Inflammation enhances Y1 receptor signaling, neuropeptide Y-mediated inhibition of hyperalgesia, and substance P release from primary afferent neurons.

Neuropeptide Y (NPY) is present in the superficial laminae of the dorsal horn and inhibits spinal nociceptive processing, but the mechanisms underlying its anti-hyperalgesic actions are unclear. We hypothesized that NPY acts at neuropeptide Y1 receptors in the dorsal horn to decrease nociception by inhibiting substance P (SP) release, and that these effects are enhanced by inflammation. To evaluate SP release, we used microdialysis and neurokinin 1 receptor (NK1R) internalization in rat. NPY decreased capsaicin-evoked SP-like immunoreactivity in the microdialysate of the dorsal horn. NPY also decreased non-noxious stimulus (paw brush)-evoked NK1R internalization (as well as mechanical hyperalgesia and mechanical and cold allodynia) after intraplantar injection of carrageenan. Similarly, in rat spinal cord slices with dorsal root attached, [Leu(31), Pro(34)]-NPY inhibited dorsal root stimulus-evoked NK1R internalization. In rat dorsal root ganglion neurons, Y1 receptors colocalized extensively with calcitonin gene-related peptide (CGRP). In dorsal horn neurons, Y1 receptors were extensively expressed and this may have masked the detection of terminal co-localization with CGRP or SP. To determine whether the pain inhibitory actions of Y1 receptors are enhanced by inflammation, we administered [Leu(31), Pro(34)]-NPY after intraplantar injection of complete Freund's adjuvant (CFA) in rat. We found that [Leu(31), Pro(34)]-NPY reduced paw clamp-induced NK1R internalization in CFA rats but not uninjured controls. To determine the contribution of increased Y1 receptor-G protein coupling, we measured [(35)S]GTPγS binding simulated by [Leu(31), Pro(34)]-NPY in mouse dorsal horn. CFA inflammation increased the affinity of Y1 receptor G-protein coupling. We conclude that Y1 receptors contribute to the anti-hyperalgesic effects of NPY by mediating the inhibition of SP release, and that Y1 receptor signaling in the dorsal horn is enhanced during inflammatory nociception.

Individual differences in the effects of chronic stress on memory: behavioral and neurochemical correlates of resiliency.

Chronic stress has been shown to impair memory, however, the extent to which memory can be impaired is often variable across individuals. Predisposed differences in particular traits, such as anxiety, may reveal underlying neurobiological mechanisms that could be driving individual differences in sensitivity to stress and, thus, stress resiliency. Such pre-morbid characteristics may serve as early indicators of susceptibility to stress. Neuropeptide Y (NPY) and enkephalin (ENK) are neurochemical messengers of interest implicated in modulating anxiety and motivation circuitry; however, little is known about how these neuropeptides interact with stress resiliency and memory. In this experiment, adult male rats were appetitively trained to locate sugar rewards in a motivation-based spatial memory task before undergoing repeated immobilization stress and then being tested for memory retention. Anxiety-related behaviors, among other characteristics, were monitored longitudinally. Results indicated that stressed animals which showed little to no impairments in memory post-stress (i.e., the more stress-resilient individuals) exhibited lower anxiety levels prior to stress when compared to stressed animals that showed large deficits in memory (i.e., the more stress-susceptible individuals). Interestingly, all stressed animals, regardless of memory change, showed reduced body weight gain as well as thymic involution, suggesting that the effects of stress on metabolism and the immune system were dissociated from the effects of stress on higher cognition, and that stress resiliency seems to be domain-specific rather than a global characteristic within an individual. Neurochemical analyses revealed that NPY in the hypothalamus and amygdala and ENK in the nucleus accumbens were modulated differentially between stress-resilient and stress-susceptible individuals, with elevated expression of these neuropeptides fostering anxiolytic and pro-motivation function, thus driving cognitive resiliency in a domain-specific manner. Findings suggest that such neurochemical markers may be novel targets for pharmacological interventions that can serve to prevent or ameliorate the negative effects of stress on memory.

Regulation of hypothalamic neuropeptide Y Y1 receptor gene expression during the estrous cycle: role of progesterone receptors.

Neuropeptide Y (NPY) stimulates the release of GnRH in an estrogen (E2)-dependent manner, which is important in generating preovulatory GnRH surges. We tested the hypothesis that E2 up-regulates NPY's actions by stimulating NPY Y1 receptor (Y1r) gene expression through a mechanism mediated by E2's ability to induce progesterone (P) receptors (PRs). In initial experiments, a specific Y1r antagonist BIBP3226 was used to confirm the involvement of Y1r in the stimulatory effects of NPY on in vivo GnRH release. Hypothalamic Y1r messenger RNA (mRNA) levels were then measured using competitive RT-PCR and were found to be significantly increased at 1000, 1200, and 1400 h on proestrus compared with other times of the day or cycle stage. Ovariectomy eliminated these increases, and E2 treatment restored them. Additional P treatment produced even larger increases in Y1r mRNA levels. To assess the role of PRs in stimulating Y1r expression, proestrous rats were treated with PR antagonist or oil vehicle and killed at 1200 h. Treatment with PR antagonist completely blocked the proestrous rise in Y1r gene expression. In parallel experiments, the same in vivo PR antagonist treatments also blocked NPY stimulation of GnRH release in vitro. Together our findings reveal that 1) Y1r mRNA levels are increased during the late morning and afternoon of proestrus; 2) Y1r mRNA levels are similarly increased by E2, and to an even greater extent by additional P; and 3) PR antagonism blocks both increased Y1r mRNA and induction of GnRH responsiveness to NPY. These observations support the idea that E2 up-regulates GnRH neuronal responses to NPY through stimulation of Y1r gene expression, and that E2's actions are mediated by the induction and subsequent activation of PRs.

Rapid photoperiod-induced increase in detectable GnRH mRNA-containing cells in Siberian hamster.

To determine whether changes in gonadotropin-releasing hormone (GnRH) neurons are early indicators of photostimulation, Siberian hamsters were placed in short days (6:18-h light-dark) at 3 (experiment 1) or 6 (experiment 2) wk of age where they were held for 3 (experiment 1) or 4 (experiment 2) wk. Hamsters were then moved to long photoperiod (16:8-h light-dark). In experiment 1, brains were collected 1-21 days after transfer from short to long days. In experiment 2, brains were collected only on the second morning of long day exposure. Long and short day controls were included in both experiments. Cells containing GnRH mRNA, as visualized by in situ hybridization, were counted. As expected, there were no differences in the number of detectable GnRH mRNA-containing cells among animals chronically exposed to long or short photoperiods. However, on the second morning after transfer from short to long photoperiod, a positive shift in the distribution of GnRH mRNA-containing cells occurred relative to the respective controls in the two experiments. Increases in follicle-stimulating hormone secretion and gonadal growth occurred days later. In conclusion, a rapid but transient increase in the distribution of detectable GnRH mRNA-containing cells is an early step in the photostimulation of the hypothalamic-pituitary-gonadal axis.

Steroid modulation of neuropeptide Y-induced luteinizing hormone releasing hormone release from median eminence fragments from male rats.

Neuropeptide Y (NPY) has been shown to stimulate hypothalamic release of luteinizing hormone-releasing hormone (LHRH) both in vitro and in vivo. In female rats, NPY facilitation of LHRH release is greatly augmented in advance of preovulatory LHRH surges, likely via the actions of ovarian steroids. However, the role of NPY in regulating LHRH release in male rats and the effects of testicular hormones on LHRH responses to NPY in males are not well understood. The objective of the present studies was to determine whether NPY stimulates LHRH release in vitro from hypothalamic tissue of male rats, and whether these effects could be modulated by testosterone (T). Mediobasal hypothalamic (MBH) or median eminence (ME) fragments from either sham-operated or castrated male rats (7 days) were placed in superfusion chambers and superfused with M199 for a 30-min baseline, 30-min challenge with NPY (10(-7)M), and a final 30-min challenge with 56 mM KCl. One-milliliter fractions were collected every 10 min and average LHRH release values over the 30-min periods were compared among groups. NPY (10(-7)M) produced a significant increase in LHRH release from the MBH and ME from intact animals. In contrast, the same dose of NPY did not stimulate LHRH release from tissues from castrated animals; only with a higher dose of NPY (10(-6)M) were the effects of NPY on LHRH release significant. Potassium challenge (56 mM KCl) significantly stimulated LHRH release from the ME of both intact and castrate male rats suggesting that all tissues were able to respond to a stimulus, and that castration did not alter the responsiveness of the LHRH neuron to a nonspecific secretagogue. To determine the extent to which T regulates the sensitivity of LHRH responses to NPY, male rats were castrated and implanted with T capsules that maintained either low (1.24 +/- 0.06 ng/ml) or high (2.17 +/- 0.31 ng/ml) physiological plasma levels of T. Treatment with the higher dose of T restored the ability of NPY to stimulate LHRH release from the ME tissues. These results demonstrate that NPY stimulates LHRH release from the hypothalamus in vitro, and that gonadal steroids, in this case T and/or its metabolites, modulate the responsiveness of the LHRH neuron to NPY. Based on these data from intact and castrate-derived tissues, it appears that steroids are necessary to maintain LHRH responsiveness to NPY receptor stimulation.

Noradrenergic activity in rat brain during rapid eye movement sleep deprivation and rebound sleep.

Noradrenergic locus ceruleus neurons are most active during waking and least active during rapid eye movement (REM) sleep. We expected REM sleep deprivation (REMSD) to increase norepinephrine utilization and activate the tyrosine hydroxylase (TH) gene critical for norepinephrine production. Male Wistar rats were deprived of REM sleep with the platform method. Rats were decapitated after 8, 24, or 72 h on small (REMSD) or large (control) platforms or after 8 or 24 h of rebound sleep after 72 h of the platform treatment. During the first 24 h, norepinephrine concentration, measured by high-performance liquid chromatography/electrochemical detection, was lower in the neocortex, hippocampus, and posterior hypothalamus in REMSD rats than in large-platform controls. After 72 h of REMSD, TH mRNA, measured by in situ hybridization, was increased in the locus ceruleus and norepinephrine concentrations were increased. Polygraphy showed that small-platform treatment caused effective and selective REMSD. Serum corticosterone measurement by radioimmunoassay indicated that the differences found in norepinephrine and TH mRNA were not due to differences in stress between the treatments. The novel finding of sleep deprivation-specific increase in TH gene expression indicates an important mechanism of adjusting to sleep deprivation.

Amplitude and frequency modulation of pulsatile luteinizing hormone-releasing hormone release.

1. A variety of neuroendocrine approaches has been used to characterize cellular mechanisms governing luteinizing hormone-releasing hormone (LHRH) pulse generation. We review recent in vivo microdialysis, in vitro superfusion, and in situ hybridization experiments in which we tested the hypothesis that the amplitude and frequency of LHRH pulses are subject to independent regulation via distinct and identifiable cellular pathways. 2. Augmentation of LHRH pulse amplitude is proposed as a central feature of preovulatory LHRH surges. Three mechanisms are described which may contribute to this increase in LHRH pulse amplitude: (a) increased LHRH gene expression, (b) augmentation of facilitatory neurotransmission, and (c) increased responsiveness of LHRH neurons to afferent synaptic signals. Neuropeptide Y (NPY) is examined as a prototypical afferent transmitter regulating the generation of LHRH surges through the latter two mechanisms. 3. Retardation of LHRH pulse generator frequency is postulated to mediate negative feedback actions of gonadal hormones. Evidence supporting this hypothesis is reviewed, including results of in vivo monitoring experiments in which LHRH pulse frequency, but not amplitude, is shown to be increased following castration. A role for noradrenergic neurons as intervening targets of gonadal hormone negative feedback actions is discussed. 4. Future directions for study of the LHRH pulse generator are suggested.

REM sleep deprivation induces galanin gene expression in the rat brain.

Rats were deprived of REM sleep for 24 h by keeping them on small platforms that were placed in a water bath (the platform method). Galanin coding mRNA was visualized using in situ hybridization, and cells expressing galanin mRNA were counted. In REM sleep-deprived animals the cell count was higher in the preoptic area and periventricular nucleus. Lesions of this area have been reported to induce wakefulness in cats and rats. Galanin administered into the lateral ventricle had no effect on sleep. We conclude that REM sleep deprivation can induce galanin gene expression in some brain areas, but galanin alone does not modify spontaneous sleep.

Gene expression in a subpopulation of luteinizing hormone-releasing hormone (LHRH) neurons prior to the preovulatory gonadotropin surge.

Gene expression in luteinizing hormone-releasing hormone (LHRH) neurons was analyzed during the periovulatory period to (1) characterize temporal patterns of LHRH gene expression and their relationship(s) to gonadotropin surges, and (2) determine if any such changes are uniform or dissimilar at different rostrocaudal levels of the basal forebrain. The number of neurons expressing mRNA for the decapeptide, and the relative degree of expression per cell were analyzed using in situ hybridization and quantitative image analysis. Rats were killed at 1800 hr on metestrus (Met), 0800 hr, 1200 hr, 1800 hr, and 2200 hr on proestrus (Pro), or 0200 hr, 0800 hr, and 1800 hr on estrus (E; n = 5-6 rats/group). All sections were processed for LHRH mRNA in a single in situ hybridization assay. Sections were atlas matched and divided into four rostrocaudal groups for analysis: vertical limb of the diagonal band of Broca (DBB), rostral preoptic area/organum vasculosum of the lamina terminalis (rPOA/OVLT), medial preoptic area (mPOA), and suprachiasmatic/anterior hypothalamic area (SCN/AHA). Plasma LH and FSH levels from all animals were analyzed by RIA. The labeling intensity per cell was similar among all time points at all four rostrocaudal levels. The number of cells expressing LHRH mRNA, however, varied as a function of time of death during the estrous cycle, and this temporal pattern varied among the four anatomical regions. At the level of the mPOA, the number of cells was highest at 1200 hr on Pro, and then declined and remained low throughout the morning of E. At the level of the rPOA/OVLT, the greatest number of LHRH neurons was noted later in Pro, at 1800 hr, dropping rapidly to lowest numbers at 2200 hr. No significant changes in LHRH cell number occurred at the DBB or SCN/AHA levels. At all anatomical levels, the secondary surge of FSH was unaccompanied by any change in the number of neurons expressing LHRH mRNA. These data demonstrate that (1) the number of detectable LHRH mRNA-expressing cells fluctuates during the periovulatory period and (2) peak numbers of LHRH-expressing cells are attained in the mPOA before the onset of the LH surge and before peak LHRH cell numbers are seen at more rostral levels. A model is proposed in which gene expression in this subpopulation of LHRH neurons may be activated by preovulatory estrogen secretion and acutely reduced following the proestrous surge of progesterone.

Neuropeptide Y gene expression in the arcuate nucleus: sexual dimorphism and modulation by testosterone.

Neuropeptide Y (NPY) peptide concentrations in the arcuate nucleus have recently been shown to be modulated by gonadal steroids in the male rat. The present study was designed to determine whether NPY messenger RNA (mRNA)-synthesizing cells in the arcuate nucleus (Arc) of the male rat are regulated by testosterone (T) and whether there is a sexual dimorphism in the expression of the NPY gene in this region. In situ hybridization and quantitative autoradiography were used to assess the level of NPY gene expression in the Arc. In the first experiment, NPY mRNA levels were measured in the Arc of intact, castrated, and castrated male rats treated with T to maintain physiological (1.3 +/- 0.1 ng/ml) and supraphysiological (5.3 +/- 0.4 ng/ml) plasma levels of T. A 2-week castration produced a modest but significant decrease in NPY mRNA levels in the Arc (P < 0.05). Replacement with either physiological or supraphysiological levels of T prevented the effect of castration on NPY gene expression, and there was no further potentiation of NPY gene expression in those animals that received high levels of T. In the second experiment, NPY gene expression was compared throughout the Arc between intact male and female rats at 1800 h on the afternoon of proestrus. Comparison of NPY gene expression throughout the rostro-caudal extent of the Arc showed that male rats had significantly more NPY mRNA-containing cells than female rats (P < 0.01). This difference was most strikingly observed in the caudal portions of the nucleus (3.80 mm caudal to bregma). No difference was detected in the mean levels of NPY gene expression in the Arc between male and female rats. These data demonstrate that 1) NPY gene expression throughout the arcuate nucleus is modulated by T in male rats, and 2) a marked regional sex difference exists in the distribution of NPY mRNA-containing cells in the caudal extremity of the Arc. It is hypothesized that gonadal hormones may exert both organizational and activational effects upon NPY neurons in the Arc.

Neuropeptide Y gene expression in the arcuate nucleus is increased during preovulatory luteinizing hormone surges.

Recent studies have suggested that neuropeptide Y (NPY) plays an important role in the induction of the preovulatory LH surge. The present study was performed in order to determine if a change in NPY gene expression within arcuate nucleus NPY neurons is associated with the generation of the preovulatory LH surge. In Exp 1, in situ hybridization was used to measure NPY messenger RNA (mRNA) levels in the arcuate nucleus of female rats at 0900 h and every 2 h from 1400-2200 h on the day of proestrus (PRO). Comparisons between groups showed a clear, stepwise increase in NPY gene expression throughout the day of PRO. At 1600 h, when LH values were significantly greater than 0900 h values, NPY mRNA labeling intensities in the arcuate nucleus were significantly greater than 0900 h levels (P < 0.01). By 1800 h, the time at which the LH surge peaked, NPY mRNA levels also peaked and were nearly 3-fold greater than levels observed at 0900 h (P < 0.01). NPY mRNA levels at 2000 h and 2200 h remained elevated above 0900 h levels (P < 0.01) but by 2000 h had decreased significantly from 1800 h levels (P < 0.05). In Exp 2, NPY mRNA levels were measured once again at 0900 h and 1800 h on PRO, and then at 0900 h and 1800 h on metestrus (MET), in order to determine if the change in gene expression seen in Exp 1 was unique to the day of PRO, or if it simply reflected a daily rhythm of gene expression in the nucleus. Analysis of mRNA levels showed no difference in NPY mRNA levels between 0900-1800 h on MET. Also, NPY mRNA levels at 0900 h and 1800 h on MET were significantly less than levels at 1800 h on PRO (P < 0.01). These results are consistent with the hypothesis that NPY neurons participate in the generation of LH surges through increased production of NPY and subsequent potentiation of the release and/or actions of LHRH.

Distribution of a renin-releasing factor in the central nervous system of the rat.

We have previously shown that the serotonergic regulation of renin secretion from the kidneys is mediated by a renin-releasing factor (RRF) that is present in both plasma and hypothalamus. The present studies were designed to determine the distribution of RRF in the brain and peripheral tissues and to test whether RRF release could be stimulated in vitro from hypothalamo-hypophyseal explants. RRF levels were determined in vitro by measuring renin release from kidney cortical slices. Addition of hypothalamic extract to rat kidney slices produced a dose-dependent increase in renin release. RRF was measurable in most brain areas with the highest renin-releasing activity in the hypothalamus, cerebral cortex, medulla oblongata and cerebellum. To determine which brain regions contain RRF cell bodies, rats received an intracerebroventricular injection of colchicine to inhibit axonal transport and concentrate RRF in the perikarya. After colchicine treatment, RRF activity in the cerebral cortex, medulla oblongata and cerebellum decreased. In contrast, the hypothalamus had increased RRF activity suggesting that RRF cell bodies are localized in the hypothalamus. Superfusion of hypothalamo-hypophyseal explants with a high potassium Krebs-Ringer solution stimulated RRF release, suggesting that depolarization of hypothalamic neurons can stimulate RRF secretion. Nephrectomy produced a significant increase in RRF concentration in the hypothalamus, suggesting that RRF neurons respond to decreased renin activity or other kidney-related substances in the circulation. The determination of RRF in peripheral tissue revealed minimal renin-releasing activity in the liver, spleen and skeletal muscle extracts. High performance chromatography of hypothalamic extract on a GPC-100 column revealed RRF activity in fractions that were estimated to have a molecular weight of 5,000. These studies suggest that RRF-containing cell bodies in the hypothalamus respond to depolarization by releasing RRF into the circulation. In addition, the hypothalamic content of RRF is regulated by the kidney. Altogether, these data suggest that RRF neurons are part of a neuroendocrine system that regulates renin secretion from the kidneys.

Age-related decline of vasopressin mRNA in the bed nucleus of the stria terminalis.

To determine whether aging influences arginine vasopressin (AVP) biosynthesis in the extrahypothalamic neurons of the bed nucleus of the stria terminalis (BNST), we used in situ hybridization and quantitative autoradiography to compare AVP mRNA in 3-month-old, 14-month-old, and 24-month-old male Fischer 344 rats. As AVP synthesis in the BNST has previously been shown to be steroid-dependent, plasma testosterone (T) was measured by radioimmunoassay. The 24-month-old animals had significantly fewer AVP-labelled cells than either the 3-month-old (p less than 0.01) or 14-month-old (p less than 0.05) animals. The cells that were present in the 24-month animals were less intensely labelled than in the other groups, as indicated by a significantly reduced number of grains per cell (p less than 0.01). Plasma T was also significantly lower in 24-month-old animals when compared with 3-month (p less than 0.01) or 14-month (p less than 0.05) groups. The results indicate that there is a marked age-related decline in vasopressin biosynthetic activity in neurons of the BNST.

Dexamethasone-induced suppression of vasopressin gene expression in the bed nucleus of the stria terminalis and medial amygdala is mediated by changes in testosterone.

Vasopressin (VP) neurons in the bed nucleus of the stria terminalis (BNST) and medial amygdala (AME) are sensitive to changes in circulating levels of testosterone (T). To determine whether these cells are responsive to changes in glucocorticoid levels, in situ hybridization and quantitative autoradiography were used to measure VP mRNA in cells of the BNST and AME in rats that were adrenalectomized (ADX; 14 days) or ADX with dexamethasone (DEX) replacement. These treatments produced the predicted changes in VP gene expression in the medial parvocellular group of the paraventricular nucleus. The VP mRNA content within cells of the BNST or AME was unaffected by adrenalectomy. Treatment with DEX significantly decreased both the number and labeling intensity of VP cells in the BNST and AME. Measurement of plasma T in these animals showed that DEX treatment significantly lowered mean T levels compared with those in either sham-operated or ADX animals. Adrenalectomy alone did not significantly alter T levels. To determine whether DEX influenced VP gene expression via a glucocorticoid action or secondarily by a suppression of T, the above experiment was repeated with groups that were castrated and implanted with Silastic capsules containing T to maintain physiological levels of T. Administration of DEX again decreased both VP cell number and labeling intensity of cells in the BNST and AME in sham-implanted animals. However, VP gene expression was unaffected in those animals that received T capsules. Administration of corticosterone did not alter T levels or the number of cells in the BNST or AME. These results suggest that, in contrast to paraventricular nucleus neurons, adrenalectomy (14 days) is not a potent stimulus in altering VP activity in the BNST or AME. The DEX-induced decrease in VP gene expression is mediated by a secondary suppression of T levels. These results support the finding that gonadal steroids are essential in maintaining the biosynthetic integrity of VP neurons in the BNST and AME.

Neuroendocrine regulation of the luteinizing hormone-releasing hormone pulse generator in the rat.

We have analyzed the mechanisms by which several known regulators of the LHRH release process may exert their effects. For each, we have attempted to determine how and where the regulatory input is manifest and, according to our working premise, we have attempted to identify factors which specifically regulate the LHRH pulse generator. Of the five regulatory factors examined, we have identified two inputs whose primary locus of action is on the pulse-generating mechanism--one endocrine (gonadal negative feedback), and one synaptic (alpha 1-adrenergic inputs) (see Fig. 29). Other factors which regulate LHRH and LH release appear to do so in different ways. The endogenous opioid peptides, for example, primarily regulate LHRH pulse amplitude (Karahalios and Levine, 1988), a finding that is consistent with the idea that these peptides exert direct postsynaptic or presynaptic inhibition (Drouva et al., 1981). Gonadal steroids exert positive feedback actions which also result in an increase in the amplitude of LHRH release, and this action may be exerted through a combination of cellular mechanisms which culminate in the production of a unique, punctuated set of synaptic signals. Gonadal hormones and neurohormones such as NPY also exert complementary actions at the level of the pituitary gland, by modifying the responsiveness of the pituitary to the stimulatory actions of LHRH. The LHRH neurosecretory system thus appears to be regulated at many levels, and by a variety of neural and endocrine factors. We have found examples of (1) neural regulation of the pulse generator, (2) hormonal regulation of the pulse generator, (3) hormonal regulation of a neural circuit which produces a unique, punctuated synaptic signal, (4) hormonal regulation of pituitary responsiveness to LHRH, and (5) neuropeptidergic regulation of pituitary responsiveness to LHRH. While an attempt has been made to place some of these regulatory inputs into a physiological context, it is certainly recognized that the physiological significance of these mechanisms remains to be clarified. We also stress that these represent only a small subset of the neural and endocrine factors which regulate the secretion or actions of LHRH. A more comprehensive list would also include CRF, GABA, serotonin, and a variety of other important regulators. Through a combination of design and chance, however, we have been able to identify at least one major example of each type of regulatory mechanism.

Detection of vasopressin mRNA in cells of the medial amygdala but not the locus coeruleus by in situ hybridization.

Vasopressin (AVP)-immunoreactive cells have been previously reported in the medial amygdala (AME) and in the locus coeruleus (LC). The present study was designed to verify the presence of AVP-synthesizing neurons in these areas using in situ hybridization histochemistry. A 35S-labelled oligonucleotide probe, complementary to the glycopeptide portion of the vasopressin-encoding mRNA, was used to label cells expressing the AVP gene in brain sections from male Wistar rats. AVP mRNA-positive cells were identified in the AME and were located throughout the anterodorsal and posterodorsal aspect of the nucleus. Cells in the LC, however, did not exhibit labelling for the glycopeptide portion of the AVP gene. The highest density of labelled cells in the medial amygdala occurred 2.30 to 2.80 mm caudal to bregma. The labelling intensity of the cells averaged 53.8 +/- 3.9 grains/cells and was constant throughout the rostro-caudal extent of the AME. These data demonstrate the presence of AVP-synthesizing cells in the AME and provide a method for quantifying their activity. In addition, these data suggest that the cells in the LC may not synthesize vasopressin.

Stress-induced renin and corticosterone secretion is mediated by catecholaminergic nerve terminals in the hypothalamic paraventricular nucleus.

Cell bodies in the hypothalamic paraventricular nucleus (PVN) mediate stress-induced increases in renin and corticosterone secretion. Since the PVN has an extensive catecholaminergic innervation, we wanted to determine the role of catecholamines in the neuroendocrine response to stress. The stressor was a conditioned emotional (fear) response paradigm (CER). The catecholamine neurotoxin, 6-hydroxydopamine (6-OHDA), was injected into the PVN 14 days before the rats were subjected to the CER procedure. Damage to noradrenergic nerve terminals was verified immunocytochemically, using an antibody against dopamine beta-hydroxylase. Injection of 6-OHDa into the PVN prevented the stress-induced increase in plasma renin activity (PRA), plasma renin concentration (PRC) and plasma corticosterone concentration, suggesting that intact catecholaminergic innervation of neurons in the PVN is necessary for the stress-induced increase in renin and corticosterone secretion. To determine if beta-adrenoceptors in the PVN mediate the effect of stress on renin and corticosterone secretion, the beta-adrenoceptor antagonist sotalol was injected into the PVN through chronically implanted bilateral cannulae. The injection was performed on the 4th day of the CER paradigm, just before the rats were placed into the CER chamber. Sotalol prevented the stress-induced increase in corticosterone concentration, but did not diminish the stress-induced increase in PRA and PRC. These results suggest that the stress-induced increase in corticosterone concentration is influenced by beta-adrenoceptors in the PVN. The stress-induced increase in PRA and PRC is mediated by different receptors whose ligands might be catecholamines acting at non-beta-receptors or other neuroactive substances colocalized in catecholaminergic nerve terminals.

Steroid dependency of vasopressin neurons in the bed nucleus of the stria terminalis by in situ hybridization.

Recent immunocytochemical studies have suggested that vasopressin (VP) neurons in the bed nucleus of the stria terminalis (BNST) of the rat are gonadal steroid sensitive. In this paper we have used in situ hybridization and quantitative autoradiography to determine whether testosterone (T) and/or its metabolites modulate the biosynthetic capacity of VP neurons in the BNST of adult male rats. In Exp 1 the number of labeled cells and the average number of grains per cell were compared in sections sampled through the BNST of intact, castrated, and castrated male rats treated with physiological levels of T (1.6 +/- 0.1 ng/ml plasma). Castration dramatically reduced the number of labeled cells (P less than 0.01) and the intensity of labeling (P less than 0.05) of cells in the BNST. T, treatment of castrated animals reversed the effect of castration on both cell number and grains per cell. In Exp 2 treatment of castrated rats with supraphysiological levels of T (7.6 +/- 0.7 ng/ml plasma) increased the number of labeled BNST cells (P less than 0.05) and the intensity of labeling (P less than 0.05) over those in castrates treated with physiological levels of T or intact rats. These results indicate that T and/or its metabolites modulate expression of the VP gene by neurons in the BNST of adult male rats.

Effect of selective serotonin (5-HT) agonists and 5-HT2 antagonist on prolactin secretion.

The present study was undertaken to determine the involvement of serotonergic 5-HT1 and 5-HT2 receptor subtypes in stimulation of the secretion of prolactin. Several 5-HT agonists were administered, in a dose-response fashion, to conscious rats and the effect on the levels of prolactin in plasma was measured. The 5-HT1A + 5-HT1B agonist RU 24969 (5-methoxy-3[1,2,3,6-tetrahydropyridin-4-yl]-1H-indole succinate) and the 5-HT1 + 5-HT2 agonist MK-212 (6-chloro-2-[1-piperazinyl]pirazine) increased levels of prolactin in plasma in a dose-dependent manner. In contrast, the selective 5-HT1A agonists 8-OH-DPAT (8-hydroxy-2-[di-n-propylamino]tetralin) and ipsapirone (2-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-1,2-benzisothiazol-3 -(2H) one-1,1-dioxidehydrochloride) did not increase levels of prolactin in plasma at any dose. The 5-HT-releasing drug, fenfluramine, also increased the concentration of prolactin in plasma. Pretreatment with the selective 5-HT2 antagonist, LY53857 (6-methyl-1-[1-methylethyl]ergoline-8-carboxylic acid, 2-hydroxy-1-methyl propyl ester (Z)-2-butenedioate [1:1]), did not significantly diminish an increase in levels of prolactin in plasma, induced by injection of fenfluramine. The antagonist LY53857 inhibited, but did not block the MK-212- and RU 24969-induced increase in the levels of prolactin in plasma. By deduction, these data suggest that 5-HT1B receptors, or as yet undefined 5-HT receptor subtypes may be involved in the stimulation of the secretion of prolactin by endogenously released 5-HT, and that 5-HT2 receptors may play a minor role in the serotonergic regulation of the secretion of prolactin.