Immunoreactive Luteinizing Hormone-releasing Hormone Research Articles

The effect of prolonged stress on the hypothalamic luteinizing hormone-releasing hormone (LHRH) in the anoestrous ewe.

A study was performed to examine the effect of the intermittent long-term (20 min/h daily during 3 consecutive days) electric stress (footshocks) on the hypothalamic LHRH in anoestrous ewe. On the third day, immediately after the last stimulation, the animals were decapitated and the hypothalami and pituitaries were taken for analysis of LHRH and LH by immunocytochemistry; LHRH was also assayed by radioimmunoassay. In the stressed animals the concentration of immunoreactive LHRH (IR-LHRH) and LHRH analysed by radioimmunoassay (RI-LHRH) increased significantly in the median eminence (ME) and the medial preoptic area (MPOA) when compared with the controls. Additionally, IR-LHRH perikarya appeared in the MPOA of stressed animals, whereas they were not seen in the controls. RI-LHRH in the medial basal hypothalamus (MBH) of stressed ewes was markedly lower than in control ones. No significant differences in RI-LHRH concentration were found in the septum between stressed and control ewes. The present study clearly indicates that prolonged stressful stimuli modify LHRH concentration in the discrete areas of the hypothalamus of the anoestrous ewe. It is suggested that these changes are attributed mainly to an inhibition of LHRH release from the ME and a suppression of LHRH transport along neural fibres within the hypothalamus.

Effect of protein deficiency on luteinizing hormone releasing hormone (LHRH), gonadotropin releasing hormone associated peptide (GAP) and luteinizing hormone (LH) immunocytochemistry in the hypothalamus and pituitary gland of prepubertal ewes.

Growing female lambs were fed diets containing 14.2% (standard) or 8.1% (protein restricted) of proteins to determine their effects on puberty and luteinizing hormone releasing hormone (LHRH), gonadotropin hormone associated peptide (GAP), luteinizing hormone (LH) hormonal system. At the end of the experiment (30-34 weeks of age), hypothalamic LHRH, GAP and pituitary LH were analysed by immunocytochemical methods using specific antibodies. Plasma LH were determined by radioimmunoassay at 21 weeks of age. It was found that lowering of the dietary proteins content decreased the concentration of basal plasma LH significantly in lambs of 21 weeks of age. None of the sheep of this group reached sexual maturity at the same time as the animals of the standard group. However, immunoreactive (ir) LHRH neuronal system of protein restricted lambs was normally developed: Numerous irLHRH perikarya, dense network of axons and abundant material stored in the nerve terminals were well visualized in the typical sites of the preoptico-septal area, hypothalamus and the median eminence (ME). Gonadotropin associated peptide (GAP) of the LHRH precursor was present in the same populations of neurons that contained LHRH in the sheep brain. The proportion of pituitary LH-cells was three fold higher in pituitaries of the nutritionally restricted group. They displayed hypertrophy and very strong immunoreaction. These results show that protein deficiency in diets of growing female sheep delays their puberty but does not impair the synthesis and processing of LHRH in the brain neurons and synthesis of LH in pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Olfactory neuroepithelioma: An immunohistochemical and ultrastructural study

Three cases of olfactory neuroepithelioma are presented in this report. Histologically, these tumors were composed of small cells with round to oval, relatively hyperchromatic nuclei and scanty cytoplasm. The tumor cells were occasionally observed in tubular formations or rosette-like arrangements. Immunohistochemically, the tumor cells showed a positive reaction for cytokeratin AE1, cytokeratin CAM5.2, Ber-EP4, antisynaptophysin and anti-S100 protein in all cases. In two cases, LH-RH was detected in the tumor cells. Ultrastructurally, the tumor cells had the differentiation features of olfactory epithelium. Olfactory neuroepithelioma is a rare occurrence and it can be very difficult to distinguish olfactory neuroepithelioma from small cell carcinoma, neuroendocrine carcinoma and so-called "olfactory neuroblastoma" on the basis of hematoxylin and eosin stained sections alone. In controversial cases, a diagnosis of olfactory neuroepithelioma must be substantiated by ultrastructural and immunohistochemical findings, particularly regarding the detection of Ber-EP4 and LH-RH immunoreactivity.

Distribution of hypothalamic, hippocampal and other limbic peptidergic neuronal cell bodies giving rise to retinopetal fibers: Anterograde and retrograde tracing and neuropeptide immunohistochemical studies

In our present work utilizing the retrograde or anterograde transport of tracers (biotinylated dextran amine and Fluorogold, respectively) we have provided direct evidence for the cells of origin of the limboretinal pathway in rats and their termination in the retina using light microscopic approach. Administration of biotinylated dextran amine into the vitreous body resulted in nerve cell body labeling in several structures: the supraoptic and paraventricular nuclei, the hippocampus (CA1, CA3), the dentate gyrus, the indusium griseum, the olfactory tubercle, and the medial habenula, all of them belong to the limbic system. We estimated that the total number of retrogradely labeled cells is 1495±516. We have seen fiber labeling in the retinorecipient suprachiasmatic nucleus and in the primary visual center, the lateral geniculate body, but labeled nerve cell bodies in these structures were never seen. Iontophoretic application of Fluorogold into the hippocampal formation, where the major part of the biotinylated dextran amine-labeled cell bodies was observed, resulted in labeled fibers in the optic nerve and in the retina indicating that the retrogradely labeled cells in the hippocampus and the dentate gyrus among others are the cells of origin of the centrifugal visual fibers. Sections showing biotinylated dextran amine labeling were stained for vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactivity using immunohistochemistry. Some biotinylated dextran amine-labeled cells also showed vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactivity. We conclude that the limboretinal pathway exists and that the cells of origin are partially vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactive.

Estrogen stimulates galanin expression within luteinizing hormone-releasing hormone-immunoreactive (LHRH-i) neurons via estrogen receptor-beta (ERβ) 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.

Reproductive activation of pine voles ( Microtus pinetorum): examination of physiological markers

We tested the hypothesis that the presence of an opposite-sex conspecific will result in time-related changes in measures of reproductive activation. We housed male–female pairs of pine voles together for 0, 2, 6, 12, or 24 h before collecting blood, reproductive organs and brains for immunocytochemical analysis of LHRH and c-fos. Control animals were never exposed to an opposite-sex conspecific. Following exposure to a male, there was a significant increase in uterine weight but not in LH levels. In males, there were no changes in peripheral indices of activation, i.e. LH levels, testes and seminal vesicle weights were not altered. Consistent with no change in circulating levels of LH, there was no change in LHRH immunoreactivity at any time. However, c-fos immunoreactivity was significantly greater in both males and females in the cingulate cortex and rostral bed nucleus of the stria terminalis (BNST) at 2 h, and in the caudal BNST at 2, 6 and 12 h. Similarly, c-fos immunoreactivity was increased in the rostral MPOA in both males and females at 2 and 6 h. However, in the caudal MPOA, there was a significant interaction between sex and time due to increased c-fos immunoreactivity in females only at 6 h. These results indicate that, in both male and female pine voles, exposure to an opposite-sex conspecific is sufficient to produce rapid, neural activation in brain areas known to be involved in reproductive activation and sexual behavior. This early activation did not occur in LHRH neurons. It is not known if this activation, particularly at early times, is due to reproductive activation or to the formation of pair bonds.

Bi-directional associations between galanin and luteinizing hormone-releasing hormone neuronal systems in the human diencephalon

Evidence suggests that galanin plays an important role in the regulation of reproduction in the rat. Galanin is colocalized with luteinizing hormone (LH)–releasing hormone (LHRH) in a subset of LHRH neurons in female rats and galanin-immunoreactive (galanin-IR) nerve terminals innervate LHRH neurons. Recent studies indicate that galanin may control gonadal functions in rats at two different levels: (i) via direct modulation of pituitary LH secretion and/or (ii) indirectly via the regulation of the hypothalamic LHRH release. However, the morphological substrate of any similar modulation is not known in human. In the present series of experiments we first mapped the galanin-IR and LHRH-IR neural elements in human brain, utilizing single label immunohistochemistry. Then, following the superimposition of the maps of these systems, the overlapping sites were identified with double labeling immunocytochemistry and examined in order to verify the putative juxtapositions between galanin-IR and LHRH-IR structures. LHRH and galanin immunoreactivity were detected mainly in the medial basal hypothalamus, in the medial preoptic area and along the diagonal band of Broca. Careful examination of the IR elements in the overlapping areas revealed close, bi-directional contacts between galanin-IR and LHRH-IR structures, which have been verified in semithin plastic sections. These galanin-LHRH and LHRH-galanin juxtapositions were most numerous in the medial preoptic area and in the infundibulum/median eminence of the human diencephalon. In conclusion, the present study is the first to reveal bi-directional juxtapositions between galanin- and LHRH-IR neural elements in the human diencephalon. These galanin–LHRH and LHRH–galanin contacts may be functional synapses, and they may be the morphological substrate of the galanin-controlled gonadal functions in humans.

Neonatal PACAP administration in rats delays puberty through the influence of the LHRH neuronal system.

The onset of puberty is a concerted action of many factors which leads to cyclic LHRH release in rats. It has been demonstrated that; in common with vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase activating polypeptide (PACAP) is also involved in the differentiation of the central nervous system. In our previous work, it was shown that a single PACAP injection into neonatal female rats delayed puberty. In the present work, neonatal administration of PACAP delayed the vaginal opening and decreased the weight of anterior pituitaries, the number of expelled ova at the first ovulation and the intensity of LHRH immunostaining in the septo-preoptico-infundibular system. PACAP antiserum had a reverse effect on LHRH immunoreactivity. The other studied parameters in the latter group remained unchanged compared to control rats. It was concluded that neonatal PACAP administration delayed the onset of puberty through the influence of the LHRH neuronal system.

Jurkat cell proliferative activity is increased by luteinizing hormone-releasing hormone

Jurkat cells were used to study the immunomodulatory role of luteinizing hormone-releasing hormone (LHRH) in immune cells. The Jurkat cell, a human mature leukemic cell line, phenotypically resembles resting human T lymphocytes and has been widely used to study T cell physiology. The data from this study demonstrate that the Jurkat cell concentration of immunoreactive LHRH was 210 +/- 36 pg/10(6) cells and that of proLHRH was 188 +/- 27 pg/10(6) cells (means +/- S.E.M.). The authenticity of this LHRH immunoreactivity is documented in two ways. First, both Jurkat LHRH and proLHRH immunoreactivity demonstrate dilutional parallelism with hypothalamic LHRH and proLHRH. Second, Jurkat lysates show LHRH bioactivity by releasing luteinizing hormone from rat anterior pituitary cells in culture. The presence of substantial amounts of LHRH in medium in which Jurkat cells were cultured for 72 h indicated that LHRH can be released from the cells. Using specific primers to exons 2 and 4 of the LHRH gene, we have found that Jurkat cells (like human T cells) express LHRH mRNA. The LHRH agonist, des-Gly10,D-Trp6-LHRH ethylamide, significantly increases the proliferative activity of Jurkat cells, as assessed by tritiated thymidine incorporation, from 15980 +/- 1491 c.p.m. in control to 28934 +/- 3395, 30457 +/- 3861 (P = 0.05 vs control) or 35299 +/- 5586 c.p.m. (P < 0.01 vs control) with 10(-11), 10(-9) or 10(-7) M agonist respectively. LHRH antagonist, [D-pGlu1,D-Phe2,D-Trp3,6]-LHRH, at a concentration of 10(-8) M decreases Jurkat cell proliferative activity form 17145 +/- 526 c.p.m. in control medium to 10653 +/- 1323 c.p.m. (P = 0.05). Co-incubation with the LHRH antagonist completely inhibits the proliferative stimulation induced by the LHRH agonist. Furthermore, applying monoclonal LHRH antibody to Jurkat cells inhibits the cell proliferative activity assessed by tritiated thymidine incorporation from 19900 +/- 2675 c.p.m. in control to 15680 +/- 2254, 15792 +/- 1854 and 9700 +/- 908 c.p.m. in media with 1:40, 1:20 and 1:10 dilution of purified antibody respectively (P < 0.01, 1:10 dilution compared with control). In addition, the cAMP level in LHRH-stimulated Jurkat cells is decreased to 74, 27 and 57% of control levels after 15, 30 and 45 min respectively of exposure to 10(-7) M LHRH agonist. In summary, Jurkat cells produce, process and release immunoreactive and bioactive LHRH, as do normal human T cells. Endogenous and exogenous LHRH increase Jurkat cell proliferative activity, and cAMP may be involved in LHRH-induced Jurkat cell proliferation. The Jurkat cell may be a useful model with which to study the role of LHRH in human T cell function.

Migration of luteinizing hormone-releasing hormone (LHRH) neurons in early human embryos.

Luteinizing hormone-releasing hormone (LHRH) neurons originate in the epithelium of the medial olfactory pit and migrate from the nose into the forebrain along nerve fibers rich in neural cell adhesion molecule (N-CAM). The present study examined the ontogenesis of LHRH neurons in early human embryos and found a similar pattern of development of these cells. Luteinizing hormone-releasing hormone immunoreactivity was detected in the epithelium of the medial olfactory pit and in cells associated with the terminal-vomeronasal nerves at 42 (but not 28-32) days of gestation. The migration route of these cells was examined with antibodies to N-CAM and antibodies to polysialic acid (PSA-N-CAM), which is present on N-CAM at certain stages of development. Neural cell adhesion molecule immunoreactivity was present in a population of cells in the olfactory placode of the earliest embryos examined (28-32 days) and later (42 and 46 days) throughout the migration route. The PSA-N-CAM immunoreactivity was not detected until 42 days and was present in a more limited distribution in nerve fibers streaming from the olfactory placode and along the caudal part of the migration route below the forebrain. Previous studies have indicated that the highly sialated form of N-CAM is less adhesive. The PSA-N-CAM may therefore facilitate the migration of these cells by lessening the adhesion between the fascicles that make up the migration route, expediting the passage of cords of LHRH cells between the nerve fibers as these cells move toward the brain.

Immunohistochemical analysis of neurotransmitters of the subfornical organ.

The localizations of peptides and putative neurotransmitters in the subfornical organ of the rabbit, rat and guinea pig were analyzed by using immunohistochemical methods. The variations that occurred in the three species were investigated. Immunoreactivities including serotonin (5-HT), neurotensin (NT), vasopressin (VP), luteinizing hormone releasing hormone (LHRH) and FMRFamide (Phe-Met-Arg-Phe-NH2) were examined in the subfornical organ. Nerve fibers that displayed 5-HT-positive immunoreactivity were observed in all species examined. Some immunoreactive perikarya were detected in guinea pigs and rabbits. Neurotensin-positive immunoreactivity was weak in the subfornical organ. LHRH immunoreactivity was detected in the rabbit only. Conspicuous vasopressin-positive immunoreactive cell bodies and fibers were detected in the subfornical organ of the rat, rabbit and guinea pig. Mild FMRFamide-positive immunoreactive fibers were observed in the rabbit and rat and no reaction was shown in the guinea pig by the PAP immunolabeling technique. Each neurotransmitter had a specific pattern of distribution in the SFO, though there were some overlapping reactive areas. Dramatic differences were demonstrated for fiber density among species.

A relative depletion of luteinizing hormone-releasing hormone was observed in the median eminence of young but not middle-aged rats on the evening of proestrus.

Computer-assisted analysis was used to examine LHRH reaction product in the median eminence of young and middle-aged rats prior to and after the expected peak of the LH surge on proestrus. The area of LHRH reaction product was analyzed in 5 rostral-caudal levels (A-E) of the median eminence. The relative depletion of LHRH in the median eminence of young females on the evening compared to the afternoon of proestrus suggested LHRH neurosecretion in conjunction with the preovulatory LH surge. The pattern of depletion observed further suggested that LHRH release may occur preferentially from restricted regions of the median eminence or in a coordinated wave-like pattern. Four of the five levels of the median eminence exhibited a relative decrease in LHRH on the evening of proestrus in young females, and this time-related difference in LHRH reaction product was statistically significant in median eminence levels B and C. In contrast, little evidence of a relative depletion in LHRH reaction product from early to late proestrus was observed in the median eminence of aging animals. Moreover, the concentration of the densest LHRH reaction product appeared diminished in the median eminence of middle-aged compared to young females at the time points examined in the present study. The age-related differences observed in LHRH reaction product in the median eminence may contribute to the attenuated LH surge documented in middle-aged female rats. Whether these changes in LHRH immunoreactivity can be attributed to age-related alterations in afferents received by LHRH neuronal cell bodies or terminals or to intrinsic deficits in signalling mechanisms within LHRH neurons remains to be determined. Computer-assisted analysis of the immunocytochemical data enabled the assessment of relative changes in reaction product within specific elements of LHRH neurons in precise regions of the median eminence.

Histochemical and immunocytochemical study of the migration of neurons from the rat olfactory placode

Immunocytochemical and histochemical methods have been used to describe the neuronal population migrating from the rat olfactory placode and to analyze the spatio-temporal evolution of this neuronal migration during development. Several neuronal markers, such as binding to the lectin Ulex europaeus (UEA I) and the presence of neuron-specific enolase (NSE), olfactory marker protein (OMP), and luteinizing hormone-releasing hormone (LHRH), have been tested in order to determine whether migrating neurons originate from both the medial and the lateral parts of the placode and whether they all express LHRH. Our data show that a large population of differentiated migrating neurons can be identified with an antibody against NSE from the 14th day of gestation and with UEA I one day later. Migrating neurons are closely associated with both the vomeronasal axon fascicles emerging from the medial pit and the olfactory axons originating from the lateral pit. However, the neuron migration from the lateral pit appears to be more discrete than that from the medial pit. No LHRH immunoreactivity has been detected among neurons migrating from the lateral pit. Some neurons accompanying the olfactory axon fascicles exhibit a high level of maturation as shown by their OMP-positivity. Numerous neurons positive for both NSE and UEA I have also been observed within the presumptive olfactory nerve layer in early embryonic stages.

Changes in the reproductive system following acute prenatal exposure to ethanol or methylazoxymethanol in the rat: I. Effects on immunoreactive LHRH cell number

It has long been recognized that ethanol (EtOH) interferes with the hypothalamo-pituitary-gonad axis in adults of many species, and recent studies have provided evidence for similar effects after prenatal EtOH exposure. Since EtOH is capable of injuring dividing cells, we investigated the possibility that a single acute in utero EtOH exposure during the period of LHRH neuron formation might change the number of immunoreactive LHRH cells in the hypothalamus. Final LHRH cell division in Long-Evans rats was determined by [3H]thymidine autoradiography to take place over a short period between gestation days 12 and 13. Subsequently, pregnant rats were treated acutely with either EtOH or methylazoxymethanol (MAM), a known neuroteratogen, and the numbers of immunoreactive LHRH cells were counted. On gestation day 22, LHRH-positive cell numbers were significantly fewer than control numbers in both EtOH- and MAM-exposed offspring. On postnatal day 60, cell numbers in EtOH-exposed offspring did not differ from control numbers, whereas cells in MAM-exposed offspring remained significantly reduced. In controls, there were 40% fewer LHRH-positive cells on postnatal day 4 than in late gestation or at maturity. We conclude that 1) acute exposure to a high dose of EtOH at a critical time in early gestation can alter the expression of LHRH in late gestation; 2) exposure to MAM in the same period alters LHRH expression before birth and in the adult; and 3) in the early postnatal period, LHRH expression decreases profoundly.

Peripheral projections of nervus terminalis LHRH-containing neurons in the tiger salamander, Ambystoma tigrinum.

The peripheral projections of the nervus terminalis (NT) have been difficult to examine due to the weak immunoreactivity of the processes to various antibodies. We performed two experimental manipulations in the tiger salamander in an attempt to increase the luteinizing hormone-releasing hormone-immunoreactive (LHRH-ir) labelling in the peripheral processes of the NT:1) the NT was sectioned centrally, or 2) a 100 mg melatonin pellet was embedded subcutaneously for 3 days prior to sacrifice. Following these manipulations, animals were sacrificed and tissue was processed with standard immunocytochemical techniques for the analysis of the distribution of LHRH-ir processes. In the nasal cavity, LHRH-ir fibers were observed projecting 1) into the rostral olfactory epithelium, 2) to Bowman's glands in the lamina propria of the rostromedial olfactory mucosa and ventrolateral mucosa between the main nasal cavity and Jacobson's organ, 3) into the naris constrictor muscle, and 4) along the palatine nerves and ganglia. These lesion and hormone manipulations have enabled the detection of peripheral projections of the NT not observed previously with immunocytochemical procedures alone. The wide distribution of LHRH-ir NT processes in the nasal cavity and cranium suggests that this nerve may influence many different cranial structures during appropriate pheromonal or neuroendocrine events.

Increase in number of LHRH neurones in septal-preoptic area of rats following chronic amitriptyline treatment: implication in antidepressant effect

Recent studies have implicated the peptide LHRH in a variety of actions including a role in modulation of affective behavior. The present study has been undertaken to determine its involvement in the action of antidepressants, if any, using amitriptyline (AMT) as the model antidepressant drug. The repeated administration of AMT (10 mg/kg/day) in rats increased the number of LHRH neurones in the septal-preoptic area. While 1 week of AMT treatment slightly augmented the number of LHRH neurones, the rise was not statistically significant, however, following 2 weeks of AMT treatment, a significant ( P < 0.05) increase (41.05%) was observed. Three and four weeks of AMT treatment further increased the number of neurones by 60.84% and 72.96% respectively; a remarkable rise in the LHRH immunoreactivity around organum vasculosum of lamina terminalis (OVLT) was also noticed. Acute AMT treatment had no effect on the number of neurons; however, the intensity of immunoreaction in the OVLT was slightly decreased. In the behavior despair test, a single dose of AMT displayed an immobility reducing effect which was also shown by a single dose of LHRH (1 mg/kg). The combination of LHRH (1 mg/kg) and AMT also reduced the immobility; the effect was the same as one produced by each drug given separately The results suggest that chronic AMT treatment may induce transcription and translation in LHRH cells and that the peptide LHRH may be involved in the mediation of the antidepressant effect, characteristic of AMT.

Migration of LHRH neurons derived from the olfactory placode in rats.

Using the olfactory placode of 12.5- and 14.5-day-old (E12.5, E14.5) rat embryos, we examined the migration of LHRH neurons by in vivo intraventricular transplantation and in vitro organotypic culture systems. In the transplantation, the olfactory placode of E12.5 embryos was co-transplanted with the cerebral cortex and also with medial basal hypothalamus (MBH). LHRH neurons that had migrated into the co-transplanted brain tissues were fusiform, but those that had moved into the neuro-mesenchymal tissue were polyhedral. The migration occurred most conspicuously in the MBH. In our in vitro studies, we used E14.5 embryos; their vomeronasal organ was cultured with MBH, the olfactory cortex, and the septum of the telencephalon in two systems (piled-culture with an intervening transferrable membrane and co-culture). Among these brain tissues, the MBH was the most effective in inducing the development and migration of LHRH neurons. We further found synaptic junctions of immunonegative nerve fibers on immunoreactive LHRH neurons located in the septum of E16.5 and 17.5 embryos. These findings suggest that the MBH may lead the intraseptal migration of LHRH neurons by yielding certain substances after introducing the neurons into the medial aspect of forebrain vesicles. The early development of the neuronal connection may further promote the migration of LHRH neurons.

LHRH neurons in the medial septal-diagonal band-preoptic area do not project directly to the hippocampus: a double-labeling immunohistochemical study.

While neurons containing immunoreactive luteinizing hormone-releasing hormone (LHRH) are scattered primarily in the medial septal-diagonal band of Broca-medial preoptic area (mS-dbB-PO) complex, autoradiographic studies have demonstrated dense concentrations of LHRH receptors in the hippocampus. The route by which LHRH is transported to its hippocampal receptors is unknown. The present study was designed to test the hypothesis that LHRH-containing neurons in the mS-dbB-PO complex project to hippocampal sites containing LHRH receptors, thereby serving as a source of innervation to these receptors. Large (0.10 microliters) or small (0.02 microliters) volumes of the retrograde tracer wheat germ agglutinin (WGA) were injected unilaterally into four separate hippocampal locations in six ovariectomized female rats. In an additional five females, a 0.15 microliter volume of the retrograde tracer fluorogold (FG) was similarly injected. After a five day survival period, the animals were sacrificed. Vibratome sections of the brain were stained for both WGA and LHRH with a dual immunohistochemical technique. Since FG is a fluorescent chromagen, brains of animals injected with FG only required processing for LHRH immunofluorescence. As a positive control, some sections containing retrogradely labeled cells filled with either WGA or FG were processed for choline acetyltransferase (CHAT) immunoreactivity. The WGA and FG injections covered targeted hippocampal sites and neurons containing retrogradely transported WGA or FG were found in abundance in the mS-dbB-PO complex. In accord with previous reports, many CHAT-positive and fewer LHRH-positive neurons were found in this complex. Approximately 5-10% of the CHAT-positive neurons also contained WGA or FG; however, no neurons were found to co-localize LHRH and either of the retrograde tracers. The results indicate that LHRH neurons in the mS-dbB-PO complex do not project directly to hippocampal sites containing LHRH receptors.

Origin of luteinizing hormone-releasing hormone (LHRH) neurons in the chick embryo: Effect of the olfactory placode ablation

A unilateral olfactory placodectomy resulted in the absence of luteinizing hormone-releasing hormone-immunoreactive (LHRH-ir) cells in the olfactory-forebrain axis of the operated side, whereas the development of the LHRH neuronal system was not disturbed on the unoperated side. In the embryos in which a fragment of the medial olfactory epithelium was spared the damage, a small number of LHRH-ir cells were detected in the nasal region of the operated side, where the lack of the central projection of the olfactory nerve caused stagnation of LHRH-ir cells, no ir-cells being found in the brain area. These results suggest that LHRH neurons originate in the olfactory placode and then migrate into the forebrain along the olfactory nerve.

Effects of in utero ethanol exposure on the development of LHRH neurons in the mouse

Prenatal ethanol exposure has been shown to result in craniofacial malformations as well as alterations of central nervous system morphology and function. Previous studies have demonstrated that acute ethanol exposure on gestational day 7 in the developing C57BL/6J mouse resulted in craniofacial abnormalities similar to that of children with fetal alcohol syndrome. We investigated the effect of ethanol on the migration and number of immunoreactive LHRH (irLHRH) neurons in this strain of mouse. Pregnant mice were intubated with 2 doses of a 25% solution of ethanol 4 h apart on gestational day 7 (G7). Control animals were intubated with water. Animals were sacrificed on G14 or G18 and immunocytochemistry was used to identify irLHRH neurons that were visualized by light microscopy. Fetal ethanol administration did not substantially affect the migration of the LHRH neurons from the medial nasal placode into the forebrain on G14 or G18. The total number of irLHRH neurons was not significantly different on G14 in ethanol-exposed animals as compared to the number in control animals. However, the total number of irLHRH neurons on G18 was significantly less ( P < 0.03) in 4 neuroanatomical regions in fetal ethanol-exposed mice compared to those in control mice; the nasal septum, the traverse area superior to the cribriform plate and ventromedial to the olfactory bulbs, the arch area which included the olfactory tubercle, medial septal nuclei and anterior hypothalamus in G18 fetuses, and preoptic area of the brain. Coronal investigation of the number of irLHRH neurons on G18 indicates that the loss of irLHRH neurons occurred predominantly in the medial region of the rostrum and brain.