Efferent projections of ProTRH neurons in the ventrolateral periaqueductal gray.

Our previous study has shown that prothyrotropin-releasing hormone (proTRH) gene expression is increased in the ventrolateral periaqueductal gray (PAG) neurons following precipitated morphine withdrawal and continues to be activated even 24 h after withdrawal. We have hypothesized that peptide products of proTRH may participate in the recovery from morphine withdrawal. To identify neuroanatomical substrates of the proposed action of proTRH-derived peptides originating from the ventrolateral PAG proTRH neurons, projections of these neurons were investigated by a series of anterograde and retrograde tract-tracing experiments. First, Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected in the ventrolateral PAG in Sprague-Dawley rats. Following transport of the tracer, simultaneous immunolabeling for PHA-L and proTRH peptides was performed and mapped in discrete brain regions. PHA-L-immunoreactive (IR) fibers showing preterminal and terminal-like arborization that contained proTRH were identified in the dorsolateral and lateral PAG, deep layer of superior colliculus (CS), parafascicular nucleus (PF), ventromedial zona incerta (ZI) and at the border of the locus coeruleus (LC) and Barrington's nucleus. Scattered double-labeled fibers were present in the lateral septal nucleus, ventromedial preoptic nucleus, lateral hypothalamus, perifornical area and in the periventricular region at the diencephalon/midbrain junction. The retrogradely transported marker, cholera toxin beta-subunit (CTb) was then injected in the dorsolateral PAG, CS, PF, ZI and medial to the LC. Double-labeled perikarya for both CTb and proTRH in the ventrolateral PAG were found for each region injected with CTb, corroborating the findings by the anterograde tracing experiment. These studies demonstrate that proTRH neurons in the ventrolateral PAG project to several regions of the brain that are involved in autonomic and behavioral regulation and thereby, may function as an integrating center to coordinate responses to opiate withdrawal.

Regional physiological adaptation of the central nervous system deiodinases to iodine deficiency.

The goal of the present investigation was to analyze the types 2 (D2) and 3 (D3) iodothyronine deiodinases in various structures within the central nervous system (CNS) in response to iodine deficiency. After 5-6 wk of low-iodine diet (LID) or LID + 2 microg potassium iodide/ml (LID + KI; control), rats' brains were processed for in situ hybridization histochemistry for D2 and D3 mRNA or dissected, frozen in liquid nitrogen, and processed for D2 and D3 activities. LID did not affect weight gain or serum triiodothyronine, but plasma thyroxine (T4) was undetectable. In the LID + KI animals, D3 activities were highest in the cerebral cortex (CO) and hippocampus (HI), followed by the olfactory bulb and was lowest in cerebellum (CE). Iodine deficiency decreased D3 mRNA expression in all CNS regions, and these changes were accompanied by three- to eightfold decreases in D3 activity. In control animals, D2 activity in the medial basal hypothalamus (MBH) was similar to that in pituitary gland. Of the CNS D2-expressing regions analyzed, the two most responsive to iodine deficiency were the CO and HI, in which an approximately 20-fold increase in D2 activity occurred. Other regions, i.e., CE, lateral hypothalamus, MBH, and pituitary gland, showed smaller increases. The distribution of and changes in D2 mRNA were similar to those of D2 activity. Our results indicate that decreases in the expression of D3 and increases in D2 are an integral peripheral component of the physiological response of the CNS to iodine deficiency.

Neuropeptide Y has a central inhibitory action on the hypothalamic-pituitary-thyroid axis.

Recent evidence suggests that neuropeptide Y (NPY), originating in neurons in the hypothalamic arcuate nucleus, is an important mediator of the effects of leptin on the central nervous system. As these NPY neurons innervate hypophysiotropic neurons in the hypothalamic paraventricular nucleus (PVN) that produce the tripeptide, TRH, we raised the possibility that NPY may be responsible for resetting of the hypothalamic-pituitary-thyroid (HPT) axis during fasting. To test this hypothesis, the effects of intracerebroventricularly administered NPY on circulating thyroid hormone levels and proTRH messenger RNA in the PVN were studied by RIA and in situ hybridization histochemistry, respectively. NPY administration suppressed circulating levels of thyroid hormone (T(3) and T(4)) and resulted in an inappropriately normal or low TSH. These alterations were associated with a significant suppression of proTRH messenger RNA in the PVN, indicating that NPY infusion had resulted in a state of central hypothyroidism. Similar observations were made in NPY-infused animals pair fed to the vehicle-treated controls. These data are reminiscent of the effect of fasting on the thyroid axis and indicate that NPY may play a major role in the inhibition of HPT axis during fasting.

Hypothalamic dorsomedial nucleus neurons innervate thyrotropin-releasing hormone-synthesizing neurons in the paraventricular nucleus.

To determine whether the hypothalamic dorsomedial nucleus (DMN) may serve as a relay center for the central actions of leptin on thyrotropin-releasing hormone (TRH)-synthesizing neurons in the paraventricular nucleus (PVN), axonal projections from the DMN to TRH-containing neurons in the PVN were studied using the anterogradely transported marker substance, Phaseolus vulgaris-leucoagglutinin (PHA-L). Stereotaxic injections of PHA-L were targeted to the mid-dorsal and mid-ventral portions of the DMN. After 10-14-day survival, the brains were prepared for immunohistochemistry and immunostained with an antibody directed against PHA-L. Focal injections confined to the DMN were identified in 14 animals and gave rise to a fiber bundle that entered the PVN at the caudal pole of the nucleus, densely innervating all parvocellular subdivisions of the PVN. In double-labeled preparations using antisera to PHA-L and preproTRH 178-199, the latter as a marker for TRH-containing neurons in the PVN, proTRH-IR neurons were observed to be enmeshed in a network of PHA-L-containing fibers. When the injection site covered the entire DMN or the mid-dorsal part of the DMN, PHA-L-containing axon varicosities were juxtaposed to approximately 97 and 90% of proTRH neurons, respectively, in all parvocellular subdivisions of the PVN, and by ultrastructural analysis were shown to be synaptic. In contrast, when the injection site was centered primarily in the mid-ventral part of the DMN, only approximately 52% of proTRH-synthesizing neurons appeared to be innervated by PHA-L-containing axons. These data demonstrate that a major projection pathway exists from the DMN, specifically to TRH-producing neurons in the PVN, and suggest that the DMN is anatomically situated to exert a regulatory effect on TRH-synthesizing neurons in the PVN.

alpha-Melanocyte stimulating hormone prevents fasting-induced suppression of corticotropin-releasing hormone gene expression in the rat hypothalamic paraventricular nucleus.

During fasting, corticotropin-releasing hormone (CRH) mRNA decreases in the hypothalamic paraventricular nucleus (PVN), but the mechanism by which this takes place is not well understood. To test the hypothesis that the melanocortin system may be involved in the regulation of CRH mRNA in the PVN during fasting, the effect of intracerebroventricularly administered alpha-melanocyte stimulating hormone (MSH) on CRH mRNA in the PVN was studied in fasted animals by in situ hybridization histochemistry. Whereas fasting suppressed CRH mRNA levels in the PVN, alpha-MSH at doses of 150 and 300 ng every 6 h for 64 h prevented the fasting-induced suppression of CRH gene expression in the PVN. These data indicate that the suppression of alpha-MSH synthesis may be responsible for the decreased CRH gene expression in the PVN during fasting.

alpha-Melanocyte-stimulating hormone is contained in nerve terminals innervating thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and prevents fasting-induced suppression of prothyrotropin-releasing hormone gene expression.

The hypothalamic arcuate nucleus has an essential role in mediating the homeostatic responses of the thyroid axis to fasting by altering the sensitivity of prothyrotropin-releasing hormone (pro-TRH) gene expression in the paraventricular nucleus (PVN) to feedback regulation by thyroid hormone. Because agouti-related protein (AGRP), a leptin-regulated, arcuate nucleus-derived peptide with alpha-MSH antagonist activity, is contained in axon terminals that terminate on TRH neurons in the PVN, we raised the possibility that alpha-MSH may also participate in the mechanism by which leptin influences pro-TRH gene expression. By double-labeling immunocytochemistry, alpha-MSH-IR axon varicosities were juxtaposed to approximately 70% of pro-TRH neurons in the anterior and periventricular parvocellular subdivisions of the PVN and to 34% of pro-TRH neurons in the medial parvocellular subdivision, establishing synaptic contacts both on the cell soma and dendrites. All pro-TRH neurons receiving contacts by alpha-MSH-containing fibers also were innervated by axons containing AGRP. The intracerebroventricular infusion of 300 ng of alpha-MSH every 6 hr for 3 d prevented fasting-induced suppression of pro-TRH in the PVN but had no effect on AGRP mRNA in the arcuate nucleus. alpha-MSH also increased circulating levels of free thyroxine (T4) 2.5-fold over the levels in fasted controls, but free T4 did not reach the levels in fed controls. These data suggest that alpha-MSH has an important role in the activation of pro-TRH gene expression in hypophysiotropic neurons via either a mono- and/or multisynaptic pathway to the PVN, but factors in addition to alpha-MSH also contribute to the mechanism by which leptin administration restores thyroid hormone levels to normal in fasted animals.

Agouti-related protein containing nerve terminals innervate thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus.

Gene expression for agouti-related protein (AGRP), an endogenous antagonist of melanocortin receptors, has been localized to the hypothalamic arcuate nucleus, where it colocalizes with neuropeptide Y (NPY). Having reported that the NPY innervation of hypophysiotropic TRH neurons in the hypothalamic paraventricular nucleus (PVN) originates primarily from NPY-producing neurons in the arcuate nucleus, here we examined the possibility that TRH neurons in the PVN are similarly innervated by AGRP nerve terminals. Using immunohistochemistry, AGRP-containing cell bodies were found almost exclusively in the arcuate nucleus, but their projections were distributed widely in the hypothalamus, most conspicuously in the paraventricular (PVN), arcuate and dorsomedial nuclei, and the posterior hypothalamic area. Ablation of the arcuate nucleus by the neonatal administration of monosodium glutamate obliterated nearly all AGRP-immunoreactivity in the hypothalamus. In the PVN, double-labeling light and electron microscopic immunohistochemistry revealed that TRH neurons receive dense innervation by AGRP nerve terminals, with the frequent occurrence of axosomatic and axodendritic synapses (mainly of the symmetrical type). These findings provide morphological basis to hypothesize a role for AGRP in the arcuato-paraventricular pathway, in the down-regulation of the hypothalamic-pituitary-thyroid axis, which occurs as an adaptive response to starvation.

Regional expression of the type 3 iodothyronine deiodinase messenger ribonucleic acid in the rat central nervous system and its regulation by thyroid hormone.

Type 3 iodothyronine deiodinase (D3) is a selenoenzyme that inactivates thyroid hormone. It is necessary for T3 homeostasis in the central nervous system. D3 activity has been identified in many regions of the brain and parallels thyroid status, but the level at which it is regulated and its specific cellular locations are not known. We evaluated the effect of thyroid status on the expression of the D3 gene within the central nervous system using in situ hybridization histochemistry. D3 messenger RNA (mRNA) was identified throughout, but with high focal expression in the hippocampal pyramidal neurons, granule cells of the dentate nucleus, and layers II-VI of the cerebral cortex. In every region, D3 mRNA abundance was correlated with thyroid status. Four different D3 transcripts were identified by Northern analyses, with evidence for region-specific processing, and D3 mRNA increased 4- to 50-fold from the euthyroid to the hyperthyroid state. D3 mRNA was not detectable in hypothyroid brain. In the central nervous system, the D3 gene is highly T3 responsive, and its focal localization within the hippocampus and cerebral cortex suggests an important role for T3 homeostasis in memory and cognitive functions.

Pituitary adenylate cyclase-activating polypeptide-nerve terminals densely innervate corticotropin-releasing hormone-neurons in the hypothalamic paraventricular nucleus of the rat.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is widely distributed in many regions of the hypothalamus including the paraventricular nucleus (PVN). In this study, using well-characterized antibodies against PACAP and corticotropin-releasing hormone (CRH), we identified numerous nerve fibers with PACAP-immunoreactivity (ir) closely apposed to CRH neurons in the medial parvocellular subdivision of the rat PVN. Electron microscopy revealed the presence of synapses between PACAP-ir containing terminals and CRH-perikarya and -dendrites. These morphological observations suggest that PACAP may modulate the activation of the hypothalamic-pituitary-adrenal axis.

Arcuate nucleus ablation prevents fasting-induced suppression of ProTRH mRNA in the hypothalamic paraventricular nucleus.

Fasting results in reduced thyroid hormone levels and inappropriately low or normal thyroid-stimulating hormone (TSH), partly attributed to central hypothyroidism due to suppression of pro TRH gene expression in the hypothalamic paraventricular nucleus. Recently, we demonstrated that the systemic administration of leptin to fasting animals restores plasma thyroxine (T4) and proTRH mRNA in the paraventricular nucleus to normal, suggesting that the fall in circulating leptin levels during fasting acts as a signal to hypophysiotropic neurons in the paraventricular nucleus to reset the set point for feedback regulation of pro TRH mRNA by thyroid hormone. To determine whether the effect of fasting on the hypothalamic-pituitary-thyroid axis is mediated through the hypothalamic arcuate nucleus where leptin receptors are highly concentrated, we studied the effect of fasting and exogenous leptin administration on plasma thyroid hormone levels and proTRH mRNA concentration in the paraventricular nucleus in adult animals with arcuate nucleus lesions induced pharmacologically by the neonatal administration of monosodium L-glutamate (MSG). In normal animals, fasting reduced plasma T4 and TSH levels and the concentration of proTRH mRNA in the hypothalamic paraventricular nucleus. In contrast, neither fasting nor leptin administration to fasting MSG-treated animals had any significant effects on plasma thyroid hormone and TSH levels and proTRH mRNA in the paraventricular nucleus. These studies suggest that during fasting, the arcuate nucleus is essential for the normal homeostatic response of the hypothalamic-pituitary-thyroid axis and may serve as a critical locus to mediate the central actions of leptin on proTRH gene expression in the paraventricular nucleus.

The arcuate nucleus is the major source for neuropeptide Y-innervation of thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus.

Neuropeptide Y (NPY) immunoreactive (-ir) nerve fibers densely innervate hypophysiotropic TRH perikarya and dendrites in the hypothalamic paraventricular nucleus (PVN). To evaluate the contribution of the arcuate nucleus (Arc) to this innervation, the effect of Arc ablation by neonatal monosodium glutamate (MSG) treatment on the density of NPY-fibers contacting TRH neurons in the PVN was investigated. After the lesioned animals and vehicle-treated controls reached adulthood, the number of contacts between NPY-ir boutons and TRH-ir perikarya in the PVN was determined in double-immunostained sections. In controls, numerous contacts between NPY-ir terminals and TRH perikarya and dendrites were observed, confirming earlier findings. MSG treatment resulted in a marked reduction of the size of the Arc and also the number of NPY-perikarya with a concomitant reduction of 82.4 +/-2.1% in the relative number of NPY terminals contacting TRH perikarya and first order dendrites in the medial parvocellular and periventricular subdivisions of the PVN. In contrast, lesioning of the ascending adrenergic bundle in the brain stem caused no statistically significant change in the number of NPY-terminals in close apposition to hypophysiotropic TRH neurons in the PVN. These data confirm earlier findings that NPY-containing axon terminals innervate TRH neurons in the PVN and further demonstrate a potentially important anatomical relationship between NPY-producing neurons in the Arc and hypophysiotropic TRH neurons.

Association between pituitary adenylate cyclase-activating polypeptide and thyrotropin-releasing hormone in the rat hypothalamus.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is present in many regions of the hypothalamus including the paraventricular nucleus (PVN). In this study the anatomical relationship between PACAP- and thyrotropin-releasing hormone (TRH)-immunoreactive neuronal elements was investigated in the rat hypothalamus. Using a well-characterized mouse monoclonal antibody against PACAP and a rabbit polyclonal antiserum against TRH, we found numerous nerve fibers with PACAP-immunoreactivity (ir) closely apposed to TRH neurons in the PVN suggesting synaptic contacts. Electron microscopy confirmed the presence of synapses between PACAP-ir terminals and TRH-ir perikarya and various dendritic profiles as well as between PACAP-ir terminals and unlabeled perikarya and small- to medium-sized dendrites. Coexistence of the two peptides in perikarya of the PVN was limited to only a few neurons in the periventricular subdivision, but PACAP-ir and TRH-ir extensively coexisted in perikarya of the perifornical cell group, medial preoptic area, lateral hypothalamus and dorsomedial nucleus. The interactions between PACAP-containing neuronal processes and TRH neurons in the PVN raise the possibility that PACAP modulates the secretion of TRH destined for regulation of anterior pituitary TSH. The more general association between PACAP and TRH in other regions of the hypothalamus suggests a further role for PACAP as a cofactor in the function of TRH neurons.

Regional distribution of type 2 thyroxine deiodinase messenger ribonucleic acid in rat hypothalamus and pituitary and its regulation by thyroid hormone.

To identify the specific locations of type 2 deiodinase (D2) messenger RNA (mRNA) in the hypothalamus and pituitary gland and determine its regulation by thyroid hormone, we performed in situ hybridization histochemistry, Northern analysis, and quantitative RT-PCR in euthyroid, hypothyroid, and hyperthyroid rats. By in situ hybridization histochemistry, silver grains were concentrated over ependymal cells lining the floor and infralateral walls of the third ventricle extending from the rostral tip of the median eminence (ME) to the infundibular recess, surrounding blood vessels in the arcuate nucleus (ARC), and in the ME adjacent to the portal vessels and overlying the tuberoinfundibular sulci. Silver grains also accumulated over distinct cells in the midportion of the anterior pituitary. In hypothyroid animals, an increase in signal intensity was observed in the caudal hypothalamus, and a marked increase in the number of positive cells occurred in the anterior pituitary. Microdissection of the hypothalamus for Northern and PCR analysis established the authenticity of D2 mRNA in the caudal hypothalamus, and confirmed that the majority of D2 mRNA is concentrated in this region. The distribution of D2 mRNA suggests its expression in specialized ependymal cells, termed tanycytes, originating from the third ventricle. Thus, the tanycyte is the source of the high D2 activity previously found in the ARC-ME region of the hypothalamus. The results indicate that tanycytes may have a previously unrecognized integral role in feedback regulation of TSH secretion by T4.

Localization and gene expression of the receptor for pituitary adenylate cyclase-activating polypeptide in the rat brain.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a recently identified member of the secretin/vasoactive intestinal polypeptide (VIP) family. There are at least two types of receptor for PACAP: type I (PACAPR), which specifically binds PACAP; and type II (VIP/PACAPR), which binds both PACAP and VIP. The localization of PACAPR in the rat brain was determined by in situ hybridization and immunocytochemistry. We raised antisera against a synthetic peptide that corresponds to the carboxy-terminal cytoplasmic domain which is found in all subtypes of PACAPR in order to localize PACAPR-like immunoreactivity (PACAPR-LI) in the rat brain. In general, the distribution of PACAPR-LI correlated well with the distribution of PACAPR transcripts. Particularly strong PACAPR mRNA expression was detected in the olfactory bulb, hippocampus, cerebellum and hypothalamus and moderate labeling was detected in other scattered regions. At the cellular level, PACAPR-LI appeared to be concentrated predominantly in neuronal perikarya and dendrites. At the ultrastructural level, strong immunostaining for the PACAPR was found in plasma membranes, rough endoplasmic reticulum, cytoplasmic matrix, and at synapses. This study provides the basis for a better understanding of the functions of PACAP in the rat brain.

Glucocorticoids inhibit stress-induced phosphorylation of CREB in corticotropin-releasing hormone neurons of the hypothalamic paraventricular nucleus.

The corticotropin-releasing hormone (CRH) gene contains a perfect palindromic motif in its promoter region that allows binding of the cyclic adenosine monophosphate response element binding protein, CREB. Since previous studies suggest that the CRH gene can be activated by cyclic adenosine monophosphate, we determined whether stress and feedback inhibition by glucocorticoids in CRH-producing neurons in the hypothalamic paraventricular nucleus could be mediated by changes in the phosphorylation of CREB. Antisera to CREB and phospho-CREB Ser133 (PCREB), the active phosphorylated form of CREB, were used for immunohistochemical studies on rat brain. In nonstressed animals CREB immunostaining was confined to the nucleus of cells ubiquitously throughout the hypothalamus, while PCREB immunostaining was discretely localized in magnocellular neurons and only a few cells in the medial parvocellular subdivision of the paraventricular nucleus. Ether and handling stress markedly increased the number of PCREB-labeled neurons in the parvocellular subdivision. Double immunolabeling with CRH antiserum revealed that the majority of hypophysiotropic CRH neurons in stressed animals expressed PCREB. Following systemic administration of dexamethasone (100 micrograms/day) for 2.5 days, PCREB immunostaining was completely abolished in parvocellular CRH-producing neurons after ether or handling stress. Dexamethasone had no apparent effect on CREB immunostaining. These results demonstrate that glucocorticoids suppress CREB phosphorylation in hypophysiotropic CRH neurons and suggest that prevention of CREB phosphorylation is a possible mechanism for feedback inhibition of CRH biosynthesis by glucocorticoids.

Leptin prevents fasting-induced suppression of prothyrotropin-releasing hormone messenger ribonucleic acid in neurons of the hypothalamic paraventricular nucleus.

Prolonged fasting is associated with a number of changes in the thyroid axis manifested by low serum T3 and T4 levels and, paradoxically, low or normal TSH. This response is, at least partly, caused by suppression of proTRH gene expression in neurons of the hypothalamic paraventricular nucleus (PVN) and reduced hypothalamic TRH release. Because the fall in thyroid hormone levels can be blunted in mice by the systemic administration of leptin, we raised the possibility that leptin may have an important role in the neuroendocrine regulation of the thyroid axis, through effects on hypophysiotropic neurons producing proTRH. Adult male, Sprague-Dawley rats were either fed normally, fasted for 3 days, or fasted and administered leptin at a dose of 0.5 microg/gm BW i.p. every 6 h. Fasted animals showed significant reduction in plasma total and free T4 and T3 levels compared with controls, that were restored toward normal by the administration of leptin. Percent free T4, but not percent free T3, increased during fasting, further suggesting a reduction in plasma transthyretin levels that did not return to fed levels after leptin administration. By semiquantitative analysis of in situ hybridization autoradiograms, proTRH messenger RNA in medial parvocellular PVN neurons was markedly suppressed in the fasting animals but was restored to normal by leptin administration [fed vs. fast vs. fast/leptin (density units x 10(8)): 8.5 +/- 0.4, 3.2 +/- 0.2, 8.1 +/- 0.8]. In contrast, proTRH messenger RNA in adjacent neurons in the lateral hypothalamus that do not have a hypophysiotropic function remained unchanged by any of the experimental manipulations. These findings indicate that leptin has a selective, central action to modulate the hypothalamic-pituitary-thyroid axis by regulating proTRH gene expression in the PVN but does not have peripheral effects on thyroid-binding proteins. We propose that the fall in circulating leptin levels during fasting resets the set point for feedback inhibition by thyroid hormones on the biosynthesis of hypophysiotropic proTRH, thereby allowing adaptation to starvation.

Calcium-sensing receptor in the rat hippocampus: a developmental study.

The extracellular Ca2+ (Ca2+(o))-sensing receptor (CaR) plays a key role in maintaining near constancy of Ca2+(o) in mammals through its presence in parathyroid gland and kidney. The CaR is also present in brain, and although its role(s) in the brain is not known, it is possible that small changes in Ca2+(o) modify essential physiological and pathological processes, since calcium is crucial for numerous neuronal functions. Northern analysis has revealed that the CaR mRNA is present in hippocampus and several other regions of the brain. The hippocampus is an important site for learning and memory, but the relevance of the CaR to these processes is unknown. Long-term potentiation (LTP), a putative in vitro analog of memory, can only be induced after 7-10 days postnatally in rat hippocampus. Therefore, in the present study we determined the time course for the developmental expression of the CaR in rat hippocampus to assess its relationship to the development of other important hippocampal functions, such as the capacity for induction of LTP. Northern and Western analyses showed that CaR mRNA and protein were expressed at low levels at 5 days postnatally but then increased markedly at 10 days. A high level of receptor expression, due primarily to an increase in a 7.5 kb transcript, persisted until 30 days, when it gradually decreased by 3-fold to reach the adult level of expression. In situ hybridization histochemistry and immunohistochemistry revealed CaR mRNA and protein in pyramidal cells of all the layers of hippocampus and in granule cells of the dentate gyrus. The results show that CaR expression rises at a time when LTP can first be induced in hippocampus and persists at high levels during the time when brain development is proceeding most rapidly. Further studies are needed to determine the role of the CaR in the development of important aspects of the function of hippocampus and other regions of brain, including LTP.

Opiate withdrawal increases ProTRH gene expression in the ventrolateral column of the midbrain periaqueductal gray.

The midbrain periaqueductal gray matter (PAG) has a critical role in the modulation of behavioral and autonomic manifestations of the opiate withdrawal syndrome. We report a nearly 5-fold increase in proTRH gene expression in neurons of the ventrolateral column of the PAG following naltrexone precipitated morphine withdrawal. The accumulation of immunoreactive proTRH-derived peptides, but not the mature TRH tripeptide was concomitantly observed in these cells. These findings indicate that proTRH-derived peptides synthesized in neurons of the ventrolateral PAG may function as modifiers of opiate withdrawal responses.

Localization of pituitary adenylate cyclase-activating polypeptide and its messenger ribonucleic acid in the rat testis by light and electron microscopic immunocytochemistry and in situ hybridization.

Pituitary adenylate cyclase-activating-polypeptide (PACAP) is a new member of the secretin/glucagon/vasoactive intestinal peptide family of peptides; it occurs as two amidated forms with 38 (PACAP38) and 27 (PACAP27) amino acids. Rabbit antisera against synthetic PACAP27 were characterized by enzyme-linked immunosorbent assay. One of the antisera, using a high antibody titer, recognized both PACAP27 and PACAP38 and was found useful for immunohistochemistry. The distribution and ultrastructural localization of PACAP-like immunoreactivity (PACAP-LI) in the rat testes at different stages of spermatogenesis were studied with this antiserum. Four oligonucleotide probes, each complementary to a different region covering a different intron-exon junction, were chosen to maximize hybridization based on the predicted secondary structure of PACAP messenger RNA. PACAP-LI was detected in the developing germ cells but not in either Sertoli or Leydig cells. Intense PACAP-LI was found in spermatids situated near the lumen of the seminiferous tubules. Lower levels of PACAP-LI were detected in spermatogonia and primary spermatocytes, but no PACAP-LI was found in mature spermatids, testicular spermatozoa, or epididymal spermatozoa. In spermatids, PACAP-LI was detected during the cap phase and acrosome phase but not in the maturation phase. At the ultrastructural level, numerous gold particles representing PACAP-LI were found in both acrosomal granules and acrosomal caps of spermatids, while a few particles were found in the Golgi complex. Very few gold particles were seen in the acrosome of mature spermatids and spermatozoa. PACAP-LI decreased and finally disappeared from spermatids during the late developmental stages. In situ hybridization indicated that most of the signal was detected near the perimeter of seminiferous tubules in early developing germ cells, especially in spermatogonia and primary spermatocytes, suggesting that transcription of the PACAP gene occurs in spermatogonia and primary spermatocytes. The processing of the prohormone appears to be slow, and mature PACAP only appears in spermatids. These morphological findings suggest that PACAP-like substances, synthesized by germ cells, participate in spermatogenesis, particularly spermiogenesis, probably by an autocrine and paracrine mechanism. However, the possibility that PACAP acts on the Sertoli and/or Leydig cells cannot be excluded.