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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. 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