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This is to certify that the

dissertation entitled

THE MATERNAL EXPERIENCE IN THE RAT: AN INVESTIGATION
INTO CELLULAR CHANGES ACCOMPANYING PREGNANCY,
PARTURITION, MOTHERHOOD (REAL OR IMAGINED), AND BEYOND

presented by

ADRIENNE KAY SALM

has been accepted towards fulfillment
of the requirements for

Ph . D. degree in Neuroscience / Psy cholo gy

 

abet/gm

Major professor

Date £3 ~Z/JL1975

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THE MATERNAL EXPERIENCE IN THE RAT: AN INVESTIGATION INTO
CELLULAR CHANGES ACCOMPANYING PREGNANCY, PARTURITION,

MOTHERHOOD (REAL OR IMAGINED), AND BEYOND.

BY
Adrienne Kay Salm

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Neuroscience Program/ Department of Psychology

1985

ABSTRACT

THE MATERNAL EXPERIENCE IN THE RAT: AN INVESTIGATION INTO
CELLULAR CHANGES ACCOMPANYING PREGNANCY, PARTURITION.
MOTHERHOOD (REAL OR IMAGINED), AND BEYOND.

BY

Adrienne Kay Salm

Supraoptic nucleus (SON) neurons produce, and release
from their axons in the neurohypophysis, the hormones
oxytocin (OK) and vasopressin (VP) during parturition,
lactation and, perhaps, concurrently with the expression of
maternal behaviors. At these times they exhibit
morphological plasticity, i.e.: ;) increases in cell-cell
apposition (two neurons with no intervening glial process),
g) dendritic bundling (two or more dendritic profiles in
apposition), and g) formation of double synapses (one
presynaptic axon terminal contacting two dendrites or
somata). Simultaneously, many of the axons, normally
enveloped in the cytoplasm of pituicytes, lose their
pituicyte ensheathment.

Six groups of female Sprague-Dawley rats- virgins
(controls), virgins in which maternal behavior had been
induced by exposure to rat pups (MBs), immediately
prepartum, immediately postpartum, 14-days lactating, and
10-days postweaning, were studied. The questions under

investigation were: 1) Does the SON change with maternal

behavior 2) What is the peptidergic content of neurons which
form cell-cell appositions during the "Motherhood
experience" and 3) Do the morphological traces of Motherhood
persist to io-days postweaning?

Electron microscopy (EM) of control and MB SONs showed
no differences in double synapse formation or soma-somatic
apposition. However, MBs displayed more dendrites, large
(9-12 dendrites) dendritic bundles, dendritic profiles, and
dendritic profile area in bundles. The dendritic zone area
was larger in Mas. In the neurohypophysis, MBs had fewer
enclosed axons per animal.

Immunoelectron microscopy of prepartum, postpartum and
14-day lactating subjects showed that OX—Neurophysin
positive cells in the postpartum group had more soma—somatic
apposition than did the controls or the prepartum group.

Conventional EM of postweaning and control SONs showed
no differences between groups in cell-cell apposition,
number of double synapses, or dendritic bundling. The areas
of single dendritic profiles and of the dendritic zone were
larger in the postweaning group, as was the number of
dendrites.

These data indicate a functional role for oxytocinergic
somatic appositions during parturition, the rapid reversal
of some traces of the "Motherhood experience", and that the

SON changes with maternal behavior.

ACKNOWLEDGMENTS

There are so many of you to thank for assistance on
this project that your assembled masses could staff a Cecil
B. De Mille epic!

First, to Glenn Hatton, who has supported my research
over the years in a style that I hope I will remain
accustomed to: thank-you for supplying me with the cadre of
workers which helped me with this project. Also, you have
permitted me risky junkets- thanks for taking a chance on
the maternal behavior experiments. Further, in retrospect,
it was an OK idea to look at neurons, they were interesting
afterall. Finally, your mentoring has produced a fledgling
scientist who feels competent to make her own way. I hope
you are pleased with the results of my being six years under
your wing. Thank-you!

My committee members have performed their duties par

excellence. Dr. Wilson, your thorough critique of this

 

dissertation and your suggestions for immunocytochemical
controls were most appreciated.

Dr. Tweedle, I have depended on you heavily for
ultrastructural guidance and good conversation about the
system we're working in. Thank-you for your extensive
consultations, moral support and access to your expertise.

Dr. Nunez, you have been a wonderful friend and adviser
in many ways. In respect to this project, I have valued

your encouragement on the maternal behavior aspect of it and
11

am grateful for your input on the data analysis for
dendritic bundling.

Hatton Labbies, past and present, you are a marvelous
group. I will always treasure our friendships. Kevin
Grant, you've mobilized your energy and your troops
countless times to supply me with everything from buffers to
photographic paper to a good laugh. Your many hours spent
assembling a computerized image analysis system for use in
this project are gratefully acknowledged.

Barb Kohn, thank-you for participating in the maternal
behavior study. You have contributed much time scoring
behavior, scoring micrographs, sampling pituicytes, and have
added to my knowledge of maternal behavior in general. Our
collaboration has been a pleasure.

Ken Smithson, true friend and confidant, never have you
denied me the benifit of your technical expertise on so many
subjects. I have especially appreciated your insights when
dealing with the vagaries of immunocytochemistry. In this
project I have especially appreciated your help in
formatting appendices and the beautiful graphs you produced
with your magic "Mac". In all, our collaborations have been
a joy.

To my student workers who have cheerfully and
faithfully aided me- I couldn't have done it without you.
Blair Petersen, darkroom magician who can correctly expose
negatives with one try, your many hours spent in the
darkroom (usually on bright beautiful days) are truly

appreciated. Inge Taubitz, budding EMer and thin sectioner
iii

extraordinaire, thank-you for keeping your sanity, and mine,

 

throughout the months of sectioning. To the two of you and
Mary Petersen, your countless hours spent with the
digitizing tablet, as well as your many other contributions
to this project, were invaluable and much appreciated.'

To Andrea Smirnes, for your wonderful drawings of
maternally behaving rats, and your steadfast moral support,
I thank you!

Other people who have helped in one fashion or another
along the way are Patricia Patterson and Dr. Karen Baker for
running excellent BM facilities. Thanks are due to Drs. H.
Gainer and W. Smith for the gift of the monoclonal
antibodies which they and their colleagues developed. Also,
Dr. M. Whitnall has been unfailingly willing to dispense
advice on EM immunocytochemistry. Mike Brown and Tim
Youngstrom have also been helpful in a variety of ways.
Thank-you for your support and your friendship. Bob
Luzinski deserves a special thanks for the development of
the image analysis software.

Finally, to my parents, words cannot express the love
and appreciation I feel for everything you've done for me -
both while in and out of school. With respect to this
project, the gift of the Apple IIe from my Father, John
Salm, expedited my progress considerably. Both he and Betty
B. Butler, as well as my Mother and Stepfather, Kay and
George Vorel, have also supplied endless amounts of love and
encouragement. Thank-you all!

This research was supported by NIH grant # NS 09140.

iv

TABLE OF CONTENTS

List of Tables ......................................... vi
List of Figures.... .................................. .vii
List of Abbreviations ................................ xiii
Introduction... ...................... . ..... ........ ..... 1
Methods..... .................................. . ....... .17
Results... ........................................... ..35
Discussion... ........................................ ..60
List of References... ....... .......... ..... ............80
Appendix A (Raw Data).. ............................... .87
Appendix B (Procedures) ......... . .................... .101
Appendix C (Supplies) ................................. 102

Table
Table
Table
Table
Table

Table

Table

Table

Table

Table
Table
Table
Table
Table

Table

10.

11.

12.

13.

14.

15.

LIST OF TABLES

Peptide content of directly apposed cells.....52

Homotypic vs heterotypic direct appositions...53

RAW DATA-SOMATA........... ....... .. ... ...... .88
RAW DATA- X SOMATA WITH DOUBLE SYNAPSES ....... 89
RAW DATA- DENDRITES...... ..... ....... ......... 90
RAW DATA- 3 DENDRITES RECEIVING DOUBLE

SYNAPSES/ AREA OF DENDRITES IN BUNDLES

AND SINGLES (unz) ...... ........... ............ 91
RAW DATA- MEAN NUMBER OF DENDRITES IN

BUNDLES 0F 1-4, 5-8 AND >= 9........ ....... ...92
RAW DATA- NEUROHYPOPHYSIS: MEAN #

ENCLOSED AXONS PER ANIMAL/ PER

PITUICYTE... ....... ...... ..................... 93
RAW DATA- VAGINAL CYTOLOGY

STAGE OF ESTROUS CYCLE........... ...... . .....94
RAW DATA- ICC...................... ......... ..95
RAW DATA- ICC APPOSITIONS... .................. 96
RAW DATA- Maternal Behavior ........ ...........97
RAW DATA— Maternal Behavior ............ .. ..... 98
RAW DATA- Maternal Behavior...................99
RAW DATA- Maternal Behavior ....... .. ......... .100

vi

LIST OF FIGURES

Figure 1. Cartoon depicting the four activities

which were considered to be maternal behaviors.. ........ 1

Figure 2. Schematic diagram depicting the
relationships of the SON somatic region (SON),
the dendritic zone (DZ), the optic tract (OT) and
the ventral glial lamina (VGL), which contains an
astrocyte cell body projecting processes into the

nucleus .......................................... .. ..... 6

Figure 3. Examples of the interrelationships

of SON somata. Panel A shows several cell bodies
which are separated from one another by neuropil,
although a small region of direct membrane
apposition (chevrons) can be seen. A dense

cored vesicle laden dendritic profile (D) can
also be seen. Panel B shows a SON neuron with
dilated endoplasmic reticulum which is directly
apposed to two other neurons (chevrons). A
double synapse (arrows) also makes contact with
two of the somata. Tissue shown in the top panel
is from maternally behaving animal 7 while that

in the bottom panel is from postweaning animal 1 ..... ...7

vii

LIST OF FIGURES- Continued

Figure 4. Examples of the interrelationships of
astrocytes (As) and dendrites in the dendritic zone
of SON. Two dendritic bundles are encircled; #1
consists of two dendrites while #2 consists of five
dendrites. Single dendritic profiles(s) are

also seen. The astrocytes, one of which contains
gliofilaments (f) can be seen sending their
processes amongst the dendrites (thin arrows).

A double synapse (thick arrow) can also be seen.

Tissue from maternal behaver #6.. ...................... 10

Figure 5. Example of a double synapse- plus one.
Three dendrites (D1, D2, and D3) in the

dendritic zone which are contacted (darts) by

a single "triple" synapse. 0f tissue examined from
42 animals this was the only instance of a triple
synapse found, double synapses being more commonly
observed. Tissue is from control animal #1.

Lead citrate - uranyl acetate stain........ ............ 11

Figure 6. Example of the interrelationships of
axons and pituicytes (P) and in the neurohypophysis.
The tissue has been immunocytochemically stained
with an antiserum raised against the
vasopressin-associated neurophysin. Specifically

stained endings (arrows) are interspersed with

viii

LIST OF FIGURES- Continued

unstained endings (*). Some "bleeding" of
reaction product onto the underlying formvar

film can be seen near intensely stained endings.
One ending (**), judged to be unstained, is
entirely enclosed by pituicyte cytoplasm.

Inset: neurohypophysial tissue which was exposed
to "Day-1" antiserum, raised against bovine
platelets, exhibits no immunocytochemical reaction
product. The darker granules seen in "unstained"
endings are likely due to the brief exposure to

osmium. Tissue from maternal behaver #2 ............... 28

Figure 7. Example of immunoreactive and
non-immunoreactive cell bodies in the SON. This
tissue was stained using an antibody to the
vasopressin-associated neurophysin. The cell

in the center (boundaries outlined with arrows)
was judged to be non-immunoreactive despite

the presence of a few densely-appearing dense
cored vesicles (DCVS), based on the prevalence

of "unstained" DCVS (right-hooked arrows) and the
robust staining seen in nearby cells.
Well-stained DCVS are marked by left-hooked
arrows. The long arrow points to non-specific
glitch. Tissue from control animal #1 ................. 31

ix

LIST OF FIGURES- Continued

Figure 8. Two electron micrographs taken at high
magnification show the appearance of
immunocytochemically stained verses unstained dense
cored vesicles (DCVS). The top panel is of tissue
which was not exposed to a primary antibody
whereas the bottom panel is of tissue
immunostained for the vasopressin-associated
neurophysin. Compare the density of the DCVS to
the density of nearby lysosomes (LY). Notice that
the immunostained DCVS are larger than their
unstained counterparts, denser than nearby
lysosomes and, in some cases, encrusted with
pentagonal peroxidase-antiperoxidase

molecules. Nuc = nucleus .............................. 33

Figure 9. Dendritic bundling in control and
maternally behaving animals. * P<.02: Tukey's test.

S.E.M.= 1.07 .......................................... 37

Figure 10. Top: Differences in the area of the
dendritic zones of control and maternally
behaving subjects. *p<.01; t-test. S.E.M.= 221
(controls) and 409 (maternal behavers). Bottom:
Differences in the number of dendrites counted in
control and maternally behaving animals.

*p<.05; t-test. S.E.M.s= 39.64 (controls)

LIST OF FIGURES- Continued

and 42.53 (maternal behavers) .......................... 39

Figure 11. Differences in the area of dendritic
profiles in the dendritic zones of controls
and maternal behavers. *p<.05;

Tukey's test. S.E.M.= 87 .............................. 42

Figure 12. Differences between maternal behavers
and controls in the number of axons/ pituicyte
totally enclosed by pituicyte cytoplasm.

*p<.035. S.E.M.s= .05 (controls)

and .04 (maternal behavers) ............................ 44

Figure 13. Serial sections of tissue from
post-partum animal #4 pass through a dendrite
adjacent to the optic tract (0T) which is
immunoreactive to a vasopressin—associated
neurophysin (VP-NP) antibody but not to an

oxytocin-associated neurophysin antibody (OX-NP) ....... 47

Figure 14. Serial sections from lactating animal
#6 through two cell bodies which have been
stained with alternate antisera show antibody
specificity and heterotypic cell-cell apposition.
The + and - indicate cells judged to be

immunoreactive and non-immunoreactive

xi

LIST OF FIGURES- Continued

respectively. The small, winged arrows point

to stained dense cored vesicles and the long,
straight arrows point to unstained dense cored
vesicles. Darts point to non-specific "glitch"
as determined at a higher magnification. Panel A
shows tissue stained with an antibody to the
oxytocin-associated neurophysin (OX-NP). The short,
fat arrows point to a region of direct membrane
apposition. The crystalline structures are of
unknown origin. Panel B shows tissue which has
been stained with an antibody to the

vasopressin-associated neurophysin (VP-NP) .............. 50

Figure 15. Top: Differences in the area of the
dendritic zone of postweaning and control group
animals. *p<.001, Mann-Whitney U test.

S.E.M.s= 220 (controls) and 642 (postweaning group).
Bottom: Differences between postweaning and control
groups in the number of dendrites observed in the
dendritic zone. *p<.02; Mann-Whitney U test.
S.E.M.s= 39.6 (controls) and

107 (postweaning group) .................... ............56

Figure 16. Differences between postweaning and
control groups in the area of dendritic profiles.

*p<.05, Tukey's test. S.E.M.=122 ....................... 58

LIST OF ABBREVIATIONS

CSF - cerebrospinal fluid

dcvs — dense cored vesicles

EM - electron microscopy

ICC - immunocytochemistry

MB - maternal behaver

MNCs - magnocellular neuroendocrine cells
ox - oxytocin

PVN — paraventricular nucleus

PW - postweaning

SON - supraoptic nucleus

VP - vasopressin

xiii

INTRODUCTION

$5.52!.-

Maternal behaviors are a constellation of activities
which have been described in many species (see Fahrbach and
Pfaff, 1982, for review) and which are comprised of various
care-giving activities which parental animals engage in with
their offspring, presumably to promote the offspring's, as
well as the species, well-being and longevity. It has
recently been shown quite dramatically (Pedersen et al.,
1982; 1984a; Fahrbach et al., 1984) that an
intracerebroventricular injection of the nonapeptide
oxytocin (0X), a hormone which promotes the milk-ejection
reflex during lactation and uterine contractions during
parturition, into ovariectomized estrogen-primed virgin
female Sprague-Dawley rats results in full maternal behavior
for that species (FMB; grouping and regrouping of pups,
licking of pups, crouching over grouped pups, nest building,
and retrieval of pups to a central location (Figure 1).
Further evidence for a role of ox in FMB is that the onset
of FMB can be blocked by the administration of an antibody
against 0X to ovariectomized estrogen-primed virgin rats
(Pedersen et al., 1984b). Two major regions of the brain
which produce oxytocin are the hypothalamic paraventricular
nucleus (PVN) and the supraoptic nucleus (SON). PVN, with

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Figure 1. Cartoon depicting the four activities which were

considered to be maternal behaviors.

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4

its extrahypothalamic projections (Swanson and Sawchenko,
1983) might be considered to be a likely way station in an
oxytocin-dependent pathway mediating FMB. However, when the
PVN is bilaterally lesioned or its lateral connections
severed, there is no disruption of FMB in already maternally
behaving rats (Numan, 1984). Eliminating PVN, other
anatomical data allow the SON to be somehow linked to the
occurrence of maternal behavior in primiparous lactating
rats as well as in ovariectomized, estrogen—primed,
nulliparous rats in which maternal behavior has been induced
by exposure to rat pups. It is fairly well established
(Fahrbach and Pfaff, 1982) that of the many brain areas
suspected of mediating maternal behaviors in the past (not
including SON) only the medial preoptic area (MPOA) appears
to be absolutely necessary for the induction and maintenance
of FMB. Because the MPOA sends projections to the SON
(Conrad and Pfaff, 1976; Swanson, 1976), the SON becomes a
strong candidate for promoting oxytocin-induced FMB. This
is particularly true in light of the recent demonstration
that some SON neurons have axon collaterals which terminate
in the nearby lateral hypothalamus (Mason, Ho and Batton,
1984), a region known to contain MPOA efferents (Conrad and
Pfaff, 1976). Thus, the influence of SON neurons now appears
to include the CNS itself.

If the SON somehow participates in the onset of
maternal behavior, might morphological changes occur in the

SON that are concurrent with this behavior? If so, what

5

form might they take? Since 1976 a growing body of work
from Batton, Tweedle, and associates (see Hatton et
al.,1984, for review, as well as Theodosia, Poulain and
Vincent (1981) and Theodosis and Poulain (1984a & b) has
established that during conditions of stimulation
(dehydration, parturition and lactation) neurons of the SON,
which produce and release vasopressin (VP) in addition to
oxytocin, display significant morphological plasticity.

This plasticity is in the form of changes in the
interrelationships of neurons and astrocytes and includes:
1) increases in cell-cell apposition (two neurons with no
intervening glial process between them), 3) dendritic
bundling (two or more dendritic profiles in direct
apposition), and g) the formation of double synapses (one
presynaptic axon terminal simultaneously contacting two
dendrites or two sonata), apparently brought about by
astroglial process retraction (Figures 2-5). Additional
complexity is indicated by the finding that of the stimuli
studied so far only the chronic stimulation associated with
gestation and lactation produce double synapse formation in
the dendritic zone (Perlmutter et al., 1984a). Ten days of
28 saline treatment produces double synapse formation in the
somatic zone (Tweedle and Hatton, 1984a), but not in the
dendritic zone (Perlmutter et al., 1984c). Indeed, evidence
for two populations of double synapses (one dendritic and
one somatic) has been presented (Tweedle and Batton, 1984b).

Based on the available circumstantial evidence it

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Figure 2. Schematic diagram depicting the relationships of
the SON somatic region (SON), the dendritic zone (02), the
optic tract (OT) and the ventral glial lamina (VGL), which
contains an astrocyte cell body projecting processes into

the nucleus.

       

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Figure 3. Examples of the interrelationships of SON somata.
Panel A shows several cell bodies which are separated from
one another by neuropil, although a small region of direct
membrane apposition (chevrons) can be seen. A dense cored
vesicle laden dendritic profile (D) can also be seen. Panel
B shows a SON neuron with dilated endoplasmic reticulum
which is directly apposed to two other neurons (chevrons).

A double synapse (arrows) also makes contact with two of the
somata. Tissue shown in the top panel is from maternally
behaving animal 7 while that in the bottom panel is from

postweaning animal 1.

Figure 4. Examples of the interrelationships of astrocytes
(As) and dendrites in the dendritic zone of SON. Two
dendritic bundles are encircled; #1 consists of two
dendrites while #2 consists of five dendrites. Single
dendritic profiles (s) are also seen. The astrocytes, one
of which contains gliofilaments (f) can be seen sending
their processes amongst the dendrites (thin arrows). A
double synapse (thick arrow) can also be seen. Tissue from

maternal behaver #6.

ix

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11

 

Figure 5. Example of a double synapse- plus one. Three

dendrites (D1, D2, and D3) in the dendritic zone which are
contacted (darts) by a single "triple" synapse. 0f tissue
examined from 42 animals this was the only instance of a
triple synapse found, double synapses being more commonly
observed. Tissue is from control animal #1. Lead citrate -

uranyl acetate stain.

12
therefore seems reasonable to ask whether maternal behavior
is accompanied by ultrastructural manifestations of hormone
production and/or release in SON neurons which are perhaps
similar to those previously documented in response to other
stimuli associated with gestation, lactation, and
dehydration. Part IA of the present study was therefore
undertaken to determine if changes occurred in SON
ultrastructure concurrent with the exhibition of maternal
behavior, without the animal experiencing pregnancy,

parturition, and lactation.

"0

art IB: Morphological correlates of maternal behavior in

 

the posterior pituitary.

Together with the examination of SON ultrastructure
under varying physiological conditions, Tweedle and Rattan
and associates have studied ultrastructural changes in the
posterior pituitary under those same conditions. They have
found that this structure, which contains axonal endings of
PVN and SON cells which release their hormones into the
fenestrated capillaries found there, also exhibits a
striking degree of plasticity when the neurohypophysial
system is stimulated. In particular, the axons of the MNCs,
many of which are normally enveloped by the cytoplasm of
pituitary neuroglia (pituicytes) have been seen to lose
their glial ensheathment under conditions of stimulation.
So far it has been shown that immediately after parturition

and during dehydration, and in response to a high osmolality

13

medium ip yippp, the number of neurosecretory axons which
are totally enclosed by pituicyte cytoplasm decreases
significantly (Tweedle and Eatton, 1980a, 1982; Perlmutter
et. al., 1984d). In Part IB of the present study the
possibility of similar changes occurring with the stimuli
associated with the induction of maternal behavior was

investigated.

Part I: Immunoelectron microscopy of SON neurons at late

 

gestation, parturition, and lactation.

Despite energetic investigation and the substantial
inroads made into understanding this complex peptidergic
system there is much yet to be learned. Many of the
questions to be resolved involve the formation of somatic
direct appositions, double synapses and dendritic bundling
over conditions of the pregnancy cycle in relation to the
peptide content of SON neurons and their dendrites. In
particular, the previous work (Hatton and Tweedle, 1982;
Perlmutter et al., 1984a) employing tissue from virgin rats,
prepartum, postpartum, and lactating rats has revealed a
different time course of events in the dendritic zone versus
at the cell body level. On the measures of percentage of
cell bodies or of dendrites directly apposed, percentage of
somatic or dendritic membrane in direct apposition, and
percentage of cell bodies or dendrites receiving double
synapses, the largest increases in the dendritic zone were

found immediately postpartum. The changes at the cell body

14

level did not reach their maximum until fourteen days of
lactation. This suggests that differences exist in the
functional demands placed on different levels of the SON
cells at parturition and lactation. Since there is evidence
(Fuchs and Saito, 1971) that both 0X and VP are released in
large quantities during parturition, whereas only oxytocin
is thought to show enhanced release during lactation
(Wakerley et al., 1973), the maximal dendritic effect at
parturition may reflect the activation of both peptidergic
cell types at this time. The same line of speculation holds
for those differences seen in the dendritic area between
pre- and postpartum groups. The peptide content of the
dendrites during parturition thus becomes of special
interest. Also, assuming that the morphological changes
seen at the level of the cell bodies reflect functional
demands, such changes during lactation would be expected
exclusively to involve OX-containing somata. Whether this
is the case remains to be elucidated. Although Theodosia
and Poulain (1984b) have reported OX- immunostained profiles
apposed to non-immunostained profiles, in tissue from
lactating subjects, methodological problems prevent easy
interpretation of these data. In Part II, the hormone
content of apposed and nonapposed neurons and, where
possible, the hormone content of dendrites participating in
bundles, was assessed by means of immunoelectron microscopy,
in tissue from control, prepartum, postpartum and lactating

rats.

15
Part II: Morphology pf SON neurons after g 19 day

 

postweaning period.

The time course of post-weaning changes is still
something of a mystery. In the SON dendritic zone area,
Perlmutter et al., (1984a) found that the lactating group
showed a trend toward a decrease from postpartum values on
the three measures of dendritic bundling, dendritic membrane
apposition, and the occurrence of double synapses. Whether
these decreases reflected an eventual return to base line is
not clear. Theodosis and Poulain (1984a) found that at one
month post-weaning, the extent of dendro-dendritic and
soma-somatic appositions was not significantly different
from that seen in virgins. Similarly, the number of double
synapses was not significantly different from virgins by one
month post-weaning. Knowledge of the time course of the
return to normal values, particularly of the interval
between 14-days of lactation and one month post-weaning,
would tell something of the lability of the system. Does
reinsertion of the glial processes occur rapidly after
weaning? How long does the system, once engaged, retain the
characteristics which might enable it to respond to the
stimulus of suckling? Further, Theodosis and Poulain
(1984a) did not distinguish between double synapses found at
the dendritic versus somatic levels, so it is unknown
whether the return to normal values differs on this measure

for the two regions. In Part III of this dissertation, the

16

morphology of neurons and dendrites in the SONs of rats
which had undergone pregnancy, parturition, 14 days of
lactation and ten days of a post-weaning interval was
studied to determine if traces of their maternal experience

were still evident at this time.

This dissertation therefore examines ultrastructural
and hormonal events which occur in the supraoptic nucleus
(SON) concurrently with the maternal experience in the
Sprague-Dawley rat. The data presented herein were gathered
to answer three basic questions: 1) Does the participation
of the SON in the events surrounding maternity include even
the promotion of the maternal behaviors which this species
engages in with its offspring7, 2) What is the peptidergic
content of the neurons which undergo morphologic
rearrangement over the course of the "Motherhood experience"
and 3) What is the lability of the system, i.e., how long do
the morphological traces of Motherhood persist?

For the sake of simplicity, these questions are
addressed here as though they are separate experiments
addressing separate issues. It should be remembered that
such a division is merely experimentally convenient. It may
be illuminating to consider the phenomena under study as
representing different facets of an underlying continuum of

events.

METHODS

Part I. Morphological correlates of maternal behavior.

Fourteen virgin Sprague-Dawley female rats were equally
divided into control and experimental groups. They were
housed in a 12:12 hr light/ dark cycle with food and water
ad libitum. Induction of maternal behavior was accomplished
with the protocol of Yogev et al. (1980): after 2 days of
habituation to 30.48 cm by 35.56 cm by 16.51 cm plastic
cages with wire tops, and to the standard light cycle,
subjects were constantly exposed to two foster young, 3-15
days old. The foster young were exchanged daily and placed
in opposite corners of the cage as far from the experimental
subject as possible. For a fifteen minute observation
period after introduction of the new pups each day, subjects
were scored on the incidence of three behaviors: 1)
retrieval of both pups to the same place in the cage, 2)
crouching over pups in a nursing posture and 3) pup-licking,
especially of the anogenital region. Subjects were also
provided with shredded paper towels and/or tissue paper and
evaluated for nest-building behavior. The behaviors were
independently scored by two observers until a consensus as
to what constituted occurrence of the behaviors was reached.
Subjects were considered to be maternal from the first of
two consecutive days that the first three behaviors were
observed and they were sacrificed on day three of FMB after
5 minutes of observation. Control subjects were housed in

17

18

plastic cages in the same nursery as were the M88, for 2
days prior to sacrifice. Control subjects were sacrificed
at age 120 days, while the experimental group ages varied:
101 days (2 animals), 104 days (2 animals), 112 days (2
animals), 138 days (1 animal). Time of sacrifice was 2 - 4
hrs after lights on. Vaginal lavage was performed to
determine the phase of the reproductive cycle at which
sacrifice occurred for both groups.

Electron microscopy.

Rats were deeply anesthetized with ether and then
transcardially perfused with physiological saline followed
by 150-200 ml of a solution of 3.5% glutaraldehyde and 1%
paraformaldehyde in a 0.1M cacodylate buffer. Brains were
removed from the skulls and placed in fixative for an
additional 2-3 hr prior to being cut into into 500 um slices
with a tissue chopper. The left and right SON were then cut
from each slice with a single edged razor, dehydrated with a
graded series of alcohols, and embedded in a water soluble
resin (L.R. White, E.F. Fullam, Inc., Latham, N.Y.).
Platinum and gold colored sections (60-90 nm) were cut with
an ultramicrotome from blocks of resin containing the middle
third of the SON, as partitioned into anterior, middle and
posterior portions, and collected on nickel grids coated
with 0.25% formvar. In rare instances it was necessary to
use tissue from the anterior or posterior SON. Thin
sections were also cut from blocks containing the posterior

pituitaries of control and experimental subjects.

19

Representative grids containing tissue from the SON or
posterior pituitary were counterstained with saturated
uranyl acetate for 30 minutes followed by lead citrate for 6
min. Grids were examined with a Philips 2010 electron _
microscope.
ngg collection- SQNp

Electron micrographs were obtained from one thin
section each from both the left and right SON of each
subject. The ventral dendritic zone of the SON was
photographed in its entirety, the criteria for inclusion
being that the dendrites were ventral to magnocellular
neurons and/ or had dendrites containing dense cored
vesicles (dcvs; presumably containing hormone) amongst them.
Grid bars were used as a guide to insure that all of the
area of the dendritic zone was photographed without overlap.
Dendrites were identified as having microtubules, rough
endoplasmic reticulum and/or free ribosomes, electron lucent
cytoplasm, at times the presence of 100-200 nm dcvs, and
often, being postsynaptic to presynaptic profiles. Care was
taken to include as many dendrites as possible in each
negative. Astrocyte cell bodies and processes were often
included in the photographs since they exist in this region.
However, the ventral glial lamina (VGL), consisting almost
exclusively of astrocyte cell bodies and processes was not
photographed except when the presence of numerous dendritic
profiles warranted its sampling. Sampling of the somatic

zone was accomplished by locating, at a magnification too

20

low to discern direct appositions (1612K, actual
calibration), 2 or more cell bodies in close proximity to
one another and then increasing the magnification for
photography. MNCs were identified primarily by their
location in the tightly packed SON adjacent to the optic
track and, at higher magnification, the presence of 100-200
nm dense cored vesicles in their cytoplasm. Pictures were
taken at a magnification of 3059K and enlarged 2.5 times
(print magnification 7590K). During the printing process,
random code numbers were assigned to the micrographs by an
associate in order to guard against observational bias
during scoring. Micrographs were then scored for 1) the
presence of direct appositions between cell bodies, 2) the
incidence and size of dendritic bundles containing two or
more dendrites, 3) length of apposed and non-apposed
membranes between dendrites or somata. and 4) the presence
of double synapses. Membrane lengths and dendritic areas
were determined by tracing membrane lengths on micrographs
which were placed on a Houston Instruments (Houston, Texas)
digitizing tablet 114 which was interfaced with a Zenith 150
personal computer (software by R. Luzenski). Data analysis
was aided, when appropriate, by the use of an Apple IIe
personal computer and statistical programs (Human Systems
Dynamics, Northridge, CA) for t-tests, 2 sample
correlations, and analysis of variance (ANOVA).

Nonparametric tests were employed when appropriate.

21

Egg; ppllection- posterior pituitary.

Without being aware of experimental group membership,
fifteen pituicytes from each neurohypophysis were randomly
sampled by a confederate (B. Kohn) at 4740K. A pituicyte
was first located on the electron microscope at low
magnification (1612K). The magnification was then increased
to 3059K and the perimeter of the pituicyte membrane
followed around its entire extent. The total number of
axonal processes completely enclosed by the pituicyte was

counted.

Part II- Immunoelectron microscopic correlation of
morphology and peptide content of SON neurons in control,
prepartum, postpartum and lactating animals.

Experimental subjects.

Twenty-one 18 day pregnant Sprague-Dawley rats were
received from Holtzman Co. (Kenosha, WI) and divided equally
into 3 groups. Animals from two of the groups, designated
prepartum and postpartum, were each housed in separate
hanging wire cages in which flooring and nesting material
were provided. The third group, designated lactating, were
each housed separately in 30.48 cm by 35.56 cm by 16.51 cm
plastic cages with wire tops. All were exposed to a 12:12
light cycle with food and water pg libitum.

On day 21 of gestation subjects in the prepartum group
were deeply anesthetized with ether and transcardially
perfused as described for the maternal behavers and the

control group. All were confirmed as being in the late

22

stages of pregnancy based on two observations: 1) the fetal
pups were well developed, to the point of being viable when
removed surgically and placed with foster mothers, and 2)
other rats determined to be sperm positive on the same date
began labor within 3 hours of the prepartum group's
sacrifice. Rats in the postpartum group were sacrificed
within 12 hours of delivering full litters of pups. Litters
of the group designated as lactating were culled or added to
in order that each subject was nursing 9—10 pups. 0n the
fourteenth day of lactation rats were sacrificed as
described for the other groups. The age at which the
various groups were sacrificed were as follows: prepartum,
112 days; postpartum, 112 days; and lactators, 128 days.
Time of sacrifice was 2 - 4 hr after lights off. Brain
tissue from all three groups, in addition to tissue from the
control group described in Part IA, was prepared for
electron microscopy as described for maternal behavers and
controls in Part 1A, except that no counterstains were

applied.

Immunocytochemistry.
Prologue. The goal of the EM immunocytochemist is to

produce tissue which has good morphology, specific
unequivocal labelling, and a paucity of excess reaction
product. For this task, he or she should have the
constitution of an Indiana Jones.

Three mouse monoclonal antibodies (MABS) were employed

23

as primary antisera. Two of these were the generous gift of
Dr. H. Gainer and were raised in his laboratory. Their
specificities have been thoroughly determined (Ben-Barak et
al., 1985). Monoclonal antibody (MAB) #38 has been
characterized as specifically binding to an as yet
undetermined portion of the oxytocin-associated neurophysin
peptide. MAB #41 is somewhat less specific, being
predominantly directed against an unknown part of the
vasopressin-associated neurophysin peptide (Russell et al.,
1980). Some cross-reactivity of this MAB with (a
presumably) oxytocin-associated peptide has been noted when
used at high concentrations (>8 1:50) in postembedding ICC.
The third MAB used, designated MAB "Day one" was the
generous gift of Dr. W. L. Smith and was raised against
bovine platelets. To date, it has only been found to bind
with certain parenchymal elements of bovine liver. This
third MAB was used to insure that the mouse MABs were not
non-specifically binding to the cells of the SON.

The ICC protocol employed in this study was a
post-embedding technique, that is, the reagents were applied
to the tissue after it had been embedded, thin sectioned,
and collected on grids. The formvar step was necessary to
prevent the rather unstable resin from disintegrating when
exposed to the electron beam in the microscope. After
sectioning, the grids were carefully inserted into squares
of Silgard (Dow Corning), a pliable, rubbery substance, into

which slits had been made with a single edged razor. Grids

24

were inserted such that each thin section would be exposed
to reagents. Reagents were applied onto the Silgard squares
where surface tension maintained their covering of all of
the grids. With this method it was thus possible to
immunocytochemically stain a great many grids at one time.
The protocol followed was:

1. phosphate buffered saline (PBS), pH 7.4- 8 minutes.

2. 18 sodium meta-periodate in PBS- 10 minutes.

3. 4 rinses with PBS 9 1 minute.

4. 10% normal goat serum (NGS) in PBS, 10 minutes.

5 Blot NGS from grids with filter paper.

6. Primary MABs #38 and #41 in PBS (1:100), MAB Day 1 in PBS
(1:50), 1 hour.

7. Reapply fresh MABs, 1 hour.

8. 6 rinses with PBS, 0 1 minute.

9. Goat anti-mouse peroxidase conjugate (Boeringer-Mannheim)
in PBS (1:20), 2 hours.

10. 6 rinses with PBS, 9 1 minute.

11. 10 mg/ 20 ml PBS 3,3'-diaminobenzidene, filtered with a
1.2 um pore size, Triton-x 100 free Millipore filter, 30
minutes.

12. 6 rinses with PBS, 8 1 minute.

13. 0.53 osmium tetroxide in PBS, 30 sec.

14. Each Silgard square was then dipped 4—8 times each in
three successive beakers of distilled water and blotted dry
with filter paper.

All incubations in sera were done on a lighted

25

histology table. Grids were variously stained on three
separate Silgard squares, one per primary MAB employed. The
Silgard squares were placed on separate pieces of filter
paper which had been dampened with PBS. They were then.
covered with the lids of glass petri dishes and placed
approximately 1.5 feet underneath a 100 watt incandescent
light bulb. All PBS was filtered prior to rinses and making
solutions with a .22 um pore size, Triton-K 100 free
Millipore filter. Step 13 was done under a safety hood.

No counterstains such as lead citrate or uranyl acetate were
applied to the tissue.

All the grids ultimately examined in this study were
stained in one session, in an attempt to reduce the
variability inherent in this technique. ICC was performed
on a rainy Tuesday afternoon in mid-April.

Examination 9; grids.

Data from a total of 48 grids, representing tissue from
28 animals (eight grids were lost in processing) was
collected on the electron microscope. Because of a lack of
contrast due to the absence of counterstain, the .253
Formvar coating on the grid, and an additional
low-contrastiness contributed by the L.R. White resin, grids
were examined with the emission control of the Philips 201c
set at 3. Setting the kv control at 40 was also employed in
an effort to improve contrast. Under these conditions it
was possible to quantitatively examine an entire SON (1

grid) in thin section in 30 - 60 min. For grids stained

26
with each MAB, either #38 (OX-NP) or #41 (VP-NP), counts
were made of the number of MNCs in close apposition, the
number unapposed, and whether they were judged to be
immunopositive or immunonegative. Efforts were also made to
identify double synapses and determine the peptide content
of the somatic or dendritic profiles they contacted.
§9:. E21211. QI m! 29.112 225 “stained?

Unlike ICC of pituitary tissue which yields
unequivocally stained or unstained axonal endings (Figure
6), ICC of MNCs, even between grids stained on the same
Silgard square, yields somewhat variable staining density of
dcvs. Therefore, it was necessary to develop, and
consistently apply, decision rules for judging dcvs to be
either stained or unstained.

Preliminary tests with various combinations of
counterstains such as lead citrate, uranyl acetate, and
osmium, applied to immunostained and control grids (primary
antisera omitted) revealed that the use of lead citrate
and/or uranyl acetate, often resulted in dark, dense cored
vesicles within MNC cytoplasm - in both control and
ICC-treated grids. Only osmium was found to leave the dcvs
in a condition which did not falsely appear "stained". The
tip-off for this phenomena was the observation that the
production of falsely -positive dcvs was accompanied by the
production of dense staining of the lysosomes within the
cytoplasm also; both apparently having a similar affinity

for the counterstains. In the absence of lead citrate and

27

Figure 6. Example of the interrelationships of axons and
pituicytes (P) and in the neurohypophysis. The tissue has
been immunocytochemically stained with an antiserum raised
against the vasopressin-associated neurophysin.
Specifically stained endings (arrows) are interspersed with
unstained endings (*). Some "bleeding" of reaction product
onto the underlying formvar film can be seen near intensely
stained endings. One ending (*t), judged to be unstained,
is entirely enclosed by pituicyte cytoplasm. Inset:
neurohypophysial tissue which was exposed to "Day-1"
antiserum, raised against bovine platlets, exhibits no
immunocytochemical reaction product. The darker granules
seen in "unstained" endings are likely due to the brief

exposure to osmium. Tissue from maternal behaver #2.

28

.o shaman

   

       

  

. .1. ii. “KM/,3. Id - .... .
.z/ wfilnnma afiX\

0...!
x .h ..H ..v..p.... \ .‘
......,.. t n ..u xi.“ 1% .IQIKAM e. «”46 0‘11..r. L ...
.- unntifixl. J ’m‘w .r .n .5 \ o' u.

.. .......H................. ....,..,:...... . ......Q... .. ..
. .... . as.

. . m. ... . 1a.! ... Q» .r m J:
a. .. ...! ..... n . .. o . , . .’
. .. .. ...... ...:c.............. 111M... u... i..- .r

29
uranyl acetate, but with the application of the .58 osmium
used in this procedure, well immunostained MNCs were found
to exhibit dcvs which were far darker in appearance than
nearby lysosomes, as well as the rest of the cytoplasm and
neuropil. This then, became a major criterion for judging
whether a cell was truly immunostained (Figures 7 and 8).
Other decision rules which evolved were based on the
following features:
1. Size. Well stained dcvs were generally seen to be
somewhat larger than normal (100-200 nm) due to being
covered by reaction product (Figure 8).
2. Numbers. Well stained cells generally appeared to
contain a wealth of dcvs because they were more visible
(Figure 7). This alone, however, was insufficient in order
to declare a cell stained, as some unequivocally stained
cells seemed to be depleted of their dcvs. Because the
VP-NP MAB #41 had been reported to somewhat cross—react with
Ox-NP when used at high concentrations (1:50), on grids
stained with that MAB, cells with only one or two "stained"
vesicles were judged to be unstained in the absence of other
information (Figure 7).
3. Staining of nearby cells. ~When there were very well
stained cells in close proximity to a cell with questionable
"staining" the former would be judged to be unstained
(Figure 7).
4. Absence of high background. Excess reaction product was

seen to distribute itself as small round, often dcv-sized

30

Figure 7. Example of immunoreactive and non-immunoreactive
cell bodies in the SON. This tissue was stained using an
antibody to the vasopressin-associated neurophysin. The
cell in the center (boundaries outlined with arrows) was
judged to be non-immunoreactive despite the presence of a
few densely-appearing dense cored vesicles (DCVS), based on
the prevalence of "unstained" DCVS (right-hooked arrows) and
the robust staining seen in nearby cells. Well-stained DCVS
are marked by left-hooked arrows. The long arrow points to

non-specific glitch. Tissue from control animal #1.

31

   

.-, .. . ....4: .. .. a .... ,.p . . ..-p .3? .fii‘a.a.. .

. r o ,u. , . . no .. 9": I... O. a e . e . h 9

..l , I 9.....‘H‘0 ‘Hfi.’ ..hs. ‘olb ale ‘1‘ u. . e . ’4' ..,-.. ' \‘ ....To 0° 1” I'Vo’l 4,”... Ha‘l..u.4’””..4‘ 0“ act as
. . . . ’21:!” A § 0 51“...» .W‘.

. . .... .J ‘o... ‘

.O
. . ..i . .
. . 3.. . . ‘.. . . ... 0.. f
., ....~.e.er 4.1;...ueuf . (.5 ..s‘.’ * “saw”. .....e‘ .
. ‘. dine l. .1 ..\. .1 ‘7 . . ....“

          
      

 

 

i .....x... .pqhai. than... ...: ... . o (a.
. . m T». if” a... x.2un..m..’.a.r?.~.,....ir e {HM/7”"
.. . .e .2. . p . - J..o-.v .’ of
......) .... .. 91...? uric. .. ,
r s M. . D 0.0- .5}. . i U
.' .. 0.....f .a )ewuufihe .h‘: .s....A~ .. Al. ...‘fi. fi
. a . . .e . . h.3.n . Ore fizua. -. .1: m..i\c¢ o . e
. .D 5" ‘ 8.1!; oozed ...- .Q'PW'IQJ’CN...;.O‘ /
. . c d .... #5, 2.1.1.1 .. .. ...»... .51
I a ...r ..‘u.anrb..\. v.‘ .0. .. .pdwofigo . IL C . .fi.

-‘

      

..

’I

ll... _ . . ......n...:..... . .. ...... .,.. 4.. . . .. .
a. . . . . : .. ... 23...... ... v.1 .. .. , ., . ., ..
. u . .. e o... l. ‘I .. ea .0“ .0 i.ed “.na* 99.. OVDPWI a... . y .. ‘o

.c .. . .. ... a \. .. .. 3f. .. ‘ lath. . ..~‘ 4 .0.) ., .’...
' C . 0.... s . C ‘o. . ‘ .e ’ ~ ' 1. ..Q. \-h 1‘35 ..s .vanfsre. ... .pfibuuose't‘J .. r We, I... the

32

Figure 8. Two electron micrographs taken at high
magnification show the appearance of immunocytochemically
stained verses unstained dense cored vesicles (DCVS). The
top panel is of tissue which was not exposed to a primary
antibody whereas the bottom panel is of tissue immunostained
for the vasopressin-associated neurophysin. Compare the
density of the DCVS to the density of nearby lysosomes (LY).
Notice that the immunostained DCVS are larger than their
unstained counterparts, denser than nearby lysosomes and, in
some cases, encrusted with pentagonal

peroxidase-antiperoxidase molecules. Nuc a nucleus.

33

 

Figure 8.

34

droplets. On occasion, it prevented unequivocal
identification of cells as stained or unstained. In such

cases data was not collected from those cells.

Part 111— Analysis of SON postweaning morphology

Seven 18-day pregnant Sprague-Dawley rats were obtained
from Holtzman Co. Upon arrival they were individually
housed in 30.48 cm by 35.56 cm by 16.51 cm plastic cages in
the laboratory animal "nursery" in conditions identical to
those described for the induced-maternal behavior
experiment. At one day postpartum their litters were either
culled or added to, such that each subject had a litter of
9-10 pups. (It was later necessary to included data from an
alternate postweaning subject who had only six pups, as some
tissue was damaged in processing). At day 14 postpartum,
pups were removed from their home cages and cross-fostered
with other non-experimental lactating mother rats. Subjects
were allowed to survive for 10 days after the removal of the
pups prior to sacrifice. Preparation of tissue for EM and
morphological analysis were as described for maternal
behaver and control tissue in Part I. Post-weaning subjects
were all 112 days old at time of sacrifice and compared with
control animals 120 days of age. Time of sacrifice was 2 -

4 hr after lights off.

RESULTS

PART IA- Morphometry of the SON of maternally behaving
virgin rats.
Behavioral observations (Figpre 1);

Experimental subjects displayed the maternal behaviors
of retrieval of pups to a central location, licking of pups.
including anogenital licking, and crouching over pups in a
nursing posture after 5 to 16 days of being housed with rat
pups. Six out of the seven rats also used the materials
provided to build distinct nests, i.e., digging depressions
in the bedding and arranging the paper towel strips around
the perimeter in a methodical fashion, where they would
deposit the rat pups after retrieval, although these did not
appear as deep or appear to employ as many paper towel
strips as those built by actual nursing mother rats with

litters of 8 or more offspring.

Morphometry.
Somatic pppositions. (Figpre p1;

A total of 298 (X=42) and 246 (E=35) magnocellular
neurons were examined from the maternally behaving and
control groups respectively. The percentage of cells seen
to be in apposition was 28.9 I 3.99% vs 19.5 I 1.788,
controls and maternal behavers (MBs) respectively, a
non-significant difference (Mann Whitney-U test, p<.08).

Likewise, the percentage of membrane in direct apposition

35

36

(5.0 I .58% versus 3.9 I .63%, controls and MBs

respectively), was not significantly different.

Double synapses-somata. (Figure 3).

Two populations of double synapses were seen to occur

 

in the somatic regions of MBs and controls; both of which
were rare events. The first consisted of one presynaptic
axon simultaneously contacting two postsynaptic somata. An
average of only 2.88 I 1.22% and 2.54 I 1.52% of the somata
in the MB and control groups were seen to receive such
synapses, a non-significant difference. The second kind of
double synapse observed was one presynaptic axon contacting
one cell body and one adjacent dendrite simultaneously. No
significant differences were found between MBs (i=2.35 I
1.54%) and controls (R=5.00 I 1.29%) on this measure.
Neither were differences detected when the two measures were
combined for analysis.

Dendritic bundling. (Figure 5);

Because previous work established that dendritic
bundles of up to four dendrites were often seen in the
dendritic zone of normal animals (Perlmutter et al., 1984a),
data were equally categorized into bundles of 1-4, 5-8 and
9-12 dendrites. A two-factor analysis of variance (ANOVA)
was employed to assess differences between control and
maternally behaving groups in the average number of
dendrites per bundle in bundles falling into each of these
categories. An overall difference (p<.025, F= 7.06, df= 1,

12) was found between the two groups for the average number

37

Dendritic Bundling: Controls vs Maternal Behavers

..a
o

v
*

V D Controls
1' Maternal Behavers

1r 113E. M.

 

..........

Mean Bundle Size per Category
C -* M 00 It 01 O) \l on 0

 

 

 

 

 

 

 

 

 

 

 

Number of Dendrites per Bundle

Figure 9. Dendritic bundling in control and maternally
behaving animals. * p<.02; Tukey's test.
S.E.MO- 1.07.

38

of dendrites per bundle. Post-hoe comparisons (Tukey's
test; Gill, 1978) revealed that whereas no differences
existed between groups in the average number of dendrites
per bundle in bundles of 1-4 and 5-8, the two groups
differed significantly (p<.025, q= 6.08, df= 2,12) in the
occurrence of bundles containing 9-12 dendrites (is = 9.26
I 1.07 and 2.71 I 1.07; MBs vs controls; Figure 9).
Percentage pf dendritic pembrapg Ag direct apposition.

Analysis of data gathered with the aid of the
digitizing tablet failed to detect any differences between
groups in either the absolute length of dendritic membrane
in direct apposition (is I S.E.M. = 232 I 31 um, controls:
269 I 36 pm, MBs) or when the percentage of membrane in
direct apposition was computed as a percentage of total
membrane (apposed membrane/ apposed membrane + unapposed
membrane). The means and standard errors for the latter
measures were 13.64 I 1.04% (controls) vs 12.48 I 1.08%
(MBs).
Number pf gendrites observed. (Figure $91;
An average of 622 I 42.53 and 511 I 39.64 dendrites were
observed per 2 thin sections per animal in the maternally
behaving and control groups respectively. By Student's
t-test, these differences were found to be significant (p <
.05, t8 1.77, df=12; since no pyknotic profiles or other
visible signs of degeneration were observed in the material,
the possibility of dendritic loss in the MB group did not
seem realistic, thus a one-tailed critical value was

accepted). The correlation (Pearson's product-moment

39 L

Area of Dendritic Zone:
Controls vs Maternal Behavers

El Controls *
7000 i E Maternal Behavers
6000 v
5000 '- T

 

4000 "
3000 n
2000 V

Dendritic Zone Area (um 2)

1000 *-

 

 

 

 

 

Number of Dendrites Observed:
Controls vs Maternal Behavers
700 1r *

600 "

 

500 "
400 "

300 l'

Number of Dendrites

200 "
100 T
0

 

 

 

 

 

Figure 10.

40

Figure 10. Top: Differences in the area of the dendritic
zones of control and maternally behaving subjects. I"p<.01:
t-test. S.E.M.= 221 (controls) and 409 (maternal behavers).
Bottom: Differences in the number of dendrites counted in
control and maternally behaving animals. *p<.05; t-test.

S.E.M.s= 39.64 (controls) and 42.53 (maternal behavers).

41

correlation coefficient) between the total number of
dendrites observed per animal and the mean number of
dendrites in bundles of 9—12 per animal was not
significantly different from zero (rs .32, p) .05).
App; pf dendritic ggpgp (Figure i9);

Due to heterogeneous variance between groups a median
test (Siegel, 1957) was used to determine that significantly
(p<.0004) more micrographs of the ventral dendritic zone
were obtained from tissue from maternally behaving animals
relative to controls. An average of 7.71 vs 5.71
micrographs were taken per animal, respectively. When
considered in terms of area, each negative, taken at the
magnification of 3,059X, encompassed an area of
approximately 835 pmz. The mean area occupied by the
dendritic zone per two thin sections per animal was 4774 I
221 um2 versus 6326 I 409 pm2 for controls and M88
respectively, a statistically significant (p<.01,t=3.092,
df=12) difference. A partial correlation analysis was
performed to determine to what extent the greater number of
micrographs obtained from the MB group contributed to the
more frequent observation of large bundles in that group.
With the amount of variance contributed by the number of
micrographs factored out, no significant correlation (r-.O8,
p>.05) was found between the number of dendrites observed in
dendritic bundles of 9 or more dendrites and the total

number of dendrites observed.

42

Area of Dendritic Zone Comprised of
Dendritic Profiles: Controls vs
Maternal Behavers

   
 

800« El Controls
700 i Maternal Behavers

600., I 1 S.E.M.
5001- -~

400 ~-
3001-
200 i-
100 i

  

Dendritic Profile Area (umz)

 

 

 

 

 

 

 

 

Single Dendrites Bundled Dendrites

Figure 11. Differences in the area of dendritic profiles
in the dendritic zones of controls and maternal

behavers. * p<.05; Tukey's test. S.E.M.- 87.

43

Area g; gendpites. (Figpre 11).

Analysis of the total area of dendritic profiles in

 

bundles and in single dendrites (ANOVA) revealed an overall
difference (F= 7.59, df = 1, 12, p< .02) between controls
and MBs in dendritic area. Tukey's test for individual
comparisons showed a significantly larger dendritic area for
those dendrites participating in bundles in the MB group (q=
3.68, df -- 2, 12, p < .05; )1 teen. - 462 i 87 m2”
controls and 785 I 87 umz, MBs). No differences were found
between the two groups in the area of single, unbundled,
dendrites.

Dendritic double syngpses.

No differences were found between the groups for the
percentage of dendrites contacted by double synapses, (1.21%
vs 0.92%; controls vs MBs).

Vaginal cytology.

Inspection of vaginal smears showed that animals from
both groups were at various points in the estrous cycle at
the time of sacrifice. As determined by this method
(Martin, 1976) MBs were distributed amongst the points of
the cycle as follows: metestrus (1), proestrus (3), diestrus
(2) and estrus (1)._ Four of the control animals were
observed to be in metestrus while the others exhibited
vaginal cytology associated with proestrus (1), estrus (1),
and diestrus (1).

Miscellaneous observations;

 

No significant correlations were found between the

number of days the MBs were exposed to rat pups and: the

44

Axons Enclosed by Pituicyte Cytoplasm
in the Posterior Pituitary:
Controls vs Maternal Behavers

 

 

0.5 v D Controls
.3 Maternal Behavers
.. b.
O a.
3 § 0.3 *r *
S s
z 5.5
5513 (AZ "
fig a:
O
E; Oler
01)

 

 

 

 

 

 

Figure 12. Differences between maternal behavers and controls
in the number of axons/ pituicyte totally enclosed
by pituicyte cytoplasm. * p(.035. S.E.M.- .05

(controls) and .04 (maternal behavers).

45

average number of dendrites in bundles of 9 - 12 (r = -.28,
df = 5): the number of dendrites (r a -.12, df = 5); or the
dendritic zone area (r a -.10, df = 5).

In a small amount of material from two maternally
behaving animals, profiles of processes containing dense
cored vesicles similar to those observed in SON dendrites
and somata, i.e., 100-200 nm in size with an electron lucent
"shell" were observed in regions approximately 600-1000 um
lateral to SON, well outside the boundaries of that nucleus.
A very few cells were seen to display other indices of
cellular activation. Nine cells displayed swollen
endoplasmic reticulum (Figure 3). Two of these were from 1
MB animal, the other eight were from 3 control animals. One
cell each from 1 control and 3 MBs displayed double

nucleoli.

PART IB-Morphological correlates of maternal behavior-
posterior pituitary.

Pituicyte enclosure 9; gggpg 1p the posterigp pituitary
(Figures g g lg);

A tally of the number of axons enclosed by pituicyte
cytoplasm revealed that the group means I standard errors
for the number of axons enclosed per pituicyte were 0.22 I
0.04 for MBs and 0.39 I 0.05 for controls, a significant

(p<.035, t=2.377, df=12) decrease of 43% for the MBs.

46
Part II- Electron microscopic immunocytochemistry of SON
neurons.
Control pgocedpres.

Examination of grids which had been exposed to MAB
Day-one as the primary antiserum, or those which had buffer
substituted for primary antiserum showed a lack of reaction
product over dcvs of SON neurons and pituitary axons. Nor
was reaction product observed over any part of the cytoplasm
or surrounding neuropil in the SON and neural lobe. Due to
the high amount of antigens concentrated in a small area,
staining of posterior pituitary tissue with anti—OX-NP or
anti-VP-NP might be expected to produce cross-reactivity
(Ben-Barak et. al., 1985; Whitnall et. al., 1985). However,
a selective staining of axonal profiles was obtained in this
region (Figure 6). Serial sections which had been
alternately stained with OX-NP and VP-NP MABs exhibited
dendritic and somatic profiles which appeared to be
specifically immunostained, i.e., immunopositive (0X—NP+ or
VP-NP+) in one section and immunonegative in the other.
(Figures 1a a 14). A total of 527 (x i S.E.M./ group = 131
I 6.38) and 705 (151 I 28.14) cells were examined in
anti-OX-NP and anti—VP-NP stained tissue, respectively.
Based on the data of Swaab et al., 1975a, it might be
expected that, at the level of the SON which was sampled,
50% of the cells observed on each grid should have been
immunopositive for either anti-OK-NP or anti-VP-NP. A tally
of the percentage of cells sampled which were immunopositive

for either antigen showed that an average of 87.53 I 3.38 %

Figure 13.

47

 

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48

Figure 13. Serial sections of tissue from post-partum
animal #4 pass through a dendrite adjacent to the optic
tract (0T) which is immunoreactive to a
vasopressin-associated neurophysin (VP-NP) antibody but not

to an oxytocin-associated neurophysin antibody (OX-NP).

 

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Figure 14.

50

Figure 14. Serial sections from lactating animal #6 through
two cell bodies which have been stained with alternate
antisera show antibody specificity and heterotypic cell-cell
apposition. The + and - indicate cells judged to be
immunoreactive and non-immunoreactive respectively. The
small, winged arrows point to stained dense cored vesicles
and the long, straight arrows point to unstained dense cored
vesicles. Darts point to non-specific "glitch" as
determined at a higher magnification. Panel A shows tissue
stained with an antibody to the oxytocin-associated
neurophysin (OX-NP). The short, fat arrows point to a
region of direct membrane apposition. The crystalline
structures are of unknown origin. Panel B shows tissue
which has been stained with an antibody to the

vasopressin-associated neurophysin (VP-NP).

51

per animal of the cells sampled were deemed immunopositive
for one antigen or the other. The percentage of
immunopositive cells/ antiserum for all groups was 49 i 2.2%
(OX-NP) and 45 t 8.4X.(VP-NP).

reproductive state. (Table ii.

A two-factor ANOVA was employed.to assess whether
OX-NP+ or VP-NP+ cells preferentially formed close
appositions during late gestation, parturition, and
lactation. The mean percentages of apposed control group
neurons immunopositive for ox-Np and VP-NP were 9.878 and
10.22% respectively. To control for possible differences in
antibody affinity, data from the experimental groups were
expressed as a percentage of the mean number of apposed
control group neurons immunopositive for the same antigen.
The analysis confirmed previous observations that the number
of cells in direct apposition varies in relation to
reproductive state (p<.02, F I 6.87, df= 3, 40). As for the
interaction between cellular peptide content and the
likelihood of forming direct appositions, no significant
interaction was found (p>.05, Fa 2.99, df= 3, 40),
indicating that one peptidergic cell type was participating
to a greater extent in cell-cell appositions. Individual
comparisons (Tukey's test) reveal that whereas VP-NP+ cells
did not differ significantly from controls in percentage of
apposition across reproductive states, the percentage of
apposed OX-NP+ cells in the postpartum group was

significantly greater (p<.05; q=5.15, prepartum vs

52

postpartum; df = 4, 16) than that seen in both control and

prepartum groups.

Table 1. Peptide content of directly apposed cells.

 

Mean x Cells in Apposition (x of Controls)

OX-NP + VP-NP +
Controls 100 i 37 100 i 37
Prepartum * 93 t 37 116 i 37
Postpartum ** 281 i 37 182 i 37
Lactating 123 t 37 225 t 37

** = different from controls (p <.05)-

a
ll

different from postpartums (p <.05).

Homotypic versus heterotypic direct appositions (Table 2).

A second analysis (two-way ANOVA) was performed to
determine if the apposition between cells of like peptide
content (homotypic) or unlike peptide content (heterotypic)
changed with reproductive state, and whether one of these
was more prevalent than the other. No changes were detected
in the type of apposition across reproductive state (F=
0.598, df= 3, 88). However, a highly significant difference
was found in the occurrence of heterotypic versus homotypic
cell-cell appositions (P<.003, F=9.40, df=1, 88).

Individual comparisons (Tukey's test) revealed that there
were significantly (p<.05, q=3.49, df= 2, 24) more

homotypic appositions in the prepartum group than

53

heterotypic appositions.

Table 2. Homotypic vs heterotypic direct appositions

 

Mean 8 Appositions

Homotypic ggtgggtypig
Control 11 t 3.0 6 i 3.0
Prepartum * 22 i 3.0 12 i 3.0
Postpartum 24 i 3.0 17 i 3.0
Lactating 22 i 3.0 19 t 3.0

*8 different from corresponding 3 heterotypic

appositions (p<.05).

Double synapses 33g bundled dendrites.

Collecting data on these items proved to be, if not
impossible, then foolhardy. Whether due to the absence of
counterstain, hence contrast, or other processes employed
for ICC, only six double synapses were seen in the
immunostained tissue- one of these was contacting one cell
body and one unstained dendrite. Two double synapses were
between cells both immunostained with anti-OX-NP. two were
between two cells stained with anti-VP-NP, and one was
between two cells which were negative for anti-VP-NP.
Similarly. dendritic peptide content was only discernible in
those dendrites containing DCVs. Further, without
counterstain it too often became an intuitive exercise

determining if dendritic membranes were indeed apposed.

54

Part III- Morphometry of postweaning group (Lead citrate -
uranyl acetate stained tissue).

Somatic appositions (Figure 3).

 

A total of 246 (X i S.E.M./animal =35.1 i 2.56) and 332
(X=47.4 t 3.14) cell bodies were examined for direct
apposition in control (N a 7; same as used in MB comparison)
and postweaning ( N = 7) animals each. No significant
differences were found on this measure between the
postweaning group and controls. The mean i S.E.M.
percentages of cell bodies in soma-somatic apposition were
29-7 i 4.5% (postweaning) and 28.9 t 4.0% (controls).
Percentage g; somatg contacted by double synapses (Figure
3);

No statistically significant differences were found in
the percentage of SON somata contacted by: 1) double
synapses which contacted two somata (i i S.E.M.. postweaning
vs control, a 6.54 t 2.12%; 2.54 t 1.52%) or 2) double
synapses which contact one soma and one dendrite (5.54 i
.99%; 5.00 i 1.29%. or 3) when values for the two kinds of
double synapses were combined.

Dendritic bundling (Figure A);

Dendritic bundles containing up to 14 dendrites were
observed in tissue from the postweaning group. Analysis of
overall differences between groups was performed as for the
maternal behavior experiment. The average number of
dendrites in bundles of 1-4 dendrites, 5-8 dendrites, and in

bundles containing 9 or more dendrites was compared for the

55

two groups with a two-factor ANOVA. The average number of
dendrites in the three categories of bundle size was not
found to be significantly greater than controls in the
postweaning group (F=4.10, df= 1, 12, p>.05). Neither were
differences found on the measure of percentage of dendritic
membrane in apposition (t=.965, df=12, p>.05). Means and
standard errors for controls and postweaning animals were
13.63 i 1.0 and 12.11 t 1.0 respectively.

Number 9; dendrites observed (Figure 15);

An average ( t S.E.M.) of 849 t 107 versus 511 t 39.6
dendrites were counted per two thin sections per animal in
the dendritic zones of postweaning and control animals,
respectively. A statistical comparison for data with
heterogeneous variance revealed this difference to be
significant (Mann-Whitney U, U = 6, p<.02).

Aggg g; dendritic ggne (Figure 15).

Due to heterogeneous variance a median test was used to
determine that significantly more micrographs of the
dendritic zone were obtained from tissue from postweaning
rats (p<.0003). An average of 5.71 versus 9.85 micrographs
were obtained from controls versus the postweaning group.
When interpreted in terms of area sampled, a mean area of
4774 i 220 pm2 versus 8235.15 1 642.53 pmz was occupied by
the dendritic zones per two thin sections per animal in
control and postweaning animals respectively. This
difference was found to be significant (U = 0, p<.001) when
tested with a Mann—Whitney U test. Data gathered with the

aid of the digitizing tablet revealed that the area of

9000 v
8000
7000
6000
5000 v
4000 v
3000 v
2000 n
1000

Dendritic Zone Area (um 2)

A
I

 

56

Area of Dendritic Zone:
Controls vs Postweaning Group
*
El Controls

I Postweaning

 

 

 

 

 

1000 -
900 -

700 v
600 ~-
500 «-
400 --
300 - -
200 - -
100 -

0

Number of Dendrites

 

Number of Dendrites Observed:
Controls vs Postweaning Group

 

 

 

 

 

Figure 15.

57

Figure 15. Top: Differences in the area of the dendritic
zone of postweaning and control group animals. *p<.001,
Mann-Whitney u test. S.E.M.s= 220 (controls) and 642
(postweaning group). Bottom: Differences between postweaning
and control groups in the number of dendrites observed in
the dendritic zone. *p<.02; Mann-Whitney U test. S.E.M.s=

39.6 (controls) and 107 (postweaning group).

900‘
800 'r
700 «-
600 «-

400 --
300 -»
200 --
100 -

Dendritic Profile Area (um2 )

 

 

 

58

Area of Dendritic Zone Comprised of
Dendritic Profiles: Controls vs
Postweaning Group

[:1 Controls
Postweaning
1 S.E.M.

 

 

 

Figure 16.

Single Dendrites Bundled Dendrites

Differences between postweaning and control
groups in the area of dendritic profiles.

p<.05, Tukey's test. S.E.M.- 122.

59

dendritic profiles was significantly greater than controls
in the postweaning group. (F=15.78, df=1, 12, p<.002;
Figure 16). Post—hoc comparisons (Tukey's test) showed that
a significantly larger area was comprised of single
dendrites, whereas no differences were found in the area
covered by bundled dendrites (q- 4.37, df= 2, 12, p<.05).
The mean area (i S.E.M., umz) of the dendritic zone devoted
to dendritic profiles was 357 i 122 (single controls), 462
i 122 (bundled controls), 793 i 122 (single postweaning),
and 818 i 122 (bundled dendrites).
Double synapses: dendritic gggg (Figures 1 g 5);

The percentage of dendrites in postweaning animals
which were contacted by double synapses was not
significantly greater than controls (Means t S.E.M. = 2.50

i 0.48% versus 1.21 i 0.30 respectively; p>.05, t=2.081, df=
12).

Discussion

Part I. Morphological correlates of induced maternal
behavior.

Once again the SON has displayed a dissociation between
events at the cell body level versus the dendritic level
(Hatton and Tweedle, 1982; Perlmutter et al., 1984a). While
no changes were found in the extent of direct apposition in
the somatic region concurrent with the induction of maternal
behavior, the stimulus or stimulus complex which elicits
such behavior appears to have promoted substantial changes
in the dendritic zone. The data presented here which
demonstrate that changes occur in SON in virgin animals in
which maternal behavior has been induced, suggest a role for
the cells of the SON which goes beyond contributing to the
well established "reflexive" hormonal events surrounding
motherhood in the rat such as the release of hormones at
parturition (Fuchs and Saito, 1971) or the milk ejection
reflex (Wakerley et al., 1973). The correlation of
ultrastructural changes in the SON with a complex behavior
in this species suggests that a hormonal mechanism may
underlie the expression of such behavior as well as
constituting a novel demonstration of neural plasticity in
adult animals.

The question arises: what stimulus or stimulus complex
was responsible, either directly or indirectly, for the

observed changes in the dendritic regions of the SON? The
60

61

necessary conditions for the establishment of maternal (or
paternal) behavior in rodents has been well studied (Beach,
1956 a & b: Benuck and Rowe, 1976). It is known that
virgin female rats, (usually ovariectomized and receiving
estrogen replacement therapy) can become maternal in the
absence of olfactory and visual cues (Herrenkohl and
Rosenberg, 1972: Rosenblatt, 1979). It appears that the
only absolutely necessary condition for the induction of
maternal behavior is that the "mother" have actual physical
contact with the pups. Because of this, it has been thought
that some substance is transferred from the pups to the
induced—behaver upon contact, perhaps pheromones in the
urine. In this study it was noticed that quite often
anogenital licking preceded the occurrence of full-blown
maternal behavior by several days. At this time, the actual
stimuli for induction of maternal behavior remains a
mystery. Also, it should not be assumed that the stimuli
which lead to maternal behavior are exactly the same as
those leading to morphological changes. In this study
control animals were housed predominantly (except for two
days prior to sacrifice) in a separate room away from the
nursery, i.e., away from non-contact kinds of pup stimuli
such as ultrasounds for example. The possibility exists
therefore, that merely being housed in the same room as pups
could lead to dendritic bundling. There is some evidence
that this is not the case, however. Most notable are the
data from the postweaning group (which will be discussed at

greater length in Part III of this discussion) which

62

indicated that the extent of bundling was not different from
controls. This group, obviously, had been extensively
exposed to both contact and non-contact pup stimuli in
addition to the morphometric analysis for dendritic bundling
for this group being identical to that performed for the MB
group. Nonetheless, in light of these issues, it is hoped
that further studies will be undertaken to more precisely
delineate those factors responsible for the morphological
changes observed in the MB group in this study. One
interesting manipulation would be to examine the SON
ultrastructure of virgin rats who had been exposed to pups
but who had failed to display maternal behavior.

Assuming that the observed changes in SON are indeed
related to the expression of maternal behavior, it is also
unknown whether the SON is a primary mediator of maternal
behavior. Given that this study represents the first time
that SON has been associated with maternal behavior the more
conservative interpretation is that it receives afferent
input from another region which is firmly established as
promoting maternal behavior, the medial preoptic nucleus
(Fahrbach and Pfaff, 1982: Conrad and Pfaff, 1976). This
notion gains credence when the necessity for estrogen in the
production of maternal behavior is considered.
Estrogen-priming is essential for eliciting of maternal
behavior in ovariectomized virgin female rats and greatly
reduces the latency to onset of the behavior in intact
females. The number of receptors for this steroid is far

greater in the medial preoptic area as compared to the SON

63

(Sar and Stumpf, 1975)

Comparison 9; SON bundles versus other CNS bundles.

 

The phenomenon of dendritic bundling (see Roney,
Sheibel, and Shaw, 1979, for review) has been reported in
the literature since 1970. Bundles have been variously
noted in the spinal cord (Anderson et al., 1976), brainstem
(Felten, 1977), cerebral cortex (Peters and Walsh, 1972),
nucleus reticularis thalami (Scheibel and Scheibel, 1972),
habenular nucleus (Iwahori, 1977), and olfactory bulb
(Scheibel and Scheibel, 1975). Some investigators have
included in bundles dendrites which are as much as 1 pm
apart although that literature seems to be based primarily
on light microscopic work. In the present study only
dendrites whose membranes appeared to be in direct
apposition were counted as being a part of any one bundle.
The number of dendrites in a bundle in the CNS varies quite
widely, from 2-3 dendrites/ bundle in layer five of the
visual cortex of the cat (Roney et al., 1979) to as many as
1200-1600 in motorneurons in the ventral horn of the rat
(Anderson et al., 1976). The extent of the bundling seen
amongst the dendrites of SON neurons (2-14 dendrites/ bundle
in this dissertation) is similar to that reported for many
regions of somatosensory cortex in a wide range of species
(Roney et al., 1979). However, functional comparisons of
the two regions based on extent of bundling would be
undoubtedly a foolhardy exercise at this juncture. Another
feature of bundling elsewhere in the CNS is an apparently

regular occurrence of bundles within any one region

64

(Scheibel et al., 1975). This certainly did not seem to be
the case in SON where bundling seemed to occur randomly in
the total population of dendrites. Finally, one feature of
SON bundling which, so far, appears to be indisputably
unique (to my knowledge) in the literature is that the
extent of the bundling is plastic, varying over
physiological (Perlmutter et al., 1984) and now behavioral
states.

The finding of significantly more dendrites and a
larger dendritic profile area in the dendritic fields of
maternal behavers was unexpected. At this juncture it is
unknown whether the increased number of profiles represent
the formation of new primary dendrites or the branching of
already existing dendrites. Given the distance of the
dendritic zone from the somatic zone, the latter explanation
seems more likely. On first inspection it might appear that
the finding of more dendrites was an artifact created by
simply taking more micrographs. This is unlikely since the
total area of the dendritic zone was sampled in each group
and the criteria for identifying dendrites in this region
held the same for both groups. The source of these
apparently new dendrites could be one of the following: 1)
sprouting of new dendritic branches from already existing
dendrites; 2) an ingrowth of dendrites from a population of
dendrites existing outside of the SON; or 3) an ingrowth of
dendrites from proliferating neurons in SON. The third

possibility can be rejected on the grounds that there are no

65

data which indicate that neurons in SON proliferate in adult
animals, even with the most robust stimulation, prolonged
dehydration for example. The second possibility may be a
bit more reasonable than the third: however Golgi studies of
this region indicate that all the dendrites present ventral
to SON emanate from SON neurons (Armstrong et al., 1982). A
development of new dendritic branches seems the most
reasonable explanation for the increased number of dendrites
in the SON of maternal behavers. Such a phenomenon is not
without precedence in other parts of the nervous system.
Buell and Coleman (1981) using a Van der Loos modification
of the Golgi method have shown that in the parahippocampal
gyrus, normal aged humans had longer and more branched
terminal apical dendrites than did younger adults or adults
with senile dementia. More related to the issues of
plasticity, Rutledge et al., (1974) found that long term
electrical stimulation of cortex in cats (thought to
represent increased neuronal activity), led to a greater
branching of the apical dendrites of contralateral cortical
pyramidal cells. With respect to the maternal behavers
examined in this study, it does not seem too radical to say
that something in these animals' environments ultimately
influenced their brain anatomy. One is reminded of the
early “enriched environment" work of Bennett, Diamond,
Krech, and Rosenzweig (1964: to cite but one of their
published reports) who found that placing toys in the cages
of rats resulted in morphological and biochemical changes in

cortex, but not in "rest of brain" (they had their

66

priorities). This line of research was extended to include
effects of an enriched environment on dendritic morphology.
Uylings et al., (1978) found that an enriched environment
consisting of toys being placed in rats' cages for thirty
days resulted in increased branching of basal terminal
dendrites of visual cortex pyramidal neurons. The addition
of pups into the cages of the maternal behavers could
conceivably be viewed as enriching the environments of these
rats. In the case of normal mother rats, it would be
particularly efficient if the appearance of pups into their
environments led to an enhanced capacity of SON neurons to
respond to incoming signals from both the environment as
well as other brain regions.

It should be noted that the increased size of the
dendritic region, as determined from the area photographed,
is likely not entirely due to a greater number of dendrites.
While an effort was made during sampling to include as many
dendrites as possible in each micrograph, astrocytes and
endothelial cells were also present in the micrographs and
it is possible that the number and/or area of these cells
increased also. Murray (1968) and Paterson and Le Blond
(1977) have both made observations suggesting that
dehydration is accompanied by astrocytic and endothelial
cell proliferation in SON, at least in young animals.
Although the validity of these results has been disputed (G.
I. Hatton, personal communication) such a proliferation
might account for part of the increase in size of the

dendritic zone observed in this study.

67

If, in fact, an increase in the extent of SON dendritic
arborization does occur in response to activation of cells
in that nucleus, what biological purpose could it serve?

One obvious possibility is that it serves to expand the area
of the neuron which is receptive to afferent input, thus
perhaps rendering the cell more responsive during times of
increased hormone demand. A second possibility is that the
new dendrites participate in bundles with dendrites of other
neurons, thereby coordinating the activity of many cells.
This notion receives some support from the finding that the
area of the dendritic zone covered by single dendrites and
the average area/dendritic profile did not differ for
controls and MBs, the "extra" dendrites apparently being
destined for bundles. If the addition of new dendrites, or
dendritic branches, were purely a random event, one would
expect to find a generalized increase in area for both
single and bundled dendrites. For the present, the purpose
of more dendrites in SON during activated states remains an
enigma.

Recent work defining the nature of the stimuli which
may produce morphological changes in SON has dichotomized
experimental manipulations into chronic (lactation or 10
days of 2% saline substitution for drinking water) and acute
(4-24 hrs water deprivation) categories. Apparently the
formation of double synapses results from chronic
stimulation, whereas dendritic bundling and somatic
appositions occur rapidly in response to short term

dehydration (Hatton et al., 1984: Perlmutter et al., 1984b)

68
as well as chronic stimulation conditions (Perlmutter et
al., 1984a & c). Based on these findings the stimulus
associated with the induction of maternal behavior might be
considered to fall into the acute category, given that there
were no changes found in the number of double synapses
either in the dendritic or the somatic region. Since the
animals used in this study were sacrificed after 3 days of
exhibiting maternal behavior it might be interesting to see
if the number of double synapses would have increased had
the animals been behaving maternally for a longer period.
Such results also once again raise the question of what
exactly is the stimulus which promotes maternal behavior.
Simple exposure to pups might be rejected as an explanation
because the experimental subjects were exposed to pups for
up to 19 days prior to sacrifice - hardly an "acute"
situation. It may be that the "acute" verses "chronic"
stimulus dichotomy is not always sufficient to predict the
morphological changes which will occur in an "activated"
SON.
Morphological changes 1p 3p; posterior pituitary.

The finding of morphological changes in the posterior
pituitaries of maternal behavers was rather suprising.
Previous reports of reduced axonal enclosure by pituicytes
have been of dehydrated (Tweedle and Hatton, 1980)
post-partum (Tweedle and Hatton, 1982) or osmotically
stimulated pituicytes ip gipgp (Perlmutter et al., 1984d).
These conditions are, or mimic, states when neurohypophysial

hormones are released into the general circulation. A

69

reduction in axonal enclosure by pituicytes at these times
is thought to allow release of hormone from the axon into
the fenestrated capillaries of the pituitary. The
biological significance of similar changes in the
pituitaries of maternal behavers is hard to fathom, as it
was supposed that any influence of SON neurons on the
production of maternal behavior would likely be exerted
centrally, via recently described collateral projections to
the nearby lateral hypothalamus (Mason et al., 1984).
Interesting data relating to this issue comes from
Jakubowski and Terkel (1980) who claimed that maternal
virgin rats were seen to lactate after 14-18 days of
exhibiting maternal behaviors. In this study, subjects were
sacrificed on the third day of exhibiting maternal behavior,
too short of an interval to induce lactation, if such is
indeed possible. One could entertain the notion that the
changes seen in the posterior pituitary in this study were
harbingers of more profound changes to come. It is hoped
that further studies will examine such phenomena. Finally,
as is the case with those morphological changes seen in the
SON, these data do not address the question of which
peptidergic cell types undergo changes under the conditions
of this study. Preliminary examination of ICC-treated
material have yielded equivocal results, largely due to a
failure to produce enough adequately stained tissue for
sampling. While the data of Pedersen et al., (1982)
indicate that oxytocin is a far more potent elicitor of

maternal behavior than vasopressin, the latter peptide was

70

not without some effect. Therefore, the possibility remains
that both peptidergic cell types participate in the changes
seen in the SON dendritic zone and pituitaries of maternal

behavers.

Part II. Immunoelectron microscopic correlation of
morphology and peptide content of SON neurons in virgin,
prepartum, postpartum and lactating animals.

Almost 50% of the cells examined in this study were
found to be immunopositive for OX-NP while a somewhat more
conservative (45%) estimation of NP-VP staining was
obtained. Based on previous work which indicates that there
are fairly even numbers of oxytocinergic and
vasopressinergic cells (Swaab et al., 1975a) at the middle
third of the SON which was sampled, and the specific
staining produced on serial sections, it appears that a
consistent and accurate estimation of peptide content was
obtained.

The results of the analysis of the peptide content of
apposed neurons as a function of reproductive state point to
a selective formation of direct appositions of oxytocinergic
cells immediately after parturition (Table 1.). Assuming
that such direct appositions reflect functional demands made
upon the cells and in regards to oxytocin at least, these
data coincide well with those of Fuchs and Saito, (1971:
rats) and Fuchs and Dawood (1980: rabbits) who reported a
significant release of oxytocin at parturition, presumably

to promote smooth muscle contractions of the uterus during

71

the birth process. The question arises as to what purpose
direct appositions between oxytocinergic cells could serve
at this time. One hypothesis is that the resulting
reduction in intercellular space could promote ephaptic
interactions between these neurons and/or result in an
increased extracellular K+ concentration, thereby increasing
overall cellular excitability. Another possibility is that
close appositions would allow the formation of gap junctions
between these neurons (Andrew et. al., 1981) again promoting
a coordinated activity of many neurons which could result in
a massive release of oxytocin at parturition (Poulain and
Wakerley, 1982).

The finding that homotypic appositions were
significantly higher than heterotypic appositions in the
prepartum group, in one respect, is not surprising given
that like-hormone containing cells tend to be grouped
together in the SON. However, because no differences were
found for this group, relative to controls, in the
percentage of cells in direct apposition the biological
significance of such events is doubtful.

The percentage of cells seen in apposition in ICC
treated control sections (20%) was lower than that seen in
controls under the conditions employed in Part I (29%),
i.e., examination of micrographs of tissue stained with lead
citrate and uranyl acetate. One likely explanation for this
is that in the absence of counterstain in the ICC treated
material, that "spot" (or tiny) appositions, commonly

observed in the counterstained material, were less likely to

72
be identified. The estimation of apposition in the control
group in ICC treated tissue (20%) was considerably higher
however, than that found by Hatton and Tweedle (1982; 3.5%)
and somewhat higher than that reported by Theodosis et al.,
1981: 12%) for virgin female rats when somatic dendritic
appositions were included in the sample. The reproductive
state producing maximal somatic appositions in this study
(postpartum) was also different from that found by Hatton
and Tweedle, (1982) who found maximal apposition with 14
days of lactation. Both results are at variance with the
work of Theodosis and Poulain (1984b) who found maximal
apposition in prepartum animals- which was equivalent to
that seen in lactating animals (they did not examine an
immediately postpartum group. These differences are hard to
reconcile, but several factors should be considered. First,
the area sampled in this study was from the mid-portion of
the SON, where the population of oxytocinergic and
vasopressinergic cells is roughly split 50-50 (Swaab et al.,
1975; 1976) whereas the sections sampled in the other three
studies were apparently obtained more randomly from
throughout the SON. Given the somewhat segregated
distribution of the two cell types in the SON where
oxytocinergic cells are found dorsally and anteriorly and
vasopressinergic cells are found more ventrally and
posteriorly, it is conceivable that different populations of
cells were sampled in the four studies. A second factor
worth considering is the time of sacrifice of the animals.

In the study of Hatton and Tweedle (1982) animals were

73
sacrificed ~2-4 hours after lights-off whereas in this study
subjects were sacrificed 2—4 hours after lights-on. It is
possible that the interaction of diurnal fluctuations in
hormone release, particularly in the case of vasopressin,
could interact with factors controlling the release of
hormones during lactation to result in the overall higher
values for neuronal apposition during lactation seen by
Hatton and Tweedle (1982).

The failure to obtain data regarding either dendritic
bundling or double synapse formation across the reproductive
cycle is unfortunate. In the case of dendritic bundles,
although many stained profiles were seen, only those
containing dcvs could be identified as such and their
numbers were small in comparison to those not containing
dcvs. It is possible that the best hope for identifying the
peptide content of bundled dendrites is the far-from-perfect
and intensively laborious preembedding technique. Such an
attempt would require large amounts of processed tissue, but
would result in reaction product being deposited in
dendritic profiles whether they had dcvs or not. Theodosis
and Poulain (1984a) used this technique to show that
oxytocinergic dendrites and somata form direct appositions
with other immunopositive, as well as immunonegative,
dendrites and somata. Further, they identified a double
synapse contacting an immunoreactive dendrite and somata.
Judging by the brevity of their report however, a
quantitative analysis of such tissue would require much time

and effort. Alternatively, the post-embedding technique,

74

coupled with colchicine injections which might prevent the
transport of dcvs out of cell bodies and dendrites, could
possibly solve the mystery of who is bundling with whom.
With respect to double synapses, none of those unequivocally
identified were found contacting one stained and one
unstained cell body, suggesting at least that such synapses
do not contact different hormone containing cells. While
not constituting an adequate sample, these findings are
consistent with the data of Perlmutter (1984d) who managed
to find 19 double synapses in her immunoelectron microscopic

study of the SON of dehydrated rats.

Part III. Postweaning group morphometry.

The portrait of the SON which was obtained by
morphological measures of postweaning group tissue is, with
one major exception, one of a nucleus not significantly
different from controls. For all of the originally proposed
measures, i.e., somatic direct apposition, dendritic
bundling, percentage of somatic or dendritic membrane in
direct apposition, and the number of somata or dendrites
contacted by double synapses, there were no differences
detected relative to controls. One could almost make the
argument that nothing much in the way of morphological
changes seemed to have occurred here were it not for one
observation: an average of roughly 300 more dendrites/
animal existed in the samples of the dendritic zones of the
postweaning animals examined. As these numbers were

gathered by counting the number of dendrites on two thin

75
sections in the mid-portion of SON, it is unknown whether
they merely hint at a far greater change which occurred
throughout the nucleus or if such changes were specific to
that part of the nucleus. Further, the size of the entire
dendritic area, including dendrites, astrocytes and
endothelial cells, was seen to be an average of 3500 um2
larger then the comparable area in controls, for the two
thin sections sampled in the postweaning group. Like the
dendritic zones of the maternal behavers discussed earlier,
the total area of dendritic profiles was significantly
greater in this group also. However, as might be expected,
since no differences were found in the extent of dendritic
bundling, the difference in dendritic profile area was
localized to single dendrites.

Comparisons of data for the postweaning group obtained
in this study with that found by Theodosis and Poulain
(1984b) show that, while the absolute values of measures
differed, they too found no significant differences from
controls on any of the variables measured. Combined
postweaning group values for double synapses contacting both
somata and dendrites was some 4.5% in this study vs 2.8% in
theirs. On the measure of percentage of somatic membrane in
apposition Theodosis and Poulain found that at one month
post-weaning, this measure was 0.4% vs 0.7% (controls and
postweaning respectively) whereas in this study at ten days
postweaning the values were 5% and 4%. Finally, they found
values of 3% vs 2%, (control vs postweaning groups) on the

measure of dendritic membrane in apposition as compared to

76

values of 13.6% vs 12.1% for the same variables measured in
this study.

The data presented here confirm the reversibility of
the somatic, dendritic, and synaptic changes that have been
shown previously to occur in the SON with gestation,
parturition and lactation. Further, the data indicate that
morphological traces of the maternal experience disappear in
a fairly short time, i.e., within 10 days postweaning. This
is in contrast to the results of Perlmutter et al., (1984c)
who investigated the reversibility of dendritic bundling in
response to dehydration. They found that even 14 days of
rehydration was insufficient to decrease dendritic bundling
to control levels. It thus appears that, in the dendritic
zone but not the somatic zone that the stimulus of
dehydration produces more long-lasting effects than does
gestation and lactation. Changes which have not been
previously noted to occur with maternity are the rather
profound differences seen in this study in the dendritic
zone: the formation of new dendrites or increased branching
of already existing dendrites as well as an overall increase
in the size of the dendritic zone which may include
astrocytic and endothelial cell changes. Some of the
biological implications of these phenomena have already been
discussed in relation to similar changes in the dendritic
zone of maternal behavers and will not be reiterated here.
In relation to the pregnancy "cycle" however, it cannot be
automatically assumed that these dendritic changes are

evident during gestation, parturition or lactation. It

77
would be interesting therefore to examine the SON during
these states using light microscopic techniques such as
Golgi staining or Golgi-like immunostaining (Sofroniew and
Glassmann (1981) to determine whether such changes occur.
The latter technique, of course, would have the advantage of
being able to identify the peptide content of any changing
dendrites.
General Discussion.

The values obtained for the control group used in this
dissertation were consistently elevated when compared to
those data reported by Theodosis et. al.(1981), Hatton and
Tweedle (1982) and Theodosis and Poulain (1984b). One
factor which could be responsible for this is the stage of
the estrous cycle at which the animals were sacrificed, as
steroid hormone levels have been shown to influence
vasopressin release (Skowsky et al., 1979). However, when
data for maternal behavers and control animals were combined
(N= 14) no significant correlation could be found between
the stage of the estrous cycle and either somatic
appositions or dendritic bundling.

A more plausible reason for the seemingly elevated
control values might be that the time of sacrifice of the
group used in this study was " 12 hours out of synchrony
with the time of sacrifice of the control group of Hatton
and Tweedle (1982). Unfortunately this information is
unavailable for the work of Theodosis and colleagues. While
animals in this study were sacrificed 2-4 hours after the

onset of the "lights-on" portion of the 12:12 hr light

78
cycle, Hatton and Tweedle sacrificed their control group 2-4
hours after "lights-off" (they had their animals on a
reversed light cycle). The data of Schwartz et al., (1983)
and Reppert et al.,(1983) showed that a diurnal fluctuation
of vasopressin exists in the cerebrospinal fluid (CSF) of
rats and that peak concentrations of the hormone occur just
prior to "lights-on" and persist to about hour 6 of
"lights-on". This is in contrast to the baseline levels
observed during almost the entire "lights-off" period.
While the source of neurohypophysial hormones in the CSF is
still a mystery, it does suggest that magnocellular neurons
are influenced by the light cycle. Some evidence also
exists for diurnal fluctuations of plasma vasopressin with
high values found four hours after "lights-on" (Mohring and
Mohring, 1975). Armstrong and Hatton (1978) also found that
multiple nucleoli were most numerous at the end of the
daylight hours, an indication of increased metabolic
activity. If diurnal fluctuation is responsible for the
elevated control values then one more stimulus complex can
be added to the growing list of stimuli which influence SON
morphology. It may be that merely a few hours difference in
the course of a diurnal cycle are necessary to effect
morphological changes in SON. (What a difference a day
makesl). That morphological changes, at least in the SON
somatic region, can occur with such rapidity has been
demonstrated by Tweedle and Hatton (1977). It would
therefore be interesting to determine if the morphological

changes described here and by others can be produced by

79

manipulations of the light-dark cycle.

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Sofroniew, M.V. and Glassmann, W. (1981) Golgi-like
immunoperoxidase staining of hypothalamic magnocellular
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Swaab, D.F. Nijveldt, F., and Pool, C. W. (1975a).
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Swaab, D.F., C.W. Pool, and Nijveldt F. (1975b)
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85
oxytocin-secreting neurones are involved in the
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86
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APPENDIX A

87

KEY TO APPENDIX A

C = Control animal

MB = Maternal Behaver

PW = Postweaning animal

Pre Prepartum animal
Pos = Postpartum animal
Lac = Lactating animal
Apps = appositions

App'd = apposed

Dbls = double synapses
Dend = dendro-

ICC = immunocytochemistry

MAB+ a immunostained cell

MAB- = nonimmunostained cell

88

APPENDIX A

Table 3. RAW DATA-SOMATA

5 Cells Observed 5 Cells App'd g Membrane App'd

 

CODE

Cl 42 21.42 6.20
C2 37 32.43 4.98
C3 44 52.27 7.94
C4 30 26.66 2.89
C5 38 28.94 4.93
C6 32 18.75 3.48
C8 23 21.73 4.69
M81A 58 22.41 4.67
M81 50 16.00 2.65
M82 41 26.82 6.28
M84 18 11.11 1.46
M85 44 20.45 2.98
M86 20 20.00 3.17
M87 67 19.40 6.02
PWl 54 44.44 10.89
PWZ 37 27.02 2.90
PW4 49 36.73 5.59
PW5 44 36.36 2.93
PW6 39 25.64 2.35
PW7 63 33.33 2.98
PWB 46 4.34 0.11

89

APPENDIX A

Table 4. RAW DATA- X SOMATA WITH DOUBLE SYNAPSES

Soma-somatic Dend-Somatic Combined Dbls.

CODE

Cl 0.00 2.38 2.39
C2 0.00 10.81 10.81
C3 9.09 4.54 13.63
C4 0.00 3.33 3.33
C5 0.00 5.26 5.26
C6 0.00 0.00 0.00
C8 8.69 8.69 17.39
M81A 3.44 12.06 15.51
M81 4.00 2.00 6.00
M82 9.75 2.43 12.19
M84 0.00 0.00 0.00
M85 0.00 0.00 0.00
M86 0.00 0.00 0.00
M87 2.98 0.00 2.98
PW1 11.11 3.70 14.81
PW2 0.00 8.10 8.10
PW4 16.32 2.04 18.36
PW5 4.54 2.27 6.82
PW6 0.00 7.69 7.69
PW7 9.52 6.34 15.87
PW8 4.34 8.69 13.04

90

APPENDIX A

Table 5. RAW DATA- DENDRITES

 

 

# DENDRITES # _lCROGRAPHS X MEMBRANE APP

CODE

01 536 4 11.96
C2 694 6 16.36
C3 604- 6 11.64
C4 383 6 9.75
C5 500 6 12.04
C6 384 6 16.94
C8 475 6 16.83
M81A 685 9 11.17
M81 543 8 14.22
M82 622 8 12.52
M84 446 7 8.85
M85 719 7 16.80
M86 540 5 8.77
M87 799 9 15.05
PW1 1264 14 9.87
PW2 635 11 8.94
PW4 537 8 15.82
PW5 507 8 10.16
PW6 977 10 13.20
PW7 838 8 10.64

PW8 1186 10 16.18

91

APPENDIX A

Table 6. RAW DATA- X DENDRITES RECEIVING DOUBLE SYNAPSES/

AREA OF DENDRITES IN BUNDLES AND SINGLES (pMz).

g DBL SYNAPSES BUNDLE AREA SINGLES
CODE
Cl 1.86 182.22 217.85
C2 1.72 516.90 562.63
C3 1.98 556.30 514.21
C4 0.00 337.07 334.11
05 2.00 690.46 419.25
C6 .52 533.42 198.74
CB .42 423.58 256.48
M81A .87 728.81 460.10
M81 .73 1061.56 389.50
M82 .90 770.11 569.79
M84 .44 611.45 641.84
M85 1.39 524.93 157.54
M86 1.11 517.94 733.42
M87 1.00 1280.58 667.14
PW1 2.37 1044.84 1377.17
PW2 1.25 859.44 925.91
PW4 4.84 735.93 478.65
PW5 .98 415.16 496.34
PW6 3.68 1549.65 614.17
PW7 1.67 927.52 745.48
PW8 2.69 1999.47 915.56

Table 7.

1-4,

BUNDLE SIZE:

CODE
01
C2
C3
C4
C5
C6
C8

M81A
M81
M82
M84
M85
M86
M87

PW1
PW2
PW4
PW5
PW6
PW7
PW8

5-8 AND >=

1-4

1.35
1.34
1.41
1.32
1.34
1.41
1.38

1.36
1.45
1.33
1.33
1.39
1.38
1.41

1.37
1.32
1.38
1.40
1.56
1.33
1.51

92

APPENDIX A

0.00
5.50
6.33
5.00
5.16
5.33
5.20

5.83
6.33
6.00
5.50
5.75
0.00
6.15

5.35
6.00
5.75
5.50
5.76
5.50
5.63

RAW DATA-MEAN NUMBER OF DENDRITES IN BUNDLES OF

93

APPENDIX A

Table 8. RAW DATA-NEUROHYPOPHYSIS: MEAN # ENCLOSED AXONS

PER ANIMAL/ PER PITUICYTE.

AXONS Egg PITUICYTE AXONS Egg ANIMAL
0033

01 .40 6
02 .20 3
03 .33 5
04 .27 4
05 .60 9
C6 .50 9
c7 .33 5
MBlA .20 3
M31 .20 3
M32 .13 2
M34 .07 1
MB5 .27 4
MB6 .27 4
M87 .40 5

Table 9.

CODE
C1
C2
C3
C4
C5
C6
08

M81A
M81
M82
M84
M85
M86
M87

METESTRUS
METESTRUS
ESTRUS
PROESTRUS
METESTRUS
METESTRUS
DIESTRUS

METESTRUS
PROESTRUS
DIESTRUS
DIESTRUS
ESTRUS
PROESTRUS
PROESTRUS

94

APPENDIX A

RAW DATA-VAGINAL CYTOLOGY
STAGE OF ESTROUS CYCLE

Table 10.

Prel
Pre2
Pre3
Pre4
Pre5
Pre6
Pre7

Pos1
Pos2
Pos3
Pos4
Pos5
P086
Pos7

Lacl
Lac2
Lac3
Lac4
Lac5
Lac6
Lac8

APPENDIX A

RAW DATA- ICC

% pf MAB+ Apposed

oi-np

 

10.52
3.84
17.39
14.28
3.33

20.00
35.48
23.33
34.78
25.00

11.11
12.50
15.38
16.27

 

VP-NP
13.15
12.50

0.00
18.18

0.00
11.11
16.66

 

24.13
25.00
0.00
0.00
5.80
9.09
19.23

25.00
22.72

8.33
20.58

3.20
21.73
28.57

32.00
23.50

6.60
35.00
30.00
16.66
17.24

95

OX-NP

106.58
38.90
176.19
144.68
33.73

155.82
63.33
110.00
0.00
135.00

202.63
359.47
236.37
352.38
253.29

112.56
126.64
155.82
164.84

57.34

% pg Control

VP-NP
128.66
122.30

0.00
177.88
0.00
108.70
163.01

236.10
244.61
0.00
0.00
56.75
88.94
188.16

244.61
222.30

81.50
201.36

31.31
212.62
279.54

313.11
229.94

64.57
342.46
293.54
163.01
168.68

96

APPENDIX A

Table 11. RAW DATA- ICC APPOSITIONS

g MAB+ Heterotypip Apps. g MAB- Homotypic.
CODE OX-NP VP-NP OX-NP
C1 13.15 .

02 6.25

C4 0.00 0.00 0.00
05 3.84 27.27 3.84
C6 0.00 0.00 0.00
C7 0.00 0.00 0.00
ca 6.66 16.66 6.66
Prel 15.51

Pre2 25.00

Pre3 30.76 0.00 0.00
Pre4 0.00 0.00 6.25
Pre5 13.04 14.70 13.04
Pre6 0.00 18.18 20.00
Pre7 0.00 23.07 30.00
Pos1 31.25

Pos2 13.63

Pos3 13.33 0.00 13.33
Pos4 19.35 26.47 25.80
Pos5 20.00 6.40 23.33
Pos6 0.00 26.08 43.47
Pos7 14.58 26.57 31.25
Laci 6.45

Lac2 20.58

Lac3 0.00 13.33 22.20
Lac4 25.00 30.00 0.00
Lac5 15.38 13.33 15.38
Lac6 32.55 33.33 9.30
Lac7 11.76 24.13 29.41

 

 

97

APPENDIX A

Table 12. RAW DATA- Maternal Behavior

Animal ID:MB-l

Behaviors
Crouching Anogenital Retrieving
Licking
222
1 0 0 0
2 0 0 0
3 1 0 0
4 1 0 0
5 15 0 0
6 12 1 1
7 16 3 1
8 5 3 1
Animal ID:MB-lA
Behaviors
Crouching Anogenital Retrieving
Licking
9.9.!
1 0 0 0
2 0 0 0
3 O 0 0
4 0 O 0
5 0 O 0
6 0 0 0
7 0 0 0
8 1 0 0
9 0 0 O
10 1 0 0
11 0 0 0
12 0 0 0
13 0 0 0
14 0 0 0
15 0 O 0
16 - - -
17 1 1 1
18 1 0 0
19 1 1 1
20 - - -
21 1 1 1

98

APPENDIX A

Table 13. RAW DATA- Maternal Behavior

Animal ID:MB-2

 

 

Behaviprs
Crouching Anogenital Retrieving
Licking
m
1 0 0 0
2 O 0 0
3 1 0 0
4 0 0 0
5 14 0 0
6 7 0 O
7 15 0 0
8 2 2 0
9 0 0 0
10 9 1 0
11 12 2 0
12 12 6 0
13 9 4 0
14 11 1 0
15 9 1 0
16 6 5 0
17 14 3 1
18 14 9 1
19 1 3 1
Animal ID:MB-5
- Behaviors
Crouching Anogenital Retrieving
Licking
29.2
1 0 2 0
2 0 0 0
3 1 O 0
4 10 1 0
5 15 0 1
6 14 5 2
7 14 2 1
8 14 2 0
9 13 9 1
10 13 8 1
11 1 4 1

99

APPENDIX A

Table 14. RAW DATA- Maternal Behavior

Animal ID:MB-4

 

Behaviors
Crouching Anogenital Retrieving
Licking
2.3.2
1 0 0 0
2 O 0 0
3 1 0 0
4 0 0 O
5 0 0 0
6 0 0 0
7 0 0 0
8 11 2 0
9 1 0 0
10 1 1 0
11 1 0 0
12 6 2 0
13 1 0 0
14 O 0 O
15 1 2 0
16 3 0 1
17 7 5 1
18 12 4 1
19 2 4 3
Animal ID:MB-6
Behaviors
Crouching Anogenital Retrieving
Licking
232
1 0 0 0
2 0 1 0
3 1 0 0
4 3 2 0
5 15 2 0
6 13 5 1
7 15 4 1
8 1 2 2

100

APPENDIX A

Table 15. RAW DATA- Maternal Behavior

Animal ID:MB-7

Behaviors
Crouching Anogenital Retrieving
Licking
Lay

1 0 0 0
2 0 0 O
3 0 0 0
4 O 0 O
5 15 9 1
6 12 4 1
7 15 2 1
8 13 6 0
9 2 9 2
10 9 7 1
11 3 3 1

APPENDIX 8

1°.

11.

Chop

101

APPENDIX 8

Procedure for L.R.White embedding

tissue with tissue chopper.

"Core" tissue with a single edged razor blade. Expose

tissue to the following solutions:

1.0%
30 %
50 %
70 %
2:1,
100%

100%

osmium tetroxide in 0.15M cacodylate buffer- 1 hr
ETOH, 0.5 hr

ETOH, 0.5 hr

ETOH, 0.5 hr

L.R.White : 70% ETOH solution, 0.5 hr

L.R.White, 0.5 hr

L.R.White, 1 hr.

Place tissue in bottom of gelatin capsules (size #00)

and fill capsule with 100% L.R. White.

Polymerize in a 50 degree oven, 48 hrs.

APPENDIX C

102

APPENDIX C
Supplies

Electron picroscopy

Electron microscopy film # 4489, 3.25" by 4" - Kodak (MSU
Stores)

Ektamatic SII activator - Kodak (MSU Stores)

Ektamatic $30 stabilizer - Kodak (MSU Stores)

Forceps, #7, curved - Ted Pella, Inc.

Formvar, 0.25% in ethylene dichloride - Ted Pella, Inc
Gelatin capsules, size #00 - Lilly

Grid Boxes, Pelco - Ted Pella, Inc.

Grids, nickel, ultra-high transmission, thin bar, 200 mesh -
Ladd Research Industries

Lead Citrate - Ladd

L.R. White Resin (acrylic) medium grade - Fullam

Osmium tetroxide, crystalline - Polysciences

Photographic paper, 8" by 10" AGFA-Gevart Rapidoprint - VWR
Scientific

Photographic paper, 8" by 10" Polycontrast Rapid II medium
weight - Kodak (MSU Stores)

Photographic paper, 8" by 10" Kodabrome II RC medium weight
- Kodak (MSU Stores)

Silgard 184 elastomer with 184 curing agent, # 1.1 kit. -

103

APPENDIX C

Supplies

Prehler Co.

Uranyl acetate - Electron Microscopy Sciences

Fixation
EM grade 20% paraformaldehyde - Polysciences
Glutaraldehyde, 25% - Electron Microscopy Sciences

Sodium cacodylate - Polysciences

immpnocytochemistry

DAB (3,3' -diaminobenzidine 4HCL) - Sigma
Filter equipment - see Master's Thesis
Goat anti-mouse IgG F(ab')2 fragment conjugated to
peroxidase - Boehringer-Mannheim

Monoclonal Antibodies - Drs. H. Gainer (NIH) and W. Smith
(Mich. St. Univ.)

Normal goat serum - Antibodies Inc.

Sodium m-periodate - Sigma