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W State
University

This is to certify that the

dissertation entitled
Effects of Photoperiod on the Hypothalamus
and on Lactotropes in the Anterior Pituitary
Gland of Holstein Bull Calves
presented by

Steven Andrew Zinn

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Animal Science

 

 

4 MAJ We;

Major professor

Datejehruaw 8 9

MS U is an Affirmative Action/Equal Opportunity Institution 0. 12771

 

 

MSU

 

 

 

RETURNING MATERIALS:
Place in book drop to

 

LjBRAfiJES remove this checkout from
—;——. your record. FINES will
be charged if book is
returned after the date
stamped below.
D
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“5E3

.3 \l LAAF‘ S72

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.0

 

 

EFFECTS OF PHOTOPERIOD ON THE HYPOTHALAMUS AND
ON LACTOTROPES IN THE ANTERIOR PITUITARY GLAND
OF HOLSTEIN BULL CALVES

BY

Steven Andrew Zinn

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Animal Science

1989

ABSTRACT

EFFECTS OF PHOTOPERIOD ON THE HYPOTHALAMUS AND
LACTOTROPES IN THE ANTERIOR PITUITARY GLAND OF
HOLSTEIN BULL CALVES

BY

Steven Andrew Zinn

To determine the effects of photoperiod on serum
prolactin, activity of tuberoinfundibular dopaminergic
(TIDA) and 5-hydroxytryptaminergic (SHT) neurons in. the
hypothalamus and secretory capacity land number of
lactotropes in the anterior pituitary gland, prepubertal
Holstein bull calves were utilized. To validate methods
the effects of euthanasia with sodium pentobarbital on
concentrations of dihydroxyphenylacetic acid (DOPAC) in the
median eminence of rats and the effects of elevated serum
concentrations of prolactin on accumulation of
dihydroxyphenylalanine (DOPA) in the pituitary stalk (P8)
of bull calves were determined.

Since euthanasia with sodium pentobarbital did not mask
stress-induced changes in activity of TIDA neurons, sodium
pentobarbital was an acceptable agent for euthanizing
animals in subsequent studies.

Concentrations of dopamine were greatest in the PS in
bulls and accumulation of DOPA in the P8 was increased
after 25 h of ha10peridol or after 25 h of infusion of

prolactin. Accumulation of DOPA in PS was increased after

9 d of infusion of PRL.

Compared with bull calves exposed to photoperiods of 8
h of light (L):16 h of dark (D) exposure to 16L:80
increased serum concentrations of prolactin 5-fold,
secretory capacity and number of lactotropes in the
anterior pituitary gland 70 and 16%, respectively and
release of PRL into the media from pituitary gland explants
57%. 16L:80 increased accumulation of DOPA in the PS 14%.

In conclusion, 16L:80-induced increases in serum
prolactin. are associated with increased number and
secretory capacity of the lactotropes. After 4 wk of
exposure to 16L:8D the elevated serum concentration may

feed back to stimulate accumulation of DOPA.

ACKNOWLEDGEMENTS

I owe thanks to many people for their contributions
during my program at Michigan State University.

First of all, I thank my guidance committee, Drs.
Werner Bergen, Robert Merkel, Ken Moore and Dale Romsos and
my surrogate member Keith Lookingland. Your participation,
contributions and suggestions certainly improved my
research program.

I thank Jimmy Neill, Jeff Mulcahey and Jeff Sellers
from University of Alabama at Birmingham for teaching me
the reverse hemolytic plaque assay.

My appreciation to all members of the Department of
Animal Science, especially chairman, Maynard Hogberg for
providing financial support and an environment conducive to
research.

I thank the workers at the Dairy and Beef Barns for
their expertise in caring for the animals in these studies.

I thank Drs. Denis Petitclerc and William Enright, both
good friends who taught me much and although each is in his
respective homeland, I still rely on their input.

To all members of the Animal Reproduction Lab, faculty
and graduate and undergraduate students, past and present

members, and actual and honorary members: I thank all of

iv

you for your contributions, assistance and friendship. I
especially thank: Ted Ferris, Trudy Hughes, Jim Ireland,
Alex Villa-Godoy, Julie Chapin, Roger Mellenberger, Marilyn
and Roy Emery, John Gill, Vasantha Padmanabhan, Joanna
Gruber, Tom Forton, Kent Refsal and Steve Lyth and I also
thank all the new people in the lab: Geoff, Brent, Alan,
Jackie, Debbie, and Faye.

A very warm thank you to Marnie Laurion for typing this
thesis and being a good friend.

My thanks to my family. Although they didn't always
know what I was doing, they were always supportive.

Four people deserve very special recognition. Each had
a tremendous and positive impact on my career at MSU.

First — my major professor R. Allen Tucker: thank you
for taking the chance on me way back when I first started
and putting up with me through the intervening years. You
are an inspiration to me and a first class role model as I
venture out on my own. I always appreciated your belt high
thumbs up when I did good and your forthright manner when I
didn't.

To Larry Chapin: you have been a great collaborator,
contributor and friend. My only regret is that you can
only win the Distinguished Staff Award once. In my
"unbiased" opinion you deserve it every year and earn it
every day.

To Dr. Roy Fogwell: thanks for the early morning

talks. Roy, you are young enough to remember the trials
and tribulations of being a graduate student but you have
been at it long enough to offer sound and soothing advice.

I am grateful to my wife, Catherine, for her love and
support and in general for putting up with me throughout my
program.

And thanks to Number 25, who imprinted the memory of

MSU in me forever.

vi

LIST OF TABLES

TABLE OF CONTENTS

0 O O O O O O O O O O O O O O O O O 0 1X

LIST OF FIGURES O O O O O O O O O O O O O O O O O O Xi

LI ST OF ABBREVIATIONS O O O O O O O O O O O O C O C Xi i 1

INTRODUCTION .

O I O O O O O O O O O O O O O O O O O l

REVI Ew 0F LITERATURE O O O O O O O C O O O O O O O O 4

A.

Chapter

Effects of Photoperiod on Hormone

Secretion . . . . . . . . . . . . . . . . . 4
1. Prolactin . . . . . . . . . . . . . . 4
2. Gonadotropins . . . . . . . . . 9
3. Other hormones related to growth . . . 13
4. Neurotransmitters . . . . . . . . . . 17
5. Pathway of light signals in

regulation of hormone secretion . . . 19
6. Summary . . . . . . . . . . . . . . . 23
Dopaminergic Regulation of Prolactin
Secretion . . . . . . . . 24
1. Inhibitory control of prolactin . . . 24
2. Intracellular mediators of dopa-

minergic action in the lactotrope . . 27
3. Tuberoinfundibular dopaminergic

neurons . . . . . . . . . . . . . . . 31
4. Summary . . . . . . . . . . . . . . . 34

5-Hydroxytryptaminergic Regulation of
Prolactin Secretion . . . . . . . . . . . . 35

1.
2.
3.
4.

1.

5-Hydroxytryptaminergic neurons . . . 36
Stimulatory control of prolactin . . . 37
Possible sites of 5-hydroxytryptaminergic

influence on prolactin . . . . . . . . 38
Summary . . . . . . . . . . . . . . . 40

Alterations in concentrations of

dihydroxyphenylacetic acid in

rats euthanized with sodium

pentobarbital . . . . . . . . . . . . 42
Introduction . . . . . . . . . . . 43
Materials and Methods . . . . . . 45

vii

Results 0 O O O O O O O O O O O O 4 6
Discussion . . . . . . . . . . . . 49

Chapter 2. Prolactin regulation of tubero-
infundibular dopaminergic neurons

in Holstein bull calves . . . . . . . 53
Introduction . . . . . . . . . . 54
Materials and Methods . . . . . . 55
Results . . . . . . . . . . . . . 63
Discussion . . . . . . .‘. . . . . 76

Chapter 3. Response of tuberoinfundibular
and 5-hydroxytryptaminergic neurons and
lactotropes to photoperiod in Holstein
bull calves . . . . . . . . . . . . 82

Introduction . . . . . . . . . . 83
Materials and Methods . . . . . . 84
Results . . . . . . . . . . . . . 92
Discussion . . . . . . . . . . . . 98

SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . 106

LIST OF REFERENCES . . . . . . . . . . . . . . . . . 111

viii

Table

Table

Table

Table

Table

Table

Table

Table

Table

8.

LIST OF TABLES

Concentrations of dopamine (DA) and
dihydroxyphenylacetic acid (DOPAC) in
brain regions of rats euthanized with
Fatal Plus, pentobarbital or

decapitation . . . . . . . . . . . . . . .

Effects of stress on concentrations of
dopamine (DA) and dihydroxyphenylacetic
acid (DOPAC) in the median eminence and
striatum of rats euthanized with
pentobarbital or decapitation . . . . .

Concentrations of dopamine in section of
pituitary stalk of bull calves . . . . . .

Concentrations of dihydroxyphenylacetic
acid in sections of pituitary stalk of
bull calves . . . . . . . . . . . . . .

Concentrations of dopamine (DA) and
dihydroxyphenylacetic acid (DOPAC) in .
brain regions of bull calves . . . . . . .

Effects of 25 h of exposure to haloperidol
on concentrations of DOPAC in pituitary
stalk of bull calves . . . . . . . . . . .

Effect of 25 h of exposure to haloperidol
on accumulation of DOPA in pituitary
stalk of bull calves . . . . . . . . . . .

Effects of 25 h of exposure to haloperidol
on accumulation of SHTP in pituitary stalk
of bull calves . . . . . . . . . . . . . .

Effects of infusion of prolactin for

25 h or 9 days on accumulation of DOPA
in pituitary stalk of bull calves . . . .

ix

48

50

64

65

66

70

72

73

77

Table

Table

Table

Table

10.

11.

12.

13.

Effects of photoperiod on accumulation
of DOPA and SHTP in the pituitary stalk
of bull calves . . . . . . . . . . . . . . 95

Effects of photoperiod on accumulation

of DOPA and SHTP in brain regions of bull
calves . . . . . . . . . . . . . . . . . . 97
Analysis of plaques formed from lactotropes

in the reverse hemolytic plaque assay in
anterior pituitary glands from bull calves
exposed to photoperiod . . . . . . . . . . 99

Release of PRL from pituitary gland
explants from bull calves exposed
to 8L:16D of 16L:8D . . . . . . . . . . . 100

Figure 1.

Figure 2.

Figure 3.

Figure 4.

LIST OF FIGURES

Schematic diagram of the dissection of

the infundibular-pituitary stalk (PS),
mediobasal hypothalamus (MBH) and
suprachiasmatic nucleus (SCN) of

bull calves . . . . . . . . . . . . . . . 58

Concentrations of prolactin in serum

of bull calves injected with haloperidol

(4}: 1 mg/kg body weight) every 6 h

( O ) or uninjected controls (. ) . All

calves received NSD 1015 (25 mg/kg body
weight;{}) 15 min before euthanasia.

Each point represents the mean of 7 or

8 samples. Standard error = 5.2 ng/ml

for haloperidol injected bulls and 1.6

ng/ml for uninjected controls . . . . . . 68

Concentrations of prolactin in serum
of prepubertal bull calves infused
with prolactin (USDA b1; 12.0 mg/d)
for 25 h (0), 9 d (A ), and non-
infused controls (0 ). Beginning of ,
infusion indicated by solid arrows

( . ) . All animals received NSD-1015
(iv; 25 mg/kg BW; O) 15 min before
euthanasia. Each point represents
the mean of 5 or 6 samples. Pooled
standard errors = 4.3 ng/ml for
periods of non-infusion and 11.2 ng/ml
for periods of infusion . . . . . . . . . 75

Photomicrograph of a plaque (zone of
hemolysis) surrounding a PRLPsecreting

cell (solid arrow.) viewed with

phase contrast optics (magnified

400X). Non-plaque forming cell indicated

by open arrow ( ) RBC membrane ghosts

can be seen in t e plaque . . . . . . . . 90

xi

Figure 5.

Concentrations of prolactin in serum

of prepubertal bull calves exposed

for 6.5 wk to 8L:16D (solid symbols),

then switched to 16L:8D (‘A ) or main-

tained on 8L:16D ((3) for an additional

4 wk. Pooled SE = 1.04 ng/ml . . . . . . 94

xii

BSA
cAMP
D

DA
DMEM
DOPA
DOPAC
FSH
GH
GnRH
HEPES

SHIAA
SHT
HPLC
INS
IP
iv
L
LH
MAO
MBH
M199
n

NE

N80 1015

oRBC
pCPA
PRL
PS
PVN
scc
SC
sen
SE
T3

T
TIDA
TRH
TSH
VS

LIST OF ABBREVIATIONS

bovine serum albumin

cyclic adenosine 3',5'-monophosphate
dark

dopamine

Delbecco's modified Eagle's medium
dihydroxyphenylalanine
dihydroxyphenylacetic acid
follicle stimulating hormone
growth hormone
gonadotropin-releasing hormone
N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic
acid

5-hydroxyindoleacetic acid
5-hydroxytryptophan

high performance liquid chromatography
insulin

inositol phospholipids

intravenous

light

luteinizing hormone

monoamine oxidase

mediobasal hypothalamus

medium 199

number

norepinephrine
3-hydroxybenzy1-hydrazine

ovine red blood cell
p-chlorophenylalanine

prolactin

pituitary stalk

paraventricular nucleus

superior cervical ganglion
subcutaneous

suprachiasmatic nucleus

standard error

triiodothryonine

thyroxin

tuberoinfundibular dopaminergic
thyrotropin-releasing hormone
thyroid hormone stimulating hormone
versus

xiii

INTRODUCTION

Photoperiod is a significant cue used by animals in
regulation of physiological function in response to
changing seasons of the year. Photoperiod can influence
reproductive activity and onset of puberty in seasonal
breeding species and onset of puberty in non-seasonal
breeding species such as cattle and pigs (Diekman and
Hoagland, 1983; Petitclerc et al., 1983a). In addition,
photoperiod affects many other physiological variables
including several production traits important to the
livestock industry. For example, exposure to long-day
photoperiods of 16 h of light (L) and 8 of dark (D),
compared with short-day photoperiods of 8L:16D, increases
milk yield 6 to 13% (Peters et al., 1978, 1981;
Stanisiewski et al., 1984a), increases mammary development
in prepubertal and postpubertal heifers (Petitclerc et al.,
1985), increases body growth rate 8 to 17% (Peters et al.,
1978; Petitclerc et al. 1983a; Zinn et al., 1986a),
increases feed efficiency (Peters et al., 1978; Petitclerc
et al., 1983a), increases percentage of protein in the
carcass 5 to 11% (Petitclerc et al., 1984; Zinn et al.,
1986b) and decreases accretion of fat in the carcass 21%

(Zinn et al., 1986b). In addition, exposure to long-day

photoperiods increases concentrations of prolactin (PRL)
two- to eight-fold compared with exposure to short-day
photoperiods (Bourne (and 'Tucker, 1975: Leining et al.,
1979; Stanisiewski et al., 1984b, 1987b).

Although hormonal mediation of photoperiod-induced
effects on lactation and growth are yet to be determined,
photoperiod-induced changes in PRL have been postulated to
play a role (Tucker et al., 1984). Indeed, PRL is an
anabolic hormone (McAtee and Trenkle, 1971). For example,
infusion of PRL increased nitrogen retention in sheep
(Brinklow and Forbes, 1983) and sheep immunized against PRL
had reduced growth rates (Ohlson et al., 1981).

Increased production and particularly increased
efficiency of production of food producing animals becomes
important as the human population increases and available
resources for food production decrease. Thus,
understanding the mechanism whereby photoperiod affects
secretion of PRL may lead to new methods to regulate and
enhance efficiency of lactation, mammary development, body
growth and carcass composition.

The primary objective of this dissertation was to
determine if exposure to photoperiods that alter
concentrations of PRL in cattle alter activity of neurons
(dopaminergic and 5-hydroxytryptaminergic) in the
hypothalamus that regulate secretion of PRL and(or) alter
the number and(or) secretory capacity of lactotropes in the

pituitary gland. Before the primary objective could be

addressed, validation of the methods was required.
Accordingly, additional objectives of this dissertation
were: 1) to determine the effects of euthanizing animals
with sodium pentobarbital on dopaminergic neurons; 2) to
locate potential sites of tuberoinfundibular dopaminergic
neurons in cattle and to determine if activity of these
neurons can be altered with pharmacological manipulations
and 3) to determine if increased activity of these neurons

can be maintained over long (9 d) periods of time.

REVIEW OF LITERATURE

A. Effects of Photoperiod 9;; Concentrations of

Hormones In Blood

Season of the year influences blood concentrations of
several hormones. For example, concentrations of
luteinizing hormone (LH) and follicle-stimulating hormone
(FSH) in blood vary with season in seasonal breeding
species (Karsch et al., 1984; Ebling and Lincoln, 1987).
Of the anabolic hormones studied, concentrations of PRL are
most responsive to changes in season (Tucker, 1982).
Season of the year also influences other hormones, such as
B-endorphins, and neurotransmitters, such as dopamine (DA)
and 5-hydroxytryptamine (SHT; Steger et al., 1985; Ebling
and Lincoln, 1987). In this section the effects of season
and more specifically the effects of'photoperiod on
concentrations of hormones and neurotransmitters are
described. In addition, the pathways of light signals in
regulation of hormone secretion are discussed.

1. Prolactin. Season of the year influences serum
concentrations of PRL. For example, elevated serum
concentrations of PRL are associated with spring and summer

and reduced PRL is associated with fall and winter in

cattle (Koprowski and Tucker, 1973: Kensinger et al., 1979;
Peirce et al., 1987), sheep (Ravault, 1976; Munro et al.,
1980; Kennaway et al., 1981; Bosc et al., 1982), goats
(Buttle, 1974), horses (Johnson, 1986; Thompson et al.,
1986), deer (Mirarchi et al., 1978; Bubenik and Schams,
1986) and wild but not domestic pigs (Ravault et al.,
1982). In these experiments, animals were exposed to
natural uncontrolled seasonal conditions; therefore, the
specific factor(s) within season that mediate season—
induced changes in PRL could not be determined.

One environmental factor that changes with season,
ambient temperature, is positively correlated with
seasonally-induced changes in PRL (r = .61; Johnson, 1986).
In addition, exposure to increased ambient temperature at a
constant photoperiod caused a rapid increase in
concentrations of PRL, while a decrease in temperature had
the opposite effect in cattle (Wettemann and Tucker, 1974;
Tucker and Wettemann, 1976) and pigs (Kraeling et al.,
1987). Concentrations of PRL were 250% greater in steers
exposed to 30'C compared with 10°C (Smith et al., 1977).
This temperature-induced increase in PRL was associated
with a 3-fold increase in secretion rate and a 36% decline
in metabolic clearance rate of the hormone (Smith et al.,
1977).

In addition to changes in temperature, length of daily
light exposure changes with season, with the longest

periods of day light occurring in summer and least in

winter in all but equatorial latitudes. Seasonal changes
in photoperiod are constant from year to year whereas
seasonal changes in ambient temperature are unpredictably
variable. Therefore, photoperiod is a more dependable cue
for signalling a change in season than ambient temperature
(Hendricks, 1956). Concentrations of PRL and length of day
within a season are positively correlated (r = .80;
Johnson, 1986). To determine the effects of photoperiod on
serum concentrations of PRL, independent of temperature,
Bourne and Tucker (1975) maintained bull calves at a.
constant temperature and varied daily light exposure. In
one experiment bulls were conditioned to photoperiods of
16L:8D and then over a 12-wk period daily light exposure
was gradually reduced to 8L:16D. A second experiment was
the reciprocal of the first; exposure began at 8In16D and
gradually increased to 16L:8D. At constant temperatures,
decreasing daily light exposure from 16 to 8 h caused an
86% decline in serum concentrations of PRL, whereas
increasing light increased PRL 300%. Thus, independent of
temperature, changes in daily light exposure altered
concentrations of PRL. Similarly, studies in cattle
(Leining et al., 1979; Stanisiewski et al., 1984b, 1987b;
Crister et al., 1987a), sheep (Forbes et al., 1975, 1979;
Fitzgerald et al., 1982; Leshin and Jackson, 1987; Poulton
and Robinson, 1987), horses (Johnson, 1987) and deer
(Abbott et al., 1984; Bubenik et al., 1987) reported

increased serum concentrations of PRL in animals exposed to

daily photoperiods of 16L:8D compared with animals exposed
to less than 12 h of light per d. The response of PRL to
16L:8D occurs regardless of the spectral properties of
light. For example, PRL was increased when the source of
light was from red, blue, Vita-Lite or cool-white
fluorescent lights, mercury vapor or high-pressure sodium
lamps or incandescent bulbs (Leining et al., 1979;
Stanisiewski et al., 1984b).

Although elevated ambient temperature and increasing
daily exposure to light increase concentrations of PRL, the
speed of response is different. In cattle, temperature-
induced changes in PRL occur within minutes whereas the
response to photoperiod requires days. In bulls
conditioned to 8L:16D, increases in serum concentrations of
PRL are not detectable for the first 4 d following an
abrupt switch to 16L:8D (Petitclerc et al., 1989) but
become significant after approximately 1 wk and reach a
maximum 4 to 6 wk after switching to 16L:8D (Leining et
al., 1979; Stanisiewski et al., 1984b). The relative
sluggishness of photoperiod-induced change in PRL in sheep
is similar to that in cattle (Pelletier, 1973; Lincoln et
al., 1978).

Once maximal or minimal concentrations of PRL are
reached with exposure to 16L:8D or 8L:16D, respectively, no
diurnal rhythmicity of concentrations of PRL exists in

cattle (Petitclerc et al., 1983b, c). In contrast, sheep

display a marked diurnal increase of PRL at the beginning
of the dark period (Lincoln, 1977; Ravault and Ortevant,
1977; Thimonier et al., 1978).

Increasing light exposure to 20 h per C! does not
increase concentrations of PRL over that obtained with 16 h
of light per d (Leining et al., 1979). Exposure to
continuous light has produced contradictory results. For
example, concentrations of PRL in animals exposed to
continuous light were intermediate to concentrations after
exposure to 8 and 16 h of light (Kenneway et al., 1983),
equal to 8L:16D (Leining et al., 1979) or equal to 16L:8D
(Stanisiewski et al., 1987a). The difference in these
results may be due to differences in duration of the
experiments. Indeed, Stanisiewski et al. (1987a) reported
that animals exposed to 24L:0D required an additional 3 wk
of exposure for PRL to increase to similar concentrations
as bulls exposed to 16L:8D.

Light does not need to be present in a continuous 16 h
block of light to increase concentration of PRL.
Photoperiod-induced increases in PRL were similar when a
short-day photoperiod (> 12 h of light per d) was coupled
with a block of light during a photosensitive phase. Fbr
example, Ravault and Ortavant (1977) exposed ewes to a
photoperiod of 16L:8D or to 7 h of light plus a 1 h block
of light 7, 11, 14, 17 or 20 h after the start of the 7 h
block of light. When the 1 h block was given 17 h after

dawn, concentrations of PRL in these ewes were similar to

PRL in ewes exposed to 16L:8D. The response was reduced
when the 1 h block of light was given at other times.
Similarly, Petitclerc et al. (1983c) reported that in bulls
the response in PRL to a 2 h block of light 14 h but not 20
h after a 6 h block of light was equivalent to 16L:8D.
Additional work has shown that photoperiods of 7L:9D:1L:7D
or 7L:10D:1L:6D in sheep or 10L:8D:2L:4D in mares increased
serum concentrations of PRL as effectively as 16L:8D when
compared with PRL in animals exposed to 8L:16D (Thimonier
et al., 1978; Schanbacher and Crouse, 1981; Brinklow and
Forbes, 1984a; Johnson and Malinowski, 1986). Moreover,
Schanbacher et a1. (1985) reported that in ewes a 1 min or
15 min block of light 16 h after dawn (7L:9D:1 min or 15
min L) increased serum PRL similar to 16L:8D.

Photoperiod-induced increments in serum concentrations
of PRL begin to wane if animals are maintained on 16L:8D
for 12 wk (Stanisiewski et al., 1987b). Similarly,
concentrations of PRL in sheep maintained on long-day
photoperiods for 16 wk begin to decline (Almeida and
Lincoln, 1984). Thus, in terms of PRL secretion, cattle
and sheep eventually become refractory to photoperiodic
stimuli.

2. Gonadotropins. Similar to PRL, photoperiod affects
serum concentrations of the gonadotropins, LH and FSH. The
direction of photoperiod-induced change in LH and FSH

typically corresponds with the breeding, season of that

10

species. That is, when seasonal breeding species are in
their reproductively quiescent period serum concentrations
of gonadotropins are low and when animals are in their
active period concentrations of gonadotropins are elevated.
For example, in sheep, a short—day breeding species, serum
concentrations of LH and FSH are low in animals exposed to
long-day photoperiods and high in ewes exposed to short-
day photoperiods (Lincoln et al., 1977). In addition,
mares, a long-day breeding species, had greatest
concentrations of LH during long-day photoperiods and
lowest concentrations during short days (Oxender et al. ,
1977). These results have been confirmed in sheep
(Lincoln, 1979; Lincoln et al., 1982) and in other short-
day breeders such as goats (Racey et al., 1975) and white-
tailed deer (Mirarchi et al., 1978; Bubenik and Schams,
1986; Bubenik et al., 1987). Similarly, long-day
photoperiod-induced increases in concentrations of
gonadotropins in long-day breeding species have been
confirmed in horses (Clay et al., 1988), golden hamsters
(Stegar et al., 1985) and Djungarian hamsters (Yellon and
Goldman, 1987). Thus, in contrast to PRL where exposure to
long <days stimulates secretion in all species studied,
exposure to identical photoperiods can have opposite
effects on LH and FSH depending on the breeding season of
that species.

In non-seasonal breeding species such as swine and

cattle, the majority of evidence indicates that photoperiod

11

does not affect secretion of gonadotropins (Ntunde et al.,
1979; Rzepkowski et al., 1982; Diekman and Hoagland, 1983;
Peirce et al., 1987; Stanisiewski et al., 1987b, 1988a).
In contrast, concentrations of LH may vary with season of
the year in ovariectomized cattle. For example, serum
concentrations of LH were highest in ovariectomized heifers
exposed to photoperiods with increasing light per d than at
other times during the year (Day et al., 1986; Crister et
al., 1987a; Stumpf et al., 1988).

In rams exposed to lighting regimens of alternating 16
wk periods of 16L:80 and 8L:16D, pulsatile activity of LH
and FSH was increased following the switch from long to
short days, and reduced following the switch from short to
long days (Lincoln, 1979). Similarly, the number of LH
pulses were greater during fall and winter than during
summer in ovariectomized ewes (5 to 6 pulses/3 h vs 3
pulses/3 h; Karsch et al., 1984). Montgomery et al. (1987)
reported similar changes in pulsatile activity of FSH in
sheep. The photoperiod-induced increases in gonadotropin
pulsatile activity may be mediated by photoperiod-induced
changes in secretion of or responsiveness of the pituitary
gland to gonadotropin-releasing hormone (GnRH; Hansen,
1985). For example, photoperiod can influence hypothalamic
content of GnRH in hamsters (Silverman and Pickard, 1979),
white-footed mice (Glass et al., 1988) and sheep (Ebling et

al., 1987). In addition, secretion of LH in response to a

12

constant dose of GnRH (1000 ng, iv) varied-with exposure to
different photoperiods in rams (Lincoln, 1977) and rusa
deer (Van Mourik et al., 1986).

Photoperiod may also influence the responsiveness of
the hypothalamic-pituitary axis to negative feedback of
gonadal steroids (Karsch et al., 1984). Concentrations of
LH and FSH in ovariectomized ewes implanted with estradiol
varied with season (Legan et al., 1977; Goodman et al.,
1982; Platt et al., 1983). For example, estradiol reduced
ovariectomy-induced elevated serum concentrations of LH to
undetectable concentrations in ewes in the summer (non-
breeding season). However, the ability of estradiol to
inhibit LH in ewes in the autumn (breeding season) was
reduced, such that serum concentrations of LH were
maintained at 10 to 20 ng/ml (Legan et al., 1977). The
seasonal effects of estradiol on concentrations of LH are
similar when sheep are exposed to controlled photoperiods
of 16L:80 or 8L:16D (Bittman et al., 1985). In contrast,
implanting ovariectomized cattle with estradiol does not
alter season-induced changes in LH secretion (Day et al.,
1986; Stumpf et al., 1988).

Similar to PRL, the response of LH and FSH to a
stimulatory’ photoperiod is sluggish. For example,
following an abrupt switch from 16L:8D to 8L:16D, serum
concentrations of LH and FSH required between 6 and 12 d to
increase and 33 to 54 d to reach maximum concentrations

(Lincoln and Feet, 1977).

13

3. the rmones e . Growth hormone
(GH), glucocorticoids, insulin (INS), thyroid hormones,
(triiodothyronine [T3] and thyroxine [T4]) and fi-endorphins
may be involved in control of body growth (Gailbraith and
Topps, 1981; Schanbacher, 1984; Ebling and Lincoln, 1987).
Although not as responsive as PRL, LH and FSH, in some
circumstances, these hormones respond to change in season
or photoperiod (Leining et al., 1980; Vaughan et al., 1982;
Terqui et al., 1984; Denbow et al., 1986; Ebling and
Lincoln, 1987). Therefore, the effects cf photoperiod on
these hormones will be discussed in this section.

In cattle, concentrations of GH are unresponsive to
changes in photoperiod. For example, serum concentrations
of GH were similar between dairy heifers exposed to 16L:8D
or natural short-day winter day lengths (Peters and Tucker,
1978). In addition, photoperiod did not affect several
measures of GH in blood; such as average concentrations,
secretion rate, metabolic clearance rate, half-life in
serum, smoothed baseline, or number or amplitude of pulses
(Leining et al., 1980; Petitclerc et al., 1983a; Zinn et
al., 1986a, b, 1989). Similarly, photoperiod did not
affect serum concentrations of GH in pigs (Kraeling et al.,
1983) but in goats that were induced to lactate,
concentrations of GH were greater in animals exposed to

15.5L:8.SD compared with 8.5L:15.5D (Terqui et al., 1984).

14

The influence of photoperiod on GH in sheep is
conflicting. For example, Barenton et al., (1987, 1988)
reported that concentrations of GH were elevated in 2-year
old rams exposed to increasing day length compared with
decreasing day length. In contrast, Forbes et al. (1979)
and Brown et al. (1979) failed to observed any significant
effects of photoperiod on GH in lambs. These differences
in photoperiod-induced change in GB may be due to
differences in composition of diet and age of the animals
used in these studies, both of which influence
concentrations of GH (Purchas et al., 1970; Sejrsen et al.,
1983).

Similar to sheep, evidence for photoperiod-induced
alterations of GH in non—domestic animals are conflicting.
Photoperiod does not alter GH in red deer (Brown et al.,
1979). However, in white-tailed deer GH is elevated during
exposure to short days and reduced during exposure to long
days (Bubenik et al., 1975). Similarly, season affects GH
in male reindeer and moose (Ryg et al., 1982; Ryg and
Jacobsen, 1982). Differences in breed, age and feed
availability may account for differences in responses of GH
to season in these different species.

Serum concentrations of glucocorticoids were reduced 29
to 58% in 10-wk old bull calves exposed to 15.7, 16 or 20
h of light per d compared with exposure to 8 h of light per
d (Leining et al., 1980). In contrast, photoperiod did not

affect serum concentrations of glucocorticoids in 4 to 6-

15

month old or 15-month old heifers (Peters et al., 1980;
Zinn et al., 1986b) or lactating cows (Peters et al.,
1981). Similarly, exposure to photoperiods of
7L:10D:1L:6D, which mimic long-day photoperiods, reduced
serum concentrations of glucocorticoids compared with
short-day photoperiods in 3-but not lo-month old lambs
(Brinklow and Forbes, 1984a, b). Additional studies
indicate that photoperiod did not alter serum
concentrations of glucocorticoids in lambs (Kennaway et
al., 1981), rams (Lincoln et al., 1982) or white-tailed
deer (Bubenik et al., 1975, 1986). (However, serum
glucocorticoids were increased in lactating sows (Kraeling
et al., 1983) and young pigs (Barnett et al., 1981) exposed
to 16L:8D compared with 8L:16D.

Differences in glucocorticoid response to photoperiod
may be the result of differences in frequency and schedule
of blood sampling relative to diurnal changes in
glucocorticoids, age or species of animal utilized or time
of feeding relative to sampling. All of these variables
may influence concentrations of glucocorticoids (Hart et
al., 1981; Thun et al., 1981; Lincoln et al., 1982:
Brinklow and Forbes, 1984b).

Herbein et al., (1985) and Denbow et a1. (1986)
reported that serum concentrations of INS were lower in
summer than in winter in lactating cows. Similarly,

crossbred beef heifers had lower serum concentrations of

16

INS after exposure to 16L:8D compared with 8L:16D (S.A.
Zinn, L.T. Chapin, H.A. Tucker, unpublished observations).
In contrast, photoperiod did not influence average
concentrations, baseline concentrations or amplitude or
number of INS pulses in prepubertal bulls, steers and sheep
(Forbes et al., 1979; Tucker et al., 1984; Zinn et al.,
1989). These differences may reflect different responses
to photoperiod between species (cattle vs sheep) or between
sexes (males vs females).

Significant photoperiod-induced changes in thyroid
hormone-stimulating hormone (TSH) or thyroid hormones, T3
and T4 have been limited to rodents. For example, rats
housed in constant darkness had significantly lower serum
concentrations of TSH and T4 than rats housed in constant
illumination (Relkin, 1978). Similarly, exposure to short-
day photoperiods compared with long-day photoperiods
reduces TSH and T4 in hamsters (Vaughan et al.,1982: Vriend
and Wasserman, 1986). However, photoperiod does not
influence T3 or T4 in cattle (Leining et al., 1980; S.A.
Zinn and K. Refsal, unpublished observations) or sheep
(Forbes et al., 1979; Lincoln et al., 1982).

In the anterior pituitary gland of rats B-endorphins
are derived from proopiomelanocortin in the corticotropes
(Weber et al., 1978). Photoperiod can influence blood
concentrations of fi-endorphins. For example, switching
sheep from a photoperiod of 16L:8D to 8L:16D results in a

20-fold increase in plasma concentrations of fl-endorphin,

17

reaching a maximum after 4 to 8 wk of exposure to 8L:16D
(Ebling and Lincoln, 1987). The reverse switch in
photoperiod led to a decline in B-endorphin (Ebling and
Lincoln, 1987). Similarly, greater concentrations of B-
endorphin were reported in the mediobasal hypothalamus
(MBH) in mice exposed to short versus long days (Glass et
al., 1988). However, plasma concentrations of fi-endorphin
in white-tailed deer were highest in summer and lowest in
winter (Bubenik et al., 1988). Although, the role of
seasonally-induced changes in fi-endorphin is unknown, these
changes have been correlated with season-induced changes in
gonadotropins in sheep (Ebling and Lincoln, 1987) and
antler growth in deer (Bubenik et al., 1988). In addition,
fi-endorphin has been linked to hyperphagia (Davis et al.,
1983) and obesity (Givens et al., 1980). Thus, season-
induced changes in fi-endorphin may be linked to
photoperiod-induced changes in body weight and carcass
composition in cattle.

4. Neurotransmitters. Neurotransmitters from the
hypothalamus influence synthesis and release of hormones
from the anterior pituitary gland (Jacobowitz, 1988).
However, only a few studies have focused on photoperiod-
induced changes in neurotranmitters. In this section the
limited data on photoperiod-induced changes in
neurotransmitters, especially DA, 5HT and norepinephrine

(NE) will be discussed. GnRH was discussed previously (in

18

section on gonadotropins) and will not be discussed
further.

Anterior pituitary glands from cattle exposed to
8L:16D, compared with cattle given 16L:8D, had greater
concentrations of DA but NE and epinephrine were not
different (Stanisiewski et al., 1984c). In addition, serum
concentrations of PRL were lower in bulls with higher
pituitary DA; indicating that photoperiod-induced changes
in DA may be a mediator of photoperiod-induced changes in
PRL.

In studies using male golden hamsters, Steger et al.
(1982, 1984) reported that changing photoperiod can alter
hypothalamic concentrations and(or) turnover of DA, 5HT and
NE. For example, after 7.5 wk, hamsters switched from
long-day (14L:IOD) to short-day (5L:19D) photoperiods had
reduced hypothalamic concentrations of DA and a-
methyltyrosine-induced turnover of DA' compared with
hamsters maintained on long days. However, after 15 wk
concentrations and turnover of DA were not different
between hamsters exposed to long and short days.
Similarly, hamsters exposed to 5L:19D had greater
hypothalamic concentrations of NE but only after 15 wk of
exposure to short days (Steger et al., 1982). One d
following a shift from short- to long-day photoperiods,
hypothalamic 5HT was increased as was the ratio of 5HT to
its metabolite 5-hydroxyindoleacetic acid (SHIAA; Steger et

al., 1984). In contrast, after 8 wk of exposure to 16L:8D

19

or 8L:16D, hypothalamic contents of DA, 5HT and NE were
similar in the white-footed mouse (Glass et al., 1988).

The conflicting results in these studies may be due to
the different species of animals utilized or to differences
in time of sampling. Indeed, Benson (1987) reported that
photoperiod-induced changes in DA, 5HT and NE are different
depending on time of sampling. Compared with exposure to
long days, hypothalamic concentrations of DA were greater
after 6 wk, lower after 9 wk and not different after 12 wk
of exposure to short days in hamsters. Changes in
concentrations of neurotransmitters in animals exposed to
the same photoperiod for long periods may reflect changes
in hormone feed back on neurotransmitter release which may
reflect development of refractoriness of the hypothalamus

to photoperiodic stimulation.

5. a wa 0 1i h s s i e ' ne
secretign. Although the exact mechanism whereby

photoperiod affects hormone secretion is not completely
understood, in most mammals the pathway is considered to
begin in the retina and travel via neural connections to
the pineal gland (Reiter, 1980). In this section of the
review, the pathway that light signals travel to influence
hormone secretion is discussed.

The retina is essential for mammals to perceive
environmental light cues (Underwood and Gross, 1982). For

example, blinding abolishes photoperiod-induced changes in

20

cattle (Petitclerc et al., 1983b), sheep (Legan and Karsch,
1983), ferrets (Herbert et al., 1978) and hamsters (Reiter,
1969). In contrast, non-mammalian species, such as birds
and amphibians, have extra-retinal photoreceptors and
therefore do not require the retina to perceive light cues
(Okshe, 1965; Van de Kamer, 1965; Follet, 1978). From the
photoreceptors in the retina, light signals are transmitted
via the retinohypothalamic tract to the suprachiasmatic
nucleus (SCN; Moore and Lenn, 1972; Takahashi and Katz,
1982). Similar to the retina, destruction of the
retinohypothalamic tract abolishes photoperiod-induced
entrainment (Klein and Moore, 1979).

The SCN is a major oscillator of endogenous circadian
rhythms in mammals (Stephan and Zucker, 1972; Groos et al.,
1983; Turek et al., 1984). In addition, coupling of
environmental light:dark cycles and endogenous circadian
rhythms occur' in the SCN (Klein and. Moore, 1979).
Destruction of the SCN disrupts seasonal changes in the
annual reproductive cycle of sheep (Przekop and Domanski,
1980; Pan and Jackson 1985) and light:dark entrainment of
the pineal gland in rats and monkeys (Moore and Klein,
1974; Klein and Moore, 1979; Reppert et al., 1981).

Autoradiographic studies indicate that fibers from the
SCN project to the paraventricular nucleus (PVN; Berk and
Finkelstein, 1981). The PVN may act as a relay station
from the SCN to the pineal gland (Klein et al., 1983). For

example, complete destruction of the PVN blocks

21

photoperiod-induced changes in secretion of melatonin and
gonadal function (Pickard and Turek, 1983; Lehman et al.,
1984; Nunez et al., 1985).

Neurons pass from the PVN through the medial forebrain
bundle and reticular formation to the intermediolateral
cell column in the spinal cord (Moore and Klein, 1974;
Klein and Moore, 1979). These projections innervate
preganglionic cells which in turn innervate the superior
cervical ganglion (SCG; Binkley, 1983). Destruction of the
SCG inhibits photoperiod-induced changes of PRL in sheep
(Lincoln, 1979; Lincoln et al., 1982) and goats (Buttle,
1977; Maeda et al., 1986).

Postganglionic noradrenergic cells in the SCG project
to the pineal gland via the inferior carotid nerve and the
nervii coronarii (Reiter, 1982; Klein‘et al., 1983).
Postganglionic fibers terminate with the perivascular
spaces near the pinealocytes within the pineal gland
(Reiter, 1982). NE, released from these nerve terminals,
in response to dark binds to B-adrenergic receptors and
initiates the synthesis and release of melatonin from
pinealocytes (Wurtman and Axelrod, 1966; Binkley, 1983).

Secretions from the pineal gland, especially melatonin,
may act as a hormonal signal an animal uses to determine
length of day. For example, secretion of melatonin from
the pineal gland is lowest during periods of light and

greatest during periods of dark (Rollag and Niswender,

22

1976; Lincoln et al., 1982). Thus, daily duration of the
nocturnal rise of melatonin signals the length of darkness
to an animal (Bittman and Karsch, 1984). Melatonin does
not appear to act directly on the anterior pituitary gland
(Bartke et al., 1978), but may act by modulating release of
hypothalamic neurotransmitters and(or) neurohormones, which
in turn affect release of hormones from the anterior
pituitary gland (Reiter, 1980; Karsch et al., 1984).
Pinealectomy suppresses photoperiod—induced changes in
sheep (Brown and Forbes, 1980; Brinklow and Forbes, 1984b;
Karsch et al., 1984) and hamsters (Reiter and Hestor, 1966;
Reiter, 1980). In addition, infusion of melatonin for 16
or 8 h in pinealectomized, ovariectomized estradiol-treated
ewes caused changes in LH secretion analagous to those in
ewes exposed to short or long days, respectively (Bittman
and Karsch, 1984). Similarly, pattern of concentrations of
melatonin in serum may mediate photoperiod-induced effects
on PRL concentrations (Kennaway et al., 1982, Symons et
al., 1983). In contrast, pattern of concentrations of
melatonin in serum does not appear to affect secretion of
PRL in cattle (Crister et al., 1987b; Stanisiewski et al.,
1988a, b). That is, regardless of pattern of melatonin in
serum, long-day photoperiods continue to stimulate
concentrations of PRL in cattle. Although patterns of
concentrations of melatonin in serum may mediate other
photoperiod-induced changes in cattle (e.g., fat accretion;

Zinn et al., 1988) they do not mediate photoperiod-induced

23

changes in PRL. Therefore, alternative pathways (in
addition to the retina-pineal axis) for perception of light
signals must exist. Indeed, lesion of the PVN destroys
photoperiod-induced gonadal function but does not affect
photoperiod-induced changes in the circadian rhythm of
activity in hamsters (Pickard and Turek, 1983; Nunez et
al., 1985) further supporting the concept that alternative
pathways for photoperiod-induced changes in physiology
exist.

6. um r . The hormone that responds most
consistently to changes in photoperiod in seasonal and non-
seasonal breeding species is PRL. Concentrations of GH,
INS and glucocorticoids are altered with changes in
photoperiod in certain species but changes in
concentrations in these hormones are not consistent.
However, the effects of photoperiod on these hormones are
not as consistent as the effects on PRL. Gonadotropins
respond to changes in daily photoperiod but are more
correlated with changes in reproductive status in seasonal
breeding species and photoperiod does not influence
gonadotropins in non-seasonal breeding species. PRL is an
anabolic hormone and therefore, may be responsible for
photoperiod-induced changes in growth, development and
lactation in cattle.

The retina is essential for perception of photic

signals in mammals. Light signals may be transformed to

24

endocrine signals via the daily pattern of melatonin
secretion. The daily pattern of melatonin may mediate
photoperiod-induced changes in gonadotropins and PRL in
seasonal breeding species, but not in cattle. Therefore
the mechanism whereby photoperiod influences secretion of
PRL is unknown.

Neurotransmitters such as DA and 5HT, affect secretion
of PRL (Jacobowitz, 1988) and photoperiod can influence
activity of DA and 5HT neurons. Photoperiod-induced
changes in DA and 5HT, therefore, may mediate photoperiod-

induced changes in serum concentrations of PRL.

B. Dopaminergic Contrgl of Prolactin Secretion

Prolactin is secreted from the lactotropes of the
anterior pituitary gland. The prevailing hypothalamic
control of secretian of PRL is inhibition (Leong et al.,
1983). The majority of the tonic inhibition of PRL by
hypothalamic factors has been attributed to DA (Clemens and
Shaar, 1980). Therefore, in this section of the review,
dopaminergic control of secretion of PRL from the
lactotropes of the anterior pituitary gland will be
discussed.

1. i 'to control of rolac i . Transplantation
of the anterior pituitary gland to under the kidney capsule

(and thus away from direct hypothalamic influence) results

25

in increased serum concentrations of PRL (Everett, 1954) .
Serum concentrations of PRL increase following:
transection of the pituitary stalk (Chen et al., 1970;
Bishop et al., 1971; Woolf et al., 1974), bilateral
destruction of the median eminence (Chen et al., 1970) and
destruction of the MBH (Arimura et al., 1972; Butler et
al., 1975). In addition, acid extracts of hypothalamic
tissue inhibited secretion of PRL in vitro (Talwalker et
al., 1963) . Furthermore, extracts from hypothalamic but
not cerebrocortical tissue decreased serum PRL when infused
into portal vessels leading to the pituitary gland (Kamberi
et al., 1971a), indicating that tonic inhibition of
secretion of PRL from the anterior pituitary gland is
direct and specific to the hypothalamus.

Infusion of DA directly into hypophyseal portal vessels
inhibits secretion of PRL (Takahara et al., 1974a). In
addition, antagonists of DA, such as haloperidol, increase
secretion of PRL whereas DA agonists, such as ergot
alkaloids, inhibit PRL (Clemens and Shaar, 1980; Seeman,
1981). In vitro, DA suppresses secretion of PRL from
explants (Takahara et al., 1974b) and dispersed cells
(Padmanabhan et al., 1979) of the anterior pituitary gland.
The influence of DA on PRL is dose dependent (Padmanabhan
et al., 1979; Yeo et al., 1979) and is reversible (Yeo et
al., 1979; Thorner et al., 1980). In addition,
pretreatment or cotreatment of pituitary cells with DA

antagonists prevents DA-induced suppression of PRL

26

(MacLeod, 1976; Caron et al., 1978; Denef and Follebouckt,
1978; Rick et al., 1979).

Concentrations of DA within hypophyseal portal vessels
are greater than in the general circulation (Ben-Jonathan
et al., 1977; Gibbs and Neill, 1978) and are altered with
physiological state and pharmacological manipulations (Ben
Jonathan et al., 1977; de Greef and Neill, 1979). In
addition, these concentrations of DA are sufficient to
inhibit secretion of PRL in rats (Gibbs and Neill, 1978)
and from bovine pituitary cells in vitro (Shaar and
Clemens, 1974; Padmanabhan et al., 1979).

Receptors for DA are present on anterior pituitary
cells (Brown et al., 1976; Calabro and MacLeod, 1978;
Cronin et al., 1978) and in particular on the plasma
membrane of lactotropes (Goldsmith et al., 1979).

D2 (Seeman, 1981) but not D1 (Cocchi et al., 1987) DA
receptors are present on lactotropes. D2 DA receptors are
negatively linked to the adenyl cyclase system (Ben-
Jonathan, 1985). The number of DA binding sites on the
anterior pituitary gland vary with changes in serum
concentrations of PRL. For example, the number of DA
binding sites increases coincident with the surge of PRL
during proestrus in the rat estrous cycle (Heiman and Ben-
Jonathan, 1982) . In addition, neonatal rats treated with
the neurotoxin monosodium glutamate which selectively

destroys retinal and arcuate nuclear neuronal perikarya,

27

had 3-fold greater concentrations of PRL and 60% more DA
binding sites than controls without change in the affinity
of receptors for DA (Ben-Jonathan, 1985). Thus, increasing
concentrations of PRL result in up-regulation of DA
receptors. Conversely, following treatment with
bromocriptine, a dopamine agonist, concentrations of PRL
are reduced as are the number of DA binding sites (Di Paolo
and Falardeau, 1984).

In summary, secretion of PRL from lactotropes in the
anterior pituitary gland is tonically inhibited and
hypothalamic DA, acting through D2 receptors directly on
the lactotrope, is the primary prolactin inhibiting factor.

2. Intracellular mediators 9f dopaminergic acrign in
rhe lactotrope. The events following DA binding to D2
receptors on the lactotrope to inhibit PRL secretion is not
completely understood. Typically extracellular factors,
such as DA, that bind to receptors on the plasma membrane
of a target cell, are coupled to an intracellular second
messenger system to elicit a specific cellular response.
Several compounds, including cyclic adenosine monophosphate
(cAMP), calcium and inosital phospholipids (IP), may act as
intracellular mediators in lactotropes. DA may influence
calcium, cAMP and(or) IP individually or in combination to
inhibit secretion of PRL (Enjalbert et al., 1988) and may
involve more than a single mechanism. Thus in this section

of the review the influence of DA on the intracellular

28

second messengers, cAMP, calcium and IP and their possible
interactions will be discussed.

DA binding to D2 receptors on lactotropes is negatively
associated. with. adenyl cyclase (Enjalbert. and. Bockaert,
1983; McDonald et al., 1984), a membrane bound enzyme that
converts adenosine triphosphate to cAMP (Stryer, 1975). DA
in nanomolar concentrations inhibits accumulation of cAMP
in dispersed pituitary cells within 1 to 5 min (Swennen and
Denef, 1982; Bockaert et al., 1988). Pretreatment with
Bordetta Pertussis toxin, which blocks receptor-mediated
inhibition of adenyl cyclase, blocks DA-induced reduction
in cAMP and PRL (Bockaert et al., 1988). In addition,
cholera toxin-induced and thyrotropin-releasing hormone
(TRH)-induced increases in cAMP and PRL are inhibited
following treatment with DA or DA agonists (Barnes et al.,
1978; Gianattasio et al., 1981). Collectively, these data
support a role for suppression of cAMP as a mechanism of DA
inhibition of secretion of PRL.

Changes in intracellular concentrations of calcium can
influence secretion of PRL. For example, exposing cells
from anterior pituitary gland to increasing concentrations
of calcium increased secretion of PRL (Thorner and MacLeod,
1980). Similarly, treatment with a calcium channel
agonist, maitotoxin, increased secretion of PRL (Schettini
et al., 1986). Conversely, removal of calcium from the
media of pituitary cells cultured in vitro or blocking

calcium channels on the lactotropes in vivo, thereby

29

preventing calcium influx, decreased secretion of PRL
(MacLeod and Fontham, 1970; Thorner and. MacLeod, 1980) .
Therefore, DA may inhibit secretion of PRL by reducing
intracellular concentrations of calcium. Indeed, DA
reduces intracellular calcium in primary cultures of
lactotropes (Shrey et al., 1986; Malgaroli et al., 1987a,
b; Login et al., 1988). Calcium may directly stimulate
secretion of PRL or it may act indirectly. For example,
treatment of dispersed pituitary cells with a calcium
channel activator, increased calcium influx and increased
secretion of PRL, and also increased intracellular
concentrations of cAMP (Schettini et al., 1986). Treatment
with DA reduced the elevated cAMP as well as the increased
PRL (Schettini et al., 1986). Thus, DA may inhibit
secretion of PRL by reducing calcium-stimulated production
of cAMP.

Hydrolysis of IP in cell membranes is an intracellular
second messenger in a number of cell systems (Joseph et
al., 1984) including the anterior pituitary gland (Bonetti
et al., 1987). IP, in turn, may stimulate release of
stored intracellular calcium pools which increases
secretion of hormone (Hawthorne, 1983). Increased
intracellular IP is associated with TRH- and angiotension
II (AID-stimulated PRL release from anterior pituitary
cells (Enjalbert et al., 1986; Law et al., 1988a). In

addition, age-related differences in incorporation of 32P

30

into IP paralleled age-related differences in secretion of
PRL (Bonetti et al., 1987). Moreover, inclusion of a IP,
hexabisphosphate, in perfusion media increases secretion of
PRL from anterior pituitary cells (Law et al., 1988b).
Thus, increased intracellular production of IP is
associated with increased secretion of PRL. DA may inhibit
secretion of prolactin by tonically inhibiting synthesis of
IP. Indeed, DA or DA agonists inhibit synthesis of IP in
vivo (Bonetti et al., 1987) and in vitro (Bockaert et al.,
1988).

Malgaroli et al., (1987a) reported that TRH causes a
biphasic increase in secretion of PRL. The initial peak is
due predominantly to redistribution of intracellular stores
of calcium and a secondary phase is due to influx of
extracellular calcium (Valler et al., 1988). Increased PI
is associated with an influx of extracellular calcium
(Valler et al., 1988). DA does not inhibit release of
calcium from intracellular stores associated with TRH-
induced rapid accumulation of IP (Valler et al., 1988).
However, DA may suppress increased synthesis of IP
associated with a secondary influx of extracellular calcium
(Valler et al., 1988) and thereby inhibit secretion of PRL
associated with this second phase of TRH-induced release.

In summary, several intracellular mechanisms may be
involved in dopaminergic inhibition of PRL secretion
including' ‘mechanisms ‘that regulate intracellular

concentrations of calcium, cAMP and IP. The extent of DA

31

inhibition of PRL may depend on the individual and(or)
interactive negative influence DA exerts on calcium, cAMP
and IP.

3. Inberoinfundibular dgnaninergic neurons. DA is the
primary inhibitor of secretion of PRL from the anterior
pituitary gland. The DA primarily responsible for
inhibition of PRL is secreted from tuberoinfundibular
dopaminergic (TIDA) neurons (Ben-Jonathan, 1985). There is
no loss of DA in the median eminence following hypothalamic
deafferentiation (Jonsson et al., 1972; Gudelsky et al.,
1978) indicating that perikarya and axons of TIDA lie
completely within the MBH of the rat (Moore and Johnston,
1982) . The TIDA neurons originate in the arcuate and
periventricular nuclei within the MBH (Ben-Jonathan, 1985;
Albenese et al., 1986). Axons of TIDA neurons are short
and project to the external layer of the median eminence
(Moore, 1987) and terminate near the perivascular spaces of
the capillary plexus of the hypophysial portal system
(Ajika and Hokfelt, 1973). Electrical stimulation of the
arcuate nucleus increases turnover and synthesis of DA in
TIDA neurons and reduces secretion of PRL (Gunnet et al.,
1987; Lookingland et al., 1987a).

Tyrosine, the precursor' of DA, is taken up by
dopaminergic neurons and hydroxylated to Irdihydroxy—
phenylalanine (DOPA) by the enzyme tyrosine hydroxylase

(Fernstrom, 1983). Tyrosine hydroxylase is the rate-

32

limiting enzyme in the synthesis of DA (Spector et al.,
1967). The enzyme, aromatic-L-amino acid decarboxylase,
rapidly decarboxylates DOPA to DA (Moore, 1987), such that
intraneuronal concentrations of DOPA are essentially zero
(Demarest and Moore, 1980). DA then feeds back to inhibit
activity of tyrosine hydroxylase (Specter et al., 1967).

Newly synthesized DA in TIDA neurons can be released
into the perivascular spaces of the primary plexus of the
hypophyseal portal system and transported via the blood to
the anterior pituitary gland to exert its influence on PRL
(Moore et al., 1980). In addition, newly synthesized DA
can be scavanged by intraneuronal monoamine oxidase (MAO)
to the predominant metabolite of DA, dihydroxyphenylacetic
acid (DOPAC; Lookingland et al., 1987b). DA can also be
stored in synaptic vesicles (Moore, 1987).

Although synthesis and release of DA in all
dopaminergic neurons may be similar, there are several
unique characteristics of TIDA neurons. For example,
unlike other dopaminergic neurons, TIDA neurons do not form
synapses but release DA directly into the portal blood
(Moore, 1987) . Intraneuronal concentrations of DOPAC are
lower in TIDA than nigrostriatal DA neurons (Umezu and
Moore, 1979), which. is :most likely' due to 'the reduced
affinity for DA of the DA-reuptake system in TIDA neurons
(Demarest and Moore, 1979; Annunziato et al., 1980). That
is, in TIDA neurons less DA is reincorporated into the

neuronal terminals and therefore less DA is available for

33

metabolism to DOPAC. In addition, TIDA activity is not
regulated directly by DA-receptor mediated mechanisms
(Demarest et al., 1985b). That is, DA agonists or
antagonists do not act directly on TIDA neurons to elicit
change in neuronal activity (Gudelsky and Moore, 1976,
1977). For example, injection of haloperidol, a DA
antagonist, into rats stimulates TIDA neuronal activity 12
or 24 h but not 2 or 8 h after injection (Demarest and
Moore, 1980; Gudelsky and Porter, 1980). 'Conversely, TIDA
activity was decreased 26 h but not 2 h after the start of
injections of bromocriptine, a DA agonist (Demarest and
Moore, 1981). Effects of haloperidol and bromocriptine on
TIDA activity were eliminated in hypophysectomized animals
(Demarest and Moore, 1980, 1981) indicating that actions of
DA agonists and antagonists on TIDA neurons are mediated
through their actions on the pituitary gland.

Since DA antagonists and agonists stimulate and inhibit
secretion of PRL from the anterior pituitary gland,
respectively, PRL is a likely choice as the mediator of
these drugs on TIDA activity (Moore, 1987). Indeed in
rats, systemic (Hokfelt and Fuxe, 1972; Gudelsky et al.,
1976) and intracerebroventricular (Annunziato and Moore,
1978; Johnston et al., 1980) injections of PRL selectively
increased TIDA activity. However, PRL must be elevated for
at least 12 h to increase TIDA neuronal activity (Demarest

et al., 1985b).

34

Although the exact mechanism whereby PRL stimulates
TIDA activity is unknown, it appears that protein synthesis
is involved, since treatment wtih cycloheximide, an
inhibitor of protein synthesis blocks the effect of PRL
(Johnston et al., 1980). Therefore, DA from TIDA neurons
tonically' inhibits. secretion. of PRL. and serum
concentrations of PRL, in turn, regulates activity of TIDA
neurons (Hokfelt and Fuxe, 1972; Gudelsky et al., 1976;
Annunziato and Moore, 1978). That is, increased
concentrations of PRL feed back to increase TIDA activity
and subsequent DA release. Then, reduced secretion of PRL
results in decreased TIDA activity and decreased DA release
(Demarest et al., 1985b).

The feedback system described above can be interrupted
during various physiological states. For example in rats,
suckling-induced and restraint stress-induced increases in
PRL are associated with decreased TIDA neuronal activity
and DA release (Selmonoff and Wise, 1981; Demarest et al.,
1985a). Int addition, estradiol treatment. increased
secretion of PRL but reduced TIDA activity by disrupting
PRL feedback on the neurons (Demarest et al., 1984). Thus,
in certain physiological states TIDA neuronal activity can
be inhibited despite high serum concentrations of PRL.

4. Snnnary. Secretion of PRL is tonically inhibited.
DA released from TIDA neurons in the MBH into the
hypophyseal portal blood, is primarily responsible for the

tonic inhibition of PRL secretion. DA binds to D2

35

receptors on the plasma membrane of the lactotrope and by
post-receptor mechanisms involving cAMP, calcium and(or)
IP, inhibits secretion of PRL. Serum concentrations of PRL
feed back to regulate TIDA neuronal activity. However, the
feedback system can be interrupted with changes in
physiological state. Therefore, it seems reasonable to
hypothesize that photoperiod may stimulate secretion of PRL
by interfering with the PRL-TIDA feedback mechanism.
Indeed, as mentioned in a previous section, photoperiod can
alter concentrations of DA in pituitaries of bulls
(Stanisiewski et al., 1984c) and concentrations of DA in
median eminence of hamsters (Steger et al., 1982, 1984,

1985).

C. 5- dro t tam'ne u 10 o rol ct'n

secretion

Many neuropeptides and neurotransmitters, including
5HT, stimulate secretion of PRL (Jacobowitz, 1988). The
5HT neurons have been localized in several areas within the
hypothalamus, including the SCN (Kent and Sladek, 1978;
Card and Moore, 1984). The SCN is a part of the neural
pathway"whereby’ light. signals are transmitted from the
retina to the hypothalamus and is implicated in circadian
rhythmicity (reviewed in the section on, the pathway of

light signals in regulation of hormone secretion) including

36

the circadian release of PRL in rats (Bethea and Neill,
1979; Dunn et al., 1980). Thus, it seems reasonable to
speculate that changes in 5HT may be involved in
photoperiodic regulation of secretion of PRL. Therefore,
in this section of the review 5HT influence on secretion of
PRL will be discussed.

1. 5-Hydrorytryntaninergig ngnrgns. The essential
amino acid tryptophan is the precursor of 5HT. Tryptophan
is transported into 5-hydroxytryptaminergic neurons and
converted to 5—hydroxytryptophan (5HTP) by the enzyme
tryptophan hydroxylase (Lovenberg et al., 1968). Aromatic-
L-amino acid decarboxylase then rapidly converts 5HTP to
5HT (Lovenberg et al., 1962), such that intraneuronal
concentrations of 5HTP are essentially zero (Fernstrom,
1983). The resulting 5HT is either released, translocated
to synaptic vessicles or converted to its predominant
metabolite, 5HIAA, in a two-step process (Baumgarten and
Schlossberger, 1984). First, 5HT is converted to 5-
hydroxyindolealdehyde acid by MAO and then to 5HIAA by an
aldehyde dehydrogenase (Fernstrom, 1983).

Tryptophan hydroxylase is not saturated at
physiological concentrations of tryptophan in the brain,
and therefore substrate (i.e., tryptophan) availability may
be an important factor in control of tryptophan hydroxylase
and subsequent synthesis of 5HT (Leathwood, 1987). For
example, increasing or decreasing concentrations of

tryptophan in the brain increases or decreases rate of 5HT

37

synthesis and accumulation of 5HIAA, respectively
(Fernstrom and Wurtman, 1971; Carlsson and Linguist, 1973;
Biggio et al., 1974; Arimanana et al., 1984). In addition,
regulation of 5HT synthesis by end-product inhibition of
tryptophan hydroxylase has been reported when
concentrations of 5HT are increased two to three-fold
following 'treatment. with. MAO inhibitors (Macon et al.,
1971),

Therefore, tryptophan hydroxylase, and thus synthesis
of 5HT, may be regulated by several different mechanisms
including end-product inhibition and(or) substrate
availability.

2. Stimulatory cgntrol or prolactin. Infusion of 5HT
stimulates secretion of PRL in vivo in a dose-dependent
manner (Kamberi et al., 1971a; Lawson and Gala, 1975, 1978;
Lawson et al., 1980; Wehrenberg et al., 1980; Thomas et
al., 1987). Similarly, treatment with 5HTP, the immediate
precursor of 5HT, stimulates secretion of PRL in vivo
(Clemens et al., 1978; Marti-Henneberg et al., 1980; King
et al., 1985; Johnson et al., 1986; Beru and Kuhn, 1987a,
b). Furthermore, treatment with 5HT agonists stimulate
secretion of PRL in vivo (Lawson and Gala, 1978; Willoughby
et al., 1982; Lewis and Sherman, 1985; Beru and Kuhn,
l987a,b).

Treatment with 5HT antagonists, p-chlorophenylalanine

(pCPA; Donoso et al., 1971; Kordon et al., 1973/74; Gallo

38

et al., 1975) or cyprohaptadine (Lewis and Sherman, 1985)
have little effect on basal secretion of PRL. However, 5HT
antagonists reduce the ability of certain factors to
stimulate secretion of PRL. For example, pCPA blocks
suckling-induced secretion of PRL (Kordon et al., 1973/74);
pCPA, ketanserin and ICS 205-930 block proestrus-induced
secretion of PRL (Jahn and Deis, 1987, 1988), metergoline
blocks stress-induced secretion of PRL (Moore et al.,
1987); and cyproheptadine blocks fenfluramine-induced
secretion of PRL (Lewis and Sherman, 1985). Therefore, 5HT
may be involved in regulation of stimulated secretion of
PRL but not basal secretion.

3. ossi e sites of 5-h d o t tam ner c nf u nce
gn nrglactin. The exact mechanism whereby 5HT influences
secretion of PRL is unknown but the site of action is
probably not directly on the anterior pituitary gland. For
example, infusion of 5HT into hypophyseal portal vessels
did not influence secretion of PRL (Kamberi et al., 1971a).
In addition, secretion of PRL from dispersed cells of the
anterior pituitary gland treated with 5HT was either not
different (Talwalker et al., 1963; Birge et al., 1970) or
decreased (Padmanabhan et al., 1979) compared with
controls. Furthermore, 5HT stimulation of PRL is prevented
in hypothalamo-pituitary disconnected sheep (Thomas et al.,
1987). Therefore, 5HT probably exerts its influence

indirectly.

39

5-Hydroxytryptaminergic neurons project from the raphe
nuclei to the SCN (Avanzino et al., 1971; Kent and Sladek,
1978; Moore et al., 1978) and since the SCN may be involved
in secretion of PRL (Pasteels, 1970; Dunn et al., 1980) 5HT
released from raphe nuclei to the SCN may be a mechanism
whereby 5HT indirectly influences PRL. For example,
microiontopheretic application of 5HT to the SCN reduces
electrical activity in this nucleus (Meijer and Good,
1988). In addition, lesions of the raphe nuclei block
suckling-induced increases in secretion of PRL, a mechanism
that involves 5HT (Barofsky et al., 1983); indicating that
5-hydroxytryptaminergic neurons may be involved in
secretion of PRL.

Alternatively, 5HT may influence DA, which, in turn,
regulates PRL. For example, 5HT inhibits dopaminergic
activity in the striatum (Ennis et al., 1981). 5HT may
also inhibit activity of TIDA neurons, thus reducing the
inhibitory influence of DA on PRL. For example,
implantation of 5HT in the arcuate nucleus, the location of
perikarya of TIDA neurons, reduces neuronal activity in
this nucleus and increases secretion of PRL (Nishihara et
al., 1986; Nishihara and Kimura, 1987). In addition, the
arcuate nucleus receives 5HT innervation (Pickel et al.,
1977; Kent and Sladek, 1978; Wosler et al., 1984).
Therefore, 5HT may influence PRL by inhibiting TIDA
neurons. Indeed, concentrations of DA in hypophyseal

portal blood (from TIDA neurons) were decreased in rats

40

receiving intraventricular 5HT (Pilotte and Porter, 1981).
In explants of rat brain, 5HT inhibits activity of tyrosine
hydroxylase, the rate limiting enzyme in synthesis of DA
(Devau et al., 1987). This inhibition was dose dependent
and reversible (Devau et al., 1987).

These two mechanisms may be connected, because axons
from the SCN project to the arcuate nucleus (Swanson and
Cowan, 1975; Rusak and Zucker, 1979). Therefore, 5HT may
affect secretion of PRL by altering activity of SCN which,
in turn, reduces dopaminergic activity in the arcuate
nucleus.

A third alternative is that 5HT may influence secretion
of PRL by positive stimulation. For example, Clemens et
al. (1978) reported that 5HT-induced secreticn of PRL was
greater than DA-inhibition of PRL. They cencluded that 5HT
stimulated secretion of PRL by causing release of a PRL—
releasing factor not by a reduction of TIDA neuronal
activity (Clemens et al., 1978).

4. Summary. Tryptophan is converted to 5HT in 5-
hydroxytryptaminergic neurons. Tryptophan hydroxylase, the
rate limiting enzyme in 5HT synthesis, can be controlled by
substrate availability or end-product inhibition. 5HT
stimulates secretion of PRL but not as a direct effect on
the anterior pituitary gland. The indirect mechanism
whereby 5HT stimulates PRL is unknown. However, 5HT may

inhibit SCN (and)or TIDA neuronal activity, which in turn,

41

increases secretion of PRL. Alternatively, 5HT may
stimulate a jprolactin-releasing factor which stimulates
secretion of PRL. Thus, photoperiod may induce changes in
5HT activity at the SCN and(or) the arcuate nucleus which
in turn may alter secretion of PRL. Indeed, as mentioned
previously, photoperiod can alter concentrations of 5HT and

5HIAA/5HT in hamsters (Benson, 1987).

Chapter 1

Alterations in Concentrations of

Dihydroxyphenylacetic Acid in Rats Euthanized with

Sodium Pentobarbital

42

43

Introduction

An objective of this dissertation was to determine if
exposure to photoperiods that alter concentrations of PRL
in cattle alter activity of TIDA and 5HT neurons. Accurate
measurement of activity of these neurons requires rapid
removal of the brain followed by dissection of the MBH
/pituitary stalk (PS), which contains these neurons. In
rats, the brains and pituitary gland are quickly removed
following decapitation but, because of body size this
method is not appropriate for cattle. Furthermore, the
traditional method of slaughtering cattle (i.e., stunning
with a captive bolt and exsanguination) is unacceptable
because it activates the hypothalamo-pituitary system
(Cooper et al., 1986) and damages the hypothalamus (Dennis
et al., 1988).

Additional requirements of this series of experiments
were that bulls be euthanized without stress and without
altering ambient temperature. Stress increases secretion
of PRL (Tucker, 1971) and restraint stress-induced
increases in PRL are associated with altered TIDA and 5HT
neuronal activity (Demarest et al., 1985a). In addition,
ambient temperature acutely affects serum concentrations of
PRL in cattle (Tucker and Wettemann, 1976). Furthermore,
cold temperatures block photoperiod-induced increases in
serum concentrations of PRL (Peters and Tucker, 1978) .

Therefore, stress or changing ambient temperatures at

44

slaughter may mask photoperiod-induced effects on serum
concentrations of PRL or TIDA and (or) 5HT neuronal
activities. Thus, a method of euthanizing cattle in these
studies was needed in which animals could be killed without
stress and at constant ambient temperature.

Sodium. jpentobarbital-based. :mixtures, available
commercially are used for euthanizing domestic animals
(McDonald et al., 1978). Large doses of pentobarbital,
given intravenously, euthanizes animals rapidly by direct
action on the medulla causing depression of the respiratory
center and cessation of heart action (McDonald et al.,
1978). Since animals die rapidly with little stress and
animals can be killed in their stalls in our
environmentally-controlled chamber, sodium pentobarbital
met the criteria for a euthanizing agent needed for these
experiments. However, the effects of a commercially
available euthanizing agent, Fatal Plus (Vortech
Pharmaceuticals, Dearborn, MI), or sodium pentobarbital on
TIDA neuronal activity were unknown. Therefore, the
primary objective of this experiment was to determine the
effects of sodium pentobarbital on activities of TIDA
neurons. Since localization of important areas in the
hypothalamus of TIDA neuronal activities have been
elucidated in rats (Moore et al., 1985), rats were utilized
in this experiment. Specifically, the objectives were: to

compare concentrations of DOPAC and DA in selected brain

45

regions of rats that were euthanized with Fatal Plus,
sodium pentobarbital or decapitation, and 2) to compare the
effects of immobilization stress on DOPAC and DA in rats

euthanized with sodium pentobarbital or decapitation.

Materials and Methods

Animals. Male Long-Evans rats (Charles River Breeding
Laboratories, Wilmington, MA) weighing 240 to 270 g were
maintained in a temperature- (22°C) and photoperiod-
(lights on between 0600 and 1800 h) controlled environment.
Food (Wayne Lablox, Continental Grain Company, Chicago, IL)
and water were provided ad libitum. Euthanizing agents
were infused iv. Three d before an experiment a
polyethylene cannula was implanted into a jugular vein in
rats that received iv injections. Before surgery, rats
were anesthetized with ether.

Irearments. 131 a. preliminary study (Experiment 1)
eight rats were euthanized with an iv injection of Fatal
Plus (equivalent to 85 mg sodium pentobarbital/kg body
weight; a dose that kills rats within 2 sec) and eight rats
were untreated and served as controls.

In subsequent studies (Experiments 2 and 3) Fatal Plus
or an aqueous solution of its active ingredient, sodium
pentobarbital (388 mg/ml; pH 8.5), were infused iv as

indicated in table legends.

46

Tlssue ggllegtion, prepararion and analysis. Following

treatment with Fatal Plus, sodium pentobarbital or no
treatment of controls, rats were decapitated and brains and
pituitary glands were removed and frozen on aluminum foil
placed directly on dry ice. Frontal sections (1 mm) were
prepared in a cryostat (-9°C) and selected brain regions
were dissected according to modifications (Lookingland and
Moore, 1985) of the method of Palkovits (1973). Tissues
were placed in 60 pl of .1 M phosphate citrate buffer (pH
2.5) containing 15% methanol and stored at -20‘C until
assayed. At the time of assay each sample was sonnicated
(3 sec), centrifuged, and contents of DA and DOPAC in the
supernatant were determined by high performance liquid
chromatography (HPLC) coupled. to an electrochemical
detector (Chapin et al., 1986). Tissue pellets were
dissolved in 1.0 N NaOH and assayed for protein (Lowry et
al., 1951). Statistical analysis of differences between
groups were analyzed by the two-tailed Students' t-test

(Gill, 1978).

Results

Experiment l. It was initially planned to measure the
concentrations of DA and DOPAC in the median eminence of
cattle that were euthanized with Fatal Plus. To determine
if this euthanizing agent per se altered the concentrations

of these compounds a preliminary experiment was conducted

47

in rats. Relative to decapitated rats, rats euthanized
with Fatal Plus had greater (P < .01) concentrations of
DOPAC (8.5 i .5 vs 16.9 i 1.6 ng/mg protein) and lower (P <
.01) concentrations of DA (110.5 1 4.6 vs 88.8 i 3.8 ng/mg
protein) in the median eminence.

Experiment 2. Since results of Experiment 1 were
unexpected, it was decided to determine if the
neurochemical effects of Fatal Plus were associated with
its content of sodium pentobarbital. Accordingly,
concentrations of DOPAC and DA determined in selected brain
regions were compared in rats that were decapitated or
euthanized with either Fatal Plus or with sodium
pentobarbital. Concentrations of DOPAC and DA in the
median eminence, striatum and SCN are summarized in Table
1. Similar' to 'the results in IExperiment. 1, Fatal Plus
increased concentrations of DOPAC 52% and reduced
concentrations of DA 28% in the median eminence. Relative
to decapitation sodium pentobarbital was without effect on
concentrations of DA but increased concentrations of DOPAC
49% in the median eminence. It was concluded that
increased concentrations of DOPAC in the median eminence of
rats euthanized with Fatal Plus in Experiment 1 were due
to its content of pentobarbital, whereas the reduction in
DA after Fatal Plus was probably due to an ingredient other
than pentobarbital. The effects of Fatal Plus and

pentobarbital on concentrations of DA‘and DOPAC were

48

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restricted to the median eminence; neither preparation
altered concentrations of DOPAC or DA in the striatum or
SCN (Table 1).

i en : Because pentobarbital increased
concentrations of DOPAC in the median eminence it was
important to determine if stimuli that normally influence
this neurochemical index of TIDA neurons are altered in
rats euthanized with sodium pentobarbital. Therefore, it
was decided to determine if the ability of restraint stress
to reduce concentrations of DOPAC in the median eminence
was masked in rats euthanized with sodium pentobarbital.
The results of this experiment are summarized in Table 2.

Consistent with the results in Experiment 2 (Table 1)
concentrations of DOPAC in the median eminence of rats
euthanized. with sodium jpentobarbital were increased
compared with DOPAC in rats that were decapitated.
Nevertheless, 15 min of physical restraint reduced
concentrations of DOPAC in the median eminence of
decapitated and pentobarbital-euthanized rats. Fifteen min

of restraint did not influence DOPAC or DA in the striatum.

Discussion

Consistent with previous reports (Umezu and Moore,
1979), concentrations of DOPAC relative to those of DA are

lower in the median eminence than in the striatum.

50

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51

However, concentrations of DOPAC in the median eminence of
animals euthanized with Fatal Plus or sodium pentobarbital
were greater than DOPAC in decapitated animals.
Concentrations of DA were reduced in animals injected with
Fatal Plus but not with sodium pentobarbital, suggesting
that this effect may be due to some constituent of Fatal
Plus other than sodium pentobarbital. Neither drug altered
concentrations of DA or DOPAC in the striatum, arcuate
nucleus, nucleus accumbens or intermediate or neural lobes
of the pituitary gland, suggesting that these effects are
selective for TIDA neurons in median eminence.

Physical restraint reduces rates of turnover and
synthesis of DA and concentrations of DOPAC in the median
eminence of decapitated rats (Demarest et al., 1985a;
Lookingland et al., 1987b); suggesting that this stressful
manipulation reduces TIDA neuronal activity. Similarly in
the current study, 15 min of restraint reduced
concentrations of DOPAC in the median eminence of
decapitated and pentobarbital-euthanized rats, but did not
alter concentrations of DOPAC in the striatum.

Results of the present study reveal that sodium
pentobarbital-induced euthanasia specifically increases
concentrations of DOPAC in the median eminence. This
effect is rapid (within 25 sec; from beginning of injection
to removal of brain) and could represent an increase in the
metabolism of DA associated with or independent of an

increase in TIDA neuronal activity. Alternatively, sodium

52

pentobarbital could decrease a post-mortem decline in
concentrations of DOPAC in the median eminence of
decapitated rats. Regardless of the mechanism, changes in
concentrations of DOPAC in the median eminence associated
with restraint stress-induced change in TIDA neuronal
activity were not masked in animals euthanized with sodium
pentobarbital. Therefore, pharmacological-induced changes
in concentrations of DOPAC in the median eminence (as an
estimation of TIDA neuronal activity) can be detected in
rats euthanized with sodium pentobarbital. Therefore, it
was decided to test if similar neurochemical measurements
made in cattle euthanized with sodium pentobarbital were

related to activity of TIDA neurons.

Chapter 2

Prolactin Regulation of Tuberoinfundibular

Dopaminergic Neurons in Bull Calves

53

54

Introduction

DA inhibits secretion of PRL in cattle in vitro
(Padmanabhan et al., 1979). In addition, treatment with a
DA agonist, bromocriptine, reduced concentrations of PRL in
cattle in vitro and in vivo (Smith et al., 1974).
Conversely, in cattle treated with drugs that disrupt
synthesis of DA (e.g., a-methyltyrosine) or interfere with
action of DA at the anterior pituitary gland (e.g.,
haloperidol) serum concentrations of PRL are increased in
vivo (Benoit, 1987). Location and characteristics of
dopaminergic neurons that regulate secretion of PRL in
mammals are based on studies conducted with the rat. The
DA primarily responsible for the tonic inhibition of
secretion of PRL in the rat is released from terminals of
TIDA neurons located in the median eminence in the MBH
(Gudelsky, 1981). DA released from TIDA neurons is
transported in the hypophyseal portal blood to the anterior
pituitary gland where it binds to receptors on the
lactotropes and inhibits secretion of PRL (Ben-Jonathan,
1985). In the bovine hypothalamus DA has been detected in
high concentrations in the infundibulum and PS (Cooper et
al., 1986), but it is not known if these dopaminergic
neurons regulate secretion of PRL.

In rats, DA released from TIDA neurons inhibits
secretion of PRL and PRL, in turn, regulates activity of

TIDA neurons (Hokfelt and Fuxe, 1972; Gudelsky et al.,

55

1976; Annunziato and Moore, 1978). Pharmacological
manipulations that increase (e.g., DA antagonists) or
decrease (e.g., DA agonists) serum concentrations of PRL
cause corresponding changes in activity of TIDA neurons
(Moore, 1987) . Furthermore, systemic (Hokfelt and Fuxe,
1972; Gudelsky et al., 1976) and intracerebroventricular
(Annunziato and Moore, 1978; Johnston et al., 1980)
injections of PRL increase activity of TIDA neurons and
decrease endogenous secretion of PRL (Selmonoff and
Gregerson, 1984), suggesting that PRL regulates its own
secretion by altering the level of inhibitory dopaminergic
tone.

In the present study, location of TIDA neurons in
cattle was determined by: 1) measuring the distribution of
DA in selected areas of the hypothalamus, and 2) examining
the ability of elevated serum concentrations of PRL to

regulate the activity of these neurons.

Materials and Methods

Erpgrimgnt l. Five prepubertal Holstein bull calves
(approximately 8 wk of age) were euthanized with sodium
pentobarbital [Sigma Chemical Company, St. Louis, MO; iv 85
mg/kg body weight in sterile water (388 mg/ml, pH 8.0)].
Skin was excised along the forehead, just above the eyes

and from the eyes to the pole on both sides of the head.

56

The skull was then cut with a reciprocating saw to expose
the dorsal portion of the brain. The optic nerve and PS
were cut at their distal junctions with the dura and brains
were removed and placed on a petri dish over ice. A block
of tissue (Figure 1A) containing the MBH and PS was
dissected with a scalpel blade, placed with the ventral
surface up on aluminum foil and frozen with dry ice.
Frontal sections (1 mm) beginning caudal to the optic
chiasm (Figure 1B) were prepared on a cryostat (-9’C) and
the PS and MBH were dissected from these frozen sections
with a scalpel blade (Figure 1C). In addition, a 2000 u
punch (12 gauge inner diameter) was used to remove the SCN
from areas indicated in Figure 1A. Although TIDA neurons
are not located in the SCN, this area was dissected and
analyzed for a comparison with the MBH and PS.

Analysis of DA and DQPAC. DA, DOPAC and protein were
determined in SCN, MBH and within each 1 mm section of PS
as described in Chapter 1.

Starisripal analysis. DOPAC and DA data were analyzed
by analysis of variance and differences among areas within
the hypothalamus and PS were determined by Bonferroni-t
procedure (Gill, 1978).

Experiment 2. Sixteen prepubertal Holstein bull calves
(approximately 8 wk of age) were assigned randomly to one
of two treatments. Eight bulls were injected so with

haloperidol (Sigma; 1 mg/kg body weight (2.5 mg

57

Figure 1. Schematic diagram of the dissection of the
infundibular-pituitary stalk (PS), mediobasal
hypothalamus (MBH) and suprachiasmatic nucleus (SCN) of
bull calves.

A. Dissected block of hypothalamus and attached
PS; III V = third ventricle OC = optic chiasm; * = area
of SCN.

B. Midsaggital view depicting the location of
sections (1 through 9) of PS.

C. Frontal views showing the first and fourth
sections; x = area of arcuate nucleus.

Dark areas represent the location of the MBH and PS

that were dissected for subsequent analysis.

.v 3.5 . _ oo=w

In: I4/..\N.. 53m I7)

33

 

 

 

 

 

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59

haloperidol/ml of .3% tartaric acid)] every 6 h for 24 h.
This dose maintains elevated serum concentrations for 6 to
8 h (S.A. Zinn and H.A. Tucker, unpublished observations).
Eight uninjected bulls were controls.

Bulls were housed in individual stalls in light
(24L:0D)- and temperature (20 i- 2°C)-controlled chambers
(four bulls/chamber). Cool-white fluorescent tubes at an
average intensity of 400 lux (measured 1 M from the floor)
were utilized as the source of light. Bulls were given 1 kg
of a commercial calf grain (Startina, Purina Mills, St.
Louis, MO), daily at 0800 and 1700 h. water was provided
ad libitum.

One d before start of treatments, each bull was fitted
with a polyvinyl cannula in a jugular vein. The next day
blood was collected and discarded every 10 min from 0700 to
0920 h, to accustom bulls to sampling procedures.
Beginning at 0930 h blood was sampled every 15 min for 1 h
before the start of haloperidol injections and then every 2
h for the next 24 h. One sample was taken just before
euthanasia (60 min after the last haloperidol injection).
Blood samples were stored at 20°C for 4 to 6 h and then
overnight at 4°C. Samples were centrifuged, sera decanted
and stored at —20°C until assayed for PRL (Koprowski and
Tucker, 1971). Bulls were euthanized with sodium
pentobarbital (iv, 85 mg/kg body weight) 60 min after the
last haloperidol injection. Following euthanasia, the

brain was dissected, frozen and concentrations of DOPAC, DA

60

and protein were determined as described in Experiment 1
(Chapter 2). Changes in concentrations of DOPAC and ratio
of DOPAC to DA were used as estimates of dopaminergic
activity in the PS (Lookingland et al., 1987b).

Statistical analysis. Treatment differences in
concentrations of DOPAC and DA were compared by analysis of
variance (Gill, 1978). PRL data were analyzed by split—
plot analysis of variance with repeated measurement (Gill
and Hafs, 1971).

Enpariment . The experimental design of Experiment 2
(Chapter 2) was repeated with an additional 16 prepubertal
bull calves (approximately 8 wk of age). Treatments,
housing, cannulations and blood sampling were as described
in Experiment 2 (Chapter 2). However, 15 min before
euthanasia an additional blood sample was collected and
then bulls were injected iv with an aromatic L-amino acid
decarboxylase inhibitor [NSD 1015 (3-hydroxybenzyl-
hydrazine, Sigma) 25 mg/kg body weight (100 mg NSD 1015/ml
.9% saline)]. NSD 1015 inhibits conversion of DOPA to DA
and 5HTP to 5HT, and acumulation of DOPA (the immediate
precursor of DA) and 5HTP (the immediate precursor of 5HT)
over time reflects in vivo rate of DA and 5HT synthesis,
respectively (Carlsson et al., 1972). Since the synthesis
and release of neurotransmitters are tightly coupled in
these neurons, the rate of DA and 5HT synthesis may be used

as indices of DA (Demarest and Moore, 1980) and 5HT

61

neuronal activity (Duda and Moore, 1985), respectively.

Following euthanasia, the brain was dissected, frozen
and concentrations of DOPA, DA and protein were determined
as described in Experiment 1 (Chapter 2) . Concentrations
of 5HTP and 5HT were determined by HPLC concurrently with
DOPA and DA (Chapin et al., 1986).

t t's ' na . Treatment differences in
concentrations of DOPA, 5HTP, DA and 5HT were compared by
analysis of variance (Gill, 1978). PRL data were analyzed
by split-plot analysis of variance with repeated
measurement (Gill and Hafs, 1971).

Enperimenr . To determine if haloperidol-induced
increases in TIDA neuronal activity were due to increased
prolactin per se and to determine if these changes in TIDA
activity could be maintained for 9 d a third experiment was
conducted. The duration of Experiment 4 was 9 d. Eighteen
prepubertal Holstein bull calves (approximately 10 wk of
age) were randomly assigned to one of three treatments.
Six bulls were infused with PRL for 9 d and six bulls were
uninfused for 8 d and then infused with PRL for the last 25
h and six uninfused bulls were controls. Housing, feeding
and photoperiod was a described in Experiment 2 (Chapter
2).

One d before (d -1) the 9 d infusion began, each bull
was fitted with a polyvinyl cannula in a jugular vein. In
addition, bulls receiving PRL infusions were fitted with a

polyvinyl cannula in the contralateral vein one d before

62

start of infusion (i.e., d -1 for the 9 d infusion group
and d 7 for the 25 h infusion group).

Prolactin (USDA b-1) for infusion was prepared in a
sterile solution containing .1% bovine serum albumin (BSA)
in .85% saline at a concentration of 1 mg PRL/ml saline.
Bulls received pulses of PRL every 3.75 min (.5 ml/h) using
a AS-ZBH Autosyringe infusion pump (Autosyringe, Inc.,
Hooksett, NH). Infusions began at 1230 h on d 0 for bulls
receiving PRL for 9 d and at 0900 h on d 8 foor bulls
receiving PRL for 25 h.

Beginning on d 0, blood was collected and discarded
every 10 min from 0930 to 1115 h, to accustom bulls to
sampling procedures. Beginning at 1130 h blood samples
were collected every 15 min for 1 h and every 6 h until
0800 h on d 8. Beginning at 0800 h on d 8 blood samples
were collected every 15 min for 1 h and then every 4 h for
the next 24 h. In addition, one sample was taken just
prior to infusion of NSD 1015 (0945 h; d 9) and one sample
was taken just prior to euthanasia (1000 h; d 9). Blood
samples were stored at 20°C for 4 to 6 h and then overnight
at 4°C. Samples were centrifuged and sera were decanted
and stored at -20°C until assayed for PRL as described in
Experiment 2 (Chapter 2).

Beginning at 0945 h on d 9 all bulls were treated with
NS 1015 as described in Experiment 2 (Chapter 2).

Bulls were then euthanized, tissue was collected and

63

prepared and concentrations of DOPA, DA and protein were
determined as described in Experiment 1 (Chapter 2).
Staristical analysis. DOPA and DA data were analyzed
by analysis of variance and differences between treatment
means were compared by Bonferoni t-test (Gill, 1978). PRL
data were analyzed by split-plot analysis of variance with
repeated measurement (Gill and Hafs, 1971) and differences
between treatments were compared by Bonferoni t-test (Gill,

1978).
Results

Erperiment 1. Among bulls, the number of slices per
animal and the concentrations of DA within each slice
varied. But greatest concentrations of DA were usually in
the PS (slice 2, 3, 4 or 5; Table 3). Concentrations of
DOPAC within the PS paralleled concentrations of DA (Table
4). Concentrations of DA and DOPAC averaged across all
slices of PS were at least 2.5-fold greater (P < .05) than
concentrations in the SCN or MBH (Table 5). Therefore,
changes in dopaminergic activity in the PS became the focus
of Experiments 2, 3 and 4 (Chapter 2).

Experiment 2. Haloperidol, injected. every 6 11
increased (P < .05) serum concentrations of PRL, relative
to control (87.3 i 6.4 vs 15.0 i 1.8 ng/ml; patterns of
serum concentrations of PRL in these calves are similar to

those shown in Figure 2).

Table 3.

64

Concentrations of Dopamine in Sections of Pituitary Stalk

of Bull Calves.

 

 

Bull
1 2 3 4 5
Slicesa
pg/ug protein

1 Proximal 3.06 1.90 1.71 5.06 1.72
2 10.00 5.42 6.99 7.86 5.52
3 12.05 7.19 9.00 8.47 12.43
4 10.40 8.81 10.23 8.36 10.36
5 9.65 5.31 10.68 7.45 10.11
6 8.45 5.62 5.04 4.51 6.58
7 6.83 6.84 1.83 3.23
8 8.83 8.90 2.47
9 Distal 3.78

 

aOne mm frontal slices beginning at the junction of the
pituitary stalk and infundibular recess (proximal)
ending at the junction of the pituitary stalk and anterior

pituitary gland (distal).

and

65

Table 4.

Concentrations of Dihydroxyphenylacetic Acid in Slices of
Pituitary Stalk of Bull Calves.

 

 

Bull
l 2 3 4 5
Slicesa
pg/uq protein

1 Proximal 2.11 1.56 .61 2.28 .77
2 4.88 3.76 2.77 3.02 1.92
3 5.08 4.08 3.31 3.59 4.17
4 3.84 4.86 5.34 2.88 3.76
5 3.60 2.61 4.13 2.73 3.40
6 2.92 2.28 2.29 1.78 2.05
7 1.44 3.24 .56 .73
8 1.94 2.26 .83
9 Distal .54

 

aOne mm frontal slices beginning at the junction of the
pituitary' stalk; and infundibular' recess (proximal) and
ending at the junction of the pituitary stalk and anterior
pituitary gland (distal).

66

Table 5.

Concentrations of Dopamine (DA) and Dihydroxyphenylacetic
Acid (DOPAC) in Brain Regions of Bull Calvesa.

 

 

Region DA DOPAC
(pg/#9 protein) (pg/#9 protein)
pituitary stalkb 5.9 1 .3° 2.72 1 .13C
Suprachiasmatic nucleus 1.6 i .4 .42 i .03
Mediobasal hypothalamus 1.9 i .4 .61 i .03

 

aValues represent means (t SE) for DA and DOPAC. Average
of five bull calves.

bAverage of all slices of pituitary stalk.

cMeans from pituitary stalk differ from means of other
regions (P < .05).

67

Figure 2. Concentrations of prolactin in serum of bull
calves injected with haloperidol (‘; 1 mg/kg body
weight) every 6 h (0) or uninjected controls (0 ).
All calves received NSD 1015 (25 mg/kg body weight;{})
15 min before euthanasia. Each point represents the
mean of 7 or 8 samples. Standard error = 5.2 ng/ml for
haloperidol injected bulls and 1.6 ng/ml for uninjected

controls.

68

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69

Similar to results in Experiment 1 (Chapter 2), the
number of slices of PS per bull and the concentrations of
DOPAC and DA within each slice varied. Concentrations of
DOPAC and DA, averaged across all slices of PS, are
summarized in Table 6. Averaged across all slices of PS,
haloperidol did not affect concentrations of DOPAC or DA or
ratios of DOPAC to DA in PS of bull calves. However, when
the statistical analysis was done on the individual section
with the greatest concentration of DA or on the second
through fourth slices regardless of concentration of DA,
the ratio of DOPAC to DA was increased (P < .05) two-fold
in bulls injected with haloperidol compared with controls.
Although, changes in the ratio of DOPAC to DA following
injections of haloperidol were in the anticipated
direction, the magnitude of response was small. Therefore,
in Experiments 3 and 4 accumulation of DOPA following a
decarboxylase inhibitor NSD 1015 was used as the index of
dopaminergic activity is PS of bull calves.

Experiment 3. Haloperidol, injected every 6 h,
increased (P < .05) serum concentrations of PRL, relative
to control (83.1 t 5.2 vs 13.3 :t 1.6 ng/ml; Figure 2).
Injection of NSD 1015 increased (P < .05) concentrations of
PRL ‘within 15 min in control animals but not in
haloperidol-treated bulls (Figure 2).

Similar to results in Experiment 1 (Chapter 2), the
number of slices of PS per bull and the concentrations of

DA within each slice varied. Concentrations of DOPA and DA

70

Table 6.

Effects of 25 h of exposure to haloperidol on
concentrations of DOPAC in pituitary stalk of bull calvesa.

 

 

Haloperidol Control
DOPACb 4.62 i .2 4.64 i .5
DAb 7.41 i .7 9.85 i 1.3
DOPAC/DA .62 i .12 .48 i .10

 

aValues represent means of 7 or 8 bulls (1- SE) for
dihydroxyphenylacetic acid (DOPAC; pg/ug protein) and
dopamine (DA; pg/ug protein).

bWithin treatment values are averages of all slices of
pituitary stalk.

71

averaged across all slices of PS, are summarized in Table
7. Concentrations of DOPA were increased and
concentrations of DA were reduced in bulls injected with
haloperidol when compared with controls. Thus, the ratio
of DOPA to DA increased in haloperidol-injected bulls
(Table 7). Inferences did not change if the data were
based on the average of all slices or in the individual
slices with the highest concentrations of DA and DOPA.
Haloperidol did not affect concentrations of 5HT or its
precursor, 5HTP, in PS (Table 8).

Erpariment . Averaged over the duration of infusion
of PRL, concentrations of PRL were increased (P < .05) in
bulls infused for 25 h (204.1 1 11.8 ng/ml) or 9 d (183.3 1
10.6 ng/ml) compared with uninfused controls (13.9 i 1.0
ng/ml; averaged over the 9-d duration of the experiment;
Figure 3) . Averaged over the non-infusion periods serum
concentrations of PRL in bulls infused for 25 h (samples
collected during the 8 d before starting infusion) and for
9 d (sample collected during the 1.11 before starting
infusion) averaged 14.7 i 1.3 ng/ml and 11.8 i 1.8 ng/ml,
respectively and were not different (P > .10) from the
control concentration (Figure 3). Injection of NSD 1015
increased concentrations of PRL within 15 min in controls
but not in bulls infused with PRL (Figure 3). Within each
day there appears to be a rhythm of secretion of PRL with a

peak at 2200 or 0400 h and a nadir 12 h later. However,

72

Table 7.

Effect of 25 h of exposure to haloperidol on accumulation
of DOPA activity in pituitary stalk of bull calvesa.

 

 

Haloperidol Control
DOPAb 1.57 1 .16C .98 i .09
DAb 6.80 1 .64C 10.71 i .54
DOPA/DA .23 1 .03C .09 i .01

 

aValues represent means of 7 or 8 bulls (t SE) for
dihydroxyphenylalanine (DOPA; pg/ug protein) and dopamine
(DA; pg/ug protein).

bWithin treatment values are averages of all sections of
pituitary stalk.

cMeans from haloperidol-treated bulls differ from means from
controls (P < .05).

73

Table 8.

Effects of 25 h of exposure to haloperidol on accumulation
of 5HTP in pituitary stalk of bull calvesa.

 

 

Haloperidol Control
surpb .29 t .03 .34 i .03
snrb 5.45 i .97 5.20 i .53
5HTP/5HT .06 i .01 .07 i .01

 

aValues represent means of 7 or 8 bulls (t SE) for 5-
hydroxytryptophan (5HTP; pg/ug' protein) and 5-
hydroxytryptamine (5HT; pg/ug protein).

bWithin treatment values are averages of all sections of
pituitary stalk.

74

Figure 3. Concentrations of prolactin in serum of
prepubertal bull calves infused with prolactin (USDA
b1; 12.0 mg/d) for 25 h (o), 9 d (A), and non-infused
controls (0) . Beginning of infusion indicated by solid
arrows (‘1). All animals received NSD-1015 (iv; 25
mg/kg EN; 0 ) 15 min before euthanasia. Each point
represents the mean of 5 or 6 samples. Pooled standard
errors = 4.3 ng/ml for periods of non-infusion and 11.2

ng/ml for periods of infusion.

75

 

 

(t.

 

 

(“"1199 I‘D/5“) NIlDV'IOHd

IO

1 0

DAY OF EXPERIMENT

76

these times correspond to the maximum and minimum
temperatures within the chambers.

Concentrations of DOPA and DA, averaged across all
slices of the PS, are summarized in Table 9. Infusion of
PRL increased concentrations of DOPA in bulls infused for
25 h or 9 d compared with controls, but infusion of PRL did
not affect concentrations of DA. However, the ratio of
DOPA to DA increased in bulls infused with PRL for 25 h or
9 d compared with controls. Concentrations of DOPA and DA
and ratio of DOPA to DA were not different between bulls

infused for 25 h or 9 d (Table 9).

Discussion

Although little is known regarding the location of
terminals of TIDA neurons in the bovine hypothalamus,
Cooper et al. (1986) reported that in steers concentrations
of DA and DOPAC in the infundibulum and infundibular stalk
were greater than in the anterior pituitary gland and zona
tuberalis. In agreement, the results from the present
study indicate that average concentrations of DA and DOPAC
in the PS of bull calves were two to three times greater
than concentrations in the MBH. Thus, TIDA neurons in the
bovine hypothalamus most likely terminate in the PS. These
conclusions are consistent with previous reports that the

PS also contains the greatest concentrations of 5HT (Piezzi

77

Table 9.

Effects of infusion of prolactin for 25 h or 9 days on
accumulation of DOPA in pituitary stalk of bull calvesa.

 

 

 

DOPAb nab DOPA/DA
Control 1.69d 11.566 .15d
25 h 3.17e 9.7d .338
9 days 3.83e 10.13d .38e
SEC .47 1.80 .04
aValues represent means of six bulls for

dihydroxyphenylalanine (DOPA; pg/ug protein) and dopamine
(DA; pg/ug protein).

bWithin treatment values are averages of all sections of
pituitary stalk.

cPooled standard error.

d'eMeans with a different superscript within a column differ
(P < .05).

78

et al., 1970) and GnRH (Kizer et al., 1976; Estes et al.,
1977).

Haloperidol, a dopamine antagonist, blocks DZ dopamine
receptors on the anterior pituitary gland thereby reducing
the inhibitory influence of DA on secretion of PRL (Gunnet
and Moore, 1988). Similar to previous reports in rats
(Dickerman et al.,1974), humans (Poland and Rubin, 1981)
and cattle (Benoit et al., 1987), haloperidol increased
serum concentrations of PRL in bulls in the present study.
In addition, elevated serum concentrations of PRL were
maintained with an injection of haloperidol every 6 h,
indicating that bulls do not become refractory to repeated
injections of haloperidol within 25 h.

NSD 1015 blocks synthesis of DA (Demarest et al., 1979)
and. reduces. concentrations. of DA in..hypophyseal portal
blood (Reymond and Porter, 1982) which results in increased
serum concentrations of PRL (Demarest et al., 1984).
Similarly in the present study injection of NSD 1015
acutely increased serum PRL in controls indicating that DA
inhibits secretion of PRL in cattle. Since treatment with
haloperidol blocks the action of DA at the pituitary gland
and bulls infused with PRL already had elevated PRL, the
acute effect of NSD 1015 on PRL in these animals was not
observed. These results are consistent with an inhibitory
role of TIDA neurons in the regulation of secretion of PRL

in cattle.

79

The accumulation of DOPA following NSD 1015
administration is an index of the in vivo rate of dopamine
synthesis in the rat brain (Carlsson, et al., 1972).
Alterations in impulse flow in TIDA neurons produce
corresponding changes in the rate of DOPA accumulation in
the median eminence (Gunnet et al., 1987), and this measure
provides a good index of TIDA neuronal activity (Demarest,
et al., 1979) . In the present study, procedures that
increased serum concentrations of PRL (i.e., haloperidol;
infusion of PRL) increased accumulation of DOPA in the PS
of bull calves in 1 d. These results are consistent with
stimulatory action of PRL on TIDA neurons in the rat
(Hokfelt and Fuxe, 1972; Gudelsky et al., 1976; Annunziato
and Moore, 1978; Johnston et al., 1980; Moore et al.,
1987). Therefore, the rate of accumulation of DOPA in the
PS reflects activity of TIDA neurons in cattle. Because of
the smaller magnitude of response to PRL, changes in
concentrations of DOPAC may not be a sensitive enough
measure of change in dopaminergic activity in the PS of
bull calves.

Elevated TIDA neuronal activity was maintained after 9
d of infusion of PRL, indicating that TIDA neurons in bulls
do not become refractory to PRL-induced stimulation for at
least 9 d.

There was no effect of haloperidol on synthesis or

storage of 5HT in PS indicating that haloperidol does not

80

alter the activity of 5-hydroxytryptaminergic neurons in
this brain region.

In summary, greatest concentrations of DA were
localized in the PS and procedures that increased serum
concentrations of PRL increased accumulation of DOPA in PS
of bull calves. Therefore, TIDA neurons in the bovine
hypothalamus most likely terminate in the PS and TIDA
neurons in cattle are responsive to the feedback actions of

PRL.

Chapter 3

Response of Tuberoinfundibular and

5-Hydroxytryptaminergic Neurons and Lactotropes

to Photoperiod in Holstein Bull Calves

82

83

Introduction

Compared with exposure to 8L:16D, exposure to 16L:8D
increased serum concentrations of PRL two- to eight-fold in
cattle (Bourne and Tucker, 1975; Leining et al., 1979;
Stanisiewski et al., 1984b, 1987b). However, the mechanism
whereby photoperiod influences concentrations of PRL in
cattle is unknown.

Several hypothalamic neurotransmitters, including DA
and 5HT influence secretion of PRL (Jacobowitz, 1988). As
described in the review of literature, DA from TIDA neurons
tonically inhibits secretion of PRL. Conversely, 5HT
stimulates secretion of PRL but not by direct action on the
pituitary gland. 5HT may inhibit TIDA neurons (Nishihara
et al., 1986; Nishihara and Kimura, 1987) or stimulate a
PRL-releasing factor (Clemens et al., 1978) which increases
secretion of PRL. Potentially, photoperiod-induced
alterations in activity of TIDA and(or) 5-
hydroxytryptaminergic neurons may be involved in
photoperiod-induced changes in serum concentrations of PRL.
Therefore, an objective of the current study was to
determine if exposure to photoperiods that alter serum PRL
alters estimates of activity of TIDA and(or) 5-
hydroxytryptaminergic neurons in cattle.

In addition, photoperiod may induce change in secretion
of PRL by causing changes in the lactotropes within the

anterior pituitary gland. For example, following lesions

84

of hypothalamic input to the anterior pituitary gland,
there is a gradual increase in the percentage of
lactotropes in the pituitary gland (Phelps and Hymer,
1986). In addition, bromocriptine, a DA agonist, reduced
the number of PRL secreting cells in vitro (Takahasi and
Kawashima, 1987). Steger et al. (1982) reported that after
20 wk of exposure to 5L:19D, secretion of PRL increased
without a change in concentrations of DA, suggesting a
decreased sensitivity of the lactotrope to DA. Thus,
photoperiod may influence the number and(or) secretory
capacity of the lactotrope or change the sensitivity of the
lactotrope to hypothalamic factors, which in turn results
in photoperiod-induced alterations in serum concentration
of PRL. Therefore, a second objective of the current study
was to determine the effects of photoperiod on number and
secretory capacity of the lactotrope and sensitivity of the

pituitary gland to DA and TRH.

Materials and Methods

Sixteen prepubertal Holstein bull calves (approximately
8 wk of age) were assigned randomly to photoperiod
treatments of 8L:16D or 16L:8D. Bulls were housed in
individual stalls in light- and temperature- (20 i 2°C)
controlled chambers (four bulls/chamber). (Cool-white

fluorescent tubes at an average intensity of 400 lux

85

(measured 1 M from the floor) were utilized as the source
of light. All bulls were exposed to 8L:16D for 6.5 wk.
Lights came on at 0900 h and were turned off at 1700 h.
Following this preliminary period, one-half of the bulls
(n=8) were exposed to 16L:8D (lights on at 0430 h and off
at 2030 h) and one-half of the bulls (n=8) remained on
8L:16D (lights on at 0430 h and off at 1230 h) for an
additional 4 wk. Bulls were fed and given water as
described in Chapter 2.

Two d before the switch in photoperiod, each bull was
fitted with a polyvinyl cannula in a jugular vein. The
next day blood was collected and discarded every 10 min
from 0900 to 1050 h to accustom the bulls to sampling
procedures. Beginning at 1100 h, blood samples were
collected every 30 min for 6 h. Cannulation and collection
of blood were repeated on all bulls 4 wk after the switch
in photoperiod. Additional blood samples were collected
from each bull 15 min before and immediately before
euthanasia. Blood samples were stored at 20°C for 4 to 6 h
and then overnight at 4°C. Samples of blood were
centrifuged, sera decanted and stored at -20°C until
assayed for PRL as described in Chapter 2.

Following collection of the last blood ‘sample, all
bulls were euthanized with sodium pentobarbital (iv; 85
mg/kg body weight). Fifteen min before euthanasia
(immediately following collection of the penultimate blood

sample) all bulls were injected iv with NSD 1015 (25 mg/kg

86

body weight) as described in Chapter 2. Accumulation of
DOPA and 5HTP was used as an index of TIDA and 5HT neuronal
activity, respectively, as described in Chapter 2.
Following euthanasia, a block of hypothalamic tissue
was removed from each calf, frozen on dry ice and the PS,
SCN and MBH were dissected as described in Chapter 2. In
addition, the arcuate nucleus was dissected with a 1000 u
punch on each side of the third ventricle just anterior to
the PS as shown in slice 1 (Figure 1C). Concentrations of
DOPA, DA, 5HTP, 5HT and protein in the SCN, MBH, arcuate
nucleus and in each slice of the PS were determined as
described in Chapter 2. The pituitary gland was removed
and transported to the laboratory on ice. Within 20 to 30
nuxl of“ euthanasia, connective tissue and. the posterior
pituitary gland were removed and the anterior pituitary
gland was weighed and bisected.
eis-e s,- .12 °v-_=- 9‘u0_ 9 tot: .: 4 .
One-half of the anterior pituitary gland was stored at room
temperature in Delbecco's modified Eagles medium (DMEM;
Gibco Chemical Co., Grand Island, NY) with .1% BSA for
determination of number and secretory capacity of
individual lactotropes. Dispersed anterior pituitary cells
were prepared by a modification of the method of
Padmanabhan et al. (1978). Briefly, one-half of anterior
pituitary gland from each hull was sliced (#1 mm in

thickness) in a Stadie-Riggs tissue slicer, then diced with

87

a scalpel blade (#1 m3) and the resulting pieces washed
four to five times in DMEM. Diced pieces were placed in a
125 ml flask with a stir bar with 30 ml of .10% collagenase
(type 1; 150 U/mg; 37°C; Sigma) in DMEM. The stir bar was
spun for 45 to 50 min causing dispersion of the pituitary
pieces. Cells were then washed three times in DMEM. Cell
clumps were dispersed further by stirring in .008% viokase
(37°C; Gibco) in DMEM for 20 min. Cells were washed four
times and resuspended in 2 ml of DMEM-.1% BSA. Cells were
counted in a hemocytometer and between .5 x 105 and 10 x
105 cells/ml were used in the reverse hemolytic plaque
assay according to a modification of the method of Smith et
al. (1986) as described below.

One ml of dispersed anterior pituitary cells in DMEM-
.1% BSA was mixed with an equal volume of ovine red blood
cells (oRBC; Colorado Serum Co., Denver, CO) linked to
staphylococcal protein-A (Sigma). Coupling of protein A to
oRBC was prepared 1 d before use as described by Smith et
al. (1986). The oRBC-pituitary cell mixture was loaded
into cunningham chambers (Cunningham and Szenberg, 1968),
coated with poly-L-lysine (380,000 MW, Sigma; .25 mg/ml
distilled water) as a monolayer. Eight to ten slides were
prepared per animal. Slides were then incubated (37°C; 95%
02:5% C02 at 100% humidity) for 45 min to permit attachment
of oRBC-pituitary cells to the slide. Unattached cells
were rinsed off with DMEM-.1% BSA. Antibody to bovine PRL

produced in rabbits (diluted 1:40 in DMEM-.1% BSA; specific

88

binding of PRL antibody was 50% at a dilution of 1:20,000
in a double antibody radioimmunoassay) was added to each
chamber. Slides were incubated for an additional 3 h.
Antibody was rinsed off with DMEM-.1% BSA and guinea pig
complement (diluted 1:40 in DMEM-.1% BSA; Gibco) was added
to each chamber and reincubated for 30 min. Excess
complement was rinsed out and cells fixed with
glutaraldehyde [2% in Sorenson's phosphate buffer (70% .1M
NazHP04 and 30% .1M KH2P04)]. Slides were stored in
Sorenson's buffer until analyzed.

A photograph of a representative plaque is shown in
Figure 4. Area of each plaque was determined by centering
the plaque in a microscopic field and selecting the ring of
best fit on a calibrated concentric ring reticle. To
identify non-plaque forming cells, cells were stained with
toluidine blue (.05 g/100 ml borate buffer) and the number
of plaque forming cells as a percentage of total pituitary
cells was calculated. A minimum of three slides and 200
cells per bull calf were analyzed. Plaques were formed
from lactotropes in 12 out of the 16 bulls (7 from 81n16D
and 5 from 16L:8D). No plaques were formed in four bulls
due to technical problems. For example, in one bull no
anterior pituitary cells were harvested and in three bulls
the oRBC dried out, preventing formation of plaques. Only
slides that had plaques were analyzed. Although plaques

were formed on slides from 12 of the 16 bulls used in the

89

Figure 4. Photomicrograph of a plaque (zone of
hemolysis) surrounding a PRL-secreting cell (solid
arrow;’) viewed with phase contrast optics (magnified
400K). Non-plaque forming cell indicated by open arrow

( (I) ). RBC membrane ghosts can be seen in the plaque.

90

 

91

study, the number of plaques formed varied among bulls and
among slides within a given bull. The biggest technical
problem with the assay was maintaining hydration of the
oRBC within the chamber. Once a chamber or part of a
chamber dries out, no plaques are formed. Maintaining damp
laboratory tissue around each slide and resoaking the
tissue following each incubation reduced the problem of
dehydration.

W. Explants were prepared by a
modification of the method of Convey et al. (1973).
Briefly, one-half of the anterior pituitary gland was
sliced and diced as described previously and the resulting
pieces washed four to five times in medium 199 (Sigma) with
25 mM HEPES (N-2-hydroxyethyl piperazine-N'-2-
ethanesulfonic acid) and .244% sodium bicarbonate (M199).
Two to three pieces were then placed into 12 x 75 mm test
tubes containing 2 ml of M199. Tubes were incubated in a
Dubnoff (Precision Scientific Co., Chicago, IL) metabolic
shaker (37°C; 95% 02:5% C02). Medium was replaced with 2
ml of M199 every 15 min for 1 h followed by a 2 h
pretreatment incubation period. Medium was then harvested
for determination of pretreatment concentrations of PRL.
Two ml of M199 were then added to each tube. Explants were
treated with 0, 10'5, 10'8 or 10'10 M DA (in M199 with .01%
ascorbic acid) or 1077, 10"9 or 10711 M TRH (in M199) and
incubated for an additional 2 h. There were two

tubes/treatment per bull. Media were harvested for

92

determination of PRL. Explants were dried and weighed and
data expressed as ng PRL/mg dry tissue.

Eraristigal analysia. DOPA, DA, 5HTP, 5HT in PS, SCN,
MBH and arcuate nucleus and area of plaques and percentage
of lactotropes data were analyzed by analysis of variance
(Gill, 1978). PRL data (serum and media) were analyzed by
split-plot analysis of variance with repeated measurement
(Gill and Hafs, 1971). In addition, regression curves
(linear and quadratic) were computed (Gill, 1978) to test

response of PRL to increasing concentrations of DA and TRH.
Results

At the end of 6.5 wk of exposure to 8L:16D,
concentrations of PRL were similar in‘both groups of
calves, averaging 5.8 and 4.9 ng/ml of serum (Figure 5).
After 4 wk, bulls exposed to 16L:8D had greater serum
concentrations of PRL than bulls that remained on 8L:16D
(35.8 vs 7.5 ng/ml; P < .05). Injection of NSD 1015
increased (P < .01) serum contractions of PRL within 15 min
to 191.1 1 35.5 from 35.8 ng/ml in bulls exposed to 16L:8D
and to 43.0 r 22.7 from 7.5 ng/ml in bulls exposed to
8L:16D.

Concentrations of DOPA, DA, 5HTP and 5HT, averaged
across all slices of PS, are summarized in Table 10.

Concentrations of DOPA were 14.7% greater than in bulls

93

Figure 5. Concentrations of prolactin in serum of
prepubertal bull calves exposed for 6.5 wk to 8L:16D
(solid symbols), then switched to 16L:8D (O) or
maintained on 8L:16D (O) for an additional 4 wk.

Pooled SE = 1.0 ng/ml.

mg...

94

 

(was uni/fin) N I13v1oad

 

95

Table 10.

Effects of photoperiod on accumulation of DOPA and 5HTP in
the pituitary stalk of bull calves“.

 

 

8L:16Db 16L:8Db SEC
DOPA 2.99 3.43 .21
DA 17.69 16.04 1.29
DOPA/DA .17 .21d .02
SHTP 1.26 1.08 .10
5HT 16.97 14.46 1.33
5HTP/5HT .07 .07 .01

 

aValues represent the means of six bull calves for
dihydroxyphenylalanine (DOPA; pg/ug protein), dopamine (DA;

pg/ug protein), 5-hydroxytryptophan (5HTP; pg/ug protein)
and 5-hydroxytryptamine (5HT; pg/ug protein).

bWithin treatment values are averages of all sections of
pituitary stalk.

cPooled standard error.

dMeans from 16L:80 differ from means of 8L:16D (P < .10).

96

exposed to 16L:8D than in bulls given 8L:16D. However,
this difference was not significant. Photoperiod did not
affect DA in PS. Exposure to 16L:8D tended (P < .10) to
increase the ratio of DOPA to DA compared with exposure to
8L:16D. There was no effect of photoperiod on
concentrations of 5HT or its precursor 5HTP in PS.

Concentrations of DOPA, DA, 5HTP and 5HT in the arcuate
nucleus, MBH and SCN are summarized in Table 11.
Concentrations of DOPA and DA in the MBH were greater in
bulls exposed to 16L:8D than values in bulls subjected to
8L:16D. However, the ratio of DOPA to DA was not different
between photoperiods. Photoperiod did not affect
concentrations of DOPA or DA in the arcuate nucleus or SCN.
Relative to 16L:8D, exposure to 81a16D increased the
concentrations of 5HTP and the ratio of 5HTP to 5HT but had
no affect on 5HT in the arcuate nucleus. Concentrations of
5HT in the MBH tended to be greater (P < .10) than in bulls
exposed to 16L:8D compared with 8L:16D. Photoperiod did
not affect 5HTP or ratios of 5HTP to 5HT in the MBH nor
were 5HTP, 5HT or ratios of 5HTP to 5HT affected.

Anterior pituitary glands from bulls exposed to 16L:8D
were heavier (P < .05) than those from bulls given 8L:16D
(595.7 1 31.4 vs 485.2 1 31.4 mg). In addition, the number
of lactotropes as a percentage of total number of secretory
cells of the anterior pituitary gland increased in bulls
exposed to 16L:80 compared with bulls on 8L:16D (Table 10).

Plaques formed around lactotropes from bulls exposed to

97

Table 11.

Effects of photoperiod on accumulation of DOPA and 5HTP in
brain regions of bull calvesa.

 

DOPA DA DOPA/DA 5HTP 5HT 5HTP/5HT

 

8222919 Nucleus

8L:16D .90 1.48 .61 .37 3.76 .10
16L:8D .94 1.72 .55 .28c 3.49 .080
SEb .16 .20 .07 .04 .31 .01
Mediobasal

DYEQLthémué

8L:16D .28 1.06 .26 .23 4.04 .06
16L:8D .66d 2.25d .29 .27 5.68c .05
ssh .04 .28 .09 .05 .56 .01
Suprachiasmatic

nucleus

8L:16D .16 1.06 .15 .41 1.98 .21
16L:8D .16 .99 .16 .42 2.11 .20
SEb .01 .10 .06 .03 .06 .02

 

aValues represent the means of six bull calves for
dihydroxyphenylalanine (DOPA; pg/u protein), dopamine (DA;
pg/u protein), 5-hydroxytryptophan (5HTP; pg/ug protein)
and 5-hydroxytryptamine (5HT; pg/ug protein).

bPooled standard error

cMeans from 16L:8D differ from means of 8L:16D (P < .10).

dMeans from 16L:8D differ from means of 8L:16D (P < .05).

98

16L:8D had 33% larger diameter and 70% larger area than
bulls given 8L:16D (Table 12); indicating that photoperiods
of 16L:8D increased the secretory capacity of lactotropes
and increased the number of lactotropes.

Across all samples from the 2 h pretreatment
incubation, pituitary gland explants from bulls exposed to
16L:8D secreted greater (P < .05) quantities of PRL into
the media than explants from bulls exposed to 81n16D
(1232.1 1- 58 vs 786 i 38 ng/mg dry tissue). However,
compared with pretreatment, concentrations of PRL in the
media were not changed following a 2 h incubation with DA
or TRH in bulls exposed to 8 or 16 h of light (Table 13).
In addition, slopes of response to increasing doses of DA
or TRH were not different from zero (P > .10) in either
group of bulls (data not shown). Thus, explants of
anterior pituitary gland from bulls given 16L:8D secreted
more PRL but explants from both groups of bulls were

unresponsive to increasing doses of DA or TRH.

Discussion

Similar to previous reports (Bourne and Tucker, 1975;
Leining et al., 1979; Stanisiewski et al., 1984b, 1987b),
in the present study prepubertal Holstein bulls exposed to
photoperiods of 16L:8D had greater serum concentrations of
PRL than bulls exposed to 8L:16D. Since exposure to 16L:8D

increased serum PRL, the objectives of the current study

99

Table 12.

Analysis of plaques formed from lactotropes in the reverse
hemolytic plaque assay in anterior pituitary glands from
bull calves exposed to photoperioda.

 

 

8L:16D 16L:8D
Animalsb 7 5
Diameter, mm .104 i .01 .139 1 .01d
Area, mm2 .0093 i .002 .0158 i .002d
Lactotropesc, % 11.2 i 2.2 17.1 r 2.68

 

aSix to 10 slides were analyzed per bull calf.

bOnly bull calves that produced plaques were included in
the analysis.

cNumber of lactotropes as a percentage of total number of
pituitary cells. A minimum of three different slides and
200 cells were analyzed per bull calf.

dValues of 16L:80 are different from 8L:16D (P < .05).

eValues of 16L:8D are different from 8L:16D (P < .10).

100

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.nH wanes

101

were to determine if photoperiods that alter PRL change: 1)
activity of TIDA and 5-hydroxytryptaminergic neurons, 2)
secretory capacity and number of lactotropes and 3)
sensitivity of the anterior pituitary gland to DA and TRH.

Photoperiod-induced increments in serum concentrations
of PRL begin to wane after exposure to 16L:8D for 12 to 16
wk in sheep and cattle (Almeida and Lincoln, 1984;
Stanisiewski et al., 1987b). Thus, in terms of PRL
secretion, sheep and cattle eventually become refractory to
photoperiodic stimuli. In the present study, bulls exposed
to 16L:8D for 4 wk tended to have greater activity of TIDA
neurons (based on increased ratio of DOPA to DA in PS) than
bulls exposed to 8L:16D. This increased activity of TIDA
neurons may act to reduce secretion of PRL and may reflect
the onset of refractoriness to photoperiodic stimulation of
PRL.

The photoperiodic-induced increases in the ratio of
DOPA to DA is most likely mediated by photoperiod-induced
increases in concentrations of PRL. Indeed, as
demonstrated in Chapter 2, elevated concentrations of PRL
feed back to stimulate activity of TIDA neurons to reduce
further secretion of PRL and the neurons remain responsive
to PRL for at least 9 d in cattle (Experiment 2 and 3,
Chapter 2).

Photoperiod—induced activation of TIDA neurons via
increased serum PRL in the present study is in agreement

with the results in Chapter 2. However, because DA from

102

TIDA neurons tonically inhibits PRL (Ben-Jonathan, 1985) it
was originally postulated that exposure to 16L:8D would
reduce activity of TIDA neurons, which in turn could permit
serum concentrations of PRL to increase. The data in the
current study do not preclude a role for TIDA neurons in
the initiation of photoperiod-induced stimulation of serum
PRL. In the present study, estimates of TIDA activity were
made at a single point in time (4 wk) relative to the start
of treatment. Measuring accumulation of DOPA in PS at
several points in time in the first 4 wk relative to the
beginning of photoperiod treatment should reflect the
ontogeny of photoperiod-induced changes in TIDA neurons.

In bulls exposed to 16L:8D, concentrations of DOPA and
DA were increased in the MBH but not in the arcuate nucleus
or the SCN. However, photoperiod did not affect the ratio
of DOPA to DA in the MBH, suggesting that although
concentrations of DOPA and DA were greater in bulls exposed
to 16L:8D there were no photoperiod-induced changes in
activity of neurons in this region.

Perikarya of TIDA neurons are located in the arcuate
nucleus (Ben-Jonathan, 1985; Albanese et al., 1986). 5HT
can inhibit activity of TIDA neurons through its action on
the arcuate nucleus (Nishihara et al., 1986; Nishihara and
Kimura, 1987) . In the current study, exposure to 16L:8D
reduced synthesis of 5HT at the arcuate nucleus indicating

reduced 5HT activityu Perhaps, photoperiod-induced

103

reductions in 5-hydroxytryptaminergic activity at the
arcuate nucleus and thus, removal of its inhibitory action
on TIDA neurons, is involved in the mechanism whereby
16L:80 increased activity of TIDA neurons in the current
study.

The reverse hemolytic plaque assay allows microscopic
visualization of hormone release at the level of a single
cell (Frawley et al., 1986). The area of the plaque that
develops around the individual cell provides an index of
the relative amount of hormone released (Neill and Frawley,
1983; Frawley et al., 1985). That is, the larger the area
of the plaque, the greater the secretion of hormone from
that cell. In the current study, the reverse hemolytic
plaque assay was used to determine secretory capacity and
relative number of lactotropes from bovine anterior
pituitary glands from prepubertal Holstein bulls exposed to
long- or short-day photoperiods.

Individual lactotropes from bulls exposed to 16L:8D
produced larger plaques and therefore, secreted greater
quantities of PRL compared with bulls exposed to 8L:16D.
In addition, exposure to 16L:8D increased the percentage of
anterior pituitary cells that secrete PRL. The origin of
these additional lactotropes is unknown. The increase may
represent a recruitment of sommatomammotropes to secrete
PRL. Treatment with estradiol causes a shift in cell types
from GH-secreting to PRL-secreting cells in rats (Boockfor

et al., 1986). Photoperiod may induce a similar shift in

104

cell type in cattle. In addition, since exposure to 16L:8D
increased weight of pituitary glands, photoperiod may also
increase hyperplasia of the lactotrope which would account
for the increase in number of PRL-secreting cells. Indeed,
following hypothalamic lesions in rats there is a gradual
increase in the number of lactotropes (Phelps and Hymer,
1986) and treatment with bromocriptine reduced number of
lactotropes (Takahasi and Kawashima, 1987).

The effect of photoperiod on PRL is a sluggish
response. For example, following a switch from short- to
long-day photoperiods, serum concentrations of PRL do not
increase significantly for at least 4 d (Petitclerc et al.,
1989) and do not reach a maximum for several weeks (Leining
et al., 1979; Stanisiewski et al., 1984b). The
sluggishness of the response of PRL to 16L:8D may be
accounted for in the time required for photoperiod-induced
increases in recruitment of or hyperplasia of lactotropes.

Pituitary explants from bulls exposed to 16L:8D
secreted more PRL into the media. Unexpectedly, neither DA
nor TRH affected release of PRL into the media. Using a
similar incubation schedule, Stanisiewski et al. (1984c)
reported a significant increase and decrease in secretion
of PRL in bovine pituitary explants treated with TRH and
DA, respectively. The reason for the failure to observe an
effect of TRH or DA in the explants of the current study

was unknown. However, Convey et al. (1973) using a similar

105

incubation schedule also failed to observe an effect of TRH
on pituitary gland explants. Since TRH and DA were
ineffective, it was not possible to determine a change in
responsiveness of the pituitary gland to these hypothalamic
factors. However, the increased serum concentrations of
PRL in all bulls following injection of NSD 1015 [which
blocks secretion of DA (Demarest et al., 1979) and reduces
concentrations of DA in hypophyseal portal blood (Reymond
and Porter, 1982)] indicate that secretion of PRL in bulls
exposed to short- or long-day photoperiods are responsive
to the inhibitory effects of DA.

In conclusion, exposure to long-day photoperiods of
16L:80 increased serum concentrations of PRL relative to
exposure to short days. The increase in serum PRL may be
mediated in part by an increased number of lactotropes and
increased secretory capacity of the lactotropes. In
addition, after 4 wk of exposure to long days, the elevated
concentrations of PRL stimulated activity of TIDA neurons,
which may indicate the start of photorefractoriness. This
process may involve a change in 5HT input to the arcuate

nucleus.

SUMMARY AND CONCLUSIONS

The objectives of this dissertation were to examine the
effects of photoperiod on serum PRL, activity of TIDA and
5HT neurons in the hypothalamus and the secretory capacity
and number of lactotropes in the anterior pituitary gland
of prepubertal Holstein bull calves. Before the objectives
could be addressed, validation of the methods was required.
Accordingly, several studies were conducted to examine the
effects of euthanasia with sodium pentobarbital on changes
in concentrations of DOPAC as an estimate of activity of
TIDA neurons in rats and the effects of elevated serum
concentrations of PRL on concentrations of DOPAC and
accumulation of DOPA as estimates of activity of
dopaminergic neurons in the PS of prepubertal Holstein bull
calves.

TIDA activity was increased in rats euthanized with
sodium pentobarbital compared with decapitated rats.
However, stress-induced decreases in activity of TIDA
neurons were not masked in rats euthanized with sodium
pentobarbital. Therefore, it was decided to test whether
similar neurochemical measurements made in cattle
euthanized with sodium pentobarbital were related to

activity of TIDA neurons.

106

107

Concentrations of DA and DOPAC were greatest in the PS
compared with MBH and SCN in Holstein bull calves
indicating that terminals of TIDA neurons may be located in
the P8 in cattle compared with the MBH in rats. Therefore,
dopaminergic activity in the P8 was the focus for
subsequent studies.

Injections of haloperidol, a DA antagonist, every 6 h
for 24 h increased serum concentrations of PRL 6-fold
compared with uninjected controls. Elevated serum PRL was
maintained for 25 h. After 25 h of haloperidol-induced
PRL or after 25 h of infusion of PRL, elevated serum
concentrations of PRL increased concentrations of DOPA 60
to 88% and increased the ratio of DOPA to DA 120 to 150% in
PS of Holstein bull calves relative to‘ controls. Thus,
greatest concentrations of DA are localized in PS and
elevated serum concentrations of PRL increased accumulation
of DOPA in PS of bull calves. Therefore, TIDA neurons in
the bovine hypothalamus most likely terminate in the PS.
In addition, TIDA neurons are responsive to feed back
actions of elevated serum concentrations of PRL in cattle.
For example, after 9 d of infusion of PRL based on
increased accumulation of DOPA, activity of TIDA neurons
was increased and therefore, TIDA neurons in bulls remain
responsive to elevated serum concentrations of PRL after 9
d.

The conclusion drawn in these experiments that TIDA

neurons most likely terminate in the P8 was based on

108

quantity of and response of concentrations of DA. However,
this conclusion has not yet been confirmed histologically.
Future investigations should examine the anatomy of the
bovine hypothalamus and identify' histologically the
location of the perikarya of TIDA neurons (arcuate nucleus)
as well as the terminals of these neurons. In addition,
the location of the MBH and SCN should be identified
histologically in the bovine hypothalamus.

Relative to exposure to 8L:16D, exposure to 16L:8D for
4 wk increased serum concentrations of PRL approximately 5-
fold in Holstein bull calves. Associated with the
increased serum PRL was increased release of PRL into the
media from pituitary explants from bulls exposed to 16L:80.
Individual lactotropes from bulls exposed to 16L:8D
produced larger plaques and therefore, secreted greater
quantities of PRL compared with bulls exposed to 8L:16D.
Exposure to 16L:8D increased the percentage of anterior
pituitary' cells that. secrete PRL indicating that bulls
exposed to long days had increased number of lactotropes.

Exposure to 16L:80 tended to increase the ratio of DOPA
to DA in PS relative to exposure to 8L:16D. Therefore, as
demonstrated with procedures that elevate serum
concentrations of PRL (i.e., haloperidol; infusion of PRL)
photoperiod-induced elevated serum PRL can feed back to
stimulate activity of TIDA neurons. Photoperiod did not

affect accumulation of DOPA in MBH, SCN or arcuate nucleus

109

nor did photoperiod alter accumulation of 5HTP in PS, MBH
or SCN. However, exposure to 16L:8D reduced accumulation
of 5HTP in the arcuate nucleus compared with 8L:16D. The
increased activity of TIDA neurons after 4 wk of exposure
to 16L:8D may indicate the start of photorefractoriness of
PRL secretion. This process may involve a change in 5HT
input to the arcuate nucleus.

Given that DA tonically inhibits secretion of PRL, the
results of the present study were unexpected. It was
originally postulated that exposure to photoperiods of
16L:8D would decrease activity of TIDA neurons which would
allow serum concentrations of PRL to increase. However,
the results of the present study do not preclude a role of
TIDA neurons in the onset of photoperiod-induced
concentrations of PRL. Measuring activity of TIDA neurons
at several points in time relative to the beginning of
photoperiod treatment should reflect the ontogeny of
photoperiod-induced changes in these neurons. I believe a
study of ontogeny is a next logical experiment in
determining the role of TIDA neurons in photoperiod-induced
changes in serum concentrations of PRL.

In conclusion, exposure to long-day photoperiods of
16L:8D increased serum concentrations of PRL relative to
exposure to short days. The increase in serum PRL may be
mediated in part by increased number of lactotropes and
increased secretory capacity of the lactotropes. In

addition, after 4 wk of exposure to long days, the elevated

110

concentrations of PRL stimulated activity of TIDA neurons,
which may indicate the start of photorefractoriness. This
process may involve a change in 5HT input to the arcuate
nucleus. Ontogeny studies are required to more completely
understand the role of TIDA and(or) 5-hydroxytryptaminergic

neurons in photoperiod-induced increases in serum

concentrations of PRL in cattle.

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