Tissue and cellular distribution of the extended family of protein kinase C isoenzymes.

Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.

Associations between the human growth hormone-releasing hormone- and neuropeptide-Y-immunoreactive systems in the human diencephalon: a possible morphological substrate of the impact of stress on growth.

Previous studies revealed that stress is a pivotal factor in the regulation of growth. Psychological harassment may result in psychosocial dwarfism with delayed puberty, short stature and depression. Growth hormone (GH) secretion is suppressed by stress, possibly via the attenuation of growth hormone-releasing hormone (GHRH) secretion. However, the morphological substrate of this phenomenon has not been elucidated yet. Since neuropeptide Y (NPY) levels in the plasma is increased by administration of various stressors, the common consensus is that NPY plays a crucial role in the stress response. In the present study, we examined the putative juxtapositions between the NPY- and GHRH-immunoreactive (IR) systems in the human hypothalamus using double-label immunohistochemistry. Our findings revealed that the majority of the GHRH-IR perikarya formed intimate associations with NPY-IR fiber varicosities. The majority of these juxtapositions were found in the infundibular nucleus/median eminence where NPY-IR fiber varicosities often covered a significant surface area of the GHRH neurons. Since the juxtapositions between the GHRH-IR perikarya and NPY-IR fiber varicosities may be functional synapses, they may represent the morphological substrate of stress-suppressed GH secretion. The large number of contacting elements indicates that NPY plays a pivotal role in GH release, and may be considered as a major factor in the attenuation of growth by stress in humans.

Gonadotropin-releasing hormone neurons express estrogen receptor-beta.

CONTEXT: Recent identification of the second estrogen receptor (ER) isoform (ER-beta) within GnRH neurons of the rodent brain has generated much enthusiasm in the field of neuroendocrine research by questioning the dogma that GnRH cells do not directly sense changes in circulating estrogens. OBJECTIVE: To address the issue of whether GnRH neurons of the human hypothalamus also contain ER-beta, we have performed dual-label immunocytochemical studies. DESIGN: Tissue sections were prepared from autopsy samples of male human individuals (n = 8; age < 50 yr), with sudden causes of death. Technical efforts were made to minimize postmortem interval (<24 h), optimize tissue fixation (use of a mixture of 2% paraformaldehyde and 4% acrolein for four tissue samples), and sensitize the immunocytochemical detection of ER-beta (application of silver-intensified nickel-diaminobenzidine chromogen). MAIN OUTCOME MEASURE: Distribution and percent ratio of GnRH neurons that also contained ER-beta immunoreactivity were analyzed under the light microscope. RESULTS: With acrolein in tissue fixative, nuclear ER-beta immunoreactivity was observed in 10.8-28.0% of GnRH neurons of the four different individuals. ER-beta-containing GnRH neurons were widely distributed in the hypothalamus, without showing a noticeable preference in regional location. CONCLUSIONS: The demonstration of ER-beta and the previous lack of detection of ER-alpha in human GnRH cells indicate that estrogens may exert direct actions upon GnRH neurons exclusively through ER-beta. In the light of differing ligand-binding characteristics of ER-beta from those of ER-alpha, this discovery offers a potential new approach to influence estrogen feedback to GnRH neurons through ER-beta-selective receptor ligands.

Three-dimensional representation of the neurotransmitter systems of the human hypothalamus: inputs of the gonadotrophin hormone-releasing hormone neuronal system.

The gonadotrophin-releasing hormone (GnRH) represents the final common pathway of a neuronal network that integrates multiple external and internal factors to control fertility. Among the many inputs GnRH neurones receive, oestrogens play the most important role. In females, oestrogen, in addition to the negative feedback, also exhibits a positive feedback influence upon the activity and output of GnRH neurones to generate the preovulatory luteinising hormone surge and ovulation. Until recently, the belief has been that the GnRH neurones do not contain oestrogen receptors and that the action of oestrogen upon GnRH neurones is indirect, involving several, oestrogen-sensitive neurotransmitter and neuromodulator systems that trans-synaptically regulate the activity of the GnRH neurones. Although this concept still holds for humans, recent studies indicate that oestrogen receptor-beta is expressed in GnRH neurones of the rat. This review provides three dimensional stereoscopic images of GnRH-immunoreactive (IR) and some peptidergic (neuropeptide Y-, substance P-, beta-endorphin-, leu-enkaphalin-, corticotrophin hormone-releasing- and galanin-IR) and catecholaminergic neurones and the communication of these potential oestrogen-sensitive neuronal systems with GnRH neurones in the human hypothalamus. Because the post-mortem human tissue does not allow the electron microscopic identification of synapses on GnRH neurones, the data presented here are based on light microscopic immunocytochemical experiments using high magnification with oil immersion, semithin sections or confocal microscopy.

Morphological substrate of the catecholaminergic input of the vasopressin neuronal system in humans.

It has been postulated that the stress response is associated with water balance via regulating vasopressin release. Nausea, surgical stress and insulin-induced hypoglycaemia were shown to stimulate vasopressin secretion in humans. Increased vasopressin release in turn induces water resorption through the kidneys. Although the mechanism of the stress-mediated vasopressin release is not entirely understood, it is generally accepted that catecholamines play a crucial role in influencing water balance by modulating the secretion of vasopressin. However, the morphological substrate of this modulation has not yet been established. The present study utilised double-label immunohistochemistry to reveal putative juxtapositions between tyrosine hydroxylase (TH)-immunoreactive (IR) catecholaminergic system and the vasopressin systems in the human hypothalamus. In the paraventricular and supraoptic nuclei, numerous vasopressin-IR neurones received TH-IR axon varicosities. Analysis of these juxtapositions with high magnification combined with oil immersion did not reveal any gaps between the contacted elements. In conclusion, the intimate associations between the TH-IR and vasopressin-IR elements may be functional synapses and may represent the morphological basis of vasopressin release modulated by stressors. Because certain vasopressin-IR perikarya receive no detectable TH innervations, it is possible that additional mechanisms may participate in the stress-influenced vasopressin release.

Estrogen stimulates galanin expression within luteinizing hormone-releasing hormone-immunoreactive (LHRH-i) neurons via estrogen receptor-beta (ERbeta) in the female rat brain.

Among the many factors that integrate the activity of the luteinizing hormone-releasing hormone (LHRH) neuronal system, estrogens play the most important role. Until recently, the belief has been that the LHRH neurons do not contain estrogen receptors and that the action of estrogen upon LHRH neurons is indirect involving several, estrogen-sensitive neurotransmitter and neuromodulator systems that regulate the activity of the LHRH neurons. Based on our recent findings that LHRH neurons of the female rat co-express galanin, that galanin is a potent LHRH releasing peptide, and that estrogen receptor-beta (ERbeta) is present in LHRH neurons, we have evaluated the effect of 17beta-estradiol on the expression of galanin within LHRH neurons. By combining immunocytochemistry for LHRH and in situ hybridization histochemistry for galanin, we demonstrate that 17beta-estradiol stimulates galanin expression within 2h following their administration to ovariectomized rats. Maximal expression, however, required a longer treatment regimen (3 days). These observations strongly suggest that estrogens stimulate galanin expression within LHRH neurons directly, via ERbeta. Moreover, ERbeta may mediate, at least in part, the positive feedback effect of estrogens during the preovulatory LHRH and subsequent LH surges.

Current status of brain hypophysiotropic factors: morphologic aspects.

Nearly 40 putative neurotransmitters and other chemical messengers, mostly peptides, are present in the median eminence that constitutes the final common pathway for signals from the brain to the pituitary. The majority of them are produced in perikarya located in different nuclei of the hypothalamus; however, some of them arise from the brainstem. The neurons contacting capillaries of the median eminence (hypophysiotropic neurons) are intermixed with neurons containing the same transmitter (hypophysiotropic factor(1)), but projecting to other areas of the brain. Depending on their site of release, the hypophysiotropic factors may function as neurohormones acting an the pituitary or neurotransmitters affecting the activity of other neurons in the central nervous system. Based on retrograde tracing studies in combination with immunocytochemistry, the origin of many nerve terminals in the median eminence has been determined.

Juxtapositions between the somatostatinergic and growth hormone-releasing hormone (GHRH) neurons in the human hypothalamus.

Somatostatin is a 14-28 amino acid peptide that is located not only in the gastrointestinal system but also in multiple sites of the human brain. The inhibitory effect of somatostatin on the growth hormone (GH) secretion of the pituitary gland is a well-established phenomenon. There is a general consensus that somatostatin is released into the hypophysial portal blood and modulates GH secretion by hormonal action. In the present study, we explored the possibility that in addition to the hormonal route, somatostatin may also influence GH secretion via influencing the growth hormone-releasing hormone (GHRH) secretion by direct contacts that may be functional synapses. Since the verification of these putative synapses by electron microscopy is virtually impossible in humans due to the long post mortem time, in order to reveal the putative somatostatinergic-GHRH juxtapositions, light microscopic double-label immunohistochemistry was utilized. By examining the slides with high magnification, we observed that the vast majority of the GHRH perikarya received contacting somatostatinergic axonal varicosities in the arcuate nucleus. In contrast, GHRH axonal varicosities rarely contacted somatostatinergic perikarya. The morphology and the abundance of somatostatin to GHRH juxtapositions indicate that these associations are functional synapses, and they represent, at least partially, the morphological substrate of the somatostatin-influenced GHRH secretion. Thus, in addition to influencing the GH secretion directly via the hypophysial portal system, somatostatin may also modulate GH release from the anterior pituitary by regulating the hypothalamic GHRH secretion via direct contacts. The rare GHRH to somatostatin juxtapositions indicate that the negative feedback effect of GH targets the somatostatinergic system directly and not via the GHRH system.

Induction of enkephalin in tuberoinfundibular dopaminergic neurons during lactation.

Single- and double-labeling immunocytochemistry was used to demonstrate that the tuberoinfundibular dopaminergic (TIDA) neurons of lactating rats, in contrast to those of male or female rats on any day of the estrous cycle, synthesize enkephalin which is colocalized with dopamine. Each enkephalin-immunopositive perikaryon in the dorsomedial and ventrolateral subdivisions of the arcuate nucleus contains dopamine; therefore, the median eminence of lactating rats contains high levels of enkephalin compared to male or female rats. It has been shown that endogenous opiates, including enkephalin, stimulate PRL secretion by reversing the inhibitory action of dopamine at the level of the TIDA neurons. The present findings suggest that enkephalin, coproduced with dopamine in TIDA neurons of lactating rats, may be an endogenous source for this action and maintain elevated PRL and milk secretion during the nonsuckling periods of lactation.

The hypophysiotropic neurotensin-immunoreactive neuronal system of the rat brain.

The present study describes the distribution of hypophysiotropic neurotensin-immunoreactive (NTi) perikarya in the rat hypothalamus. After peripheral administration of the retrograde tracer Fluoro-Gold, NT immunoreactivity was demonstrated with fluorescence immunocytochemistry using Texas red-labeled avidin. The results indicate that approximately 70% of all NTi neurons that are connected to the hypophysial portal system are located in the arcuate nucleus. Approximately 30% of these hypophysiotropic NTi neurons reside in the parvocellular subdivisions of the paraventricular nucleus. Our results also show that both the arcuate and the paraventricular nuclei contain NTi perikarya that do not project to the median eminence.

Evidence for a negative ultrashort loop feedback regulating galanin release from the arcuate nucleus-median eminence functional unit.

Recent studies from our laboratory have demonstrated that galanin (GAL) is a member of the hypothalamic-hypophysiotropic hormone family. Most of the hypothalamic hormones regulate their own secretion rate by ultrashort loop feedback mechanisms. The purpose of these studies was to evaluate the possibility that hypothalamic GAL could regulate its own release through a similar mechanism. Galanin secretion from median eminence (ME) fragments incubated in vitro increased exponentially with time, whereas GAL release from arcuate nucleus-ME (AN-ME) fragments depicted a secretory profile consisting of an initial exponential rising phase, followed by a plateau phase in which GAL secretion was apparently abolished. Moreover, preexposure of AN-ME fragments to porcine GAL (pGAL) increased tissue responsiveness to K(+)-induced depolarization, suggesting that pGAL reduced the gain of the system. Thus, after pGAL removal, AN-ME fragments appear to be more sensitive to the depolarizing stimulus. In addition, blockade of GAL biological activity in vivo by administration of a sheep antirat GAL serum increased GAL release from AN-ME fragments in vitro, whereas this treatment did not affect GAL release from ME terminals. These results indicate that GAL neurons may diminish their own activity, establishing, therefore, a negative ultrashort loop feedback that controls the firing of the AN galaninergic network and maintains a balanced physiological status. By means of electron microscopy, we demonstrated that GAL-containing perikarya and proximal dendrites receive synapsing axons immunoreactive for the same peptide in the AN, which provides the anatomical basis for interactions between galaninergic neurons. In conclusion, our data support the notion that the galaninergic system, as other peptidergic neurotransmitters, is able to regulate its own release via a negative ultrashort loop feedback control mechanism that is operative at the level of the AN.

Glucagon-like peptide-1 receptor (GLP1-R) mRNA in the rat hypothalamus.

GLP-1 has been shown to dramatically reduce food intake in fasted rats and is thought to exert its effects by modulating neuronal function in the hypothalamus. To date, little is known about the distribution of GLP1-R and its mRNA in the rodent hypothalamus. The purpose of the present study was to utilize in situ hybridization histochemistry to determine the anatomical distribution of GLP1-R mRNA in the rat hypothalamus. The results of these studies revealed an extensive distribution of GLP1-R mRNA throughout the rostral-caudal extent of the hypothalamus; with a dense accumulation of labeled cells in the supraoptic, paraventricular, and arcuate nuclei. Additional labeled cells were also detected in medial and lateral preoptic areas, periventricular nucleus, ventral division of the bed nucleus of the stria terminalis, lateral hypothalamus, and dorsomedial nucleus. The results of these in situ hybridization histochemical studies have provided detailed and novel information about the distribution of GLP1-R mRNA in the rat hypothalamus. In addition, this morphological data provides important information about the neuronal systems modulated by GLP-1 and their potential role in feeding behavior.

Regulation of progesterone receptor messenger ribonucleic acid in the rat medial preoptic nucleus by estrogenic and antiestrogenic compounds: an in situ hybridization study.

Progesterone receptor (PR) messenger RNA (mRNA) is concentrated in neurons of the preoptic area and other regions of the rat hypothalamus where it is colocalized with the estrogen receptor and regulated by changes in the steroid hormonal milieu. To date, little is known about the regulation of PR mRNA by estrogens and whether antiestrogenic compounds are capable of modulating its expression. The present studies used in situ hybridization to ascertain the time course of PR mRNA regulation in the medial preoptic nucleus by 17beta-estradiol, determine the effective dose required to elicit a response, and compare the efficacy of 17beta-estradiol with a variety of estrogenic or antiestrogenic compounds. The first series of studies revealed that the treatment of ovariectomized rats with 17beta-estradiol resulted in an increase in PR expression within 2 h, after which it remained elevated until 10 h postinjection and then returned to baseline levels. When ovariectomized rats were injected with 25-1000 ng/kg of 17beta-estradiol and euthanized 6 h later, a dose-dependent increase in the level of PR mRNA was observed, with a maximal response at 1000 ng/kg and an EC50 of 93.5 ng/kg. Subsequent studies evaluated the efficacy of a variety of estrogenic and antiestrogenic compounds in the rat preoptic nucleus. 17Beta-estradiol, diethylstilbestrol, and 17alpha-estradiol all significantly increased the level of PR mRNA, although the degree of induction varied with each compound. The injection of tamoxifen, raloxifene, toremifene, droloxifene, clomiphene, GW 5638, or ICI 182,780 had no significant estrogenic effect on PR gene expression at the dose evaluated. In contrast, when tamoxifen or raloxifene, but not ICI 182,780, was administered in the antagonist mode, a significant dose-related decrease in the estradiol-induced level of PR mRNA was seen in the preoptic area. The results of these studies clearly demonstrate that PR mRNA expression in the rat preoptic area is rapidly stimulated by a small dose of 17beta-estradiol. Moreover, the present report has also shown that the estrogenic nature of compounds such as tamoxifen, raloxifene, toremifene, droloxifene, clomiphene, and GW 5638 cannot be predicted by their activity in peripheral tissues. Together, the results of these studies provide important information about the central activity of estrogens and provide evidence for their tissue-specifc actions in the rat.

Evidence for the colocalization of estrogen receptor-beta mRNA and estrogen receptor-alpha immunoreactivity in neurons of the rat forebrain.

Estrogen receptor beta (ER beta) mRNA is expressed in several rat brain regions where ER alpha is abundant. In vitro studies have shown that ER alpha and ER beta can heterodimerize and that the activity of this complex may be different than an ER alpha or ER beta homodimer complex. The purpose of the present study was to ascertain if ER alpha and ER beta are co-expressed by certain neuronal populations using a double label in situ hybridization/immunocytochemistry method. The results revealed that neurons in the bed nucleus of the stria terminalis, medial amygdala and preoptic area contain both ERs, with the vast majority of the neurons being double labeled. In other brain regions including the arcuate nucleus, cortical amygdaloid nuclei and ventromedial nucleus, only a few double-labeled cells were detected, while neurons in the paraventricular nucleus, supraoptic nucleus, and cerebral cortex expressed only ER beta mRNA. The results of these double label experiments provide the first evidence that ER alpha and ER beta coexist in neurons under in vivo conditions and suggest that estrogens may differentially modulate the activity of certain neuronal populations depending on whether the cells expresses ER alpha, ER beta or both ERs.

Biologically active estrogen receptor-beta: evidence from in vivo autoradiographic studies with estrogen receptor alpha-knockout mice.

Estrogen receptor-1 (ER beta) messenger RNA (mRNA) has been detected in the brain of wild-type and estrogen receptor-alpha knockout (ER alphaKO) mice. The present study used in vivo autoradiography to evaluate the binding of 125I-estrogen, a compound with a similar affinity for both ERs to ascertain whether ER beta mRNA is translated into biologically active receptor. Mice were injected with 125I-estrogen, and sections were mounted on slides and opposed to emulsion. After exposure, labeled cells were seen in ER alphaKO brain regions where ER beta is expressed (preoptic and paraventricular nuclei of the hypothalamus; bed nucleus of the stria terminalis; amygdala; entorhinal cortex; and dorsal raphe). Competition studies with 17beta-estradiol eliminated binding in the ER alphaKO brain, whereas 16alphaIE2, an ER alpha selective agonist and dihydrotestosterone had no effect. In contrast, competition studies with 16alphaIE2 in wild-type mice eliminated 125I-estrogen binding to ER alpha and resulted in a pattern of residual binding comparable to that seen in the ER alphaKO brain. The results demonstrate that residual estrogen binding sites are present in regions of the ER alphaKO brain where ER beta is expressed, brain regions that were also seen after eliminating binding to ER alpha in wild-type mice. These data provide the first evidence that ER beta mRNA is translated into a biologically active protein in the rodent brain.

Sexual differences in the distribution of neurons coexpressing galanin and luteinizing hormone-releasing hormone in the rat brain.

We have recently reported that a subpopulation of galanin (GAL)-immunoreactive perikarya in the preoptic area near the organum vasculosum of the lamina terminalis (OVLT) has morphological characteristics similar to those of LHRH-containing neurons. In fact, both peptides are colocalized in those neurons in the male rat brain. In these studies we describe sexual differences in the incidence of neurons colocalizing GAL and LHRH in this region. In male rats, about 20% of LHRH-immunoreactive cells in the diagonal band of Broca and the medial preoptic area are also immunoreactive for GAL. In contrast, in female rats, about 65% of LHRH-containing perikarya near the OVLT are immunostained for GAL. In addition, GAL and LHRH levels were measured in tissue extracts containing either the OVLT and surrounding areas or the median eminence. The data indicate that the preoptic area, including the OVLT, contains a significantly higher amount of GAL in females killed in proestrus than in those killed in estrus. The amount of GAL measured in males is lower than that in the proestrous females, but somewhat greater than that present in the estrous females. There were no significant differences among the three groups in LHRH content in this region. The GAL content of the median eminence was the highest in proestrous females, followed by estrous females and males. The LHRH content in the median eminence was not significantly different among the three groups. In conclusion, these observations indicate that the coexpression of GAL with LHRH in a discrete subpopulation of LHRH-producing neurons is a sex-related phenomenon. The fact that changes in GAL levels in specific regions can be seen at different times during the estrous cycle suggests that gonadal steroids, possibly estrogens, may physiologically regulate the expression of GAL in this subpopulation of LHRH neurons.

Galanin-immunoreactive axons innervate somatostatin-synthesizing neurons in the anterior periventricular nucleus of the rat.

The anterior periventricular nucleus (PeN) regulates GH secretion by synthesizing and releasing somatotropin release-inhibiting factor (SRIF) into the portal circulation. This territory of the diencephalon is heavily innervated by axons of galanin (GAL)-immunoreactive (IR) neurons. The connections between GAL-IR fibers and hypophysiotropic SRIF neurons were studied by means of immunocytochemical double labeling at the light and electron microscopic levels. Retrograde axonal labeling with Fluoro-Gold revealed the anterior PeN as the main site of hypophysiotropic SRIF-synthesizing neurons. These cells were densely surrounded by GALergic axons that made contacts with their cell bodies and dendrites. At the ultrastructural level, diaminobenzidine-labeled SRIF neurons received synapsing GAL-IR axons marked with silver-gold particles. Both axo-somatic and axo-dendritic forms of connections were observed. These morphochemical data revealed an interaction between GAL- and SRIF-synthesizing neurons in the anterior PeN. Furthermore, the presence of synaptic connections between these neuronal systems suggests a role for GAL in the control of SRIF secretion and, in turn, in the regulation of GH release.

Identification and immunocytochemical localization of a new thyrotropin-releasing hormone precursor in rat brain.

Antisera were raised to a tridecapeptide, Ser-Asp-Val-Thr-Lys-Arg-Gln-His-Pro-Gly-Arg-Arg-Phe, that was synthesized based on the sequence (residues 166-178) of a proposed cDNA for pro-TRH reported by Lechan et al. With this antiserum, immunostaining of Western blots of rat brain extracts revealed two major proteins with mol wt (Mr = 39,000 and 52,000) considerably larger than that of the largest protein (Mr = 29,000) that could be encoded by the cDNA of Lechan et al. Because these observations suggested the possibility of novel TRH precursors, we studied the immunocytochemical distribution of pro-TRH (39-52K) in rat brain. Our anatomical findings were 4-fold. 1) The distributions of 29K pro-TRH and 39-52K pro-TRH are not identical. 2) TRH is found only in regions containing 29K pro-TRH, 39-52K pro-TRH, or both. 3) There are regions that contain both 29K pro-TRH and 39-52K pro-TRH, but no TRH. 4) Regions containing only 39-52K pro-TRH do not contain 29K pro-TRH mRNA as mapped by Segerson et al. From these electrophoretic and anatomical observations, we postulate the existence of at least one and possibly two additional precursors that can be processed to TRH in rat brain.

Aging impairs galanin expression in luteinizing hormone-releasing hormone neurons: effect of ovariectomy and/or estradiol treatment.

In the medial preoptic area and the diagonal band of Broca, a subset of LHRH neurons coexpresses galanin at a 4- to 5-fold higher rate in female than male rats, suggesting that estradiol (E2) plays a key role in galanin gene expression within LHRH neurons. In the present studies we investigated the incidence of colocalization of these peptides in different age groups, i.e. 2-, 10-, 18-, and 24-month-old intact female Fisher rats; 24-month-old E2-treated rats; 24-month-old ovariectomized (OVX) rats; and 24-month-old OVX E2-treated rats with single or double labeling immunocytochemistry. For cell counting, we took advantage of the typical fusiform morphology of galanin-immunoreactive neurons that colocalize LHRH. The presence of both peptides in the same perikaryon was substantiated by double staining representative sections from each brain. Our observations indicate that the number of galanin/LHRH-coexpressing perikarya dramatically decreased with age. Although in 18-month-old rats a moderate decline was observed, in 24-month-old female rats no, or only a few, faintly stained, fusiform galanin-immunopositive perikarya were present. Although galanin was absent from LHRH neurons of aged rats, their LHRH content was not altered. E2 treatment of intact 24-month-old rats had no effect on the low incidence of colocalization. However, when OVX 24-month-old rats were E2 treated, the incidence of colocalization increased to the level seen in 2- or 10-month-old estrous animals. Our observations on the presence of colocalizing perikarya in intact and E2-treated aged animals provide further evidence for the key role of E2 in galanin gene expression within LHRH neurons and emphasize that some ovarian factor(s) may blunt this effect.

Sexual dimorphism in copackaging of luteinizing hormone-releasing hormone and galanin into neurosecretory vesicles of hypophysiotrophic neurons: estrogen dependency.

Hypophysiotrophic neurons projecting to hypophyseal portal vessels in the median eminence of the hypothalamus maintain the operation of the master gland, the pituitary, by secreting releasing and release-inhibiting hormones into the bloodstream. LHRH, synthesized in neurons of the rat prosencephalon, is one of the key substances that governs the anterior pituitary-gonadal axis. Recently, it has been shown that the peptide galanin (GAL) is coproduced in a subpopulation of LHRH neurons and is a potent modulator of central processes regulating reproduction. A better understanding of the secretory mechanisms involved in pulsatile hormone release from LHRH axons of the median eminence requires exploration of the organelle domain that displays the cosynthesized peptides in terminal boutons. This study shows that LHRH- and GAL-immunoreactive axons overlap heavily in the lateral part of the median eminence. Double fluorescent labeling revealed colocalization of the peptides at the level of single axon terminals. By means of dual colloidal gold immunolabeling, LHRH and GAL were detected in the same secretory vesicles at the ultrastructural level. The incidence of colocalizing vesicles was high in the female (45%) and low in the male (3%) rat. Ovariectomy resulted in a dramatic decline in the number of LHRH/GAL-coexpressing vesicles (23%), which was reversed (55%) by the administration of estradiol. The observations indicate a sex-related difference in the packaging of LHRH and GAL and suggest that the events are estrogen dependent. Furthermore, the simultaneous release of GAL and LHRH from the colocalizing vesicles provides a mechanism that might ensure the potentiating effect of GAL on LHRH by synchronizing events at the receptor sites in the anterior pituitary.