
the areas they innervate dramatically declines after castration, but not if testosterone
replacement is given (Lonstein and De Vries, 1999b; Wang and De Vries, 1993).

Furthermore, vasopressinergic ﬁber density increases in females given estradiol (Lonstein

l6

and De Vries, 1999b). Thus, similar to other rodents, the prairie vole BST and MeA are

sensitive to hormones in adulthood.

Hormonal inﬂuences on sociosexual behaviors in prairie voles

Hormone levels also inﬂuence the display of sociosexual behaviors in prairie
voles. Similar to other rodents, the ability to copulate is under gonadal hormone control
in male and female prairie voles (Carter et al., 1987a, b, 1988; Cushing et al., 2004;
Dluzen and Carter, 1979). The role of gonadal hormones in the post-copulatory
formation of pair bonds has not yet been investigated, but other steroid hormones, such as
glucocorticoids, modulate partner preference formation (DeVries et al., 1995, 1996a, b).
Finally, gonadal hormones also inﬂuence parental behaviors in prairie voles; the effects
of castration on paternal behavior are controversial (Lonstein and De Vries, 1999b; Wang
and De Vries, 1993), but virgin females given testosterone or estradiol in adulthood show
much higher levels of parental behavior than untreated females (Lonstein and De Vries,
1999b; Lonstein et al., 2002). These hormone-mediated changes in sociosexual
behaviors may result from changes in morphology or neurochemistry of cells in the pBST
or MeApd, as these regions also contain many androgen and estrogen receptors in prairie
voles (Cavanaugh and Lonstein, 2010a; Cushing et al., 2004; Hnatczuk et al., 1994;
Northcutt and Lonstein, 2008). Indeed, high levels of vasopressin immunoreactivity in
the BST and MeA are only seen when testosterone levels are high (Bamshad et al., 1993,
1994; Wang and De Vries, 1993; Wang et al., 1994b), which may affect paternal
behavior (Wang and De Vries, 1993; but see Lonstein and De Vries, 1999b). Other

neurochemicals regulated by hormones in adult prairie voles, such as DA (Lansing and

17

Lonstein, 2006), may also contribute to alterations in the expression of sociosexual
behaviors.

Although most research to date has examined neurochemistry or behavior after
drastic hormone manipulations, gonadal hormones do, indeed, ﬂuctuate in adult male and
female prairie voles. Female prairie voles are induced ovulators and, in the absence of an
unfamiliar male, their ovaries are quiescent and release very low amounts of estradiol and
progesterone (Carter et al., 1989). Within 48 hours of male exposure, serum estrogen
levels increase to about ﬁve times that of unexposed females (Cohen-Parsons and Carter,
1987), and progesterone levels rise dramatically after about 72 hours of mating (Carter et
al., 1989). In contrast, the testes secrete signiﬁcant amounts of testosterone even in the
absence of unfamiliar females (Wang et al., 1994b), but testosterone levels may slightly
increase after initial exposure to a female and after a male’s partner gives birth and
undergoes a postpartum estrus (Bamshad et al., 1994; Gaines et al., 1985; Wang et al.,
1994b). These natural changes in hormone levels likely affect neurochemistry and

sociosexual behaviors across different reproductive states.

Overview of dissertation chapters

Groups of TH-ir cells in the pBST and MeApd have never before been described
in other vertebrates, and are likely to inﬂuence the monogamous behaviors of prairie
voles, given the role of DA in pair bonding and parental behavior and the importance of
the pBST and MeApd in sociosexual behaviors in all mammals. The projects in this
dissertation further explore whether these cells could be speciﬁc to prairie voles and

characterize the hormone regulation, IEG responses, and neuroanatomical projections of

18

TH-ir cells in the prairie vole pBST and MeApd. Chapter 2 describes species differences
in TH-ir expression in these regions among prairie voles and three non-monogamous
rodents, as well as sex differences and adult gonadal hormone regulation of these cell
groups in prairie voles. Chapter 3 investigates possible functions of these TH-ir cell
groups in male prairie voles by examining IEG expression after one of several social
interactions. Finally, Chapter 4 details the anatomical projections of TH-ir cells in the
pBST and MeApd, and proposes ways that these neurons may interact with cells of other
phenotypes to facilitate prairie vole social behaviors. These experiments provide insight
into the possible role of these species-speciﬁc TH—ir cells and expand the understanding

of how they may be integrated within other social behavior networks in the brain.

19

CHAPTER 2

Northcutt, K. V., Wang, Z., and Lonstein, J. S. (2007). Sex and species differences in
tyrosine hydroxylase-synthesizing cells of the rodent olfactory extended
amygdala. J Comp Neurol 500(1), 103-115.

20

CHAPTER 2:
Sex and species differences in tyrosine hydroxylase-synthesizing cells of the rodent

extended amygdala

Abstract

The bed nucleus of the stria terrninalis (BST) and the medial amygdala (MeA) are
anatomically connected sites necessary for chemosensory regulation of social behaviors
in rodents. Prairie voles (Microtus ochrogaster) are a valuable model for studying the
neural regulation of social behaviors because, unlike many other rodents, they are
gregarious, pair bond after copulating, and are biparental. In this chapter, sex and species
differences in immunoreactivity for tyrosine hydroxylase (TH), the rate-limiting enzyme
for catecholarnine synthesis, in the BST and MeA are described. Virgin male prairie
voles had a large number of TH-immunoreactive (TH-ir) cells in areas analogous to the
rat principal nucleus of the BST (pBST) and the posterodorsal medial amygdala
(MeApd). Virgin female prairie voles had far fewer TH-ir cells in these sites (~17% of
the number of cells as males in the pBST, ~35% of the number of cells in the MeApd). A
few TH-ir cells were found in the BST of male and female hamsters and meadow voles,
but not in rats. The MeApd also contained a few TH-ir cells in male and female hamsters
and male meadow voles, but not rats. Castration greatly reduced. the number of TH-ir
cells in the male prairie vole pBST and MeApd, an effect that could be reversed with
testosterone. Furthermore, treating ovariectomized females with testosterone substantially
increased TH-ir cells in both sites. Therefore, a species-speciﬁc sex difference in TH

expression is found in a chemosensory pathway in prairie voles. Expression of TH in

21

these sites is inﬂuenced by circulating gonadal hormones in adults, which may be related

to changes in their display of social behaviors across the reproductive cycle.

22

Introduction

Neurons expressing tyrosine hydroxylase (TH), the rate-limiting enzyme for
catecholamine synthesis, are widespread throughout the vertebrate brain. In most species,
cells producing epinephrine or norepinephrine are invariably found in the brainstem,
whereas cells producing dopamine (DA) are located in both the brainstem and the
forebrain (Smeets and Gonzalez, 2000). The speciﬁc distribution of these
catecholaminergic cells, however, differs between vertebrates (Smeets and Gonzalez,
2000; Tillet and Kitahama, 1998). In fact, differences are even found within the order
Rodentia. For example, the anterior and posterior medial amygdala (MeA) and
posteromedial bed nucleus of the stria terrninalis (BSTpm) of Syrian hamsters
(Mesocricetus auratus) contain catecholaminergic cells not found in rats, mice, or
Siberian hamsters (Asmus et al., 1992; Asmus and Newman, 1993; Shi and Bartness,
2000; Wommack and Delville, 2002). Furthermore, species-speciﬁc TH-immunoreactive
(TH-ir) cells are found in the Syrian hamster diagonal band of Broca, lateral preoptic
area, and cortex (Vincent, 1988). There also may be differences between laboratory mice
and rats in the distribution and density of TH-expressing cells in the preoptic area of the
hypothalamus (Ruggiero et al., 1984).

Not only are there differences between rodent species, but sex differences in
forebrain TH expression can be found within a species. TH-expressing cells of the
anteroventral preoptic area (AVPV), necessary for gonadotropin release and ovulation
(Weigand and Terasawa, 1982), are two to four times more numerous in gonadally intact
female rats and mice than in intact males (Simerly et al., 1985a, b, 1997; Simerly, 1989;

Zup et al., 2003). A sex difference in TH immunoreactivity in the AVPV also exists in

23

prairie voles (Microtus ochrogaster), although subjects must be gonadectomized for this
sex difference to be revealed (Lansing and Lonstein, 2006).

Other areas of the rat, mouse, and prairie vole forebrain have not been reported to
be sexually dimorphic in the number of TH-ir cells (Simerly et al., 1985a, b; Simerly,
1989; Lansing and Lonstein, 2006). During a previous examination of sex differences in
the number of TH-ir cells in the hypothalamus of prairie voles (Lansing and Lonstein,
2006), however, two unexpected populations of TH-ir cells that appeared sexually
dimorphic were noticed (but not reported). One population was found in an area
analogous to the principal bed nucleus of the stria terrninalis (pBST) of the rat or the
posteromedial bed nucleus of the stria terrninalis (BSTpm) of the hamster (Swanson,
1998; Wood and Swarm, 2005). The other population of TH-ir cells was found in the area
analogous to the posterodorsal medial amygdala (MeApd) of both rats and hamsters. In
both cases, a large number of TH-ir cells were found in males, but relatively few in
females.

The pBST/BSTpm and MeApd have dense reciprocal connections in male rats
and hamsters (Canteras et al., 1995; Coolen and Wood, 1998; Wood and Swarm, 2005)
and are involved in chemosensory processing necessary for their sociosexual behaviors
(for reviews see Hull et al., 2002; Newman, 1999; Wood, 1998). The presence of TH-ir
cells in the pBST/BSTpm and MeApd of male, but not female, prairie voles is intriguing
given the complex and unique social structure of this species (for review see Carter etal.,
1995). Unlike many male rodents, male prairie voles form life-long pair bonds with their
mates and later display high levels of parental care toward their offspring. Pair bonding

and paternal behavior in male prairie voles require processing of olfactory inputs, insofar

24

as either olfactory bulbectomy or lesions encompassing the MeApd disrupt these
behaviors (Kirkpatrick et al., 1994a, c). Furthermore, impeding DAergic
neurotransmission also impairs these social behaviors in male prairie voles (Lonstein,
2002; Wang and Aragona, 2004). Here, populations of TH-ir cells in the pBST/BSTpm
and MeApd of male prairie voles are described, and the effects of gonadal hormones on
the TH-ir cells are examined in these sites. It may be that these relatively unique
populations of catecholaminergic cells in the male prairie vole brain are involved in the

chemosensory control of their pair bonding, parental, or other social behaviors.

Methods

Subjects

Male and female prairie voles (Microtus ochrogaster) were born and raised in the
colony, which was established in 2002 at Michigan State University, from breeding stock
that originated from offspring of voles captured in 1994 in Urbana, Illinois, provided by
Dr. Betty McGuire (Smith College, Northampton, MA) and Dr. Zuoxin Wang (Florida
State University, Tallahassee, FL). These voles were outbred in 2000 at the University of
Massachusetts with voles of Illinois origin provided by Dr. C. Sue Carter (University of
Illinois at Chicago) and brought to Michigan State University in 2002. Animals were
mated by socially isolating adult virgin female and male prairie voles for 4 days, after
which females were placed in the cage of an unfamiliar male. Animals were maintained
on a 14:10-hour light:dark cycle with an ambient temperature maintained at ~21°C. At all
ages, animals were housed in clear plastic cages (48 X 28 X 16 cm) containing wood

chips, wood shavings, and a substantial hay covering. Water and a food mixture

25

containing cracked corn, whole oats, sunﬂower seeds, and rabbit chow (Teklad rodent
diet No. 2031) in a ratio of 1:1:2:2 were freely available. Pups were weaned from their
parents at 20 days of age, placed in same-sex sibling groups of two or three animals/cage
between 50 and 60 days of age, and housed in these groups until they were killed at least
6 weeks later. The comparative study used eight voles of each sex, whereas the hormone
manipulation study (see below) used 24 voles of each sex.

The eight male and ﬁve female meadow voles were born in the breeding colony
of the Wang laboratory. Voles were weaned at 21 days of age and placed in same-sex
sibling pairs in clear plastic cages (29 X 18 X 13 cm) containing cedar chip bedding until
the time of death during adulthood. Food (Purina rabbit chow No. 5326), sunﬂower
seeds, and water were provided ad libitum. All cages were maintained in a 14:10-hour
lightzdark cycle while the ambient temperature was maintained at about 21°C.

Six male and six female Long-Evans rats (Rattus norvegicus) were purchased
from Harlan Laboratories (Indianapolis, IN) and sent to the laboratory when
approximately 75 days of age. Rats were housed in same-sex groups of two or three
animals per cage in clear polypropylene cages (48 X 28 X 16 cm) with wood shavings for
bedding. Food (Teklad rodent diet No. 8640) and water were continuously available,
lights were on a 12:12-hour lightzdark cycle with onset at 0800 hours daily, and the
ambient temperature was maintained at ~21°C. Rats were maintained in the laboratory
for at least 3 weeks before death.

Seven male and seven female Syrian hamsters (Mesocricetus auratus) were also
purchased from Harlan Laboratories and sent to the laboratory when approximately 75

days of age. Hamsters were singly housed in clear polycarbonate cages (12 X 4 X 8 in.)

26

with food (Teklad rodent diet No. 8640) and water freely available. Animals were
exposed to a 14:10-hour lightzdark cycle, and the ambient temperature was maintained at

~21°C. Hamsters were maintained in the laboratory for at least 2 weeks prior to death.

Gonadectomy and hormone replacement

To determine the effects of circulating gonadal hormones on TH expression in
prairie voles, voles of both sexes were weighed and anesthetized with an IP injection of
an anesthetic cocktail containing ketamine (62.5 mg/kg), xylazine (7.5 mg/kg), and
acepromazine (0.8 mg/kg). Males were either castrated or received a sham surgery in
which an incision was made in the scrotum, but the testes were not removed. Similarly,
female prairie voles were anesthetized and either were ovariectomized through a single
rrridline ventral incision or received a sham surgery in which the ovaries were visualized
but not removed. Half of the castrated males and ovariectomized females were
subcutaneously implanted with a 2.5-cm-long Silastic capsule containing crystalline
testosterone (Sigma, St. Louis, MO). This capsule provides supraphysiological levels of
testosterone that maintain masculine behavior and neurochemistry in male prairie voles
(e.g., Wang and DeVries, 1993; Lonstein et al., 2005). The other half of the castrated
males and ovariectomized females, and the sham surgery controls of both sexes, received
an empty Silastic capsule. Four weeks after surgery, subjects were killed and their brains
collected as described below. Each of the six groups of prairie voles contained eight
animals. All procedures were in accordance with the Institutional Animal Care and Use

Committees at Michigan State University and Florida State University.

27

Pm“) a

inur‘ :.._._

' R -
”4“. 0
tn. 3 m

i I
.._‘ ‘
“.0;- ‘
b;~‘jl‘l;\l~
T'F ‘r"
11.,1‘_~>\ ‘

16
I h
' " Til
HT"!
‘., l l
~~~~ :1.
5

Perfusion, tissue collection, and immunocytochemistry

All voles, rats, and hamsters were weighed, overdosed with sodium pentobarbital,
and perfused through the heart with 0.9% saline (100 ml for voles, 150 ml for hamsters
and rats), followed by 4% paraformaldehyde in sodium phosphate buffer (NaPB; pH 7.6,
100 ml for voles, 150 ml for hamsters and rats). Brains were removed, postﬁxed for 1-4
hours (species comparison) or overnight (sex and hormonal condition comparison) in 4%
paraformaldehyde in NaPB, and then submerged in a 20% sucrose/NaPB solution for at
least 3 days before sectioning into 40-um coronal sections with a freezing microtome.
Immunocytochemistry for TH was performed on every other section through the brain for
the prairie voles, meadow voles, and hamsters and on every third section through the rat
brains. Immunocytochemical procedures were identical to a procedure described
previously (Lansing and Lonstein, 2006). Brieﬂy, sections were rinsed three times for 5
minutes each in Trisma-buffered saline (TBS; pH 7.6), incubated in 0.1% sodium
borohydride for 15 min, rinsed three times in TBS, incubated in 0.3% Triton X-100 and
1% hydrogen peroxide in TBS for 10 min, rinsed three times in TBS, blocked with 20%
normal goat serum in 0.3% Triton X in TBS for 15 min, and then incubated with a rabbit
anti-TH polyclonal primary antiserum (A8152; 122,000; Chemicon, Temecula, CA) in
0.3% Triton X and 2% NGS in TBS at room temperature for approximately 18 hours.
According to the manufacturer, the immunogen for this primary antibody is denatured
TH from rat pheochromocytoma and has been shown by Western blot analysis not to
recognize other monoaminergic synthetic enzymes. Indeed, labeling was found for all

four species in the sites previously described to express TH in rodents, including the

28

periventricular hypothalamus, zona incerta, substantia nigra, and ventral tegmental area.
Furthermore, in rats and hamsters, no labeling was seen in any site not previously
described to express TH in these species. Sections were then rinsed three times in TBS,
incubated in a biotinylated goat anti-rabbit secondary antibody (1:500; Vector
Laboratories, Burlingame, CA) in 0.3% Triton-X and 2% NGS, rinsed three times in TBS
for 5 minutes each, and incubated with avidin-biotin complex (Vectastain Elite, Vector
Laboratories) for 60 minutes. After rinsing three times with TBS, visualization of TH
immunoreactivity occurred with the use of Vector SG chromagen (Vector Laboratories)
according to the manufacturer's instructions, which provided a light blue cytoplasmic
label. Sections were mounted on microscope slides, dehydrated, and coverslipped.
Immunocyotchemical control procedures included omission of the primary or secondary
antiserum, which abolished any speciﬁc labeling. A single immunocytochemical run
including the tissue from all four species was used for the species comparison, and a
separate immunocytochemical run was used for the gonadectomy and hormone-
replacement study in prairie voles.

To determine whether TH-ir cells in the pBST and MeApd of prairie voles might
be DAergic or noradrenergic, the expression of DA-B-hydroxylase (DBH), the enzyme
that converts DA into norepinephrine, was examined in these sites. An additional series
of brain sections that included the forebrain and brainstem of two gonadally intact male
and two intact female prairie voles was processed immunocytochemically as described
above but incubated for 3 days at 4°C in a rabbit polyclonal anti-DBH primary antiserum
(ABlSSS; 1:500; Chemicon). According to the manufacturer, the immunogen for this

primary antibody is DBH obtained from bovine adrenal glands and has been shown by

29

ruthr

'1L'\

3"
a.
'1r.. ,
‘4‘“

a 1‘ -, .
“q“ Md:
5: v

Western blot analysis to be speciﬁc for DBH. Indeed, many DBH-ir cells were found in
the locus coeruleus of both the male and the female prairie vole brainstem, but none were
found in pBST or MeApd, or in the hypothalamus and zona incerta on these same

sections. Omission of the primary or secondary antiserum abolished all speciﬁc labeling.

Analyses

To compare the presence of TH immunoreactivity in the BST and MeA of prairie
voles, meadow voles, hamsters, and rats of both sexes, the entire rostrocaudal extents of
the BST and MeA were examined for each subject. The presence or absence of TH-ir
cells at any rostrocaudal level within these areas was noted.

Slides for the gonadectomy and hormone-replacement study in prairie voles were
masked and coded for analysis, which was performed by only one observer (K.V.N.). The
three areas examined were analyzed bilaterally from consecutive sections in a one-in-two
series, with the number of TH-ir cells totaled from these sections. The number of cells
containing detectable TH immunoreactivity was counted by eye with the use of a Nikon
E400 microscope at X200 magniﬁcation with the aid of a reticle placed in one of the
ocular lenses. The area of the BST analyzed included the area of the prairie vole brain
that had the densest cluster of TH-ir cells and corresponded to the dorsal principal BST of
the rat or the dorsal posteromedial BST of the hamster. The total number of TH-ir cells
was quantiﬁed from four consecutive sections in a one-in-two series through the middle
and caudal pBST, roughly corresponding to plates 20-22 of Swanson's (1998) atlas of the
rat brain. Males rarely had TH-ir cells in the dorsal pBST rostral or caudal to these four

sections. The area of the MeApd analyzed began with the section roughly corresponding

30

to plate 28 of Swanson's rat atlas, where TH-ir cells ﬁrst appeared for most subjects, and
continued for six consecutive sections in a one-in-two series through the MeApd, ending
approximately at the level corresponding to Swanson's atlas plate 30. Subjects reliably
had six MeApd sections represented, but not necessarily more, because the amygdala was
often lost in the most caudal sections. In addition, a small cluster of TH-ir cells in the
dorsal reticular nucleus of the thalamus was also found, at the level roughly
corresponding to Swanson's atlas plate 27. These cells are also found in the hamster brain
(Asmus et al., 1992). These thalamic cells were found only in two sections of the one-in-
two series for each subject and did not appear to differ between the sexes, so this was
used as a control site not likely to be affected by the hormone manipulations. Average
soma size of TH-ir cells in the pBST and MeApd was evaluated for each subject by
nonsystematically choosing for each subject one hemisection through dorsal pBST and
MeApd and then tracing the perimeter of every TH-ir soma under X200 magniﬁcation
with the use of an image analysis system (Image Pro Plus; Media Cybernetics).
Stereology was not used to analyze these TH-ir cells for numerous reasons. First, these
TH-ir populations were found on only a small number of sections through each brain
region. Second, it was possible to analyze every other section through these sites. Third,
the TH-ir cells were not very densely packed, and, fourth, the total number of TH-ir cells
per section was relatively small and easily quantiﬁed. Cell counts were subjected to an
Abercrornbie correction factor of 0.83 based on the ﬁnding of a z-plane average soma
diameter of ~8 pm and are presented as corrected counts. Photomicrographs were

arranged with Adobe Photoshop 6.0, with the Dodge tool used to reduce any

31

inconsistencies in illumination within a given panel, and the brightness and contrast were
adjusted to maintain consistency across panels.

Data from the sex and hormone treatment comparison in prairie voles were
analyzed for each site with a 2 (sex) X 3 (treatment) ANOVA, followed by Fisher LSD
post-hoe tests on main effects. Separate Fisher LSD post-hoe tests were then used as
planned comparisons to compare treatment groups within each sex. Because a single
hemisection was analyzed for somal area, the number of subjects included in this analysis
was greater for some groups than the analysis of the total number of TH-ir cells, which
required many intact sections for us to include a subject in the analysis. Statistical

signiﬁcance was indicated at P = 0.05.

Results

Species comparison

All male prairie voles had a distinct cluster of intensely TH-ir cells in the dorsal
pBST, with the majority of cells appearing at the rostrocaudal level where the third
ventricle appears both dorsal and ventral to the anterior commissure. Many fewer cells
were found in sections of the male dorsal pBST anterior or posterior to this dense cluster,
although some TH-ir cells were found adjacent to the stria medullaris and fomix in more
caudal sections, as the pBST extended ventromedially. In contrast to males, female
prairie voles had many fewer TH—ir cells in the dorsal pBST (Figure 2.1), and the
intensity of immunoreactive labeling in most of these cells was relatively weak. Some
females also had a few weakly immunoreactive cells in the ventromedial pBST more

caudally. Small numbers (one to eight cells) of very weakly TH-ir cells were also found

32

in each section of the dorsal pBST of most male and female meadow voles, and one to
four moderately immunoreactive cells per section were found in the dorsal pBST of male
and female hamsters (Figure 2.2). No TH-ir cells were found in the dorsal pBST of any
male or female rat (Figure 2.2). Although relatively few or no TH-ir cells were found in
the dorsal pBST of female prairie voles or meadow voles, hamsters, and rats of both
sexes, the periventricular hypothalamus on these same sections contained a large number
of intensely TH-ir cells, as previously described (e.g., Chan-Paley et al., 1984; Vincent,

1988; Lansing and Lonstein, 2006).

33

 

egg ‘

,h:r.“XT-;‘M ' T

Figure 2.1. Photomicrographs of TH-ir cells in the pBST of representative prairie voles.
A: Male prairie vole at X100 magniﬁcation. B: Male at X200 magniﬁcation. CI Female at
X200 magniﬁcation. LV, lateral ventricle; ac, anterior commissure. Scale bars = 100 um
111A35()lmlinBandC.

34

'a‘
1'
-

4&9"

 

Figure 2.2: Photomicrographs (X200 magniﬁcation) of TH-ir cells in the dorsal PBST 0f
Icpresentatrve male meadow voles, hamsters, and rats. Males of these SPCCieS were
sunrlar to females. Scale bars = 50 um.

35

All male prairie voles showed a large number of highly TH—ir cells throughout the
entire rostrocaudal extent of the MeApd, and some males also had a few TH-ir cells in
the posteroventral MeA (MeApv; Figure 2.3). The MeApd of all female prairie voles also
contained TH-ir cells, but there were far fewer compared with males, and many of these
cells were only weakly immunoreactive (Figure 2.3). Some (one to 12 cells/section) very
weakly TH-ir cells were also found in the MeApd of most male and female meadow
voles. The MeApd of all hamsters contained a smaller number (one to four cells/section)
of moderately TH-ir cells, with no obvious difference between the sexes in the number of

cells. No TH-ir cells were found in the MeApd of male or female rats (Figure 2.4).

36

 

Figure 2.3. Photomicrographs of TH-ir cells in the MeApd of representative prairie
voles. A: Male prairie vole at X100 magniﬁcation. B: Male at X200 magniﬁcation. C:
Female at x200 magniﬁcation. opt, Optic tract. Scale bars = 100 pm in A; 50 pm in B,C.

37

hilt:

‘C 12R:

. 1

”'1’

\ll'l

ET

 

Figure 24 Photomicrographs (x200 magniﬁcation) of TH-ir cells in the MeApd of
representatrve male meadow voles, hamsters, and rats. Males Of these SPCCIeS were
s1milar to females. Scale bars = 50 pm-

38

Sex difference and effects of gonadal hormones on TH immunoreactivity in prairie voles
In the dorsal pBST, there were signiﬁcant main effects of sex (F(l,33) = 11.63, P
< 0.002) and treatment (F(2,33) = 18.79, P < 0.0001) on the number of TH-ir cells. The
interaction between sex and treatment was not signiﬁcant (F (2,33) = 1.69, P > 0.2). Post-
hoc analysis on the main effects revealed that males had signiﬁcantly more TH-ir cells
than females and that testosterone-treated animals had signiﬁcantly more TH-ir cells than
gonadally intact and gonadectomized animals, which did not signiﬁcantly differ from
each other (Figures 2.5, 2.6). Planned comparisons within each sex revealed that
castration of males signiﬁcantly reduced the number of TH-ir cells, whereas testosterone
treatment maintained the number of cells in castrated males at a level similar to that of
sham males. Ovariectomy had no signiﬁcant effect on the already low number of TH-ir
cells in the dorsal pBST of female prairie voles, whereas treating ovariectomized females
with testosterone signiﬁcantly increased the number of cells compared with the other two
groups of females (Figure 2.5). The average cross-sectional area of these TH-
immunoreactive somata did not differ between the sexes (F (1,39) = 0.35, P > 0-3) 01’ by
treatment (F(2,39) = 2.05, P > 0.1), and there was no signiﬁcant interaction between

these factors (F(2,39) = 2.40, P > 0.1; group averages = 109 a 4 to 135 i 5 m2)-

39

[:1 Sham
250 CZZ] GDX
* — GDX+T

 

 

N
O
O

 

 

 

 

150

 

100 -

Number of TH-ir Cells
0'!
O

 

 

 

 

 

 

 

 

 

Males Females

Figure 2.5. Number (Mean at SEM) of TH-ir cells in every other section of the dorsal
pBST of male and female prairie voles that were gonadally intact (Sham),
gonadectomized (GDX), or gonadectomized and implanted with a capsule ﬁlled with
testosterone (GDX + T). *Signiﬁcant difference between the sexes, collapsed across
treatment. or, GDX males signiﬁcantly different from other groups of males; B, GDX + T
females signiﬁcantly different from other groups of females.

40

 

Figure 2.6. Photomicrographs (X200 magniﬁcation) of TH-ir cells in the dorsal pBST of
male (top) and female (bottom) prairie voles that were gonadally intact (Sham),
gonadectomized (GDX), or gonadectomized and implanted with a capsule ﬁlled with
testosterone (GDX + T). Scale bar = 50 pm.

41

In the MeApd, there were signiﬁcant main effects of sex (F(l,35) = 25.35, P <
0.0001) and treatment (F(2,35) = 22.47, P < 0.0001), but no signiﬁcant interaction
between them (F(2,35) = 2.17, P > 0.12). Post-hoe analysis on the main effects revealed
that the number of TH-ir cells was greater in males than females and that all three
hormone treatments signiﬁcantly differed from each other, with the number of TH-ir cells
lowest in gonadectomized animals, intermediate in intact animals, and highest in
testosterone-treated animals (Figures 2.7, 2.8). Similar to the case in the dorsal pBST,
planned comparisons of the treatment groups within each sex revealed that castration of
males signiﬁcantly reduced TH-ir cells in the MeApd, whereas testosterone treatment
maintained a high number of these cells. Ovariectomy had no signiﬁcant effect on the
number of TH-ir cells in the MeApd of females, whereas treating ovariectomized females
with testosterone signiﬁcantly increased it (Figure 2.7). The average cross-sectional area
of the TH-ir somata did not differ between the sexes (F(l,37) = 0.22, P > 0.64) or by
treatment (F(2,37) = 0.42, P > 0.65), and there was no signiﬁcant interaction between

these factors (F(2,37) = 0.01 , P > 0.99; group averages = 132 :1: 7 to 143 i 9 umz).

42

1::I Sham
. . IE] GDX

300 _ * - GDX'I'T
250 ‘

350

J

 

 

200 -
150

100

 

Number of TH-ir Cells

L

50

 

 

 

 

 

 

 

Males Females

Figure 2.7. Number (Mean :15 SEM) of TH-ir cells in every other section of the MeApd of
male and female prairie voles that were gonadally intact (Sham), gonadectomized
(GDX), or gonadectomized and implanted with a capsule ﬁlled with testosterone (GDX +
T). *Signiﬁcant difference between the sexes, collapsed across treatment. or, GDX males

Signiﬁcantly different from other groups of males; B, GDX + T females signiﬁcantly
drfferent from other groups of females.

43

 

Figure 2.8. Photomicrographs (X200 magnification) of TH-ir cells in the MeApd of male
(top) and female (bottom) prairie voles that were gonadally intact (Sham),
gonadectomized (GDX), or gonadectomized and implanted with a capsule ﬁlled with
testosterone (GDX + T). Scale bar = 50 um.

44

In the small cluster of TH-ir cells in the dorsal reticular nucleus of the thalamus,
there were no signiﬁcant main effects of sex (F(l,34) = 1.31, P > 0.26) or treatment
(F(2,34) = 0.33, P > 0.71), and no signiﬁcant interaction between them (F(2,34) = 0.38, P

> 0.69; Figure 2.9).

I:I Sham
[ZZ] GDX
- GDX-I-T

.3
0|
0

100 ~

 

 

 

Number of TH-ir Cells
0|
0
___.|
,.__4

 

 

 

 

 

 

 

 

Males Females

Figure 2.9. Number (mean i SEM) of TH-ir cells in every other section of the
dorsolateral thalamus of male and female prairie voles that were gonadally intact (Sham),

gonadectomized (GDX), or gonadectomized and implanted with a capsule ﬁlled with
testosterone (GDX + T).

45

Discussion

These experiments demonstrate that large numbers of TH-ir cells were found in
the dorsal pBST and MeApd of male prairie voles. Female prairie voles and meadow
voles, hamsters, and rats of both sexes had relatively few or no TH-ir cells in these sites.
Furthermore, these populations of cells were sensitive to manipulations in gonadal
hormones in adult prairie voles, in that castration reduced the number of TH-ir cells in
males, an effect that could be prevented by chronic treatment with testosterone. Females
treated with testosterone during adulthood also showed a sharp increase in the number of
TH-ir cells. Differences between the sexes in TH-ir cells within a known chemosensory
pathway, and the sensitivity of these cells to circulating hormones, suggest that
catecholamine release from these sites may be involved in regulating sex differences in
social behaviors in prairie voles as well as changes in these social behaviors across the

reproductive cycle at times when hormones dramatically ﬂuctuate.

Species differences in TH immunoreactivity

There seems to be notable species differences in TH expression in the pBST and
MeApd. However, the absence of large numbers of TH-ir cells in the BST and MeA of
meadow voles, hamsters, and rats of both sexes does not necessarily indicate that TH-
producing cells do not exist in the BST and MeA of these species. In fact, Syrian
hamsters of both sexes can express a large number of TH-ir cells in these sites, apparently
almost as many as were found for male prairie voles, if treated with colchicine 48 hours
prior to sacriﬁce (Asmus et al., 1992; Asmus and Newman, 1993). The “appearance” of

these immunocytochemically identiﬁable cells in hamsters likely is due to increased

46

accumulation of TH in the somata after colchicine treatment, rather than a de novo
increase in TH synthesis that can occur as a result of colchicine (Cortes et al., 1990). This
is suggested by the ﬁnding that these cells are present in high numbers when TH mRNA
is visualized with in situ hybridization in hamsters not treated with colchicine (Asmus
and Newman, 1993). Nonetheless, under normal conditions, only male prairie voles
express high enough levels of TH in the dorsal pBST and MeApd to be visualized with
immunocytochemistry, although other rodents may express relatively low levels of TH in
these sites. The comparison between species in TH immunoreactivity in the pBST and
MeApd also revealed that male prairie voles, meadow voles, and hamsters have at least
some TH-ir cells in both sites, whereas rats do not. All of the rodents examined herein are
within the superfamily Muroidea. However, based on recent genetic examination, voles
and hamsters are both members of the family Cricetidae, whereas Old World (laboratory)
rats are within the family Muridae (Steppan et al., 2004). Hamsters and voles are more
phylogenetically related to each other than either species is to laboratory rats, and cells
expressing TH in these regions of the brain may be relatively speciﬁc to Cricetidae.
Nonetheless, there are some notable differences between voles and hamsters. TH-ir cells
were rare in the anterior MeA of male voles, but are quite dense in both the anterior and
the posterior MeA of colchicine-treated male hamsters (Asmus et al., 1992), indicating
that the distribution of TH-ir cells in these sites is similar, but not identical, among
relatively closely related species.

Possibly more surprising, based on degree of relatedness, is the difference in the
level of TH expression between male prairie and male meadow voles. The brains of these

two very closely related species differ in numerous ways, including expression of

47

receptors for oxytocin (Insel and Shapiro, 1992), vasopressin (Insel et al., 1994),
corticotropin-releasing factor (Lim et al., 2005), estrogen (Fowler et al., 2005), and DA
(Curtis, Fowler, Lonstein, and Wang, in preparation). Male prairie and meadow voles
also differ in how social interactions alter neurochemistry (Bamshad et al., 1993; Wang et
al., 1994; Curtis et al., 2003). This divergent neurochemistry has often been suggested to
be related to differences between these species in their social organization, with prairie
voles highly gregarious and socially monogamous and meadow voles nongregarious and
polygamous (Young et al., 2001). An examination of TH expression in the pBST and
MeApd of males from other monogamous species, such as California mice (Peromyscus
californicus), Djungarian hamsters (Phodopus campbelli), or Mongolian gerbils
(Meriones unguiculatus), would be useful to determine whether very high TH expression

in these sites is typical of monogamous male rodents.

Sex diyfkrences and hormone sensitivity of TH-immunoreactive cells

There was a large sex difference in the number of TH—ir cells in the prairie vole
dorsal pBST and MeApd. As noted above with regard to differences between species, the
absence of TH-ir cells in untreated female prairie voles does not necessarily mean that
TH-synthesizing cells do not exist, but rather that levels of TH are below the threshold of
immunocytochemical detection. It is valuable to note that males do not have greater
levels of TH-ir cells everywhere in the brain; sex differences in detectable cells are not
found in the dorsal reticular thalamus (present results) or hypothalamus and zona incerta

(Lansing and Lonstein, 2006). Furthermore, the sex difference in TH expression in the

48

AVPV is the reverse of that found in the pBST or MeApd (Lansing and Lonstein, 2006).
The sex differences in the dorsal pBST and MeApd are, therefore, quite unique.

These data also demonstrate that these TH-ir populations are sensitive to gonadal
hormones. When treatment groups were compared within each sex, castration of male
prairie voles demasculinized (i.e., reduced) the number of TH-ir cells in both sites,
whereas exogenous testosterone maintained the numbers of these cells at a level similar
to that of gonadally intact males. Conversely, the number of TH-ir cells was masculinized
(i.e., increased) in females given testosterone. The lack of an effect of ovariectomy on
these cells in females was expected, insofar as prairie voles are induced ovulators and, in
the absence of sensory cues from males, have little circulating estradiol (Cohen-Parsons
and Carter, 1987). This does not necessarily mean that estradiol is not responsible for
modulating TH expression in these sites. Many effects of testosterone on sex differences
in the rodent brain are mediated through its aromatization to estradiol and subsequent
activity of the estradiol receptor (DeVries and Simerly, 2002; Wallen and Baum, 2002).
The pBST and MeApd are extremely steroid sensitive and express high levels of estradiol
receptors in prairie voles of both sexes (Cushing and Wynne-Edwards, 2005; Fowler et
al., 2005; Hnatczuk et al., 1994). Aromatization of testosterone into estradiol may be
largely responsible for testosterone's effects on TH-ir cells in these sites, a detail that
should be examined in future experiments.

The effects of hormone manipulations in male prairie voles were somewhat
dissimilar to those found in colchicine-treated male hamsters. Castration of hamsters
reduces TH immunoreactivity only in the anterior MeA, not the posterior MeA or BSTpm

(Asmus and Newman, 1993). Furthermore, although exogenous testosterone fully

49

maintained of the number of TH-containing cells in the MeApd of male prairie voles, it
does not in the hamster anterior MeA. Asmus and Newman (1993) also found that the
decrease in TH immunoreactivity in the hamster anterior MeA after castration was
transient, insofar as high numbers of TH-ir cells were again found in males examined 12
weeks after castration. Similar long-term castration effects may occur in the MeApd of
male prairie voles. It appears that differences in TH expression between members of
Cricetidae are apparently not only in distribution but also in sensitivity to gonadal
hormones.

Even though castration greatly reduced TH expression in males and testosterone
greatly increased it in females, the sex difference was not completely eliminated by
equating adult circulating hormones. Indeed, adult female prairie voles treated with
testosterone still had ~25% fewer TH-expressing cells in the dorsal pBST than
testosterone-treated males. Conversely, castrated males still had almost twice as many
cells as ovariectomized females. However, the magnitude of the sex difference is greatly
reduced when gonadal hormones are equated in adult animals. In the dorsal pBST, the
magnitude of the sex difference changed from an almost 600% difference in intact
animals to a 25% difference when animals were similarly treated with testosterone. The
sex difference in the MeApd went from almost 300% to 25%. A similar reduction in the
magnitude of the sex difference in MeA volume occurs when adult male and female rats
are given the same testosterone treatment (Cooke et al., 1999), so prairie voles are not the

only species in which adult gonadal hormones can greatly alter the magnitude of a neural

sex difference.

50

Given that the sex differences in TH expression were not completely eliminated,
gonadal hormones acting on these sites during perinatal development also probably
inﬂuence this sex difference to some extent. Perinatal exposure to testicular hormones
may render adult males more sensitive to a given amount of testosterone, leading to the
persistence of a sex difference in TH immunoreactivity even when adult males and
females receive the same hormone treatment. Adult female prairie voles have greater
estrogen receptor expression in these sites than males (Hnatczuk et al., 1994), so reduced
effectiveness of testosterone in adult females may instead be due to reduced intraneuronal
aromatization of testosterone to estradiol (Roselli et al., 1985). It is also possible that
testosterone actions on the androgen receptor, which is sexually dimorphic in these sites

(Wood and Newman, 1999), also contributes to the sex differences (Morris et al., 2005).

Functional considerations

Differences in TH immunoreactivity between individual cells within a given
structure reﬂect functional and anatomical heterogeneity (Bayer and Pickel, 1990; Benno
et al., 1982; Weiss-Wunder and Chesselet, 1990, 1991; Zigmond and Ben-Ari, 1977). It
is reasonable to believe that differences between animals in TH immunoreactivity in a
given site also reﬂects such heterogeneity. The presence of a large number of TH-ir cells
in the pBST and MeApd of male prairie voles is presumably associated with unique
anatomy and neural processing compared with female prairie voles, as well as meadow
voles, hamsters, and rats of both sexes. Indeed, these populations in male prairie voles do
not contain an insigniﬁcant number of cells. The quantiﬁcation of all TH-ir cells in every

other section suggests that the male prairie vole dorsal pBST contains over 200 TH-ir

51

Mr

'P

(3’

 

 

-,_ ~
.’1. ‘
‘~ El,

 

 

cells, and the MeApd contains at least twice that many. The absence of DBH
immunoreactivity the pBST and MeApd indicates that these cells may be DAergic, rather
than noradrenergic, which is consistent with what is found in colchicine-treated hamsters
(Asmus et al., 1992; Asmus and Newman, 1993; Vincent, 1988).

In hamsters, lesions encompassing the BSTpm reduce olfactory investigation of
estrous females, although many lesioned males continue to copulate, albeit at a reduced
level (Powers et al., 1987). Lesions of the MeA that include the MeApd, particularly its
rostral component, also severely reduce males’ olfactory investigation of females and
virtually eliminate copulation (Lehman et al., 1980; Powers et al., 1987). Various
impairments in copulation are also found in male laboratory rats after pBST or MeApd
lesions (Claro et al., 1995; Emery and Sachs, 1976; Kondo, 1992; McGregor and Herbert,
1992; Valcourt and Sachs, 1979). Not much is known about the prairie vole pBST and
MeApd, but the anatomy and function of these areas in other rodents suggests that these
TH-ir populations in male prairie voles could be related to the chemosensory control of
copulation, as well as to the more unique social behaviors they display, such as pair
bonding and paternal care.

Both male and female prairie voles pair bond and show parental care,‘though,
which complicates the suggestion that pBST/MeApd TH expression in males is involved
in their ability to display these behaviors, as one might expect that the sexes employ
similar neural mechanisms to achieve these similar behavioral endpoints. De Vries (2004)
has suggested that the opposite may be possible, such that sex differences in the brain,
rather than similarities, produce similar behavioral or physiological endpoints by

compensating for sex differences in circulating gonadal hormones. A sex difference in

52

TH expression in the pBST and MeApd may allow males to show monogamous
behaviors even in light of their higher circulating androgens, which are sometimes
regarded as inconsistent with monogamous behaviors (Wingﬁeld et al., 2001).
Nonetheless, any function for the dorsal pBST and its catecholaminergic cells in
the formation and expression of pair bonds in male prairie voles remains to be examined.
The pBST and surrounding area show increased activity of the immediate early gene c-
fos when male prairie voles copulate with females (Lim and Young, 2004), which
normally is necessary for their pair bonding (see Carter et al., 1995). Furthermore,
peripheral injection of the DA antagonist haloperidol eliminates males' social preferences
for their mates (Aragona et al., 2003). Conversely, injection of the DA agonist
apomorphine promotes males' social preferences for familiar females in the absence of
mating (Aragona et al., 2003). Areas of the brain where changes in DAergic
neurotransmission produce these effects include the mesolimbic system (Aragona et al.,
2003, 2006; Curtis and Wang, 2005), but, as with other social behaviors, multiple
catecholaminergic systems are likely to be involved (Dominguez and Hull, 2005; Miller
and Lonstein, 2005). A function for the dorsal pBST in parental behavior in prairie voles
is also unknown. It may be sex-speciﬁc, in that paternal behavior in prairie voles requires
intact olfaction, whereas maternal behavior does not (Kirkpatrick et al., 1994c; Williams
et al., 1992c), suggesting that olfactory inputs to the pBST may be more important for
paternal care than maternal care. In partial support, lesions of the entire dorsal BST
(including the dorsal pBST) of lactating female rats do not signiﬁcantly affect their

maternal behavior (Numan and Numan, 1996).

53

In contrast to the pBST, the MeApd has been investigated for roles in both pair
bonding and paternal behavior in male prairie voles. Although large lesions of the
corticomedial amygdala (which include the MeApd) reduce contact with a familiar
female, lesions speciﬁcally of the medial amygdala do not (Kirkpatrick et al., 1994a).
Such lesions do reduce their paternal behavior, though (Kirkpatrick et al., 1994a).
Inhibiting DAergic neurotransmission with peripheral injection of haloperidol also
impairs their paternal behavior (Lonstein, 2002), and the MeApd may be one source of
critical DAergic neurotransmission.

In summary, species—speciﬁc catecholaminergic activity originating from cells in
the pBST and MeApd of male prairie voles may be involved in the display of their unique
social behaviors. Direct examination of how catecholarnines originating from these two

sites inﬂuence any behavior in male prairie voles remains to be determined.

Acknowledgements

The authors thank Kyle Gobrogge and Ray Figueira for their assistance with

components of this project.

54

CHAPTER 3

Northcutt, K. V. and Lonstein, J. S. (2009). Social contact elicits immediate-early gene

expression in dopaminergic cells of the male prairie vole extended olfactory
amygdala. Neuroscience 163(1), 9-22.

55

.._
11:1

r

‘1“,,.

"ro§.‘

CHAPTER 3:
Social contact elicits immediate-early gene expression in catecholaminergic cells of

the male prairie vole extended amygdala

Abstract

Male prairie voles (Microtus ochrogaster) are a valuable model in which to study
the neurobiology of sociality because, unlike most mammals, they pair bond after mating
and display paternal behaviors. Research on the regulation of these social behaviors has
highlighted dopamine (DA) neurotransmission in both pair bonding and parenting.
Recently, large numbers of tyrosine hydroxylase-immunoreactive (TH-ir) cells were
described in the male prairie vole principal nucleus of the bed nucleus of the stria
terrninalis (pBST) and posterodorsal medial amygdala (MeApd), but such cells were very
few in number or absent in the non-monogamous species that were examined, including
meadow voles. This suggests that TH-ir cells in these sites may be important for
sociosexual behaviors in male prairie voles. To gain some insight into the function of
these TH-ir neurons in male prairie voles, expression of the immediate-early genes
(IEGs) Fos and Egr-l were examined in TH-ir cells of the pBST and MeApd after males
interacted (or not) with one of several social stimuli. IEGs were constitutively expressed
in some TH-ir neurons under any social condition, but that the number of cells expressing
IEGs decreased after a 3.5-h social isolation. Thirty-minute mating bouts (but not 6- or
2441 bouts) that included ejaculation elicited more TH-ir cells expressing IEGs than did
non'eiaculatory mating, interactions with a familiar female sibling, or interactions with

pUps, Furthermore, Fos expression in TH-ir cells was positively correlated with the

56

U“ 0'
r

display of copulatory, but not parental, behaviors. These effects of mating were not found
in other DA-rich sites of the forebrain (including the anteroventral periventricular
preoptic area, periventricular anterior hypothalamus, zona incerta, and arcuate nucleus).
Thus, activity in TH-ir cells of the male prairie vole pBST and MeApd is inﬂuenced by
their social environment, and may be particularly involved in mating and its

consequences, including pair bonding.

57

’Ir’ ‘
.1\'-:

r,»-
.N-

51“

.1
Mg

Introduction

Prairie voles (Microtus ochrogaster) are an invaluable rodent model to study the
neurobiology of sociality because they form lifelong pair bonds after mating and show
biparental behavior after the birth of pups (Carter et al., 1995). Release of numerous
neurochemicals, including dopamine (DA), is required for the display of these social
behaviors (Young et al., 2008). D2 receptor antagonism prevents the formation of partner
preferences after mating in both sexes of prairie voles, while D2 stimulation induces
partner preference formation in the absence of mating (Aragona et al., 2006; Wang et al.,
1999). DA receptor antagonism also impairs parental responding in both sexes of prairie
voles, with the mixed D1/D2 receptor antagonist haloperidol reducing voles' motivation
to make contact with pups and lick them (Lonstein, 2002). The nucleus accumbens
(ACB) is one site where dopaminergic (DAergic) activity is critical for pair bond
formation in prairie voles (Aragona et al., 2003; Gingrich et al., 2000), and this is
probably also true for their biparental behaviors (see Numan and Stolzenberg, 2009).

The ventral tegmental area (VTA) is likely a major source of DA to the ACB and
other forebrain sites necessary for social behaviors in prairie voles (Curtis and Wang,
2005), but the involvement of other regions that may provide DAergic signaling
necessary for sociality has not been investigated. Novel populations of neurons
eXPTGSSing high levels of tyrosine hydroxylase (TH), the rate—limiting enzyme in
catecholamine synthesis, were recently described in the prairie vole principal nucleus of
the bed nucleus of the stria terrninalis (pBST) and posterodorsal medial amygdala
(MeApd) (Northcutt et al., 2007). These cells do not contain DA-beta-hydroxylase and,

““18, may be DAergic (Northcutt et al., 2007). Few or no TH-immunoreactive (TH-1r)

58

1‘) \

5V“-
'_ .
”like
,-.

DJ 1’

«M

\y
u g,

' I
., '1),
I. (M

cells were found in these sites in three non-monogamous species that were examined,
including the closely related meadow vole, suggesting that these cells may be involved in
how DA inﬂuences social behaviors relatively unique to prairie voles. In addition, male
prairie voles have three- to ﬁve-fold more TH-ir cells in these sites than do female prairie
voles, indicating particular importance of these cells for social behaviors in males.

The pBST and MeApd have dense connections with the main and accessory
olfactory systems, as well as with each other (Aldheid et al., 1995; Coolen and Wood
1998; Davis et al., 1978; Scalia and Winans 1975). They process and transmit olfactory
information essential for conspeciﬁc identiﬁcation and appropriate social responding in
laboratory rats, hamsters, and mice (Newman, 1999). The same is true for male prairie
voles, as indicated by the increased expression of the immediate-early gene (IEG) Fos in
both the pBST and MeApd after male prairie voles cohabitate with a female, mate with
her, or interact with pups (Cushing et al., 2003; Kirkpatrick eta1., 1994b; Lim and Young
2004). Furthermore, lesions of the entire MeA decrease males' paternal behaviors
(Kirkpatrick et al., 1994a). How the TH-ir cells of the pBST and MeApd are speciﬁcally
involved in these effects is unknown.

To begin investigating potential roles for the TH-ir cells of the male prairie vole
PBST and MeApd, Fos expression in TH-ir neurons was examined in these brain sites
after males interacted for 30 min with one of several social stimuli, including a sexually
reeel-"five female prairie vole or conspeciﬁc pups. In addition, Fos in other forebrain TH-
ir cell groups was examined to determine the selectivity of F 03 expression in TH-ir cells
in the PBST and MeApd after sociosexual interactions. Because many, but not most, TH-

1r cells 1n these two sites contained Fos immunoreactivity after social interactions, a

59

ll}?

subsequent experiment was designed to determine if even more of these TH-ir cells
would contain another IEG, Egr-l, after particular sociosexual interactions. Egr-l was
chosen because it is expressed in many brain sites after the display of sexual or maternal
behaviors, including in cells that do not express Fos (Numan et al., 1998; Polston and
Erskine 1995; Wersinger and Baum 1996). Because the formation of partner preferences
in male prairie voles requires rather long sexual interactions with a female (up to 24 h;
Insel et al., 1995; Winslow et al., 1993), and surprisingly prolonged Fos expression can
be seen after acute or chronic social or other stimulation (Bullitt et al., 1992; Matsuda et
al., 1996; Quattrochi et al., 2005; Ricci et al., 2007; Stack and Numan 2000; Wrynn et al.,
2000; Xu et al., 2006), Fos expression was also examined in TH-ir cells of the pBST and

MeApd after males cohabitated with a sexually receptive female for 6 or 24 h.

Methods

Subjects

Subjects were adult prairie voles (Microtus ochrogaster) that were born and
raised in the colony at Michigan State University. The colony originated with voles
originally captured in Urbana, IL, USA, in 1994, and since maintained in laboratory
environments and interbred with voles from the colonies of Drs. C. Sue Carter, Zuoxin
Wang, Geert De Vries, and Betty McGuire. This stock was brought to Michigan State
University in 2002. Animals were housed in clear plastic cages (48X28X16 cm),
Containing wood chips, wood shavings, and a layer of hay. Food and water were provided
ad libitum; food consisted of a mixture of cracked corn, whole oats, sunﬂower seeds, and

rabbit chow (Teklad rodent diet # 2031, Harlan, Madison, WI, USA) mixed in a 1:1:2:2

60

-v.
t.

r” .

it.

ratio. The colony room was maintained with an ambient temperature of approximately
21°C and a 14:10-h light/dark cycle. Litters were weaned from breeding pairs at 20 days
of age and housed with their siblings in mixed-sex groups consisting of the entire litter
until adulthood (60—120 days old) when they were used in one of the experiments. While
exposure to male siblings affects sensitivity to estradiol in female prairie voles (Cushing
and Carter, 1999), the effects of the postvveaning social environment on males'
reproductive potential are less pronounced (Mateo et al., 1994). In fact, the sex ratio was
retrospectively determined for 75% of the subjects' natal litters, and found that neither the
total number of siblings nor the litter sex ratio was signiﬁcantly correlated with any
sociosexual behavior or the IEG expression found in any site examined in the present
experiments (data not shown). The experiments were carried out in accordance with the
National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH
Publication No. 80—23), as well as the Institutional Animal Care and Use Committee at
Michigan State University. The minimal number of subjects per group that would provide
sufficient statistical power was used, and every effort was made to ensure that subjects

did not experience any pain during any of the behavioral tests or procedures.

Experiment Ia: effects of 3 0—min social interactions on F as expression

Sexually na’r‘ve male prairie voles were placed individually into a clean cage
(48x28X16 cm) with bedding, food, and water, and transported to a nearby room
illuminated with dim ambient light (20 1x) for behavioral testing. After a 3-hour
habituation to the testing cage and room, each subject was presented with a social

Stilnulus (or nothing) for 30 min. After 30 min, the stimulus animal was removed and the

61

a P...

 

male subject remained in the cage and testing room for 1 h before being sacriﬁced. An
additional control group was not placed in a novel cage or brought to the testing room,
but inStead sacriﬁced directly after removal from their home cage in the colony room.

The social stimuli presented were:

Groups 1 and 2: sexually receptive female
Males (n = 40) were allowed to interact with an adult female that was ovariectomized,
allowed to recover for at least 2 weeks, and injected so with estradiol benzoate (10 ug in
50 ul sesame oil; Sigma, St. Louis, MO, USA) 48 and 24 h before testing to induce
sexual receptivity (Carter et al., 1987a; Northcutt and Lonstein 2008). Stimulus females
were tested for receptivity with a sexually experienced male from the colony immediately
before testing, and only females displaying lordosis when mounted were used. Because
Fos expression in the pBST and MeApd of male rats is greatly increased after ejaculation,
but this increase does not necessarily occur after mating bouts that do not include
ejaculation (Coolen et al., 1997, 1998; Veening and Coolen 1998), male prairie voles
interacting with receptive females were screened until two groups were ﬁlled: males that
mated to ejaculation within the 30-min test (EJAC; n = 8) and males that mated but did
not ejaculate (NON EJ AC; n = 8). Previous work demonstrated that up to one third of the
sexually inexperienced males in the colony ejaculate during a 30-min sexual behavior test
(Northcutt and Lonstein, 2008). After eight NON EJAC males were obtained, the
subsequent males that were screened but did not ejaculate within 30 min were

unnecessary, so were not sacriﬁced or included in the experiment.

62

(41'1"!

lo a:

U-

”I;

l? Iv

.31.

IL; 1

ill j

\J“

Group 3: pups

To examine the effects of other unfamiliar social stimuli on Fos expression, a group of
parentally na'r've adult virgin males was allowed to interact with two 2—3 day old
conspeciﬁc pups (PUPS; n = 9). Pups were taken from breeding pairs in the colony just
prior to testing, and were wiped clean with distilled water before being placed in the
testing cage with the subject for 30 min. One subject attacked the pups, so testing was

terminated and this male was not included in the study (ﬁnal n = 8).

Group 4: familiar female sibling

To control for the effects of general social interaction on Fos expression, a fourth group
of males interacted for 30 min with a familiar female sibling that he had been housed
with until 3 hours before testing (SIBLING; n = 9). Female prairie voles are induced
ovulators and do not come into behavioral estrus unless exposed to an unfamiliar male
(Carter et al., 1980, 1987b). Incest is avoided in this species because females do not sniff
the anogenital region of their familiar male siblings (Carter et al., 1980), which prevents

estrus induction, so expectedly none of these males attempted to mate with his sibling.

Group 5: alone control

To determine the effects of handling and habituation to a clean cage and the testing room,
these males were removed from the colony but remained alone in the testing cage and
room (ALONE; n = 8). The cage lid was removed and a hand placed into the cage after

the 3 h habituation, and again 30 min later, to mimic inserting and removing a social

stimulus.

63

 

f4»

" l

(.1

«EVA.

\\

Group 6: colony control
To determine baseline Fos expression in socially housed males, these subjects were
removed from their home cages in the colony room, immediately overdosed with

anesthetic, and perfused (COLONY; n = 8).

All behavioral interactions for the socially stimulated groups were continuously
recorded using a Panasonic low-light-sensitive camera connected to a Panasonic VCR
(Panasonic, Secaucus, NJ, USA). Sexual behaviors, paternal behaviors, social contact,
and non-social behaviors (including feeding, drinking, and exploring) were scored
similarly to what the laboratory has previously described (Lonstein 2002; Northcutt and
Lonstein 2008).

One hour after the stimulus was removed, or 4.5 h after ALONE males were
placed in the novel cage, subjects were overdosed with an anesthetic cocktail containing
ketamine (62.5 mg/kg; Butler, Dublin, OH, USA), xylazine (7.5 mg/kg; Butler), and
acepromazine (0.8 mg/kg; Butler). They were perfused transcardially with 100 ml of
0.9% saline and 100 ml of 4% paraformaldehyde in 0.1 M sodium phosphate buffer.
After perfusion, brains were postﬁxed overnight in 4% paraformaldehyde, and then
cI'y0protected in a 20% sucrose solution for at least 2 days. Brains were sectioned into
35-um coronal sections on a freezing microtome and stored in cryoprotectant at —20 °C
until further processing. One series containing every other section throughout each brain

Was processed immunocytochemically for both TH and Fos.

o4

1'— .
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r L:

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last

'I
slid

T

I
11.

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I

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If]

F as and TH dual immunocytochemistry

Sections were rinsed three times in Tris-buffered saline (TBS; pH 7.6) between
each step. First, sections were incubated in 0.1% sodium borohydride for 15 min, and
then incubated in 1% hydrogen peroxide and 0.3% Triton X-100 in TBS for 10 min.
Sections were blocked in 20% normal goat serum (NGS) and 0.3% Triton X-100 in TBS
for 30 min before being placed in rabbit anti-Fos polyclonal primary antiserum (1 :10,000;
cat. # sc52; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in 2% NGS and 0.3%
Triton X-100 in TBS at 4 °C for 48 h. Next, sections were incubated in a biotinylated
goat anti-rabbit secondary antiserum (1:500; Vector Laboratories, Burlingame, CA, USA)
in 2% NGS and 0.3% Triton X-100 in TBS for 1 h, followed by a l-h incubation in
avidin—biotin complex (Vectastain Elite, Vector Laboratories). Fos was visualized by
incubating sections in 3,3 ’-diaminobenzidine (Sigma) in TBS, which produced a brown
nuclear reaction product. Brains were then blocked again in 20% NGS for 30 min, and
were incubated in a mouse anti-TH monoclonal primary antiserum (1:2000; cat. #
MAB318; Chemicon, Temecula, CA, USA) in 2% NGS and 0.3% Triton X-100 in TBS
at room temperature overnight for ~18 h. Sections were then incubated in goat anti-
mouse secondary antiserum (1:500; Vector Laboratories) in 2% NGS and 0.3% Triton X-
100 in TBS for 1 h, followed by a l-h incubation in avidin—biotin complex. TH was
visualized with Vector SG (Vector Laboratories) diluted in TBS, which resulted in a blue
cytoplasmic reaction product. Cross-reactivity was not expected, given that the primary
antisera were obtained from different species, and visual inspection of the tissue revealed
that numerous single-labeled cells of both types were clearly visible in many brain areas.

1mmunocytochemical controls that included omitting one or both of the primary and

65

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11.»;-

MI
Ji.‘

n,
\a
li)

{ﬂ
"Ml

secondary antisera eliminated the corresponding speciﬁc labeling. After processing, brain
sections were mounted onto slides, dehydrated, and coverslipped. Two

immunocytochemical runs were performed, with a similar number of subjects from each

group represented in each run.

Analysis of F as and T H-ir cells

Slides were randomized and coded for analysis. Sections were analyzed by a
single observer blind to subject condition (K.V.N.). The number of Fos-ir, TH-ir, and
dual-labeled cells was counted in each site examined (see below). All cells expressing TH
were counted at 200X magniﬁcation using a Nikon E400 light microscope (Nikon Inc.,
Melville, NY, USA) with the aid of a reticle in one ocular lens. A cell was counted if it
contained any blue cytoplasmic labeling. Fos-expressing cells were also examined at
200X magniﬁcation, and included in the analysis if they contained brown nuclear
labeling. To determine the number of dual-labeled cells, TH-ir cells were re-examined at
400X magniﬁcation and were considered to also contain Fos immunoreactivity if they
clearly contained a brown nuclear label surrounded by a blue cytoplasmic label.

Cells were examined bilaterally from four consecutive sections containing the
pBST (roughly corresponding to Swanson's plates 21—22; Swanson, 1998) and from
seven sections containing the MeApd (Swanson's atlas plates 28—30). To determine if the
effect of social stimulation on IEG expression within TH-ir cells of the pBST and MeApd
was unique, numerous other forebrain regions were examined where TH-ir cells are
found in prairie voles and other rodents (Bjorklund and Dunnett 2007; Lansing and

Lonstein 2006; Smeets and Gonzalez 2000). This involved bilaterally examining two

66

sections containing the anteroventral periventricular nucleus of the preoptic area (AVPV)
and ventrolateral region of the anteroventral preoptic area (AVP) (Swanson's atlas plate
19), periventricular anterior hypothalamus (PVa; atlas plate 24), zona incerta (ZI; atlas
plate 28), and arcuate nucleus of the hypothalamus (ARH; atlas plate 30). For all regions,
cells were counted from the entire area containing TH-ir cells, with the size of the area

examined remaining constant across all subjects.

Statistical analyses

Behavioral differences between EJAC and NON EJAC males were compared
with unpaired t-tests. The duration of time that socially stimulated groups spent with their
stimulus was compared with a one-way ANOVA, followed by Fisher's LSD post-hoc

tests to compare differences between individual groups.
To analyze TH and IEG expression in each brain site, the Shapiro—Wilk test was
used to determine whether the data were normally distributed. The TH immunoreactivity
data from every site were normally distributed, so one-way ANOVAs were used to
compare groups in the number of TH-ir cells. The IEG measures were not normally
distributed, so data were subjected to a square root transformation, and normality was
assessed again. If the transformation resulted in a normal distribution, one-way ANOVAs
were used to analyze the transformed data. This was the case for Fos immunoreactivity in
the AVP, ZI, and ARH, the number of cells containing both TH immunoreactivity and
Fos immunoreactivity in the pBST, MeApd, AVP, PVa, and ZI, and the percentage of
‘H-ir cells containing Fos immunoreactivity in the pBST, MeApd, AVP, PVa, and ZI. If

rese omnibus ANOVAS were signiﬁcant, F isher's LSD post-hoe tests were then used to

67

V
LL.

rrr

 

”n-
L\

.1
o

n;

compare differences between individual groups. If square root transfermation did not
result in a normal distribution, Kruskal—Wallis tests were used to compare treatment
groups, followed by Mann—Whitney tests to determine which individual groups differed
from each another. This was the case for Fos immunoreactivity in the pBST, MeApd,
AVPV, and PVa, the number of dual-labeled cells in the AVPV and ARH, and the
percentage of TH-ir cells expressing Fos in the AVPV and ARH. Pearson's r was used to
correlate the percentage of TH-ir cells expressing Fos in the pBST and MeApd with the
latencies, frequencies, and durations of sexual behaviors displayed by EJAC and NON
EJ AC males, paternal behaviors in PUPS males, and total duration of all social behaviors
in males tested with a stimulus animal. Based on the previous studies (Coolen et al.,
1997, 1998; Veening and Coolen 1998), EJAC males were expected to have greater IEG
expression than NON EJAC males, so one-tailed post-hoe tests were used to compare
IEG expression between these two particular groups (signiﬁcance indicated by P S 0.10).
In all other cases, statistical signiﬁcance was indicated by P S 0.05. Tissue from the
AVPV and AVP of one NON EJAC male was damaged and could not be analyzed,

resulting in a sample size of seven subjects for this group for these sites.

Experiment 1b: effects of 3 0-min social interactions on E gr-I expression in T H-ir cells
Egr-1 and TH immunocytochemistry and analysis

Because mating to ejaculation was the most effective stimulus for eliciting Fos in
many, but still not most, TH-ir cells in the pBST and MeApd (see Results), an additional
eXperiment determined if mating-induced modulation of even more TH-ir cells could be

reﬂected by expression of another IEG, Egr-l. As noted above, Egr-l is expressed in

68

I

many brain sites after the display of social behaviors (Numan et al., 1998; Polston and
Erskine 1995; Wersinger and Baum 1996), including in cells that do not express Fos. To
do this, the alternate series of brain sections for ALONE, SIBLING, NON EJAC, and
EJ AC males from Experiment Ia were examined. Egr-l and TH immunocytochemistry
was performed identically to that described above for Fos and TH, except that the Fos
primary antiserum was replaced with a rabbit polyclonal primary antiserum against Egr-l
(121000; cat. # sc189; Santa Cruz Biotechnology). Immunocytochemical controls that
omitted the primary or secondary antisera abolished speciﬁc labeling. The number of
cells containing TH immunoreactivity, the number containing Egr-l immunoreactivity,

and the number containing both TH immunoreactivity and Egr-l immunoreactivity were

counted bilaterally from the two sections through the pBST and three sections through

the MeApd that contained the most TH-ir cells in these sites.

Statistical analyses

Statistical analyses of the number of TH-ir cells, the number of Egr—l-ir cells, and
the percentage of TH-ir cells also expressing Egr-l were performed as described for
Experiment la. The number of TH-ir cells among groups was normally distributed, so
one-way ANOVAs were used to analyze these data. The number of Egr-l-ir cells was
normally distributed after square root transformation of the data, so one-way ANOVAs

were performed on these transformations. All other data sets were not normally
distributed even after square root transformation, so Kruskal—Wallis analyses were used
to compare groups. Some tissue processed for TH and Egr-l was damaged during

immunocytochemical processing, resulting in ﬁnal sample sizes for the pBST of ALONE

69

___—ll=:

and SIBLING n = 8, NON EJAC n = 6, EJAC n = 5 and for the MeApd of ALONE n = 8,

SIBLING n = 6, NON EJAC n = 5, and EJAC n = 6.

Experiment 11: effects of 6 or 24-h social interactions on F as expression

Prolonged bouts of mating up to 24 h in duration are typically necessary to induce
partner preferences in male prairie voles (Insel et al., 1995; Winslow et al., 1993). To
determine if prolonged sexual interactions could induce persistently high numbers of TH-
ir cells expressing Fos in the pBST and MeApd that might reﬂect establishment of a pair
bond, sexually naive male prairie voles were exposed to sexually receptive females or a
control stimulus for 6 or 24 h. Males were placed in a novel cage and moved to a testing
room as described in Experiment Ia. After a 3-h habituation, one group of males
interacted with a sexually receptive female (MATED; 6 h: n = 12, 24 h: n = 9).
Receptivity in females was induced and veriﬁed before testing as described in
Experiment Ia. Another group of males interacted with a familiar female sibling
(SIBLING; 6 h: n = 8; 24 h: n = 7). Behavioral interactions were recorded at 5:1 time-
lapse using a Panasonic video recorder, and were viewed to ensure that males mated with
sexually receptive females within 2 h after the beginning of testing, but did not mate with
their siblings. It was observed that ﬁve males in the MATED groups (four 6-h males, and
one 24-h male) failed to mate in the ﬁrst 2 h, and were excluded from the study, but the
remaining males began mounting the stimulus female within 40 min of the beginning of
the test (ﬁnal n = 8 for both time points). None of the SIBLING males attempted to mate

with their sisters. A third group of males remained alone in the observation cage

(ALONE; 6 h: n = 7, 24 h: n = 8), but had the cage lid removed and a hand placed inside

70

the cage after habituation to imitate inserting a stimulus animal. In the 6-h groups, 6
hours aﬁer the stimulus was introduced to the cage, it was removed (or a hand was placed
into the cage for the ALONE group) and the male remained alone in the cage for l h
before perﬁjsion. In the 24-h groups, the male subject was perfused 24 h after the
stimulus was placed in the cage. Tissue collection, processing, and analysis, as well as
staixsﬁxcal analyses, were identical to that described in Experiment Ia. TH
immunoreactivity data were normally distributed, so one-way ANOVAs were used to
analyze these data. All other data were not normally distributed, even aﬁer square root

transformation, so Kruskal—Wallis tests were used instead.

Results
Experiment Ia: F as and TH expression after 30-min social interactions
Behavior
All males in the NON EJAC and EJAC groups mounted the stimulus female with
a latency and frequency that did not signiﬁcantly differ between the two groups (latency
to mount: NON EJAC = 346 :t 78 s, EJAC = 182 i 35 s, t(14) = —1.93, P = 0.075;
frequency of mounts: NON EJAC = 19 i 6, EJAC = 34 i 10, t(14) = 1.36, P = 0.194).
However, EJAC males displayed more thrusting bouts than did NON EJAC males (110 i
19 vs. 30 i 13, t(14) = 3.50, P = 0.004). Males in the EJAC group ejaculated a mean of
1.9 i 0.3 times, with the ﬁrst ejaculation occurring 389 :t 182 s after the ﬁrst mount and
383 i 182 s aﬁer the ﬁrst thrust.

All males in the PUPS group licked and hovered over pups. Males spent 777 :1: 72

slick'mg pups, and began licking 64 i 27 s after the beginning of testing. They spent

\ 71

1061 :b l 56 s huddled over pups, with an average latency of 99 2|: 20 s after they began
licking. Males retrieved pups an average of 2 :t 1 times, but 37% of males did not retrieve
at all, which was not surprising given that prairie voles rarely retrieve pups (see Lonstein
and De Vries, 1999a).
The duration of time that males spent in physical contact with their stimulus
(mduding the durations of snifﬁng, grooming, and huddling) signiﬁcantly differed
among groups (F (3, 29) = 4.47, P = 0.011), and was greatest in the PUPS group (1207 i
133 s). This was signiﬁcantly more time spent with the stimulus than the duration of time
that SIBLTNG and EJAC males spent in physical contact with their stimulus females
(STBLTNG: 682 i 137 s, EJAC: 725 :l: 53 s), but it did not differ from NON EJAC males
(951 :t 103 s). SIBLING, NON EJAC, and EJAC males spent a similar amount of time in

contact with their respective stimuli.

TH expression
No signiﬁcant differences were found between groups in the number of TH-ir
cells in the pBST, MeApd, AVPV, AVP, PVa, or 21. Unexpectedly, EJ AC and SIBLIN G
males had signiﬁcantly more TH-ir cells in the ARH (by approximately 30%) than the

number of TH-ir cells found in the ARH of ALONE and NON EJ AC males (Table 3.2).

72

Table 3.1. The number of cells expressing TH and the number of dual-labeled cells
(Mean :1: SEM) in the pBST and MeApd after males interacted with one of several social

stimuli for 30 min. Groups with any of the same superscript letters do not signiﬁcantly
differ from each other.

# of TH-ir cells # of Dual-labeled cells

 

 

pBST COLONY 154 :t 20 4 :1: 1b
ALONE 184 i 31 1 1 0a
SIBLING 175421 441b
PUPS 143426 21:1b
NON EJAC 167 :t 22 5 1 1b
EJAC 169i 12 9420
F(5, 43) 0.425 5.72
p 0.829 <0.001
MeApd COLONY 336 i: 33 9 2: 3b°
ALONE 330 i 33 1 4 1a
SIBLING 334 i: 27 13 :t 3"d
PUPS 343 :t 52 4 a: 1ab
NON EJAC 289 :t 34 6 i 2bc
EJAC 333 i 42 23 :1: 6d
F(5, 43) 0.278 7.26
p 0.926 <0.001

 

73

Table 3.2. The number of cells expressing TH, Fos, both Fos and TH, and the
percentage of TH-ir cells also expressing Fos (Mean 4 SEM) after subjects interacted
with one of several social stimuli for 30 min. Groups with any of the same superscript
letters do not signiﬁcantly differ from each other.

 

 

 

% of TH-ir
cells
# of TH-ir # of Fos-ir # of Dual- expressing
cells cells labeled cells Fos
b Ta
AV?V COLONY 342 4 15 25 4 9a 3 +- 1 1 4 0
ALONE 276418 1846a 140a 04021
b
SIBLING 293 4 29 19 4 2a 1 4 0a 0 4 0ab
PUPS 297435 125423b 341b 140bc
NON EJAC 293 427 181440b 743b 241bc
EJAC 277419 3734 57c 742b 3 41c
F(5, 42) =
0.916 H(5) = 35.72 H(5) = 12.33 H(5) = 13.40
p 0.480 <0.001 0.031 0.020
a
AVP COLONY 185415 95i22 643 341
a
ALONE 166418 114416 241 140
SIBLING 176422 1144153l 341 241
PUPS 188415 384445C 741 441
b
NON EJAC 163 4 25 242 4 46 3 4 1 2 4 1
be
EJAC 202418 314448 642 241
F(5, 42) 0.551 11.85 1.54 1.58
p 0.736 <0.001 0.199 0.186
T ab a a
Na COLONY 508419 117449 542 140
b
ALONE 449 4 30 92 4 Isa 5 41a 14 0a
b b d b
SIBLING 498 4 20 162 4 26 8 4 1 c 2 4 0a c
d be b
PUPS 477 4 37 472 4 58c 7 4 1a 1 4 02‘
d be
NONEJAC 430431 410444c 1042c 240
d
EJAC 480427 576491 1443d 3 41C
F(5, 43) = H(5) = 30.84 F(5, 43) = F(5, 43) =
1.138 4.03 3.95
p 0.355 <0.001 0.004 0.005

74

Table 3.2 (cont’d)

 

 

% of TH-ir
cells
# of Fos-ir # of Dual- expressing
# of TH-ir cells cells labeled cells Fos
21 COLONY 732 4 53 172 4 38a 20 4 5a 3 4 1a
ALONE 586 4 58 208 4 27a 21 4 4a 4 4 1a
b
SIBLING 585 4 46 242 4 17a 24 4 6a 4 4 la
b b
PUPS 647429 347441b 4443 741
NON EJAC 575 4 41 362 4 62b 37 4 4b 7 4 1b
EJAC 6104 33 266444ab 244 5a 441a
F(5, 43) 1.800 3.08 4.50 5.60
p 0.133 0.004 0.002 0.001
abc a
ARH COLONY 194:1:19 31:1:7 1i] liO
ALONE 152415a 66412ab 341 241
ab
SIBLING 230412c 62413 241 141
PUPS 208:1: 12bc 762t10b 3:1: 1 1:1:0
b b
NON EJAC 17742521 105425 643 342
b
EJAC 2404200 103 426 743 341
F(5, 43) = 3.51 F(5, 43) = 2.83 H(5) = 3.98 H(5) = 3.94
p 0.010‘ 0.027 0.552 0.558

 

75

F as expression
In the pBST and MeApd, as well as the AVPV and PVa, the number of Fos-ir
cells signiﬁcantly differed among groups of males (Figure 3.1, Table 3.2; pBST, H(5) =
39.96, P < 0.001; MeApd, H(5) = 38.46, P < 0.001; AVPV, H(5) = 35.72, P < 0.001;
PVa H(5) = 30.84, P < 0.001). In the pBST, MeApd, and AVPV, EJAC males had 15—18
tunes more Fos-ir cells than ALONE, SIBLING, and COLONY males, while NON EJAC
and PUPS males had approximately eight times more Fos-ir cells than these three control
groups. In the PVa, EJAC and PUPS males had approximately ﬁve times more Fos-ir
cells, and NON EJAC males had approximately four times more Fos-ir cells, than that

found in the three control groups.

For the other forebrain sites examined, males exposed to any novel social
stimulus (EJAC, NON EJAC, and PUPS groups) generally had more Fos-ir cells
compared to SIBLING, ALONE, and COLONY control males, with the pattern of group

differences in Fos expression different in each brain site. Notably, the pattern of Fos
expression seen in the pBST, MeApd, AVPV (i.e. EJAC males having more Fos-ir cells
than all other groups) was not found in any of the other brain sites that were examined

(see Table 3.2).

76

‘. - In” ‘1 ll

 

'1.
«Km:

4.31.31

(,5. 7.
j .

1600 -1

d
pBST - 30 min

1400 -

 

1 200 -
1 000

l

800 -
600 -

400 -
200 -

 

colony alone sibling pups non ejac ejac

3000 l

MeApd - 30 min d

 

2500 -
2000 -
1500 l

1000 4

# Fos-ir cells

500 - b
3 3b

 

 

 

 

 

 

colony alone sibling pups non ejac ejac

Figure 3.1. Number of Fos-ir cells (Mean 4 SEM) in four sections through the pBST and
seven sections through the MeApd aﬁer male subjects interacted with one of several

Stimuli for 30 min. Bars with any of the same letters above them do not signiﬁcantly
d1ffer from each other.

77

...\

 

1‘
4n
.
.
.

 

 

Fos expression within TH-ir cells

The percentage of TH-ir cells in the pBST and MeApd that also expressed Fos
signiﬁcantly differed among groups (Figure 3.2 and Figure 3.3; pBST, F(5, 43) = 5.44, P
= 0.001; MeApd, F(5, 43) = 7.29, P < 0.001). In both sites, EJAC males had a greater
percentage of TH-ir cells expressing Fos (~6% of all TH-ir cells) than did ALONE,
PUPS, and NON EJAC males. This pattern was unique, because EJAC males did not
have a greater percentage of TH-ir cells expressing Fos than NON EJAC males in any of
the other forebrain regions analyzed, including the AVPV and PVa (see Table 3.2). In
addition, the percentage of TH-ir cells in the pBST and MeApd also expressing Fos was
higher in all groups exposed to an adult social stimulus (COLONY, SIBLING, NON
EJAC, and EJAC males) when compared to ALONE males.

The number of dual-labeled cells in the pBST and MeApd (Table 3.1) showed a
very similar pattern as the percentage Of TH-ir cells expressing Fos, with EJAC males
having signiﬁcantly more dual-labeled cells than all other groups in the pBST and more

than the COLONY, ALONE, PUPS, and NON EJAC males in the MeApd.

78

 

co
4]

pBST - 30 min

 

°/o TH-ir cells expressing Fos
.h

 

o _
colony alone sibling pups non ejac ejac

 

(D

E 8, MeApd-30 min d
U}

.E ‘
3’)

<1: 6‘

E-

Q

5‘:

2 4‘

'0')

o l .

'; 24 ab

I- a

o\° oJ

 

 

colony alone sibling pups non ejac ejac

Figure 3.2. Percentage of TH-ir cells expressing Fos (Mean :t SEM) in the pBST and
MeApd after male subjects interacted with one of several stimuli for 30 min. Bars with
any of the same letters above them do not signiﬁcantly differ from each other.

79

 

 

 

 

PBST ,1 .J , J _
.y, 7‘ ‘ ‘ ‘ " ‘ 9
~ , , . ‘ ~ . .
‘ .' Q .I ‘4 i I -.
Q // I \ " /
1‘ I. ’l - \
it 4’ . / Y / . "
liLoNE no»: EMF: .1” ‘
lMeAW 7.. ' R I l
I ‘ “y-
r .> L .I ‘0 ‘I . ‘ ' ‘ .4 ,
I‘ I I g ‘ ‘_ ' I ‘4‘;
..- t . , V ‘ . ‘
.v k ,. i 3»;
.g y 2 . § 0 . .
ALONE s. __ you EJAC '\ L EJAC 5 "I 1 .4 ,*‘<,

 

 

 

 

 

Figure 3.3. Photomicrographs of the pBST (top row) and MeApd (bottom row) from
representative males in the ALONE, NON EJAC, and EJAC groups in Experiment Ia.
Small arrows in top left panel indicate cells that are single-labeled with TH-
immunoreactivity (blue cytoplasmic label). Large arrows in top middle panel indicate
cells that are single-labeled with Fos-immunoreactivity (brown nuclear label). Dual-
labeled cells are highlighted at higher magniﬁcation in the two panels showing EJAC
males. Scale bar in bottom left panel = 50 um. Scale bar in bottom right panel inset = 10
um.

80

Correlations between the percentage of TH-ir cells expressing F as and behavior

The percentage of TH-ir cells expressing Fos in the pBST and MeApd was
signiﬁcantly correlated with the expression of numerous sexual behaviors (see Figure 3.4,
Table 3.3). In the pBST, the percentage of TH-ir cells expressing Fos was positively
correlated with the number of mounts, number of thrusting bouts, number of ejaculations,
duration of time spent mounting, and total time engaged in all sexual behaviors. In the
MeApd, the percentage of TH-ir cells expressing Fos was positively correlated with the
number of thrusting bouts, duration Of time spent mounting, duration Of time spent
thrusting, and total amount of time engaged in sexual behaviors. The percentage of TH-ir
cells in the pBST that expressed Fos was also positively correlated with the latency to
lick pups in PUPS males, but there were no signiﬁcant correlations between the

percentage of TH-ir cells expressing Fos in the MeApd and any paternal behaviors.

81

1, 1 MEApd

10«

 

   
   

 

r(14)=.583,p=.016 2 . r(14)=.564,p=.021

% TH-lr cells expressing Fos

 

 

 

 

   
  

 

 

 

I O
0 . T % 4 4 4 . o, . c.’ 4 4 . .
0 100 200 300 400 500 0 100 200 300 400 500
time mounting without thrusting (s) time mounting without thrusting (s)
'1’ 12
.2 1 .
g 10 «
'8
2 3 ‘
1%
o 5 ‘
£
'6 4 ‘
3 2
'17 ' r114) = 493110 = -051 2 414) = .610, p = .011
1- I 0
32 0 . . e, . . 4 0 T f . . .
0 50 100 150 200 250 0 50 100 150 200 250
time thrusting (s) time thrusting (s)
0 _
'2 12 .
3’ 10 < '
'3
a 8 *
a
5 6~
2
3 4 .
1: ‘ o
g 0 _ 1114) = .573, p = .019 2 ' r114) = .601, p = .012
0

32 o - 4 0 4L . 4

 

 

 

 

0 100 200 300 400 500 600 700 0 100 200 300 400 500 600 700

total time engaged in sexual behavior (3) total time engaged in sexual behavior (3)

Figure 3.4. Correlations between the percentage of TH-ir cells in the pBST (left) or
MeApd (right) that also expressed Fos and the duration of time spent mounting and
thrusting by EJAC males (squares) and NON EJAC males (circles) during a 30-min test
With a sexually receptive female. Time engaged in male sexual behavior (bottom row) is
the summed duration of mounting, thrusting, and ejaculating. See Table 3.3 for

gonelations between other behaviors and the percentage of TH-ir cells that also expressed
08.

82

Table 3.3. Correlations between the percentage of TH-ir cells expressing Fos and the
expression of sexual behavior, paternal behavior, and all social behaviors during 30-min
interactions with a sexually receptive female, pups, or a familiar female sibling. See
Figure 3.4 for additional correlations associated with the duration of sexual behaviors.

 

 

 

 

 

 

% TH-ir cells in pBST % TH-ir cells in
expressing Fos MeApd expressing Fos
Sexual behaviors
Latency (s)
Mount -.481 -.184
Thrust from ﬁrst mount -.063 .112
Ejaculation from 1St thrust .469 .434
Frequency
Mounts .515* .473
Thrusting bouts .517* .646**
Ejaculations .516* .410
Paternal behaviors
Latency (s)
Lick pups .753* -.328
Huddle from ﬁrst lick -.112 .390
Frequency
Retrieve pups -. 160 .210
Duration (3)
Lick pups -.390 .537
Huddle over pups -.309 .016
Time in contact with stimulus (s) .141 .116
* p S 0.05, ** p S 0.01

83

Experiment lb: Egr-l expression after 3 0-min social interactions

TH expression
The number of TH-ir cells in the pBST or MeApd did not differ among groups

(pBST, F(3, 23) = 0.119, P = 0.948; MeApd, F(3, 21) = 0.915, P = 0.451) (Table 3.4).

Table 3.4. The number of cells expressing TH, and the number of cells containing both
TH and Egr-l (Mean 4 SEM) in two sections through the pBST and three sections
through the MeApd after males interacted with one Of several stimuli for 30 min. Groups
with any of the same superscript letters do not signiﬁcantly differ from each other.

# of TH-ir cells # of Dual-labeled cells

 

 

a
PBST ALONE 88 4 6 1 4 0
b
SIBLING 82 4 10 4 4 1
b
NONEJAC 92418 341
b
EJAC 87 4 16 9 i 4
F(3, 23) = 0.119 H(3) = 10.31
p 0.948 0.016
a
MeApd ALONE 113 4 12 1 2t 0
b
SIBLING 80 4 12 8 4 3
b
NONEJAC 112419 741
b
EJAC 97 4 22 13 4 4
F(3, 21) = 0.915 H(3) = 15.04
p 0.451 0.002

 

84

Egr-I expression

All groups of males had more Egr-l-expressing cells in the pBST and MeApd
than the number of Fos-expressing cells they had in Experiment Ia. In both sites, Egr-l
expression differed among groups (Figure 3.5; pBST, F(3, 23) = 53.52, P < 0.001;
MeApd, F(3, 21) = 26.11, P < 0.001), with all groups being signiﬁcantly different from
one another. SIBLING males had approximately 1.5 times the number of Egr-l-ir cells
than did ALONE males, and NON EJAC males had almost twice the number found in
ALONE males. EJAC males had signiﬁcantly more Egr-l expressing cells than all other

groups, including 1.5 to two times more than that seen in NON EJAC males.

85

# Egr-1-ir cells

 

 

alone sibling non ejac ejac

40001 MeApd

# Egr-1-ir cells

 

alone siblingnon ejac ejac

Figure 35. Number of Egr-l-ir cells (Mean 4 SEM) in two sections of the pBST and
three sections through the MeApd after male subjects interacted with one of several
stimuli for 30 min. Bars with any of the same letters above them do not signiﬁcantly
dlffer from each other.

86

Egr-I expression within TH-ir cells

The percentage of TH-ir cells in the pBST and MeApd that also contained Egr-l
immunoreactivity signiﬁcantly differed among groups (Figure 3.6; pBST, H(3) = 9.58, P
= 0.023; MeApd, H(3) = 18.96, P < 0.001). In these regions, EJAC males had nine and
23 times the percentage of TH-ir cells expressing Egr-l than did ALONE males and
twice that of NON EJAC males. Notably, the total percentage of TH-ir cells in these sites
that also contained Egr-l was higher than the percentage that contained F 03 in
Experiment 1a, but it was still quite moderate (up to ~10%—15% Of all TH-ir cells). The
raw number of dual-labeled cells also differed among groups in both sites (Table 3.4),

with a generally similar pattern as the percentage of TH-ir cells that were dual-labeled.

Correlations between the percentage of T H—ir cells expressing Egr-l and behavior

The percentage of TH-ir cells in the pBST that also contained Egr-l
immunoreactivity was not signiﬁcantly correlated with any measure of males' sexual or
other social behaviors. In the MeApd, the percentage Of TH-ir cells expressing Egr-l was
positively correlated with the number of ejaculations (r(9) = 0.853, P < 0.001) and

negatively correlated with the latency to ejaculate after the ﬁrst thrust (r(4) = -0.814, P =

0.049).

87

18-

pBST

14-
12-

% TH-ir cells expressing Egr-1
8

 

 

alone sibling non ejac ejac

MeApd %

% TH-ir cells expressing Egr—1

 

alone sibling non ejac ejac

Figure 3.6. Percentage of TH-ir cells (Mean 4 SEM) in the pBST and MeApd that also
expressed Egr-l after male subjects interacted with one of several stimuli for 30 min.
Bars with any of the same letters above them do not signiﬁcantly differ from each other.

88

Experiment 11: F os expression after 6- or 24-h social interactions
TH expression

The number of TH-ir cells in the pBST and MeApd did not signiﬁcantly differ

among groups at either the 6- or 24-h time point (Table 3.5).

F as expression

After 6-h interactions, the number of Fos-ir cells in the pBST and MeApd
signiﬁcantly differed among groups (Figure 3.7; pBST, H(2) = 15.05, P = 0.001; MeApd,
H(2) = 14.71, P = 0.001), with MATED males having ﬁve-fold more Fos-ir cells in both
brain sites than did ALONE and SIBLING males.

Fos expression in the pBST and MeApd also differed among groups after 24-h
interactions (Figure 3.7; pBST, H(2) = 15.85, P < 0.001; MeApd, H(2) = 16.73, P <
0.001). In both brain sites, MATED males had twice the number of Fos-ir cells than did
SIBLING males, and SIBLING males had two to three times as many Fos-expressing

cells as did ALONE males.

89

Table 3.5. The number of cells expressing TH and the number of cells containing both
TH and Fos (Mean :1: SEM) in four sections through the pBST and seven sections through
the MeApd after males interacted with one of several stimuli for 6 or 24 h.

# of TH-ir cells

# of Dual-labeled cells

 

 

 

 

pBST ALONE 236 4 29 0 4 0a
6 h SIBLING 226 4 22 1 4 0a
MATED 266 4 18 6 4 3b
F(2, 20) = 0.851 H(2) = 13.27
p 0.442 0.001
pBST ALONE 221 4 19 0 4 0a
24 h SIBLING 221 4 17 4 4 2b
MATED 285 4 24 3 4 1b
F(2, 20) = 3.23 H(2) = 6.21
p 0.567 0.045
MeApd ALONE 426 4 12 0 4 0a
6 h SIBLING 378 4 25 6 4 2b
MATED 363 4 50 4 4 2b
F(2, 20) = 0.888 H(2) = 9.94
p 0.427 0.007
MeApd ALONE 387 4 18 0 4 0a
24 h SIBLING 351 4 32 5 4 3b
MATED 413432 241b
F(2, 20) = 1.31 H(2) = 6.98
p 0.292 0.031

 

90

  

 

 

   

 

 

16°° pBST - 6 h 15°“ pBST - 24 h

1400 14oo l

1200 « 1200 «
2
E 1000 « 1ooo -
0
37' 600 — coo -
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12 600 - b 600 4
* aoo — 400 ~ c

200 4 a a 200 4 a bi -

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3000 3000 1
MeApd - 6 h MeApd - 24 h

2500 2500 J
2
B 2000 2000 4
U
94' 1500 1500 ‘
in
u‘.’
:14 1000

c
500 ‘ b
a ._a_—-
o _
alone sibling mated alone sibling mated

Figure 3.7. Number of Fos-ir cells (Mean 4 SEM) in four sections through the pBST and
seven sections through the MeApd after male subjects mated, interacted with a familiar
female sibling, or remained alone for 6 or 24 h. Note that the Y-axis scales for both sites
are the same as that in Figure 3.1. Bars with any of the same letters above them do not
Signiﬁcantly differ from each other.

91

Fos expression within T H—ir cells

The percentage of TH-ir cells in the pBST and MeApd that also contained Fos
immunoreactivity signiﬁcantly differed among groups after 6-h interactions (Figure 3.8;
pBST, H(2) = 12.77, P = 0.002; MeApd, H(2) = 10.35, P = 0.006), with MATED males
having a signiﬁcantly greater percentage than that found in ALONE males, although this
percentage was very low (~2% of all TH-ir cells).

A similar pattern of Fos expression within TH-ir cells was seen after 24 h
interactions with the stimuli. The percentage of TH-ir cells expressing Fos differed
among all groups in the pBST and MeApd (Figure 3.8; pBST, H(2) = 6.40, P = 0.041;

MeApd, H(2) = 7.17, P = 0.028), but was very low (less than 2% in all groups).

92

 

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Figure 3.8. Percentage Of TH-ir cells also expressing Fos (Mean 3: SEM) in the pBST
and MeApd after male subjects mated, interacted with a familiar female sibling, or
remained alone for 6 or 24 h. Note that the Y-axis scales for both sites are the same as
that in Figure 3.2. Bars with any of the same letters above them do not signiﬁcantly
differ from each other.

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Discussion
IEG expression aﬁer sociosexual interactions

The pBST and MeApd are necessary for sexual and parental behaviors in many
rodents (reviewed in Newman 1999; Numan and Insel 2003). Consistent with this, and
similar to other studies of male prairie voles (Cushing et al., 2003; Kirkpatrick et al.,
1994b; Lim and Young 2004), there were more cells expressing IEGs in these brain sites
after male prairie voles interacted with either a sexually receptive female or pups. These
data are the ﬁrst evidence in prairie voles, though, that mating to ejaculation induces even
greater Fos and Egr-l expression in these sites than do mating bouts that do not result in
ejaculation. Similar results have been found in male rats, with Fos expression higher aﬁer
ejaculation compared to that observed after only intromissions, even when the amount Of
mating stimulation required for ejaculation is drastically reduced (Baum and Everitt
1992; Coolen et al., 1997; Veening and Coolen 1998). This suggests that some cells in
the pBST and MeApd may express IEGs only after males ejaculate and possibly
approach sexual satiety.

The effects of social interactions on Fos expression in the two rostral
periventricular regions that were examined (AVPV and PVa) were very similar to what
was found in the pBST and MeApd. This is similar to ﬁndings that Fos expression
increases in the AVPV of other rodents after mating, and particularly after ejaculation
(Simmons and Yahr, 2002). Because the AVPV receives projections from cells in the
MeApd that also express Fos after ejaculation (Simmons and Yahr, 2002), the AVPV and
Possibly the PVa may work with other sites to regulate physiological or behavioral events

occurring after ejaculation, such as hormone secretion or the sexual refractory period. Of

94

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the forebrain sites examined, only in the pBST, MeApd, AVPV, and PVa did mating to
ejaculation increase the number of cells expressing F 05 above and beyond mating without
ejaculation, so these sites may be part of a neural network regulating ejaculation-related
events in male prairie voles, as is true in other rodents. Because mating greatly facilitates
partner preference formation in male prairie voles (Insel et al., 1995; Winslow et al.,
1993), these four neural sites may be particularly inﬂuential in the formation of social

bonds with mates.

IEG expression within T H—expressing cells after sociosexual interactions

The behavioral or physiological functions of the hundreds of TH-ir cells in the
male prairie vole pBST and MeApd are unknown. Because cells containing
immunocytochemically detectable levels of TH are few or non-existent in the pBST and
MeApd of the non-monogamous species that have been examined (including meadow
voles), these cells were hypothesized to contribute to the characteristic social behaviors
of prairie voles (Northcutt et al., 2007). These data support this hypothesis, although they
further suggest that these cells may have a somewhat non-speciﬁc role in their sociality.
Interaction with any social partner maintained at least baseline levels of Fos or Egr-l
Within the TH-ir cells of the pBST and MeApd. In fact, social isolation (ALONE group)
Caused a signiﬁcant decrease in the number of TH-ir cells of these sites expressing IEGs
compared to that found in COLONY and SIBLING controls. These results highlight the
importance of including a group of completely unmanipulated COLONY controls in
Studies such as these, because this group allowed us to discover the constitutive Fos

expression in TH-ir cells of the pBST and MeApd of socially-housed prairie voles.

95

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Unexpectedly high constitutive expression of IEGs within TH-ir cells has also been found
in the male zebra ﬁnch brain, although in zebra ﬁnches the number of dual-labeled cells
decreases after social interactions rather than being maintained or increasing (Bharati and
Goodson, 2006). A similar example of high constitutive expression of IEGs is provided

by the visual cortex, where expression of these transcription factors decreases after
adaptation to the dark (Worley et al., 1991). The pBST and MeApd were the only brain
sites examined where both COLONY and SIBLING males differed from ALONE males
in Fos expression within TH-ir cells, which highlights these regions as possibly unique
among brain sites containing TH-ir cells. These cells may be part of a neural network
monitoring the ongoing status of a vole's social environment; their decreased activity
resulting from even a few hours of social isolation may increase the motivation to seek
out a social partner, or if unsuccessful, contribute to the stressful effects of social
isolation in this highly gregarious species (Grippo et al., 2007; Kim and Kirkpatrick

l 996; Klein et al., 1997).

While any social contact maintained at least baseline numbers of TH-ir cells of
the pBST and MeApd expressing IEGs, mating to ejaculation was particularly effective
in inducing further IEG expression in additional cells. The pBST and MeApd were the
only forebrain sites examined where EJAC males had more TH-ir cells expressing Fos
than NON EJAC males. As noted above, EJAC males had more Fos-ir cells in the AVPV
and PVa than did NON EJAC males, but not speciﬁcally within TH-ir cells. Thus, of the
forebrain DA-rich sites examined, IEG expression preceding or resulting from ejaculation
Was speciﬁc to the TH-ir cells of the pBST and MeApd. Furthermore, the percentage of

TH-ir cells expressing Fos in both regions was positively correlated with males' sexual

96

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behaviors, but not males' paternal behaviors or total duration of social contact. In other
rodents, the pBST and MeApd receive olfactory information from both the main and
accessory olfactory systems, and process this information in a manner that promotes the
appropriate expression Of sociosexual behaviors (eg. Newman 1999; Numan and Insel
2003). TH-ir cells in these sites might help males establish olfactory-based memory of
mates, which is necessary for the formation and maintenance of partner preferences
(Curtis et al., 2001; Kirkpatrick et al., 1994c).

Mating bouts extending up to 24 h are typically necessary for establishing social
bonds in prairie voles (Insel et al., 1995; Winslow et al., 1993). Because of this,
prolonged sexual interactions were hypothesized to be even more effective than 30-min
mating bouts in eliciting F 03 expression in TH-ir cells of the pBST and MeApd. This was
not the case, as males mating for 6 or 24 h had even less Fos expression in TH-ir cells
than did males sacriﬁced after a 30-min bout (although MATED males did have a greater
percentage of TH-ir cells expressing Fos than did ALONE males at both extended time
- points). Thus, mating elicits Fos expression in these TH-ir cells soon after initial mating
and ejaculation, but does not appear to produce chronic or repeated waves of Fos
eXpression, although it remains possible this might occur at times points that were not
investigated. TH-ir cells in the pBST and MeApd may be involved in males' display of
COpulatory behaviors and initiating the neural cascades that lead to the development of
social bonds after males ejaculate. Additional mating interactions over the following 24 h
may instead have a greater impact on downstream targets in this pathway, such as the

ACB and ventral pallidum, which may be involved in the solidiﬁcation, rewarding

qualities, and later expression of the social bond (Lim and Young, 2004).

97

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Ejaculating males had the greatest percentage of TH-ir cells in the pBST and
MeApd expressing IEGs, but it is clear that only a relatively small percentage of all TH-ir
cells in either site was dual-labeled. This is completely consistent with studies in birds
and rats investigating neural Fos expression after mating, which report that fewer than
5%—10% of TH-ir cells in any site also express Fos (Balfour et al., 2004; Bharati and
Goodson 2006; Charlier et al., 2005). In addition, it is clear that the majority of IEG-
expressing cells in the pBST and MeApd of ejaculating males do not synthesize TH and
are not DAergic. Therefore, many neurons in the pBST and MeApd of yet unknown
phenotype are also modulated in response to sociosexual interactions in male prairie
voles. If prairie voles are similar to gerbils, these other cells are probably glutamatergic

or GABAergic (Simmons and Yahr, 2003).

DA and prairie vole sociosexual behaviors

These data demonstrate that mating to ejaculation is more potent than some other
social stimuli in eliciting IEG expression in TH-ir cells of the prairie vole olfactory
extended amygdala. In addition to being convenient markers for cellular modulation
including depolarization (Hoffman and Lyo 2002; Morgan and Curran 1991), both Fos
and Egr-l can induce TH transcription, which may restore DA stores after cells have
been active (Ghee et al., 1998; GuO et al., 1998; Nakashima et al., 2003; Stefano et al.,
2006). It is possible these cells contribute to DAergic regulation of pair bonding, as D2
receptor activation is necessary for inducing partner preferences (Aragona et al., 2006).
In addition, changes in DA release from these cells may help upregulate D1 receptors in

the ACB and elsewhere after pair bond formation to prevent the formation of extra-pair

98

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bonds (Aragona et al., 2006). DAergic neurons in the VTA may be the predominant
source of DA release in the ACB during mating (Curtis and Wang, 2005), but very little
Fos expression was found in either TH-ir or non-TH-ir cells of the VTA (or the substantia
nigra; data not reported) in any group of males. This is similar to what is found in the
VTA after lactating female rats display maternal behaviors (Lonstein and Stern, 1997),
and indicates that not all DAergic cells involved in behavioral displays show increased
IEG expression. Ascending projections from the VTA to the ACB are surely involved in
DAergic control of social behaviors in prairie voles, but it is intriguing to consider that
that TH—ir cells in the pBST and MeApd may also project to the ACB and other sites (e.g.
lateral septum, ventral pallidum, MPO, each other) to modulate sociosexual behaviors in
prairie voles.

These studies provide initial insight into how TH-ir cells of the male prairie vole
pBST and MeApd are inﬂuenced by, and might inﬂuence, social interactions. DA affects
olfactory processing and increases olfactory discrimination in rats, mice, and primates
(Miwa et al., 2004; Pavlis et al., 2006; Tillerson et al., 2006; Yue et al., 2004).
Furthermore, DA is involved in learning about Olfactory stimuli (Kruzich and Grandy
2004; Rosenkranz and Grace 2002; Weldon et al., 1982), including learning about
socially-relevant odors (Comwell-Jones and Bollers, 1983). Thus, a system of DAergic
cells existing within the male prairie vole pBST and MeApd may be a unique neural
mechanism through which these animals process socially relevant olfactory information,
and then transmit this information to DA-sensitive areas of the brain necessary for social
bonding. These could include sites essential for memory, reward, and emotional

regulation. Current studies in the laboratory are examining the anatomical projections of

99

:"‘l

I
:1 (ur-
elk-Ml

TH-ir cells of the male prairie vole pBST and MeApd, which will determine if there is an

anatomical basis to support this hypothesis.

Acknowledgments
We would like to thank Drs. Lyn Clemens and Tony Nunez for the use of their
electronic video recording equipment, and Dr. Juli Wade for providing the Egr-l primary
antiserum. This research was supported by NSF grant # 0515070 to J.S.L., and an NSF

graduate research fellowship to K.V.N.

100

[niq

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063311

CHAPTER 4:
Unique catecholaminergic projections from the extended olfactory amygdala to the

medial preoptic area in male prairie voles

ABSTRACT

The principal nucleus of the bed nucleus of the stria terrninalis (pBST) and
posterodorsal medial amygdala (MeApd) are densely interconnected sites that transmit
olfactory information via parallel projections to many brain areas mediating sociosexual
behaviors in mammals. In male prairie voles (Microtus ochrogaster), the pBST and
MeApd each contain hundreds of cells intensely immunoreactive for tyrosine
hydroxylase (TH). These large populations of TH-immunoreactive (TH-ir) cells do not
exist in other rodents examined, and studies from the laboratory suggest these cells could
be part of a unique catecholaminergic network necessary for mating and pair bonding in
monogamous prairie voles. To gain information about how TH-ir cells in the prairie vole
pBST and MeApd communicate with other brain areas involved in social behaviors, and
with each other, anterograde tracing from the pBST was performed using biotinylated
dextran amine (BDA) and analyzed where these pBST projections overlapped with TH-ir
ﬁbers. Only in the medial preoptic area (MPO) and MeApd did dense plexuses of BDA-
labeled ﬁbers co-exist with TH-ir ﬁbers. To examine whether these sites receive input
from TH-ir cells of the pBST, the retrograde tracer FluoroGold was infused into the MP0
or MeApd, and the location of cells throughout the brain containing both FG-ir and TH-ir
determined. Approximately 77% of all TH-ir projections to the MPO originated from the

pBST or MeApd, and these projections involved over 40% of all TH-ir cells in the latter

101

_t\.~

two sites. The remaining TH-ir projections to the MPO originated in the
supramammillary nucleus, arcuate nucleus, anteroventral periventricular nucleus, and
posterior hypothalamus. In contrast, the MeApd received almost no input from TH-ir
cells in the pBST, and instead received most of its TH-ir input from the ventral tegmental
area (VTA). Retrograde tracing from the pBST itself revealed substantial input from TH-
ir cells of the MeApd, and some additional input from TH—ir cells in the VTA and caudal
periventricular hypothalamus. Thus, the male prairie vole brain contains a unique
catecholaminergic network involving the pBST, MeApd, and MP0. This species-
speciﬁc catecholaminergic network connecting brain sites involved in olfaction, social
behaviors, and motivation (through input from the VTA) may be essential for the

monogamous behaviors of prairie voles.

102

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Introduction

The principal nucleus of the bed nucleus of the stria terrninalis (pBST) and
posterodorsal medial amygdala (MeApd) are highly interconnected brain sites processing
chemosensory and hormonal information necessary for sociosexual behaviors in a
number of mammalian species (Coolen and Wood, 1998; Kling, 1972; Newman, 1999;
Numan and Insel, 2003; Wood and Swann, 2005). The chemical phenotype of neurons in
the pBST and MeApd is heterogeneous, and includes cells expressing GABA, glutamate,
vasopressin, cholecystokinin, galanin, opioids, and substance P (De Vries et al., 1984;
DeVries et al., 1985; Malsbury and McKay, 1989; Miller et al., 1993; Muganini and
Oertel, 1985; Poulin et al., 2009; Simerly and Swanson, 1987; Simmons and Yahr, 2003;
Swarm and Newman, 1992). In male prairie voles (Microtus ochrogaster), the pBST and
MeApd also contain hundreds of neurons intensely immunoreactive for tyrosine
hydroxylase (TH), and because these TH-immunoreactive (TH-ir) cells do not contain
dopamine-B-hydroxylase (Northcutt et al., 2007), they could be dopaminergic (DAergic).
These large populations of densely immunoreactive TH-ir cells do not exist in the pBST
and MeApd of any non-monogamous rodents examined to date (Albanese et al., 1986;
Kalsbeek et al., 1992; Northcutt et al., 2007), and this species difference has led us to
suggest that these cells may inﬂuence species-speciﬁc social behaviors in prairie voles,
including high gregariousness, pair bonding, and biparental care (Northcutt and Lonstein,
2009; Northcutt et al., 2007).

Recently, social interaction with familiar conspeciﬁcs was found to maintain
immediate-early gene (IEG) expression in TH-ir cells of the male prairie vole pBST and

MeApd, and that the number of cells expressing IEGs is even further increased by mating

103

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and ejaculation (Northcutt and Lonstein, 2009). These results suggest that TH-ir cells in
the pBST and MeApd may be part of the neural networks required for high levels of
afﬁliation in prairie voles, as well as for mating and its consolidation of pair bonds.
Although IEG experiments provide insight into the possible ﬁmction of these cells, the
absence of anatomical information about this system’s placement within the brain’s
social behavior network prevents a more comprehensive understanding of their potential
roles (Young et al., 2008; Young et al., 2005).

The anatomical projections of the pBST and MeApd have been well studied in
laboratory rats, hamsters, and mice. They have tremendous reciprocal projections to each
other and a very similar termination pattern throughout the rest of the brain (Coolen and
Wood, 1998; Dong and Swanson, 2004; Gomez and Newman, 1992; Gu et al., 2003;
Usunoff et al., 2009; Wood and Swann, 2005). In the forebrain, both sites have
prominent projections to the medial preoptic area (MPO), medial hypothalamus, lateral
septum (LS), and locally within the BST and amygdala. The MP0 and LS are implicated
in social behaviors in many rodents (Clarke and File, 1982; Heimovics et al., 2009;
Korzan et al., 2006; Sheehan et al., 2004), including prairie voles; IEG expression in the
MPO increases dramatically alter male prairie voles mate or interact with pups (Lim and
Young, 2004) and the LS is involved in their pair bonding and parenting (Liu et al.,
2001). The TH-ir cells in the prairie vole pBST and MeApd may provide a unique
catecholaminergic input to the MP0 or LS, and may also contribute novel projections to
areas of the social behavior network that receive no or only sparse innervation from the

pBST and MeApd in other rodents. One intriguing possibility is that these cells terminate

104

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in the nucleus accumbens (ACB), where DA release and D2 receptor activity is required
for pair bonding in male prairie voles (Aragona et al., 2003; Aragona et al., 2006).

In the experiments presented here, biotinylated dextran amine (BDA) was used to
ﬁrst determine the efferent projections of the male prairie vole pBST, and visually
compare those projections with the distribution of TH-ir ﬁbers in their brain. The pBST
was chosen for anterograde tracing because in other rodents it projects to all of the areas
innervated by the MeApd, but has some additional projections, including to the ACB and
VTA (Wood and Swann, 2005). Sites receiving input from TH-ir cells were expected to
have a moderate to extremely dense plexus of BDA-labeled ﬁbers because of the
hundreds of TH-ir cells located in the pBST (N orthcutt et al., 2007). Two sites (the MPO
and MeApd) were found to have greatly overlapping BDA-labeled ﬁbers and TH-ir
ﬁbers, so a second experiment investigated whether TH-ir neurons of the pBST
speciﬁcally projected to these sites. To accomplish this, the retrograde tracer FluoroGold
(FG) was injected into the MP0 or MeApd in separate groups of males and neurons in
the pBST and elsewhere in the brain that contained both TH-ir and FG-ir were identiﬁed.
Lastly, because the rat, hamster, and mouse pBST and MeApd are so densely
interconnected (Coolen and Wood, 1998; Dong and Swanson, 2004; Gomez and
Newman, 1992; Gu et al., 2003; Usunoff et al., 2009; Wood and Swann, 2005),
retrograde tracing was used to determine if the MeApd sends TH-ir input to the pBST.
These results demonstrate a unique catecholaminergic network among the pBST, MeApd,
MPO, and VTA that is likely essential for the monogamous social behaviors of this

SPecies.

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Experiment 1: Anterograde projections of the prairie vole pBST

Methods
Subjects

Subjects were adult (90-140 day old) male prairie voles (Microtus ochrogaster)
born and raised in the colony at Michigan State University. The colony originated with
voles captured in Urbana, IL and animals were last outbred in 2000 with voles from
Illinois. The colony was maintained on a 14:10 lightzdark cycle with an ambient
temperature of 21°C. After weaning at 20 days of age, subjects were housed with male
and female littermates in clear plastic cages (48 x 28 x 16 cm) containing wood chips,
wood shavings, and a covering of hay. Water and a food mixture containing cracked
corn, whole oats, sunﬂower seeds, and rabbit chow (Teklad rodent diet No. 2031; Harlan,
Madison, WI) in a 1:1:2:2 ratio were provided ad libitum. All procedures were in
accordance with the Institutional Animal Care and Use Committees at Michigan State

University.

Stereotaxic surgery and tissue collection

Subjects (n = 19) were anesthetized with an i.p. injection of a mixture of ketamine
(44 mg/kg; Butler Co., Dublin, OH), xylazine (5 mg/kg; Butler), and acepromazine (0.6
mg/kg; Butler). Biotinylated dextran amine (BDA; 40 ml of 10% solution in dHZO;
Invitrogen, Carlsbad, CA) was slowly injected over the course of 10 min into the right

pBST (A/P +0.3 mm, M/L -0.9 mm, DN -5.0 mm) via a 0.5 111 Hamilton syringe.

106

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Tissue collection and immunohistochemistry

Seven days after surgery, males were overdosed with the anesthetic mixture (see
above), and perfused through the heart with 100 ml 0.9% saline, followed by 100 ml 4%
paraformaldehyde in 0.1 M sodium phosphate buffer (NaPB; pH = 7.6). Brains were
removed and postﬁxed overnight in 4% paraformaldehyde, and then submerged in 20%
sucrose in NaPB for at least two days before being sectioned into 35 um sections on a
freezing microtome. During sectioning, a small hole was made in the cortex on the side
contralateral to the injection to help maintain section orientation during tissue histology
and microscope analysis.

BDA histochemistry and tyrosine hydroxylase (TH) immunohistochemistry were
performed on every third section throughout the brain. On the ﬁrst day of processing,
sections were rinsed three times with 0.1 M NaPB (pH = 7.4) between incubations. First,
sections were incubated in 1% hydrogen peroxide in NaPB for 10 min, followed by 1 h in
0.5% Triton X-100 in NaPB. Then, sections were incubated in avidin—biotin complex
(1:250; Vectastain Elite; Vector Laboratories, Burlingame, CA) and 0.5% Triton X-100
in NaPB for 1 h. BDA-labeled cells and ﬁbers were visualized by incubating sections in
Ni-enhanced 3,3’-diaminobenzidine (Sigma; 0.05% DAB and 0.4% nickel ammonium
sulfate) and 0.01% hydrogen peroxide in Tris-buffered saline (TBS; pH = 7.6), which
produced a dark blue reaction product in cell bodies at the injection site and in processes
of these cells. From this point on, sections were rinsed in TBS between incubations.
Sections were blocked with 20% normal goat serum (NGS) and 0.3% Triton X-100 in

TBS for 30 min, and were incubated for 18 h in mouse anti-TH primary monoclonal

107

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antiserum (1 :2000; cat.# MAB318; Chemicon, Temecula, CA) in 2% NGS and 0.3%
Triton X-100 in TBS at room temperature. Next, sections were incubated in a goat anti-
mouse secondary antiserum (1:500; Vector Laboratories) in 2% NGS and 0.3% Triton X-
100 in TBS for 1 h, followed by 1 h in avidin-biotin complex in TBS. TH was visualized
with a solution of hydrogen peroxide (0.09%) and DAB (0.02%) in TBS, which produced
a light brown cytoplasmic label. Omission of the primary or secondary antisera abolished
speciﬁc labeling. After tissue processing, sections were mounted onto slides, dehydrated,

and coverslipped.

BDA Microscope Analysis

Seven subjects were chosen for analysis because their BDA injection included
most or all of the region where TH-ir cells are found in the prairie vole pBST, but did not
extend far beyond this region. The entire forebrain and midbrain of these subjects were
examined for the presence of BDA-labeled ﬁbers, which were mapped onto images from
an atlas of the laboratory rat brain (Swanson, 1998); an atlas of the vole brain does not
exist. Dense collections of parallel ﬁbers were assumed to be ﬁbers of passage, and are
not reported in the results. The location of TH-ir ﬁbers on the contralateral side of the
brain was compared to the location of BDA-labeled ﬁbers on the injected side to

determine the areas most likely to receive TH-ir input from the pBST.

Results

Injection site

108

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Based on the pattern of somata labeled with BDA, seven subjects had injections that
covered almost all of the area of the pBST where TH-ir cells are found (Figure 4.1). In
two subjects (1, 2), no cells containing only TH-immunoreactivity were visible,
indicating that the BDA injection covered every TH-ir cell in that region. In the
remaining ﬁve subjects (3-7), a small number (4-20 cells) of TH-ir cells in the pBST
were still visible that did not contain BDA labeling. The pBST was the center of the
injection site in all cases, but there was minimal spread to the anterodorsal BST (adBST)
in all subjects. Additionally, some BDA was found in the transverse nucleus of the BST
(trBST) of ﬁve subjects (1, 2, 4, 6, 7), the interfascicular nucleus of the BST (ifBST) of
four subjects (1, 2, 4, 7), the reticular thalamus (RT) of one subject (2), and in very small
portions of the anterolateral (avBST), anteroventral (alBST), and rhomboid (thST)
nuclei of the BST of one subject (4). Projections are detailed below in rostrocaudal

order.

Projections to the lateral septum

The projections of the pBST were almost exclusively ipsilateral to the injection
site, with the few exceptions noted below. pBST projections to the LS were primarily
ventral (LSv; Figure 4.2). However, some labeled ﬁbers were also seen in the ventral
part of the rostral LS (LSr.v), and a few ﬁbers were found bilaterally in the dorsal LSr
(LSr.d) and the ventrocaudal LS (LSc.v) in every subject. In all subjects, the dorsal LSc

contained few or no BDA-labeled ﬁbers.

109

 

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pBST

 

Figure 4.1. Representation of BDA injection sites (gray shading) in the pBST for the
seven subjects (1-7) mapped onto atlas plate 21 from Swanson’s rat brain atlas (Swanson,
1998), and a photomicrograph of the BDA injection site (dark blue staining on right)
compared to TH-ir cell distribution (brown staining on leﬁ) of subject 1 (1a).

 

 

 

 

110

 

 

 

 

 

 

 

 

 

Figure 4.2. Relative distribution of BDA-labeled ﬁbers throughout the forebrain in one
representative subject receiving a BDA injection into the pBST, mapped onto plates 13-
37 (A-Y) of Swanson’s rat brain atlas (Swanson, 1998).

111

 

 

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Projections to the preoptic area and hypothalamus

The pBST provided very dense projections to the ipsilateral MPO and
anteroventral periventricular nucleus (AVPV) in every subject, with some minor
projections contralaterally to both sites (Figure 4.3). Hypothalamic regions receiving
dense innervation from the pBST included the lateral hypothalamus (LHA),
paraventricular hypothalamus (PVH), anterior hypothalamus (AHN), and tuberal nucleus
(TU). The lateral preoptic area (LPO), arcuate nucleus (ARH), dorsomedial nucleus
(DMH), ventromedial nucleus (VMH), and posterior hypothalamus (PH) contained some
sparse labeling in all subjects. No labeled ﬁbers were found in the supraoptic or

suprachiasmatic nuclei of the hypothalamus.

Projections to other regions of the BS T

The pBST also densely projected to subregions within the anterior BST, including
the anterodorsal (adBST), anterolateral (alBST), anteroventral (avBST), rhomboid
(thST), dorsolateral (dlBST), magnocellular (mgBST), and ventral (vBST) divisions. A
few labeled ﬁbers were found in all subjects in the dorsomedial (deST), fusiform
(ﬁrBST), and oval (ovBST) nuclei, as well as in the subcommissural zone (scBST), but
none were found in the juxtacapsular nucleus (juBST) of any subject. Extensive labeling
was observed throughout the entire posterior BST of all subjects, including the ventral

portion of the pBST that was outside the injection site.

117

 

 

 

Figure 4.3. Photomicrograph showing BDA-labeled ﬁbers (dark blue staining) and TH-ir
(brown staining) in the MPO (A) and MeApd (B). Both regions received dense
innervation from the BST that overlapped with the distribution of TH-ir ﬁbers.

118

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Projections to the amygdala

The pBST of all seven subjects projected densely to the amygdala (Figure 4.2),
particularly the MeApd (Figure 4.3), but also to the anterodorsal MeA (MeAad) and
posterior amygdala (PA). All subjects also had some BDA-labeled ﬁbers in the
posteroventral MeA (MeApv). In a few subjects (1-4), ﬁbers extended into the central
and medial portions of the central nucleus of the amygdala (CEA), and most subjects (1-
4, 7) had labeled ﬁbers in the cortical and basomedial nuclei of the amygdala (COA and

BMA).

Projections to mammillary bodies

The premammillary nucleus (PM) and ventral tuberomammillary nucleus (TMV)
each contained many BDA-labeled ﬁbers in every subject. The supramammillary (SUM)
and medial mammillary (MM) nuclei of a few subjects (2-5) contained labeling, but no

BDA-labeled ﬁbers were found in any other subdivision of the mammillary bodies.

Other projections

A moderate number of BDA-labeled ﬁbers were found in the nucleus accumbens
(ACB) of only two subjects (1, 3), primarily in the medial portion of the shell. Two other
subjects (2, 7) had only sparse BDA-containing ﬁbers in the ACB, and BDA-labeled
ﬁbers were non-existent in the ACB of the remaining three voles. There was a sparse to
moderate projection to the substantia innominata (SI) in all subjects. In the ventral
pallidum (VP) region of the SI, two subjects (1, 7) had a moderate number of BDA-

labeled ﬁbers, four had a few labeled fibers (2-5), and one subject had none (6). Finally,

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the paraventricular thalamus (PVT) and medial and lateral habenula (MH and LH) had

many BDA-labeled ﬁbers in all subjects, and these ﬁbers occasionally extended into the

mediodorsal thalamus (MD); a few labeled ﬁbers were seen in the contralateral PVT.

Overlap between BDA -labeled ﬁbers and TH-irfibers

Two areas, the MeApd and MPO/AVPV region, stood out as receiving very dense
projections from the pBST and also containing many TH-ir ﬁbers. Other areas of the
amygdala, BST, and hypothalamus/preoptic region receiving input from the pBST had
very few or no TH-ir ﬁbers and, thus, may be unlikely to receive meaningﬁil input from
TH-ir cells in the pBST. Although the LS contained a dense plexus of TH—ir ﬁbers, these
were found in the dorsal portion of the caudal LS (LScd), while BDA-labeled ﬁbers
coming from the pBST existed ventrally (Figure 4.4). The ACB contained a tremendous
number of TH-ir ﬁbers, but the number of BDA-labeled ﬁbers was very sparse or non-
existent in most subjects. Finally, some subjects had a sparse to moderate number of
BDA-labeled ﬁbers in the VP, but there was very little overlap with the few TH-ir ﬁbers

in this region.

120

 

Figure 4.4. Photomicrograph showing BDA-labeled ﬁbers (dark blue staining) and TH-ir
(brown staining) in the ACB (A) and LS (B). The ACB contained a dense plexus of TH-
ir ﬁbers, but few BDA-labeled ﬁbers. In the LS, BDA-labeled ﬁbers were primarily
found in the LSv and LSr subregions, and did not overlap with TH-ir ﬁbers in the LSc
(B).

121

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Experiment 2: Retrograde analysis of TH-ir projections to the MPO, MeApd, and
pBST

Methods
Subjects and stereotaxic surgery

The details of subjects and anesthesia are identical to those described for
Experiment 1. FluoroGold (PG; 40 n1 of 4% solution in saline; Fluorochrome, Denver,
CO) was slowly infused over 10 min via a 0.5 ul Hamilton syringe into the right MPO
(A/P +0.7 mm, M/L -O.4 mm, DN -6.1 m; n = 10), pBST (n = 14), or MeApd (A/P -0.9
mm, M/L -2.7 mm, D/V -6.1 m; n = 10). The MP0 and MeApd were chosen because
Experiment 1 revealed that they both receive extensive projections from the pBST, and
that this innervation was in the same regions containing TH-ir ﬁbers. Based on the
results of tracing studies in other rodents demonstrating that the pBST receives the
majority of output from the MeApd (Coolen and Wood, 1998; Gomez and Newman,
1992), possibility that the pBST receives input from TH-ir cells in the MeApd was also

investigated.

Tissue collection and immunohistochemistry

Brains were collected, postﬁxed, and sectioned similar to that described in
Experiment 1. F G and TH immunohistochemistry was performed on every other section
throughout the brain similar to previous work from the laboratory (Miller and Lonstein,
2009). Sections were rinsed with TBS between each incubation. First, sections were
incubated with 0.1% sodium borohydride in TBS for 15 min, 1% hydrogen peroxide and

0.3% Triton X-100 in TBS for 10 min, and 20% NGS and 0.3% Triton X-100 in TBS for

122

 

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30 min. Sections were then incubated for 18 h in a rabbit anti-FG polyclonal antiserum
(1:3000; cat.# AB153; Chemicon, Temecula, CA) in 2% NGS and 0.3% Triton X-100 in
TBS at room temperature. Next, sections were incubated with a goat anti-rabbit
secondary antiserum (1:500; Vector Laboratories) in 2% NGS and 0.3% Triton X-100 in
TBS for 1 h, and avidin-biotin complex in TBS for 1 h. FG was visualized with a
solution of hydrogen peroxide (0.09%) and DAB (0.02%) in TBS, which produced brown
puncta in the cytoplasm. Sections were then blocked and incubated in anti-TH primary
antiserum, goat anti-mouse secondary antiserum, and avidin-biotin complex as described
in Experiment 1. TH was visualized with Vector SG (Vector Laboratories), which
produced a dark blue cytoplasmic reaction product. Omission of any of the primary or
secondary antisera abolished speciﬁc labeling for the antigen. After tissue processing,

sections were mounted onto slides, dehydrated, and coverslipped.

F G Microscope Analysis

Five subjects with accurately placed FG injections in the MPO, six with
accurately placed injections in the pBST, and four with accurately placed injections in the
MeApd were chosen for detailed analysis. In all cases, the injection covered the entire
area of interest and did not extend much beyond the intended site. The number of TH-ir
cells was counted bilaterally in four sections of the pBST and bilaterally in seven sections
of the MeApd, excluding the site of injection, at 200x on a Nikon E400 light microscope.
These areas were then examined at 400x, and the number of cells containing both TH-
and FG-labeling were counted. Cells were considered double-labeled if they clearly

contained both dark-blue (TH) and brown (FG) reaction products. The number and

123

 

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percentage of TH-ir cells containing FG-ir was then calculated for each side of the brain
in each brain area. Finally, TH-ir cells in other brain sites, including the anteroventral
periventricular nucleus (AVPV), periventricular nucleus (PV), zona incerta, arcuate
nucleus (ARH), posterior hypothalamus (PH), other hypothalamic regions containing
scattered TH-ir cells, supramammillary nucleus (SUM), ventral tegmental area (VTA),
substantia nigra, and periaqueductal gray (PAG), were scanned at 400x, and double-
labeled cells were counted in each site. To estimate the total number of double-labeled
cells in each site, the number of double-labeled cells counted was multiplied by two for
each subject, and an Abercrombie correction factor of 0.78 was applied (Abercrombie,
1946). This correction was calculated as has previously been done (Northcutt et al.,
2007) using an average soma] diameter of 10 pm, which is a conservative estimate given

that TH-ir cells across these regions had average diameters of 8-10 pm.

Results
Injection sites

Most injections targeting the pBST or MeApd extended to other nearby
subregions, but never included other areas containing TH-ir ﬁbers, such as the lateral
portions of the BST (including juBST, alBST, and thST) or basolateral amygdala
(Asan, 1998; Brummelte and Teuchert-Noodt, 2006; Kozicz, 2001; Phelix et al., 1992;
Pinard et al., 2008). Injections extended into adjacent regions of the central amygdala,
but no TH-ir ﬁbers were observed on the contralateral side of the brain in the
corresponding location as the injection site. Furthermore, there was no spread of FG to

the pBST after injection into the MP0 or vice versa.

124

F G injection into MPO

In the ﬁve animals receiving accurately placed FG injections in the MPO (Figure
4.5), an average of 204 :h 34 TH-ir cells projected to the MPO, and 77% of these
originated in the pBST or MeApd. Many double-labeled cells were found in the
ipsilateral pBST (50 :1: 10 cells, 38 i 5% of TH-ir cells in the ipsilateral pBST; Table 4.1;
Figures 4.6 and 4.7) and MeApd (107 2t 19 cells, 42 i 3% of TH-ir cells in the ipsilateral
MeApd), although a very small number were also found in the contralateral pBST (3 i 1
cells, 2 i 0.5% of TH-ir cells in the contralateral pBST) and MeApd (8 d: 2 cells, 3 d:
0.4% of TH-ir cells in the contralateral MeApd). In all subjects, the remaining TH-ir/FG-
ir cells were found bilaterally in the supramammillary nucleus (SUM; 20 i 7 cells) and
arcuate nucleus (ARH; 5 d: 2 cells), and contralaterally in the anteroventral
periventricular nucleus (AVPV; 7 :t 1 cells). A small number of double-labeled cells (3 i
1) were also found in the posterior hypothalamus (PH) of three subjects (2, 4, 5), in the
medial mammillary nucleus of one subject (1; nine TH-ir cells), and in the lateral

hypothalamus of one subject (3; three TH-ir cells).

125

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Table 4.1. Estimated number of TH-ir neurons (i SEM) in regions throughout the
forebrain that project to the MPO, pBST, or MeApd in all subjects examined (also see
Figure 4.7).

Projecting to:

 

TH-ir neuron in: 'M—PO- BEEF. M
pBST 50 i 10 - 8 i 4
MeApd 107i19 112i18 -
AVPV 7i] 6i3* 3i2*
(contralateral)
ARH 5 i 2 0.3 i 0.3* 0
SUM 20 i 7 0 1 i 1*
PV (bilateral) 0 11 i 4 0
VTA 0 40 i 9 19 j: 5

* indicates that double-labeled cells were not found in that site in every subject, but the
number of double-labeled cells was averaged across all subjects (see text for details).

126

 

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Figure 4.5. Representation of the center of F G injection sites (gray shading) in the MP0
for the ﬁve subjects mapped onto plates 20 (1-4) and 21 (5) of Swanson’s rat brain atlas
(Swanson, 1998), and a photomicrograph of the FG injection site (brown staining on
right) in subject 1 (1a). TH-ir cells and ﬁbers (blue staining) can also be seen on the left.

127

 

Figure 4.6. Photomicrographs of TH-ir (blue labeling) and F G-ir (brown labeling) cells
and ﬁbers in the pBST (panels A, B) and MeApd (panels C, D) in voles with FG injected
into the MPO (scale bars = 20 or 100 um). Some double-labeled cells are indicated by
arrows. Photomicrographs of TH-ir cells in the pBST after FG injection in the MeApd
(panels E, F), and TH-ir cells in the MeApd aﬁer FG injection in pBST (panels G, H)
(scale bars = 20 or 100 um). Note the lack of double-labeled cells in panels E and F.

128

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MeApd

 
 

   

® MP0

Figure 4.7. Diagram of TH-ir cells throughout the forebrain and midbrain that project to
the MPO, pBST, and MeApd. Different arrow sizes represent the number of TH-ir cells
connecting the two regions (see Table 4.1).

129

‘II 'J‘ .
I =

 

PG int"

liter

Eabe

F G injection into pBST

In animals receiving accurately placed F G injections in the pBST (Figures 4.8 and
4.9), there were an average of 172 i 28 TH-ir cells projecting to the pBST, with most
found in the ipsilateral MeApd (112 :1: 18 double-labeled cells; 49 d: 4% of all TH-ir cells
in the ipsilateral MeApd). Similar to what was observed in MPO-injected subjects, a
very small number of double-labeled cells were found in the contralateral pBST (1 :t 1
double-labeled cells, 0.6 :t 0.3% of TH-ir cells in the contralateral pBST) or MeApd (12
i 7 double-labeled cells, 3 i 2% of TH-ir cells in the contralateral MeApd). Other TH-ir
cells containing FG-ir were found for all subjects in the ipsilateral ventral tegmental area
(V TA; 40 i 9 cells) and bilaterally in the caudal periventricular region (PV; 11 i 4 cells).
Four subjects also had some cells containing TH-ir and FG-ir in the periaqueductal gray
(PAG; 12 d: 6 cells; subjects 2-5) and in the substantia nigra (SN; 7 :h 2 cells; subjects 1-
3, 5). Lastly, three subjects (2, 4, 6) had an average of 6 i 3 double-labeled cells in the
paraventricular nucleus of the hypothalamus (PVH), three other subjects (3, 4, 6) had an
average of 11 i l double-labeled cells in the AVPV, and one subject (4) had two double-

labeled cells in the ARH.

130

 

 

 

 

 

 

 

 

Figure 4.8. Representation of PG injection sites in the pBST (gray shading) for the six
subjects (1-6) mapped onto plate 21 of Swanson’s rat brain atlas (Swanson, 1998).

 

Figure 4.9. Photomicrograph of the FG injection site in the pBST of subject 4 (Figure
4.8) showing FG-Iabeled cells (brown staining) and TH-ir cell and. ﬁber distribution (blue
staining).

131

IEG sin-ft

4.101.

dl‘dl‘l:

more
Furtl
121 -
4L 2

14)

had

F G injection into MeApd

Four subjects received accurately placed F G injections in the MeApd (Figure
4.10). An average of 52 :t 16 TH-ir cells projected to the MeApd, but relatively few
double-labeled cells were found in the ipsilateral pBST (8 3c 4 cells, 4.5 :t 1.2% of all TH-
ir cells in the ipsilateral pBST) or contralateral MeApd (2 :t 1 cells, 0.5 :t 0.3% of all TH-
ir cells in the contralateral MeApd); no double-labeled cells existed in the contralateral
pBST. However, there was extensive FG labeling in other pBST cells of unknown
phenotype (see Figure 4.6E), indicating that terminals in the MeApd did take up the PG
and retrogradely transported it to the soma. Instead of the pBST, all four subjects had
more cells containing TH-ir and FG-ir in their ipsilateral VTA (19 :h 5 cells).
Furthermore, dual-labeled cells were found bilaterally in the PAG in three of the subjects
(21 :t 7 cells; subjects 1-3), bilaterally in the PVH of three subjects (3 :1: 1; subjects 1, 3,
4), and in the contralateral AVPV of two subjects (5 i 2 cells; subjects 1, 3). One subject
(4) had ﬁve double-labeled cells in the SUM and two in the SN, and another subject (2)

had two double-labeled cells in the PH.

132

 

 

 

 

 

 

 

 

 

Figure 4.10. Representation of MeA and surrounding regions at plates 28 (A), 29 (B),
and 30 (C) of Swanson’s brain atlas (Swanson, 1998), and FG injection sites in the
MeApd that were centered on corresponding plate (1, 2, 3, 4). 2a and 4a are
photomicrographs of the PG injection site (brown staining on right) in subjects 2 and 4.
TH-ir cells and ﬁbers (blue staining) can also be seen on the left.

133

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lH-i
nucl

A l 0

mm

Volt

Me;

Dot
hat
"~11

301

Discussion

The results of these experiments demonstrate a species-speciﬁc network of
catecholaminergic projections involving the pBST, MeApd, MPO, and VTA. Similar to
projections of the entire pBST and MeApd in other rodents (Dong and Swanson, 2004;
Gu et al., 2003; Wood and Swarm, 2005), many TH-ir cells in both sites terminated in the
MPO. In fact, over 75% of the TH-ir input to the male prairie vole MPO originated in the
pBST and MeApd, and almost all (91%) of the TH-ir cells in the MeApd projected to
either the pBST or the MPO. In addition, there was substantial input to this system from
TH-ir cells of the VTA as well as other areas (posterior hypothalamus, supramammillary
nucleus, and ventral PAG) containing TH-ir cells considered to be from the “extended”
A10 and All groups (Albanese et al., 1986; Hokfelt et al., 1984). This unique network
(see Figure 4.7) may process information about olfaction, social recognition, and
motivation to regulate the gregarious and monogamous behaviors found in male prairie

voles but not most other mammals.

Methodological considerations

In the anterograde tracing experiment, minimal tracer spread to other subregions
of the BST adjacent to the pBST was seen, but this was not likely to dramatically affect
these results, as only areas containing FG-ir ﬁbers across all subjects were considered as
potential targets of TH-ir cells. Furthermore, these results are very similar to what others
have reported using other anterograde tracers and injections in various parts of the pBST,
with a few exceptions discussed in detail below (Dong and Swanson, 2004; Gu et al.,

2003; Wood and Swann, 2005). Previous investigations in rats and hamsters have used

134

 

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projcctl
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a slow

ear: 1?
AL>J L.‘

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'/
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a1

the anterograde tracer PHA-L, but this was not likely to contribute to subtle differences in
projection patterns. PHA-L and BDA typically produce similar patterns of labeled ﬁbers
(Raju and Smith, 2006; Reiner et al., 2000; Wouterlood et al., 2002), but BDA does have
a slower rate of transport (Raju and Smith, 2006). This is unlikely to have impacted these
results, however, because BDA can travel 15-20 mm a week (Renier et al., 2000), which
is ample time for it to be transported throughout the prairie vole forebrain and midbrain.
Some BDA-labeled cells were found in the LS in most subjects, and it is possible that
tracer was deposited in the LS as the syringe was lowered into the pBST. However, brain
sites receiving projections from the LS and not the pBST in other rodents, such as CA1
and CA3 subﬁelds of the hippocampus, VTA, anterior cingulate cortex, olfactory
tubercle, and PAG (Meibach and Siegel, 1977; Staiger and Numberger, 1991; Tsukahara
and Yamanouchi, 2001), had minimal or no BDA-labeled ﬁbers in these subjects.
Furthermore, BDA-labeled LS cells were widely distributed, suggesting that they were
likely labeled through retrograde transport, which does occur to some degree with BDA
(Reiner et al., 2000). Spread to the nearby fomix is also not likely, given that there were
few or no labeled ﬁbers in the hippocampus. Finally, BDA probably did not spread to the
MPO, because no labeled ﬁbers were seen in the lateral parabrachial nucleus, zona
incerta, VTA, pedunculopontine nucleus, PAG, or laterodorsal tegmental area, which
receive input from the MPO (Finn et al., 1993; Simerly and Swanson, 1988; Swanson et
al., 1987). In the retrograde tracing experiment, almost all of the MPO, pBST, or MeApd
were included in the injections, but there are very few TH-ir ﬁbers in areas adjacent to
the PG injection sites, therefore even if the tracer spread to outlying areas, it is unlikely

that it would have been taken up by TH-ir ﬁbers not of interest in the current study.

135

 

These it

it tells of the

  
   
    

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lemming no la
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these cells pl'm
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Comparison (it

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3004: Gu ct al
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that were uniq
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Some sparse i n‘

labeled ﬁbers

 

These retrograde tracing studies have accounted for much of the output from TH-
ir cells of the male prairie vole pBST and MeApd. This is particularly true for the
MeApd, with 91% of the TH-ir cells in this site targeting either the pBST or MP0
(assuming no collateralization). The termination site(s) of the remaining TH-ir cells in
the MeApd is unknown, but would be very minor compared to the dense innervation
these cells provide to the pBST or MP0. On the other hand, just under half of the TH-ir
cells in the pBST project to either the MP0 or MeApd, and the projections of the
remaining cells are unknown. No other brain sites contained notable comingling of
innervation from the pBST and TH-ir ﬁbers, so it is probably unlikely that one site
receives the other half of TH-ir input from the pBST. Instead, an array of brain sites that
have not yet identiﬁed probably each receive a small amount of input from this

population of cells.

Comparison of pBS T projections in prairie voles and other rodents

Overall, the pattern of projections from the prairie vole pBST was extremely
similar to what has been reported from the pBST of laboratory rats (Dong and Swanson,
2004; Gu et al., 2003; Wood and Swann, 2005) and the homologous BSTpm of hamsters
(Wood and Swann, 2005). This included that the densest projections from the pBST
terminated in the MPO and MeApd. In fact, there were no projections from the pBST
that were unique to prairie voles, and some minor projections reported in other species
were not found in prairie voles. For example, the PAG is consistently reported to receive
some sparse input from the pBST or BSTpm in rats and hamsters but there were no BDA-

labeled ﬁbers in prairie voles (Dong and Swanson, 2004; Gu et al., 2003; Wood and

136

Swann, 2005). There were also fewer BDA-labeled ﬁbers in the ARH and PV than what
has been found in rats and hamsters. These minor differences are likely due to variation
among studies in tracer injection site; two previous studies examined projections of the
ventral portion of the pBST or BSTpm (Dong and Swanson, 2003; Wood and Swarm,
2005), Gu et al. (2003) examined projections from the center of the pBST, and the
present study involved primarily the rostral dorsal pBST (where most TH-ir cells are
found) (N orthcutt et al., 2007). It is possible that the ventral part of the prairie vole pBST

' does project to the PAG, and more heavily than the rostrodorsal pBST to the ARH and
PV.

Wood and Swarm (2005) describe differences between the hamster BSTpm and
the rat pBST, including more elaborate projections to the ACB and 81, as well as
projections to the entire LS, medial septum (MS), nucleus of the diagonal band (NDB),
supraoptic nucleus (SON), and VTA that are not found in the rat brain or in the present
results. These differences are particularly notable given the small tracer injection site in
their experiment. Again, variation in tracer injection site may account for these
differences; their injections were caudal and ventral to these and to those of Gu et a1.
(2003), but were similar to Dong and Swanson’s (2004). Wood and Swarm (2005) also
propose that the hamster BST has only three divisions, as opposed to the four described
in rats (Aldheid et al., 1995), because a different division of the posterior BST, the BSTtr,
innervates many of these sites in rats. Interestingly, these results suggest that the prairie
Vole BST is more similar to that of rats than hamsters, as there was no visible innervation

0f any of these sites, at least from the dorsal portion of the pBST.

137

 

 

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Implications of species-speciﬁc catecholaminergic circuit
The ﬁnding that many TH-ir cells in the prairie vole pBST and MeApd project to
the MP0 is particularly interesting because DAergic neurotransmission in the MPOA
inﬂuences the expression of sociosexual behaviors in other species (Dominguez and Hull,
2005; Miller and Lonstein, 2005). In male rats, DA neurotransmission within the MP0 is
necessary for consummatory and motivational aspects of male sexual behavior
(Dominguez and Hull, 2005; Hull and Dominguez, 2007). Furthermore, DA receptor
activity in the MP0 is required for maternal behavior in female rats, with D1 receptor
antagonism in the MPO impairing retrieval and licking of pups (Miller and Lonstein,
2005), whereas D1 agonists facilitate the onset of these behaviors (Stolzenberg et al.,
2007). Previous work in the Lonstein laboratory has described the DAergic projections
to the MP0 of female rats (Miller and Lonstein, 2009), and found that most of it arises
from the posterior hypothalamus (PH), supramammillary bodies (SUM), and VTA
(Miller and Lonstein, 2009). Surprisingly, two of these sites, the PH and VTA, did not
consistently provide DAergic innervation in male prairie voles. This could be attributed
to a species difference, although sex or reproductive state differences could also be
responsible. Some TH-ir cells in the SUM did project to the MPO, and the pBST and
MeApd both received TH-ir input from the VTA, which may provide the same
information that is relayed directly to the MPO in postpartum rats. Of course, because
laboratory rats do not have TH-ir cells in their pBST and MeApd, input from these areas
to the MPO may be unique to prairie voles. Even so, the rat MeA interacts with other
Sites to enhance DAergic activity in the MPO necessary for sociosexual behaviors; MeA

stimulation increases DA release in the MPO, and DA receptor activation in the MP0

138

SIC IT:

acme

tales.

som

restores sexual behavior in male rats after MeA lesions (Dominguez et al., 2001;
Dominguez and Hull, 2001). This may also occur in male prairie voles, but at least some
of the catecholaminergic tone in the MPO appears to arise directly from the MeA itself
and may allow faster communication between the two sites or may increase DA release
above and beyond what is found in rats to facilitate behaviors that are speciﬁc to prairie
voles.

In prairie voles, direct catecholaminergic input to the MP0 from the pBST and
MeApd may facilitate high gregariousness and initiate pair bonding after mating. Given
the role of DA release in the MPO in two very different sociosexual behaviors in rats, it is
interesting to consider that DAergic input may be necessary for social motivation,
whereas other networks regulate the speciﬁc behaviors displayed. If this is the case, TH-
ir cells in the pBST and MeApd may relay olfactory information to the MPO through DA
release during all social interactions in male prairie voles, in addition to the DA release
that occurs only in speciﬁc situations (mating or maternal care) in a number of rodent
species. Thus, TH-ir cells in the prairie vole pBST and MeApd may enhance
catecholaminergic neurotransmission to the point that prairie voles will initiate social
interactions with many individuals, including pups and partners, but also siblings and in
some cases unfamiliar conspeciﬁcs (Bowler et al., 2002; Firestone et al., 1991; Olazabal
and Young, 2005; Roberts et al., 1996). Furthermore, DA release in the MPO may
contribute to speciﬁc prairie vole social behaviors. Many neurons in the rat pBST and
MeApd that express IEGs after mating project to the MPO (Coolen et al., 1998), and TH-
ir cells expressing IEG expression after ejaculation in prairie voles may also project to the

MPO. If so, additional DA input to the MPO may motivate males to initiate the next

139

 

sung
Gent 1

bonds

nude
syner
tsee ‘
pint

Ctll’lr'lt

but]
if Ce
l-‘t tl

lhis

mating bout, as prairie voles have protracted mating encounters of 1-2 days (Carter and
Getz, 1985), or may directly initiate neural cascades involved in the formation of pair
bonds.

The MP0 has widespread connections throughout the forebrain (Anderson and
Shen, 1980; Chiba and Murata, 1985; Coolen et al., 1998; Simerly and Swanson, 1988),
projects to many brain sites involved in social behavior, and may link input from TH-ir
cells in the pBST and MeApd with networks already known to be involved in regulating
male prairie vole sociosexual behaviors. The extra-hypothalamic vaSOpressin (AVP)
system and the mesolimbic DA system are both critical for the formation of pair bonds
(see Young et al., 2008), and the MPO may integrate information from these networks
with catecholaminergic input from the pBST and MeApd. Indeed, the MPO has
connections with the BST and MeA that contain AVP cell bodies (Chiba and Murata,
1985; Coolen and Wood, 1998; Wood and Swann, 2005), and their targets in the VP and
LS (Anderson and Shen, 1980; Chiba and Murata, 1985; Firm et al., 1993; Grove, 1988a,
b; Staiger and Numberger, 1991; Varoqueaux and Poulain, 1999). In addition,
projections between the MPO and ACB or VTA may allow multiple catecholaminergic
networks to communicate with one another (Chiba and Murata, 1985; Fahrbach et al.,
1986; Groenewegen and Russchen, 1984).

A similar percentage of TH-ir cells in the pBST and MeApd project to the MPO,
but there was a remarkably uneven projection between these two sites; about half of TH-
ir cells in the MeApd project to the pBST, but very few TH-ir cells in the pBST terminate
in the MeApd. Generally, overall projections from these two brain sites seem to follow

this trend (Coolen et al., 1998; Dong and Swanson, 2004; Gu et al., 2003; Wood and

140

 

C '00

Son

cell

smz

and

MM

pro j

~

Coolen, 2005), but there were numerous non—TH-ir pBST cells projecting to the MeApd.
Some TH-ir cells in the MeApd communicate directly with the MPO, but others may
indirectly enhance catecholamine release via connections with TH-ir cells in the pBST, or
may coordinate sociosexual behaviors by inﬂuencing non-TH-ir cells. On the other hand,
the pBST may communicate with the MeApd through other neurotransmitters, likely
GABA given that the majority of pBST neurons in other rodents are GABAergic
(Sirmnons and Yahr, 2003; Stefanova, 1998), but possibly any of the numerous other
neurotransmitters found in the pBST (De Vries et al., 1984; DeVries et al., 1985;
Malsbury and McKay, 1989; Miller et al., 1993; Muganini and Oertel, 1985; Poulin et al.,
2009; Simerly and Swanson, 1987; Simmons and Yahr, 2003; Swann and Newman,
1992). In the present experiment, the termination site of approximately 45% of TH-ir
cells in the pBST was accounted for, and it is likely that the remainder have an array of
small projections throughout the forebrain, and possibly also connections with nearby
BST cells that were within the injection site in the retrograde tracing experiment (Dong
and Swanson, 2004; Gu et al., 2003; Wood and Coolen, 2005).

The VTA may also provide motivational information to the pBST and MeApd,
and could inﬂuence catecholamine release from these sites. DA release in the ACB,
probably ﬁom the VTA (Chronister et al., 1980; Curtis and Wang, 2005), increases
during mating in male prairie voles, and activation of D2 receptors is critical for the
consolidation of pair bonds (Aragona et al., 2003; Aragona et al., 2006). The VTA may
simultaneously release DA in the pBST and MeApd to alter catecholamine release in the
MP0 or to inﬂuence pBST and MeApd cells of other phenotypes. Interestingly, TH-ir

projections from the VTA to the pBST and MeApd are also unique to prairie voles, as the

141

 

pBST 2
\l'ood.
and an
teceix'
Noodt
from

rets 8.1
the p

to pr

(on.

VT:

Volt

pBST and MeApd of other rodents receive very little input from the VTA (Coolen and
Wood, 1998; Herbert et al., 1997; Wood and Swarm, 2005). Other regions of the BST
and amygdala, including the lateral BST and basolateral and central amygdala, may also
receive some DAergic innervation from the VTA (Asan, 1998; Brummelte and Teuchert-
Noodt, 2006; Kozicz, 2001; Phelix et al., 1992; Pinard et al., 2008). DAergic projections
from the VTA to the ACB are part of complex networks mediating motivation and
reward (Carlezon and Thomas, 2009; Ikemoto, 2010; Kiyatkin, 2002), and projections to
the prairie vole pBST and MeApd may link motivational information with olfactory input

to promote social interactions in this highly social species.

Conclusions

A unique catecholaminergic network consisting of the pBST, MeApd, MPO, and
VTA has been discovered that probably affects the unique social behaviors of prairie
voles. DA input from the VTA may be integrated with olfactory information in the pBST
to MeApd to inﬂuence social motivation. Projections between TH-ir cells in the
pBST/MeApd and MP0 may then inﬂuence the desire for all types of social interaction,
but also impact mating and the initiation of pair bonding. This unique catecholaminergic
network may also interact with AVP and other DA systems to facilitate high levels of

gregariousness and monogamous behaviors in this species.

142

 

\te'i‘
5‘“
.

EXpr
Vtitle

SEES

it is

CHAPTER 5:

Conclusions

This dissertation describes a species-speciﬁc, steroid hormone sensitive,
catecholaminergic system that likely inﬂuences monogamous social behaviors
characteristic of male prairie voles. This network probably integrates olfactory,
endocrine, and motivational input necessary for males’ social behavior, and interacts with

other brain areas already implicated in pair bonding in this species.

Signiﬁcance of sex difference in T H—ir cells

Given the relative sexual monomorphism of prairie vole behaviors, it is at ﬁrst
surprising that males have many more TH-ir cells than do females. However, De Vries
(2004) proposed that neural sex differences sometimes function to compensate for sex
differences in physiology, such as circulating gonadal hormones, to eliminate sex
differences in behavior. Indeed, gonadal hormone activity has been associated with the
expression of vole social behaviors; males of non-social vole species or less social prairie
vole populations have more estrogen receptors in the BST and MeA and are more
sensitive to testosterone than males from highly social species and populations (Cushing
et al., 2004; Cushing and Wynne-Edwards, 2006; Kramer et al., 2006). High numbers of
TH-ir cells may also compensate for high testosterone levels in male prairie voles to
allow social interactions.

These studies have focused on characterizing TH-ir cells in male prairie voles, but

it is important to note that female prairie voles have more TH-ir cells than rats, hamsters,

143

 

or meadow voles of either sex (Northcutt et al., 2007), and these cells may be important
for the expression of some female social behaviors. Given the ability of exogenous
testosterone and estrogen to increase the number of TH-ir cells in the pBST and MeApd
of females (Cavanaugh and Lonstein, 2010a; Northcutt et al., 2007), dramatic changes in
hormone levels throughout different reproductive states were hypothesized to change TH
expression in females (Northcutt et al., 2007). Indeed, the number of TH-ir cells does
slightly increase at times when estrogen levels are normally high (such as aﬁer pairing
with a male), but ovarian hormones are surprisingly not required, as the number of TH-ir
cells is greater after pairing even in ovariectomized females (Cavanaugh and Lonstein,
2010b). Despite this increase, the number of TH-ir cells never approaches the number
found in males (Cavanaugh and Lonstein, 2010b), and they are likely more critical for the

expression of monogamous behaviors in males.

Species comparison between T H-ir cells and others in p88 T and MeApd

Although non-monogamous rodents studied to datedo not have large numbers of
TH-ir cells in the pBST and MeApd (Albanese et al., 1986; Hokfelt et al., 1984; Kalsbeek
et al., 1992; Northcutt et al., 2007), the regulation and projections of TH—ir cells in these
regions in prairie voles is remarkably similar to that of other cell groups found in the BST
and MeA of other rodents. In particular, the male-biased sexual dimorphism in TH
expression and its maintenance by testosterone in adulthood parallels morphological
(overall volume, soma size, astrocyte number and complexity) and neurochemical
(expression of vasopressin, CCK, and substance P) features of these two brain sites in

rats, hamsters, and mice (Cooke et al., 1999, 2003; De Vries et al., 1984, 1985; Malsbury

144

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19931.
inﬂuence
mnes a
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and McKay, 1994; Morris et al., 2008; Simerly and Swanson, 1987; Swann and Newman,
1992). Recent data demonstrate that testosterone must be aromatized to estradiol to
inﬂuence TH expression in prairie voles; estradiol maintains TH expression in castrated
males and increases TH expression in females, whereas testosterone’s androgenic
metabolite DHT does not have these effects (Cavanaugh and Lonstein, 2010a).
Similarly, estrogenic metabolites of testosterone are largely responsible for the regulation
of many neurochemicals in the rat and mouse BST and MeA (De Vries et al., 1986, 1994;
Pak et al., 2009; Wang and De Vries, 1995), though often the activation of both estrogen
and androgen receptors is required to fully maintain a masculine phenotype (Cooke et al.,
2003; De Vries et al., 1986; Durazzo et al., 2007; Johnson et al., 2008; Morris et al.,
2005, 2008; Plumari et al., 2002; Scordalakes and Rissman, 2004). The projections of
the prairie vole pBST are also very similar to what has been reported in rats and hamsters
(Dong and Swanson, 2004; Gu et al., 2003; Wood and Swarm, 2005), and TH-ir cells in
the pBST and MeApd terminate in areas receiving the densest innervation from these
areas in rats, mice, and hamsters (Coolen and Wood, 1998; Dong and Swanson, 2004;
Gomez and Newman, 1992; Gu et al., 2003; Usunoff et al., 2009; Wood and Swarm,
2005). These remarkable similarities suggest that prairie voles may not have more cells
in the pBST and MeApd than non-social rodents (which could be determined through a
stereological analysis), but that some of the cells common to all rodents began to express
TH at some point in the evolutionary history of the prairie vole. In environments with
low population density and many predators, voles with random mutations causing TH to
be expressed in the pBST and MeApd may have had more reproductive success due to

their ability to be highly social and adopt a cooperative breeding strategy. Because

145

c3161

hen;

catecholaminergic input to the MP0 is necessary for a wide variety of sociosexual
behaviors (Dominguez and Hull, 2005; Miller and Lonstein, 2005; Stolzenberg et al.,
2007), additional catecholaminergic projections that originate in areas receiving olfactory
input and involved in male sexual behavior could promote the formation of social bonds
aﬁer mating.

One of the few differences between TH-ir cells in the male prairie vole pBST and
MeApd and cells of all phenotypes in these sites in rats and mice is their IEG production
in response to social stimuli. In male rats, IEG expression in the pBST and MeApd
remains at baseline levels when they are exposed to anestrous females (Coolen et al.,
1997), and in prairie voles, increases in IEG expression after interactions with siblings
were speciﬁc to TH-ir cells in the pBST and MeApd (Lim and Young, 2004; Northcutt et
al., 2009). TH-ir cells in the pBST and MeApd may receive very speciﬁc input from
olfactory regions that do not innervate other cell types in the pBST and MeApd to
communicate information about non-reproductive social contexts. Alternatively,
enhanced signaling from areas involved in processing of goal-oriented behaviors and
motivation may facilitate responses in these cells. The latter possibility is especially
intriguing given the species-speciﬁc innervation of the pBST and MeApd by TH-ir cells
in the VTA; however, it is unknown whether TH-ir cells receive this input.

Although male Syrian hamsters do contain a few TH-ir cells in the BSTpm and
MeApd (Asmus et al., 1992; Asmus and Newman, 1993; Davis and Macrides, 1983;
Northcutt et al., 2007; Vincent, 1988; Wommack and Delville, 2002), the distribution and
number of cells, as well as their hormone sensitivity and IEG expression, is very different

from what has been seen in the present experiments in prairie voles. First, hamsters have

146

 

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TH-ir cells throughout the MeA and some scattered in lateral regions of the BST (Asmus
and Newman, 1993), whereas TH-ir cells are largely conﬁned to the pBST and MeApd in
prairie voles. In addition, axonal transport must be blocked to see more than ~10 TH-ir
cells in hamsters (Northcutt et al., 2007; Vincent, 1988; Wommack and Delville, 2002).
The hormonal regulation of these cell groups is also dramatically different, further
supporting the idea that these are ﬁrnctionally different groups of cells. Approximately
70% of TH-ir cells in the male hamster BSTpm and MeApd contain androgen receptors
(Asmus and Newman, 1993), similar to what has been shown in prairie voles (Cavanaugh
and Lonstein, 2010a), but the number of TH-ir cells in the BSTpm and MeApd does not
change after castration, and only transiently decreases in the anterior MeA (Asmus and
Newman, 1993). Experiments examining IEG expression also suggest that the cell
groups have very different functions in the two species. Approximately 60% of the TH-ir
cells in the male hamster MeApd express Fos in males that have been socially housed or
leﬁ alone until sacriﬁce, and mating does not change F 05 expression (Asmus and
Newman, 1994). In contrast, less than 1% of TH-ir cells expressed IEGs when male
prairie voles were tested alone, and dramatic increases in IEG expression were seen after
mating (Northcutt and Lonstein, 2009). These drastic differences between the two
species provides further evidence that TH-ir cells in the male prairie vole pBST and
MeApd are involved in their unique social behaviors.

The function of TH-ir cells in the pBST and MeApd in hamsters is unknown, but
chronic social stress increases TH expression and these neurons may affect aggression
(Wommack and Delville, 2002). In addition to previous results implicating their role in

afﬁliative, mating, and/or bonding behaviors, other investigations in prairie voles

147

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demonstrate that TH-ir cells are unlikely to be involved in at least some forms of
aggression because IEG expression in TH-ir cells in the pBST and MeApd is similar
among pair bonded males that ﬁght with an intruder male or female and bonded males
that remain with their partner (Gobrogge et al., 2007). It is interesting to consider that
two neurochemically similar, but ﬁmctionally different groups of cells exist in similar
sites in these two species. A similar number of TH-ir cells was found in meadow voles
and hamsters, and meadow voles may provide an interesting model in which to study the
diversity in function in TH-ir cells in the pBST and MeApd. Meadow voles are relatively
non-social during the summer, but become more gregarious in short day environments
(Beery et al., 2008, 2008; Parker and Lee, 2001, 2002), and seasonal plasticity in TH

expression and function would be fascinating.

Phenotype of T H-ir cells of the male prairie vole pBST and MeApd

The absence of dopamine-B-hydroxylase in the pBST and MeApd (Chapter 1) led
us to tentatively conclude that TH-ir cells in these sites could be dopaminergic (DAergic).
TH is the rate-limiting enzyme in DA synthesis and is very often used as a marker for
DAergic cells in the vertebrate brain (Smeets and Gonzalez, 2000). However, not all TH-
ir cells in the rat brain contain L-amino acid decarboxylase (AADC), which is the
enzyme required to convert L-DOPA to DA (Jaeger et al., 1984; Ugrumov, 2009). In
fact, cells expressing TH but not AADC are found in many sites throughout the brain in
rats, hamsters, mice, guinea pigs, monkeys, and humans, and have a number of functions

that have only recently been explored (reviewed in Ugrumov, 2009).

148

 

Clev
fest
HUT
det
AA
AA

em

Although often considered to only be a DA precursor, L-DOPA itself can act as a
neurotransmitter and neuromodulator itself when released from cells that do not contain
the AADC required to convert it into DA (Misu et al., 2002, 2003; Opacka-Juffry and
Brooks, 1995). L-DOPA is released in the ventrolateral medulla, striatum, and
hippocampus in a calcium-dependent manner and acts at postsynaptic targets or nearby
presynaptic terminals (reviewed in Misu et al., 2003; Opacka-Juffry and Brooks, 1995).
A speciﬁc receptor for L-DOPA has yet to be identiﬁed, but L-DOPA can induce
depolarization and glutamate release from striatal neurons (Goshima et al., 1993). L-
DOPA also acts on presynaptic terminals to inﬂuence DA release in the striatum (low L-
DOPA concentrations enhance release via actions at B-adrenergic receptors while high L-
DOPA concentrations inhibit it via D2 DA receptors), and also potentiates postsynaptic
D2 receptors (Hume et al., 1993; Misu et al., 1986). In other neural networks, L-DOPA
can inﬂuence the release of norepinephrine, acetylcholine, and glutamate, as well as the
postsynaptic response to GABA (Goshima et al., 1986, 1991, 1993; Honjo et al., 1999;
Misu et al., 1985; Ueda et al., 1995).

In some brain sites, TH-ir cells only express AADC and produce DA in speciﬁc
developmental periods or in response to environmental cues. In the arcuate nucleus, very
few cells express both TH and AADC in embryonic and early postnatal rats, but the
number of bi-enzymatic neurons increases dramatically throughout postnatal
development (Ershov et al., 2002a, b). Additionally, environmental cues can affect
AADC expression; hamsters with short photoperiods have approximately half of the
AADC-ir cells of their long photoperiod counterparts in the arcuate nucleus and median

eminence (Krajnak and Nunez, 1996). Finally, in some brain sites L-DOPA can be taken

149

 

up by neighboring neurons expressing AADC, “and sometimes dopamine-B-hydroxylase,
and converted to DA or norepinephrine (Ugrumov, 2009). Preliminary experiments show
that many AADC-it cells exist in the BST, and these may produce DA from L-DOPA
that is released in this site from the MeApd, VTA, and possibly pBST.

This is particularly relevant to this dissertation, because there is now preliminary
evidence that most of the TH-ir cells in the pBST and MeApd of virgin male prairie voles
do not express immunocytochemically-detectable levels of AADC. AADC may be found
in the terminals of these TH-ir cells or at low levels that are unable to be detected using
immunocytochemical techniques, but this recent ﬁnding opens up fascinating possibilities
about the phenotype of these cells. TH-ir cells in the pBST and MeApd may inﬂuence
MPO neurons through direct actions at postsynaptic receptors and/or modulations of the
release of other neurotransmitters, including DA from other sites. Another possibility is
that TH-ir in the pBST and MeApd neurons only produce AADC in response to speciﬁc
developmental, environmental, or acute physiological cues, and that the enzyme is
nonexistent or below detectable levels in adult virgin males. It is particularly intriguing
to consider that AADC might be produced only when a male ﬁrst encounters a female
and that subsequent DA release in the MPO during mating would then facilitate the
consolidation of a pair bond. Although this is not part of this dissertation, current

experiments are exploring this possibility.
Possible role of pBST and MeApd T H-ir cells in social motivation or ability

The results of the IEG experiments suggest that some TH-ir cells in the pBST and

MeApd are tonically active in any social situation. This is not surprising given the role of

150

 

 

 

the pBST and MeApd in a wide range of social behaviors, including mating, parental
care, and social recognition, in other rodents (Newman, 1999; Numan and Insel, 2003;
Petrulis, 2009). Many investigations of prairie vole social behavior have focused on the
role of neurotransmitter systems in single social behaviors, typically pair bonding or
parental behavior (see Young et al., 2008). In addition to these speciﬁc behaviors that
have received much attention, prairie voles are highly gregarious (Bowler et al., 2002;
Firestone et al., 1991; Olazabal and Young, 2005; Roberts et al., 1996). When left
undisturbed in the laboratory, they spend the majority of time in close contact with
siblings, either grooming one another or huddling together (Chapter 2; Firestone et al.,
1991), and virgin males and females will often show high levels of afﬁliative behavior
towards unfamiliar same-sex voles (Bowler et al., 2002; Firestone et al., 1991; Olazabal
and Young, 2005). The high levels of social behavior in many settings indicate that there
may be neural mechanisms promoting all social interactions in addition to networks
speciﬁc for some behaviors. These initial experiments suggest that the TH-ir cells in the
pBST and MeApd may be playing this role, and it will be very interesting to know if
disrupting L-DOPA or DA release from these cell groups disrupts the motivation or
ability to engage in all social interactions.

Similarly, Newman (1999) proposed that the BST and MeA are part of an
“integrated social behavior circuit” along with the LS, MPO, anterior hypothalamus,
ventromedial hypothalamus, and periaqueductal gray. She suggests that all of these areas
are important for the expression of all mammalian sociosexual behaviors, but the relative
activity among the regions differs depending on the behavior (see Figure 5.1). When

applying this model to prairie voles, it seems necessary to expand the network to include

151

 

 

 

other areas critical for social behavior expression, particularly the ACB, VP, and VTA,
and the integration across regions is likely to be more complex given the greater array of
social interactions. Furthermore, TH-ir cells in the pBST and MeApd may act as a
separate node, as their IEG responses are very different than those of the entire pBST and
MeApd to some social stimuli, particularly siblings and pups (Chapter 3). Responses in

TH-ir cells would be integrated with input from the other nodes to determine the

 

appropriate behavior.

Male Sexual Behavior

  
 
 
 

 

 
 

CMNBST

   

 

MPO

 
  
 

Ant Hyp

  

Figure 5.1. Hypothetical representation of neural activity in Newman’s “integrated
social behavior circuit” during male sexual behavior. Higher peaks indicate greater
neuronal activity in that brain site. Figure from Newman, 1999.

152

An alternative explanation is that two functionally different, but anatomically
overlapping groups of TH-ir cells exist in the male prairie vole brain. One group of TH-
ir cells may be active after any social situation, while a second group is active only aﬁer
ejaculation. This would be interesting given that subregions of the pBST and MeApd are
differentially activated after male rats mount, intromit, or ejaculate (Coolen et al., 1996,
1997). IEG expression appeared to be scattered throughout TH-ir cells in the entire
pBST and MeApd, but this does not rule out the possibility of two overlapping, but
functionally separate groups. This may also explain why males have many more TH-ir
cells than do females; females may have a moderate number of TH-ir cells because they
only need one of these groups. Females form partner preferences more readily than
males and their preference formation is less dependent on mating (DeVries et al., 1996b;
DeVries and Carter, 1999; Insel et al., 1995; Winslow et al., 1993), and this may be
independent of pBST and MeApd TH-ir cells in females.

Both of these possibilities are interesting, and future work will elucidate more
about the role of these cells in monogamous behaviors. Initial attempts to lesion these
cells by infusing a DA-speciﬁc toxin, 6-hydroxydopamine, into the pBST were
unsuccessful, likely due to a lack of DA transporter in the cell bodies of TH-ir cells in the
pBST and axon terminals from TH-ir cells in the MeApd, or because they are resistant to
oxidative stress as are some other catecholaminergic cell groups in the forebrain
(Behrouz et al., 2007). As an alternative, others in the laboratory are currently exploring
the effects of intra-MPO infusions of a DA transporter (DAT) antibody conjugated to
saporin toxin, which will be retrogradely transported to the soma and destroy DAT-

synthesizing cells projecting to the MPO (Wiley et al., 2003). This work suggests that

153

 

 

this could result in the death of approximately 40% of TH-ir cells in the pBST and
MeApd. Results of the current and future experiments on these TH-ir populations will
provide insight into the neural networks that evolved to allow monogamous behaviors

and cooperative breeding

Conclusions

The experiments in this dissertation describe a unique network in the prairie vole
brain that is likely required for the high gregariousness and pair bonding characterisitic of
this species. These species-speciﬁc groups of TH-ir cells probably work in conjunction
with other “social network nodes” to motivate all social interactions and regulate mating
and pair bonding. Now that functional possibilities and the anatomical projections of
these cells are known, future experiments can determine the precise role of TH-ir cells in
prairie vole sociosexual behaviors, and determine how they interact with other
neurotransmitter systems that inﬂuence both general and speciﬁc social behaviors.
Future studies that can now be performed because the projections of pBST and MeApd
TH-ir cells are known, and will reveal the role of these cell populations in the complex

sociosexual behaviors typical of male prairie voles.

154

 

 

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