Download Molecular Mechanisms of Signal Integration in Hypothalamic

AMER. ZOOL., 33:244-254 (1993)
Molecular Mechanisms of Signal Integration in
Hypothalamic Neurons1
2
R. THOMAS ZOELLER A N D NISREEN KABEER
Department of Anatomy and Neurobiology, University of Missouri,
School of Medicine, Columbia, Missouri 65212
AND
H. ELLIOTT ALBERS
Laboratory of Neuroendocrinology and Behavior, Departments of Biology and Psychology,
Georgia State University, Atlanta, Georgia 30303
SYNOPSIS. The purpose of this paper is to describe our studies focused
on the mechanisms by which hypothalamic neurons process multiple
signals and produce an integrated response. We illustrate our research
strategy by reviewing our work on two separate neural systems: the hypothalamic paraventricular nucleus (PVN) and the suprachiasmatic nucleus
(SCN). We have focused on different peptidergic subpopulations within
these nuclei to address two issues. In the PVN, we concentrate on the
population of neurons containing thyrotropin-releasing hormone (TRH).
These neurons are inhibited by thyroid hormones, but activated by cold
exposure. Using a molecular approach, we have demonstrated that these
conflicting signals simultaneously act on the same population of TRH
neurons. This system will continue to be a productive model to study the
mechanisms by which neurons process multiple signals. In the SCN, we
concentrate on the population of neurons containing vasoactive intestinal
peptide (VIP), peptide histidine isoleucine (PHI) and gastrin releasing
peptide (GRP). We have demonstrated that injection of all three peptides
into the SCN of hamsters mimics the phase-delaying effects of light on
circadian wheel running behavior. In addition, the genes encoding these
peptides exhibit different 24-hour profiles of changes in neurons of the
SCN. These data support the hypothesis that one mechanism by which
these neurons produce an integrated response is by changing the concentration ratio of co-released peptides.
INTRODUCTION
The brain coordinates behavioral, endocrine and autonomic functions by processing a variety of internal and external stimuli,
Stimuli from both internal and external
sources can converge on a single neuron
which must process these signals and produce an integrated response. Our work is
focused on the mechanisms by which hypothalamic neurons process information they
receive from both internal and external
sources, and how they may produce an integrated response. Considering that external
stimuli reach the brain through sensory systerns that synapse on specific neurons, and
that internal stimuli can communicate with
the brain in the form of hormones, we have
identified a model system that will allow us
1
From the Symposium Neural Aspects of Repw- to examine how a single neuron can co-productive Endocrinology: Old Questions, New Approaches cess trans-synaptic and endocrine signals to
presented at the Annual Meeting of the American Sod- regulate neuroendocrine function. In addiety of Zoologists, 27-30 December 1990 at San Anto- ^
t Q d e t e r m i n e h o w hypothalamic neu-
nio, 1 cx3.s.
*
•
*
= Present address: Department of Biology, Univer- r o n s m a y produce an integrated response to
sity of Massachusetts at Amherst, Morrill Science Cen- multiple Signals, we have identified a group
ter, Amherst, MA 01003.
of neurons that contains three different pep244
HYPOTHALAMIC INTEGRATION
tides; we are investigating the functional
consequences of these co-localized peptides
and whether the biosynthesis of these peptides by external and internal stimuli might
represent a mechanism by which these neurons produce an integrated response.
The purpose of this paper is to illustrate
our research strategy by describing these two
separate projects. These projects are focused
on well-defined subpopulations of neurons
in two hypothalamic nuclei of rats known
for their role in integrating multiple signals
and in coordinating behavioral and physiological processes: the paraventricular
nucleus (PVN) and the suprachiasmatic
nucleus (SCN). We particularly emphasize
the necessity of validating certain characteristics of a neural system to be the focus
of studies investigating mechanisms of signal processing, and the experimental utility
of looking first at mechanisms regulating
cellular levels of neuroendocrine peptide
mRNAs.
IDENTIFICATION OF A MODEL SYSTEM TO
EXAMINE MOLECULAR MECHANISMS BY
WHICH HYPOTHALAMIC NEURONS
PROCESS MULTIPLE SIGNALS
We have been guided by four criteria to
identify a "model" neuroendocrine system
that would be accessible to the kinds of
experiments we felt were required to understand the molecular and cellular mechanisms by which neurons can integrate multiple signals. First, the neuronal system
should be spatially restricted; that is, the
neurons under study should be anatomically clustered so that they can be "harvested" from adult tissue for biochemical
and molecular analyses, as well as for culturing. Second, there should be a background of information describing the physiological systems regulating this group of
neurons, and these systems should be easily
manipulated under laboratory conditions.
Third, all neurons within this "cluster" must
be regulated by the same set of physiological
stimuli, so that mechanisms of integration
can be reasonably interpreted as occurring
within the same neuron. Finally, the neurons should be viable in vitro to allow access
to experiments focused on signal transduction mechanisms. In this section, we describe
245
how these criteria have guided our approach
to defining a neuroendocrine system that
will be accessible to experimental manipulation.
Information on TRH neurons of the PVN
TRH was the first releasing factor chemically defined (Folkers et al, 1969; Burgus
et al, 1970; Nair et al, 1970); thus, there
is a wealth of information about its role as
a regulator of thyrotropin synthesis and
secretion (Morley, 1981; Koenig et al, 1984;
Taylor et al, 1986, 1990; Shupnik et al,
1986), as well as the location of TRH-containing neurons within the brain. Although
TRH is found widespread throughout the
brain (Jackson and Lechan, 1985; Liao et
al, 1985; Lechan et al, 1986), TRH in the
median eminence is produced exclusively
by a cluster of parvocellular neurons in the
PVN (Brownstein et al, 1975, 1982; Palkovits et al, 1982; Ishikawa et al, 1988).
Thus, TRH neurons of the PVN met the
first criterion: They are restricted in their
distribution, and they appear to represent a
homogeneous population regulating the
pituitary-thyroid axis. In addition, an abundance of work has demonstrated that TRH
neurons of the PVN integrate multiple signals; we have focused on two—thyroid hormone (T3) and cold exposure.
External and internal stimuli
interact to regulate TRH
neurons of the PVN
Several recent studies have demonstrated
that T3 exerts a negative feedback effect on
TRH neurons of the PVN. T3 reduces TRH
concentration in pituitary-portal blood
(Rondeel et al, 1988), TRH content of the
median eminence and paraventricular
nucleus (Mori and Yamada, 1987; Yamada
et al, 1989), and ultrastructural characteristics of neuronal activity in TRH-containing neurons of the PVN (Hisano et al, 1986).
In contrast, acute exposure to cold produces
a rapid (within 15 min) increase in TRH
release from median eminence (Rondeel et
al, 1988; Arancibia et al, 1989) as well as
in plasma TSH and T3 (Jobin et al, 1975,
1976; Hefco et al, 1975; Mannisto, 1983).
This effect of cold is blocked by PVN lesions
246
R. T . ZOELLER ET AL.
(Ishikawa et al, 1984), passive immunization with TRH antisera (Szabo and Frohman, 1977; Szabo et al, 1978; Mori et al,
1978; Prasad et al, 1980), and local (to PVN
or ME) or systemic administration of a-adrenergic antagonists or inhibitors of catecholamine synthesis (Onaya and Hashizume, 1977; Krulich et al, 1977; Schettini
et al, 1979; Terry, 1986; Arancibia et al,
1989). Considering that TRH neurons of
the PVN receive adrenergic synapses
(Liposits et al, 1987), these findings provide
strong evidence that cold exposure produces
a neural reflex that stimulates TRH neurons
of the PVN through adrenergic afferents
despite elevated levels of T3. The detail of
this background information and the relative ease with which ambient temperature
and circulating levels of T3 can be regulated
established our third criterion for a "model"
system.
the activity of the same population of neurons, then TRH mRNA levels should be
elevated by cold exposure in the same neurons in which T3 reduces TRH mRNA levels.
As predicted, TRH mRNA levels are regulated by T3: cellular levels of TRH mRNA
in the PVN are elevated in hypothyroid rats,
and daily injections of T3 reduce TRH
mRNA levels in the PVN of both hypothyroid and euthyroid rats (Koller etal, 1987;
Segerson et al, 1987) (Fig. 1). This effect
was observed only in neurons of the PVN,
not in TRH-containing cells of the ventrolateral hypothalamus or in the reticular
nucleus of the thalamus. In contrast, cold
exposure increased TRH mRNA levels in
the PVN (Zoeller et al, 1990), but not in
TRH-containing neurons of the reticular
nucleus of the thalamus. Hence, we have
used well-developed and well-characterized
methods of manipulating the activity of
Neural and endocrine factors interact
TRH neurons of the PVN to confirm that
within the same TRH neurons
TRH mRNA levels are regulated in a manIn principle, cold exposure must either ner predicted by the hypothesis that changes
activate a subset of TRH neurons in the in TRH expression are coupled to changes
PVN that are not suppressed by T3 or atten- in neuronal activity (Fig. 1).
uate the inhibitory effect of T3 within each
To approach the analysis of possible subTRH neuron. Our approach to discrimi- sets of TRH neurons differentially regulated
nating between these possibilities was based by cold exposure and T3, we postulated that
on the observation that many genes encod- if cold-activated TRH neurons in the PVN
ing neurotransmitter enzymes and peptides are separate from T3-inhibited TRH neuare regulated in parallel with neuronal activ- rons, TRH mRNA levels should become
ity. There is a rapidly growing literature elevated only in a subset of TRH neurons
demonstrating that cellular levels of neu- in an animal exposed to cold, and reduced
ropeptide mRNAs are regulated by trans- only in a subset of TRH neurons in an animembrane mechanisms initiated by the mal treated with T3. We tested this hypothsame stimulus {e.g., synaptic activity) that esis by performing single-cell analysis of
depolarizes the membrane and leads to TRH mRNA levels in animals with differtransmitter release (see reviews by Young ing thyroid status and exposed to different
andZoeller, 1987; Comb etal, 1987; Good- ambient temperatures (Zoeller et al, 1990).
man, 1990; Habener, 1990; Uhl and Nishi- We found no evidence of functional subsets
mori, 1990; Van Nguyen et al, 1990). This of TRH neurons in the PVN (Fig. 2).
coupling between transmitter secretion and
These studies demonstrate that cellular
cellular levels of mRNAs provides a molec- levels of TRH mRNA are elevated by cold
ular correlate of neuronal activity as well as exposure in the same PVN neurons that
a pivotal justification for our approach. We TRH mRNA levels are reduced by T . Furreasoned that if changes in TRH mRNA ther, because previous studies have so 3clearly
levels mirror changes in TRH neuronal demonstrated that T inhibits, and cold
3
activity, then T3 will reduce TRH mRNA exposure stimulates, TRH
release from neulevels in neurons of the PVN while cold rons whose perikarya reside in the PVN, our
exposure will elevate TRH mRNA levels. studies indicate that changes in cellular levFurther, if these two signals are regulating els of TRH mRNA in neurons of the PVN
247
HVPOTHALAMIC INTEGRATION
B
200
RNPVN —<
Cont PTU
T3
Cont PTU
Treatments
T3
FIG. 1. Quantitative in situ hybridization reveals that TRH mRNA levels in the rat PVN are reduced by T3
and elevated by environmental temperature. Techniques for manipulating circulating levels of T3 and ambient
temperature, and for in situ hybridization and quantitative analysis of resultingfilmautoradiograms are described
in detail by Zoeller et al. (1990). Film autoradiograms are shown in (A) for an animal exposed to 25°C, and in
(B) for an animal maintained at 5°C. The hybridization signal appears as an area of film darkening over the
reticular nucleus of the thalamus (RN), paraventricular nucleus (PVN), and ventrolateral hypothalamus (V).
Note that the density of the signal over the PVN in (B) is much greater than that in (A). The graph (C) displays
the results of an experiment designed to establish the interaction of T3 and cold exposure on TRH mRNA levels
in the rat PVN. Cont- (untreated), PTU- [treated with 6-n-propyl-2-thiouracil (0.05% wt/wt in chow) for 12
days], T3- (PTU-treated animals given a daily injection of 20 Mg/kg T3 for the final 4 days of the experiment)
treated animals (n = 10) were maintained at 25°C (left panel) or transferred to an adjacent environmental
chamber maintained at 5°C (right panel) for 6 hr beginning at 0900 hr. Note that PTU (which decreases circulating
levels of T3) increases (a) and T3 decreases (b) TRH mRNA levels in the PVN of animals at both temperatures.
The observation that cold exposure significantly elevated TRH mRNA levels (c) only in euthyroid animals
(untreated) suggests that T3 is a primary regulatory signal while environmental temperature can attenuate the
efficacy of T3 negative feedback. Defining the cellular processes by which environmental temperature produces
this effect on TRH neurons of the PVN may reveal a molecular mechanism by which the "set point" around
which negative feedback functions can be modulated by environmental factors.
mirror changes in the activity of these neurons. Therefore, as we study the molecular
mechanisms by which trans-synaptic and
endocrine signals interact to regulate cellular levels of TRH mRNA, we will gain
insight into how TRH neurons process these
convergent signals to produce an integrated
response. To further investigate these mechanisms, we must characterize the second
messenger systems activated by cold-stimulated afferents synapsing on TRH neurons,
and determine whether TRH mRNA levels
are being regulated transcriptionally or posttranscriptionally. The observation that TRH
neurons of the PVN remain viable and
aggregated in culture (Fig. 3) will be essential to these experiments.
Application to comparative endocrinology
There are several approaches to measuring mRNA levels, including northerns, dotand slot-blot, and solution hybridization
(Sambrook et al., 1989). Each of these
approaches presents a specific set of strengths
and weaknesses which must be considered
when designing a particular experiment. For
example, we decided to map the distribution of TRH expressing cells in Xenopus
laevis brain to determine whether the TRH
neuronal system in Xenopus is organized in
a manner similar to that of mammals
(Zoeller and Conway, 1989). Hence, we
designed a 48-base oligomer complementary to the TRH mRNA originally described
by Richter et al. (1984), and used this probe
to visualize the widespread TRH-expressing neuronal system by in situ hybridization. However, we found by northern analysis that the probe hybridizes to several size
classes of mRNAs (Fig. 4). Kuchler et al.
(1990) have confirmed this observation and
have demonstrated that these different size
classes of mRNA encode a precursor containing seven copies each of the TRH tripeptide. Thus, to clarify the physiological
significance of these multiple size classes of
TRH mRNA in Xenopus, we must determine 1) whether these transcripts are differentially expressed; that is, whether they
are produced by a subset of TRH neurons,
248
R . T . ZOELLER ET AL.
Increasing mRNA Level
50
D
40
g & 30
r-l
f!!^i
•..*••,
Increasing mRNA Level
n „
Increasing mRNA Level
Increasing mRNA Level
FIG. 2. Single cell analysis of TRH mRNA levels demonstrate that T3 and cold exposure are acting on the
same population of TRH neurons in the rat PVN. A) Bright field micrograph of the in situ hybridization signal
in Kodak NTB3 emulsion for TRH mRNA in the PVN from an animal exposed to cold. The autoradiographic
signal appears as dense clusters of grains over individual parvocellular elements along the border of the third
ventricle (III). Note that grain density over individual neurons varies within a single PVN. This variation can
arise from differences in TRH mRNA levels among neurons, and from differences in the amount the neuron
contained within this 12 jim-thick section. B) Histogram relating the proportion of labeled neurons (frequency;
ordinate) to grain density (abscissa; a measure of TRH mRNA level). Single cell analysis was performed on 100
cells/animal for the 10 animals exposed to cold. C) Brightfieldmicrograph of the PVN from an animal maintained
at 25°C after in situ hybridization for TRH mRNA and exposure to Kodak NTB3 nuclear track emulsion. The
autoradiographic signal appears as clusters of grains over individual parvocellular elements along the border of
the third ventricle (III). Note that grain density over individual neurons in C is less than that over individual
neurons in A. This is reflected in the frequency histogram plotted for control animals in D. The purpose of this
exercise was to test whether cold exposure increased TRH mRNA levels in a subset of TRH neurons in the rat
PVN that is separate from that in which T3 reduces TRH mRNA levels. Hence, we compared the frequency
histograms following single cell analysis of TRH mRNA levels in animals maintained at 25°C (open bars) with
those of animals exposed to cold (hatched bars) (E), and in animals treated with PTU (open bars) with those of
animals treated with T3 (hatched bars) (F). If cold exposure increased TRH mRNA levels in a subset of PVN
neurons, cold exposure would produce a bimodal distribution in the frequency histogram. Similarly, if T, acted
on a subset of PVN neurons, we expected to see a bimodal distribution for both groups in F. We found no
evidence of a bimodal distribution in E or F, and thus conclude that T3 and cold exposure are acting on the
same population of TRH neurons within the PVN. (Data redrawn from Zoeller et ai. 1990.)
HYPOTHALAMIC INTEGRATION
249
while the TRH molecule is identical among
vertebrates, and the organization of the
mRNA (Richter et al., 1984; Lechan et al.,
1986) and the neuronal system (Zoeller and
Conway, 1989) is remarkably similar
between Xenopus and mammals, there are
clear differences in the mechanisms regulating TRH expression among vertebrates
that require the use of different technical
approaches.
IDENTIFICATION OF A MODEL SYSTEM TO
EXAMINE THE ROLE OF CO-LOCALIZED
NEUROTRANSMITTERS AS A CELLULAR
MECHANISM OF HYPOTHALAMIC
INTEGRATION
Neurons processing multiple signals must
possess flexibility in their signalling mechanism so that they can produce an integrated response. In principle, the signalling
potential of a neuron containing several
transmitters may be affected by the concentration ratio of co-released factors (FischerColbrie et al., 1988). Hence, differential regulation of co-localized transmitter synthesis
may be an important mechanism by which
neurons can integrate multiple signals.
However, despite an abundance of anatomical data documenting cellular co-localization of multiple neurotransmitters in the
central and peripheral nervous systems
(Hokfelt et al, 1986), little is known about
the functional consequences of neurotransmitter co-release.
To address this issue experimentally, we
FIG. 3. In situ hybridization for TRH mRNA in have focused on a population of neurons in
organotypic cultures of rat PVN demonstrate that TRH the suprachiasmatic nucleus (SCN) that
neurons of the PVN remain aggregated after 21 days
of culture. The signal on film (left panel) appears as an contain two peptides derived from the same
area of darkening in the center of the culture which gene, VIP and peptide histidine isoleucine
appears as a circular region of background staining, (PHI) (Nishizawa et al., 1985) as well as a
while in emulsion (right panel) the signal appears as third peptide derived from a different gene,
grains clustered over individual cells aggregated in the gastrin-releasing peptide (GRP) (Okamura
center of the cultured tissue. Coronal sections of 5 dayold rat pups were cultured according to the procedures et al., 1987; Albers et al., 1991). We condescribed in Wray et al. (1989). This culture was pro- sidered these neurons a useful "model,"
vided by Dr. Susan Wray, Laboratory of Neurochem- using the criteria we applied to the TRH
istry, NINDS, Bethesda.
system of the PVN. VIP-containing neurons
of the SCN are clustered along the ventrolateral portion of the SCN (Card et ai, 1981),
2) whether potential regulators {e.g., T3 or and there exists a large literature of backenvironmental temperature) exert an effect ground information about the function of
specifically on a particular transcript, and the SCN. These data support the view that
3) whether these regulatory effects are the SCN acts as a circadian clock that both
observed in specific brain regions. Hence, generates rhythms in behavior and physi-
250
R . T . ZOELLER ET AL.
VIP PHI GRP
V/P V/G P/G
Treatment
V/P/G
FIG. 5. The full effect of microinjection of VIP, PHI,
and GRP into hamster SCN requires the co-injection
of all three peptides. Open bars represent the mean
phase delay in the onset of wheel running in animals
injected with VIP, PHI, or GRP alone; hatched bars
represent the mean phase delay in animals injected
with a combination of VIP + PHI (V/P), VIP + GRP
(V/G), and PHI + GRP (P/G); the cross-hatched bar
represents the mean phase delay in animals injected
with VIP, PHI and GRP.
FIG. 4. Northern hybridization of the TRH 48-base
probe to total RNA extracted from Xenopus brain.
RNA was extracted by the method of Chomczynski
and Sacchi (1987) and electrophoresed on a 1.2% agarose formaldehyde denaturing gel. The 48-base probe
was labeled with [32P]-dATP and terminal transferase.
After transfer by capillarity, the RNA blot was hybridized and washed under the same conditions as we had
performed the in situ hybridization (Zoeller and Conway, 1989). Horizontal bars indicate the position of
18 and 28 S ribosomal RNAs.
ology, and synchronizes those rhythms with
the environmental light-dark (LD) cycle
(reviewed by Meijer and Rietveld, 1989).
Considering that VIP/PHI/GRP-containing neurons of the SCN receive synapses
from neurons carrying information about
light:dark cycles (Kiss et al, 1984; Bosler
and Beaudet, 1985; Hisano et al, 1988;
Wyatt et al, 1988; Ibata et al, 1989), we
considered the possibility that synchronization of circadian rhythms with the LD
cycle may depend on the co-release of VIP,
PHI and GRP. If so, the combined effect of
these peptides on circadian rhythms should
be different from the effects of each peptide
given alone, or in combination with one of
the other two peptides. In fact, we found
(Albers et al, 1991) that the combined
injection of VIP, PHI and GRP into the
SCN mimics the phase delaying effects of
light on circadian control and firing rate in
cells in the SCN, while injection of VIP, PHI
or GRP alone or in any combination of two
produces only small shifts in circadian phase
(Fig. 5).
Since VIP, PHI, and GRP appear to be
co-expressed in a subset of SCN neurons,
and to interact within the SCN to phase shift
circadian rhythms, it is possible that the
concentration ratio of peptides in these neurons, and hence the signalling potential,
might change throughout the LD cycle. If
so, changes in peptide biosynthesis may be
reflected by changes in cellular levels of the
mRNAs encoding these peptides. We (Stopa
et al, 1986; Albers et al, 1990) and others
(Gozes et al, 1989) have shown that VIP
mRNA levels in the SCN change throughout the LD cycle. We followed this with a
detailed study to determine whether GRP
mRNA levels also exhibit a 24-hour profile
of changes, and whether these two genes are
regulated in parallel or out of phase with
each other. As had been shown earlier, we
found that VIP mRNA levels were higher
251
HYPOTHALAMIC INTEGRATION
130
120
techniques are available to study cellular
mechanisms in the SCN.
T
100
90
80
70
60
0
V
CONCLUSIONS
V
4 8 12 16 20 24
Circadian Tine (Hrs)
FIG. 6. Twenty-four hours profile of changes in cellular levels of GRP (solid circles) and VIP/PHI (open
circles) mRNA in the SCN of rats. Points represent
mean (±SEM) mRNA levels standardized with respect
to the value observed at circadian time 4 (CT-4).
during the dark phase than during the light
phase (Fig. 6); however, GRP mRNA levels
were higher during the light phase than during the dark phase.
These data support the hypothesis that
the synchronization of circadian rhythms
with the LD cycle may depend on the corelease of VIP, PHI and GRP, and that the
concentration ratio of these co-localized
peptides, and hence the signalling potential,
might change throughout the LD cycle.
Clearly, a complete test of this complex
hypothesis must include experiments
designed to test questions about 1) the relationship between VIP and GRP mRNA levels and the biosynthesis of their respective
peptides, as well as 2) the physiological
effects of different concentration ratios of
these peptides. However, this system is
amenable to experimentation at both levels.
The VIP/PHI/GRP neurons are spatially
restricted so that they can be harvested for
biochemical analyses and culturing. There
is a wealth of information about the function of the SCN; its role in the control of
circadian activity rhythms, tracked by measuring wheel running behavior, provides a
convenient assay for SCN activity and the
role these neurons may play in the control
of that activity. This assay for SCN activity
will allow us to determine whether all VIP/
GRP neurons in the SCN are regulated by
the same set of stimuli. And finally, culture
Our view of the mechanisms by which
the brain processes the myriad internal and
external stimuli to coordinate behavioral,
endocrine and autonomic processes is being
greatly enhanced by progress in molecular
biology and the development of the kinds
of hybridization techniques (e.g., in situ
hybridization) with the quantitative sensitivity and anatomical resolution required
for application to the central nervous system. As we apply these techniques to learn
more about the cellular mechanisms by
which neurons integrate a variety of information, we will gain a clearer understanding
of the underlying biology we observe in the
life histories of animals, including humans.
However, to relate the molecular and cellular events occurring in the nervous system
to behavioral, endocrine and autonomic
phenomena, we must combine these molecular techniques with the "traditional" techniques which have allowed us to so clearly
define the major issues of today.
ACKNOWLEDGMENTS
We thank Dr. Sandra Petersen, Professor
Frank L. Moore and members of his laboratory for helpful comments made on the
initial drafts of this manuscript.
REFERENCES
Albers, H. E., E. G. Stopa, R. T. Zoeller, J. S. Kauer,
J. C. King, J. S. Fink, H. Mobtaker, and H. Wolfe.
1990. Day-night variation in prepro vasoactive
intestinal peptide/peptide histidine isoleucine
mRNA within the rat suprachiasmatic nucleus.
Mol. Brain Res. 7:85-89.
Albers, H. E., S. Y. Liou, E. G. Stopa, and R. T. Zoeller.
1991. Interaction of co-localized neuropeptides:
Functional significance in the circadian timing system. J. Neurosci. 11:846-851.
Arancibia, S., L. Tapia-Arancibia, H. Astier, and I.
Assenmacher. 1989. Physiological evidence for
alpha 1-adrenergic facilitatory control of the coldinduced TRH release in the rat, obtained by pushpull cannulation of the median eminence. Neurosci. Lett. 100:169-174.
Bosler, O. and A. Beaudet. 1985. VIP neurons as
prime synaptic targets for serotonin afferents in rat
suprachiasmatic nucleus: A combined radioautographic and immunocytochemical study. J.
Neurocytol. 14:749-763.
252
R . T . ZOELLER ET AL.
Brownstein, M. J., R. D. Utiger, M. Palkovits, and J.
S. Kizer. 1975. Effect of hypothalamic deafferentation on thyrotropin-releasing hormone levels
in rat brain. Proc. Natl. Acad. Sci. U.S.A. 72:41774179.
Burgus, R., T. F. Dunn, D. Desiderio, D. N. Ward, W.
Vale, and R. Guillemin. 1970. Characterization
of ovine hypothalamic hypophysiotropic TSHreleasing factor. Nature 226:321-323.
Card, J. P., N. Brecha, H. J. Karten, and R. Y. Moore.
1981. Immunocytochemical localization of vasoactive intestinal polypeptide-containing cells and
processes in the suprachiasmatic nucleus of the
rat: Light and electron microscopic analysis. J.
Neurosci. 1:1289-1303.
Chomczynski, P. and N. Sacchi. 1987. Single-step
method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform extraction. Anal.
Biochem. 162:156-159.
Comb, M., S. E. Hyman, and H. M. Goodman. 1987.
Mechanisms of trans-synaptic regulation of gene
expression. Trends Neurosci. 10:473-478.
Fischer-Colbrie, R., A. Iacangelo, and L. E. Eiden.
1988. Neural and humoral factors separately regulate neuropeptide Y, enkephalin, and chromagranin A and B mRNA levels in rat adrenal
medulla. Proc. Natl. Acad. Sci. U.S.A. 85:32403244.
Folkers, K., F. Enzmann, J. Boler, C. Y. Bowers, and
A. V. Schally. 1969. Discovery of modification
of the synthetic tripeptide-sequence of the thyrotropin releasing hormone having activity. Biochem. Biophys. Res. Commun. 37:123-126.
Goodman, R. H. 1990. Regulation of neuropeptide
gene expression. Annu. Rev. Neurosci. 13:111127.
Gozes, I., Y. Shani, B. Liu, and J. P. H. Burbach. 1989.
Diurnal variation in vasoactive intestinal peptide
messenger RNA in the suprachiasmatic nucleus of
the rat. Neurosci. Res. Commun. 5:83-86.
Habener, J. F. 1990. Cyclic AMP response element
binding proteins: A cornucopia of transcription
factors. Mol. Endocrinol. 4:1087-1094.
Hefco, E., L. Krulich, P. Illner, and P. R. Larsen. 1975.
Effect of acute exposure to cold on the activity of
the hypothalamic-pituitary-thyroid system. Endocrinology 97:1185-1195.
Hisano, S., H. Ishizuka, T. Nishiyama, Y. Tsuruo, S.
Katoh, and S. Daikoku. 1986. Immunoelectron
microscopic observations of hypothalamic TRHcontaining neurons in rats. Exp. Brain Res. 63:
495-504.
Hisano, S., M. Chikamori-Aoyama, S. Katoh, Y.
Kagotani, S. Daikoku, and K. Chihara. 1988.
Suprachiasmatic nucleus neurons immunoreacti ve for vasoactive intestinal peptide have synaptic
contacts with axons immunoreactive for neuropeptide Y: An immunoelectronmicroscopic study
in the rat. Neurosci. Lett. 88:145-150.
Hokfelt, T., K. Fuxe, and B. Pernow. 1986. Coexistence of neuronal messengers: A new principle
in chemical transmission. Progress Brain Res. 68:
3-404.
Ibata, Y., Y. Takahashi, H. Okamura, K. Fumio, H.
Terubayashi, T. Kubo, and N. Yanaihara. 1989.
Vasoactive intestinal peptide (VlP)-like immunoreactive neurons located in the rat suprachiasmatic nucleus receive a direct retinal projection.
Neurosci. Lett. 97:1-5.
Ishikawa, K., Y. Taniguchi, K. Inoue, K. Kurosumi,
and M. Suzuki. 1988. Immunocytochemical
delineation of the thyrotrophic area: Origin of thyrotropin-releasing hormone in the median eminence. Neuroendocrinology 47:384-388.
Jackson, I. M. D., P. Wu, and R. M. Lechan. 1985.
Immunohistochemical localization in the rat brain
of the precursor for thyrotropin-releasing hormone. Science 229:1097-1099.
Jobin, M., L. Ferland, J. Cote, and F. Labrie. 1975.
Effect of exposure to cold on hypothalamic TRH
activity and plasma levels of TSH and prolactin
in the rat. Neuroendocrinology 18:204-212.
Jobin, M., L. Ferland, and F. Labrie. 1976. Effect of
pharmacological blockade of ACTH and TSH
secretion on the acute stimulation of prolactin
release by exposure to cold and ether stress. Endocrinology 99:146-151.
Kiss, J., C. Leranth, and B. Halasz. 1984. Serotoninergic endings of VIP-neurons in the suprachiasmatic nucleus and on ACTH-neurons in the arcuate nucleus of the rat hypothalamus. A combination
of high resolution autoradiography and electron
microscopic immunocytochemistry. Neurosci.
Lett. 44:119-124.
Koenig, R. J., D. Senator, and P. R. Larsen. 1984.
Phorbol esters as probes of the regulation of thyrotropin secretion. Biochem. Biophys. Res. Commun. 125:353-359.
Koller, K. J., R. S. Wolff, M. K. Warden, and R. T.
Zoeller. 1987. Thyroid hormones regulate levels
of thyrotropin-releasing hormone mRNA in the
paraventricular nucleus. Proc. Natl. Acad. Sci.
U.S.A. 84:7329-7333.
Krulich, L., A. Giachetti, A. Marchlewska-Koj, E.
Hefco, and H. E. Jameson. 1977. On the role of
the central noradrenergic and dopaminergic systems in the regulation of TSH secretion in the rat.
Endocrinology 100:496-505.
Kuchler, K., K. Richter, J. Trnovsky, R. Egger, and G.
Kreil. 1990. Two precursors of thyrotropinreleasing hormone from skin ofXenopus laevis. J.
Biol. Chem. 265:11731-11733.
Lechan, R. M., P. Wu, and I. M. D. Jackson. 1986.
Immunolocalization of the thyrotropin-releasing
hormone prohormone in the rat central nervous
system. Endocrinology 119:1210-1216.
Liao, N., M. Bulant, P. Nicolas, H. Vaudry, and G.
Pelletier. 1985. Immunocytochemical distribution of neurons containing a peptide derived from
thyrotropin-releasing hormone precursor in the rat
brain. Neurosci. Lett. 85:24-28.
Liposits, Z., W. K. Paull, P. Wu, I. M. D. Jackson, and
R. M. Lechan. 1987. Hypophysiotrophic thyrotropin releasing hormone (TRH) synthesizing
neurons. Ultrastructure, adrenergic innervation and
putative transmitter action. Histochemistry 88:110.
HYPOTHALAMIC INTEGRATION
Mannisto, P. T. 1983. Central regulation of thyrotropin secretion in rats: Methodological aspects,
problems and some progress. Med. Biol. 61:92100.
Meijer, J. H. and W. J. Rietveld. 1989. Neurophysiology of the suprachiasmatic circadian pacemaker
in rodents. Physiol. Rev. 69:671-707.
Mori, M. and M. Yamada. 1987. Thyroid hormones
regulate the amount of thyrotropin-releasing hormone in the hypothalamic median eminence of
the rat. J. Endocrinol. 114:443-448.
Mori, M., I. Kobayashi, and K. Wakabayashi. 1978.
Suppression of serum thyrotropin (TSH) concentrations following thyroidectomy and cold-exposure by passive immunization with antiserum to
thyrotropin-releasing hormone (TRH) in rats.
Metabolism 27:1485-1490.
Morley, J. E. 1981. Neuroendocrine control of thyrotropin secretion. Endocr. Rev. 2:396-436.
Nair, R. M., J. F. Barrett, C. Y. Bowers, and A. V.
Schally. 1970. Structure of porcine thyrotropin
releasing hormone. Biochemistry 9:1103—1106.
Nishizawa, M., Y. Hayakawa, N. Yanaihara, and H.
Okamoto. 1985. Nucleotide sequence divergence and functional constraint in VIP precursor
mRNA evolution between human and rat. FEBS
Lett. 183:55-59.
Okamura, H, Y. Takahashi, H. Terubayashi, S.
Hamada, N. Yanaihara, and Y. Ibata. 1987.
Coexistence of vasoactive intestinal peptide (VIP)-,
peptide histidine isoleucine amide (PHI)-, and gastrin releasing peptide (GRP)-like immunoreactivity in neurons of the rat suprachiasmatic nucleus.
Bio. Res. 7:295-299.
Onaya, T. and K. Hashizume. 1977. Effects of drugs
that modify brain biogenic amine concentrations
on thyroid activation induced by exposure to cold.
Neuroendocrinology 20:47-58.
Palkovits, M., R. L. Eskay, and M. J. Brownstein.
1982. The course of thyrotropin-releasing hormone fibers to the median eminence in rats. Endocrinology 110:1526-1528.
Prasad, C, J. J. Jacobs, and J. F. Wilber. 1980.
Immunological blockade of endogenous thyrotropin-releasing hormone produces hypothermia in
rats. Brain Res. 193:580-583.
Richter, K.., E. Kawashima, R. Egger, and G. Kreil.
1984. Biosynthesis of thyrotropin-releasing hormone in the skin of Xenopus laevis: Partial sequence
of the precursor deduced from cloned cDNA.
EMBOJ. 3:617-621.
Rondeel, J. M. M., W. J. deGreef, P. van der Schoot,
B. Karels, W. Klootwijk, and T. J. Visser. 1988.
Effect of thyroid status and paraventricular area
lesions on the release of thyrotropin-releasing hormone and catecholamines into hypophysial portal
blood. Endocrinology 123:523-527.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989.
Molecular cloning. A laboratory manual. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor.
Schettini, G., A. Quattrone, G. Di Renzo, G. Lombardi, and P. Preziosi. 1979. Effect of
6-hydroxydopamine treatment on TSH secretion
253
in basal and cold-stimulated conditions in the rat.
Eur. J. Pharmacol. 56:153-157.
Segerson, T. P., J. Kauer, H. C. Wolfe, H. Mobtaker,
P. Wu, I. M. D. Jackson, and R. M. Lechan. 1987.
Thyroid hormone regulates TRH biosynthesis in
the paraventricular nucleus of the rat hypothalamus. Science 238:78-80.
Shupnik, M. A., S. L. Greenspan, and E. C. Ridgway.
1986. Transcriptional regulation of thyrotropin
subunit genes by thyrotropin-releasing hormone
and dopamine in pituitary cell culture. J. Biol.
Chem. 261:12675-12679.
Stopa, E. G., N. Minamitani, J. A. Jonassen, J. C. King,
H. Wolfe, H. Mobtaker, and H. E. Albers. 1988.
Localization of vasoactive intestinal peptide and
peptide histidine isoleucine immunoreacti vity and
mRNA within the rat suprachiasmatic nucleus.
Mol. Brain Res. 4:319-325.
Szabo, M. and L. A. Frohman. 1977. Suppression of
cold-stimulated thyrotropin secretion by antiserum to thyrotropin-releasing hormone. Endocrinology 101:1023-1033.
Szabo, M., N. Kovathana, K. Gordon, and L. A. Frohman. 1978. Effect of passive immunization with
an antiserum to thyrotropin (TSH)-releasing hormone on plasma TSH levels in thyroidectomized
rats. Endocrinology 102:799-805.
Taylor, T., N. Gesundheit, and B. D. Weintraub.
1986. Effects of in vivo bolus versus continuous
TRH administration on TSH secretion, biosynthesis, and glycosylation in normal and hypothyroid rats. Mol. Cell Endocrinol. 46:253-261.
Taylor, T., F. E. Wondisford, T. Blaine, and B. D.
Weintraub. 1990. The paraventricular nucleus
of the hypothalamus has a major role in thyroid
hormone feedback regulation of thyrotropin synthesis and secretion. Endocrinology 126:317-324.
Terry, L. C. 1986. Regulation of thyrotropin secretion by the central epinephrine system. Neuroendocrinology 42:102-108.
Uhl, G. R. and T. Nishimori. 1990. Neuropeptide
gene expression and neural activity: Assessing a
working hypothesis in nucleus caudalis and dorsal
horn neurons expressing preproenkephalin and
preprodynorphin. Cell. Mol. Neurobiol. 10:73-98.
Van Nguyen, T., L. Kobierski, M. Comb, and S. E.
Hyman. 1990. The effect of depolarization on
expression of the human proenkephalin gene is
synergistic with cAMP and dependent upon cAMPinducible enhancer. J. Neurosci. 10:2825-2833.
Wray, S., R. T. Zoeller, and H. Gainer. 1989. Differential effects of estrogen on luteinizing hormone-releasing hormone gene expression in slice
explant cultures prepared from specific rat forebrain regions. Mol. Endocrinol. 3:1197-1206.
Wyatt, L. M., R. B. Norgren, and M. N. Lehman.
1988. Retinal and neuropeptide Y innervation of
the hamster suprachiasmatic nucleus: Light and
electron microscopic observations. Soc. Neurosci.
Abstr. 14:50.
Yamada, M., D. Rogers, and J. F. Wilber. 1989.
Exogenous triiodothyronine lowers thyrotropinreleasing concentrations in the specific hypothalamic nucleus (paraventricular) involved in thy-
254
R . T . ZOELLER ET AL.
rotropin regulation and also in posterior nucleus.
Neuroendocrinology 50:560-563.
Young, W. S., HI and R. T. Zoeller. 1987. Neuroendocrine gene expression in the hypothalamus: In
situ hybridization histochemical studies. Cell. Mol.
Neurobiol. 7:353-366.
Zoeller, R. T. and K. M. Conway. 1989. Neurons
expressing thyrotropin-releasing hormone-like
messenger ribonucleic acid are widely distributed
in Xenopus laevis brain. Gen. Comp. Endocrinol.
76:139-146.
Zoeller, R. T., N. Kabeer, and H. E. Albers. 1990.
Cold exposure elevates cellular levels of messenger
ribonucleic acid encoding thyrotropin-releasing
hormone in paraventricular nucleus despite elevated levels of thyroid hormones. Endocrinology
127:2955-2962.