Central nervous system neurons acquire mast cell products via transgranulation.

Resting and actively degranulating mast cells are found on the brain side of the blood-brain barrier. In the periphery, exocytosis of mast cell granules results in the release of soluble mediators and insoluble granule remnants. These mast cell constituents are found in a variety of nearby cell types, acquired by fusion of granule and cellular membranes or by cellular capture of mast cell granule remnants. These phenomena have not been studied in the brain. In the current work, light and electron microscopic studies of the medial habenula of the dove brain revealed that mast cell-derived material can enter neurons in three ways: by direct fusion of the granule and plasma membranes (mast cell and neuron); by capture of insoluble granule remnants and, potentially, via receptor-mediated endocytosis of gonadotropin-releasing hormone, a soluble mediator derived from the mast cell. These processes result in differential subcellular localization of mast cell material in neurons, including free in the neuronal cytoplasm, membrane-bound in granule-like compartments or in association with small vesicles and the trans-Golgi network. Capture of granule remnants is the most frequently observed form of neuronal acquisition of mast cell products and correlates quantitatively with mast cells undergoing piecemeal degranulation. The present study indicates that mast cell-derived products can enter neurons, a process termed transgranulation, indicating a novel form of brain-immune system communication.

Early expression of chicken gonadotropin-releasing hormone-1 in the developing chick.

Gonadotropin-releasing hormone (GnRH), which is essential for reproductive function, is made by neurones that migrate from the nasal region into the brain during early embryonic development. This migration begins in chick when the olfactory pit is formed. This is approximately the time that GnRH neurones can be detected immunocytochemically. The present study investigated (i). how early in development the GnRH gene is expressed and (ii). the sites of its expression. Accordingly, reverse transcriptase-polymerase chain reaction (PCR) and in situ hybridization were performed on chick embryos before gastrulation up until the stage by which GnRH neurones have begun to migrate into the central nervous system. Primers were made to the 5'- and 3'-UTR region of the message for cGnRH-I, the form of the peptide that is essential for reproductive function in the chicken. PCR product was found in all stages and the sequences of products from all stages were identical. Thus, the GnRH gene is expressed continuously throughout embryonic development. In situ hybridization with a digoxygenin labelled riboprobe revealed staining along the primitive streak immediately before gastrulation. In later stages, cGnRH-I gene expression was seen in association with the anterior neural ridge. The expression was subsequently restricted to a narrow, clearly defined region, which is associated with the presumptive nasal cavity and olfactory placode. Later, GnRH neurones could be seen in their migratory routes by both in situ hybridization and immunocytochemistry. Expression of the GnRH gene has been described in preimplantation stages in mammals and there is evidence that the neuropeptide plays a role in formation and maintenance of the placenta. What role (if any) it may play in early avian development remains unknown. The demonstration of sites of GnRH expression during the early period of neurulation suggests that GnRH neurones arise before olfactory placode formation.

Distribution of gonadotropin-releasing hormone neurones in the chick forebrain is independent of lineage relationships among cells of the early nasal placode.

The regulation of reproduction depends upon the successful migration of gonadotropin-releasing hormone (GnRH) neurones from the nasal placode to the ventral forebrain during embryogenesis. Within the central nervous system (CNS), these neurones migrate to stereotyped, highly reproducible locations in septal, preoptic and hypothalamic nuclei. We postulated that lineage relationships (descent from a common precursor) might predict the final location of these neurones. To test this hypothesis, a complex retroviral library was used to label dividing cells in the placode and subsequently to identify them by the presence of the alkaline phosphatase marker. GnRH was detected immunocytochemically and lineage relationships determined by single cell polymerase chain reaction and sequencing of the degenerate oligonucleotide component of the retrovirus. GnRH-positive and GnRH-negative neurones were confined to the side ipsilateral to the injection; many cells derived from the placode that entered the CNS did not contain GnRH. This precise method of identifying and mapping the progeny of single neurones revealed that GnRH cells in any given area were derived from multiple precursors. This developmental pattern may contribute to assuring that all CNS locations critical to the orchestration of reproductive events will be populated by GnRH neurones.

Direct innervation of GnRH neurons by encephalic photoreceptors in birds.

In nonmammalian vertebrates, photic cues that regulate the timing of seasonal reproductive cyclicity are detected by nonretinal, nonpineal deep brain photoreceptors. It has long been assumed that the underlying mechanism involves the transmission of photic information from the photoreceptor to a circadian system, and thence to the reproductive axis. An alternative hypothesis is that there is direct communication between the brain photoreceptor and the reproductive axis. In the present study, light and confocal microscopy reveal that gonadotropin releasing hormone (GnRH) neurons and processes are scattered among photoreceptor cells (identified by their opsin-immunoreactivity) in the lateral septum (SL). In the median eminence (ME), opsin and GnRH immunoreactive fibers overlap extensively. Single and double label ultrastructural immunocytochemistry indicate that in the SL and preoptic area (POA), opsin positive terminals form axo-dendritic synapses onto GnRH dendrites. In the ME, opsin and GnRH terminals lie adjacent to each other, make contact with tanycytes, or terminate on the hypophyseal portal capillaries. These results reveal thatbrain photoreceptors communicate directly with GnRH-neurons; this represents a means by which photoperiodic information reaches the reproductive axis.

Gonadal steroids regulate the number and activational state of mast cells in the medial habenula.

While mast cells in connective tissues have long been associated with allergic reactions, it is now clear that they are also present within the central nervous system under normal physiological conditions. The mast cell population increases 10-fold in the medial habenular region of the brain within 2 h after pairing in doves. The first study explored whether this increase was due to exposure to gonadal steroids. Light microscopic immunocytochemistry indicates an increased number of brain MC following exposure to either testosterone (T) or dihydrotestosterone (DHT) in the male, or 17beta estradiol (E) in the female, but not in cholesterol-treated controls. Thus, the increased habenular MC population is produced by gonadal hormones in the absence of sexual behavior, is not sexually dimorphic, and does not require aromatization of androgen. In the next study, MC activational state was determined using electron microscopy. Cells were categorized into five states: (I) resting; (II) initiation of degranulation; (III) fully degranulated; (IV) piecemeal secretion; and (V) resynthesizing. Hormone treatment (T, DHT, or E) resulted in a significant increase in the percent of cells in activated states. MC granules contain a wide range of biologically active molecules. The release of these granule contents into the neuropil of the central nervous system is likely to have wide ranging effects at multiple levels including vascular permeability and neuronal excitability. In that steroid treatment is known to result in such effects, the present demonstration of a hormonally induced shift in MC secretory state is one avenue by which these effects are mediated.

Mast cells migrate from blood to brain.

It is well established that mast cells (MCs) occur within the CNS of many species. Furthermore, their numbers can increase rapidly in adults in response to altered physiological conditions. In this study we found that early postpartum rats had significantly more mast cells in the thalamus than virgin controls. Evidence from semithin sections from these females suggested that mast cells were transiting across the medium-sized blood vessels. We hypothesized that the increases in mast cell number were caused by their migration into the neural parenchyma. To this end, we purified rat peritoneal mast cells, labeled them with the vital dyes PKH26 or CellTracker Green, and injected them into host animals. One hour after injection, dye-filled cells, containing either histamine or serotonin (mediators stored in mast cells), were located close to thalamic blood vessels. Injected cells represented approximately 2-20% of the total mast cell population in this brain region. Scanning confocal microscopy confirmed that the biogenic amine and the vital dye occurred in the same cell. To determine whether the donor mast cells were within the blood-brain barrier, we studied the localization of dye-marked donor cells and either Factor VIII, a component of endothelial basal laminae, or glial fibrillary acidic protein, the intermediate filament found in astrocytes. Serial section reconstructions of confocal images demonstrated that the mast cells were deep to the basal lamina, in nests of glial processes. This is the first demonstration that mast cells can rapidly penetrate brain blood vessels, and this may account for the rapid increases in mast cell populations after physiological manipulations.

Distribution and local differentiation of mast cells in the parenchyma of the forebrain.

Mast cells are found in the brain of many species. Although a considerable body of information is available concerning the development and differentiation of peripheral mast cells, little is known about brain mast cells. In the present study, the ontogeny of mast cells in the dove brain was followed by using three markers: acidic toluidine blue, alcian blue/safranin, and an antiserum to gonadotropin-releasing hormone (GnRH). Mast cells first appear in the pia on embryonic day (E)13-14 in ovo, then along blood vessels extending from the pia into the telencephalon on posthatch day 4-5, and in the medial habenula at week 3. Medial habenular mast cell numbers increase during development, peaking in peripubertal birds, and declining thereafter. Several measures indicate that mast cells mature within the medial habenula: there is an increase in the intensity of metachromasia, a switch from alcian blue granules in young animals to mixed alcian blue and safranin granules in older animals, and an increase in GnRH-like immunoreactivity. These results were extended by using electron microscopy. The architecture of mast cell granules evolved from electron lucent with small electron dense deposits at E15 to more electron dense granules with complex patterns of internal structure by 2 months. Ultrastructural immunocytochemistry for the GnRH-like peptide at 1 month revealed both immunopositive and negative cells, suggesting that the acquisition of this phenotype is not simultaneous across the population. Thus, immature mast cells infiltrate the central nervous system and undergo in situ differentiation within the neuropil.

Development of the chick olfactory nerve.

Gonadotropin releasing hormone (GnRH) is produced and secreted by neurons dispersed throughout the septal-preoptic and anterior hypothalamic areas in adult birds and mammals. These neurons, essential for a functional brain-pituitary-gonadal axis, differentiate in the olfactory placode, the superior aspect of which forms the olfactory epithelium. To reach their final placement within the brain, GnRH neurons migrate out of the epithelium and along the olfactory nerve to the CNS. This nerve is essential for the entrance of GnRH neurons into the CNS. Due to the importance of the nerve for the proper migration of these neurons, we have used immunocytochemistry, DiI labeling and 1 microm serial plastic-embedded sections to characterize the nerve's earliest development in the embryonic chick (stages 17-21). Initially (stage 17) the zone between the placode and prosencephalon is a cellular mass contiguous with the placode. This cluster, known as epithelioid cells, is positive for some but not all neuronal markers studied. The epithelium itself is negative for all neuronal and glial markers at this early stage. By stage 18, the first neurites emerge from the epithelium; this was confirmed at stage 19 by examination of serial 1 microm plastic sections. There is sequential acquisition of immunoreactivity to neuronal markers from stage 18 to 21. The glial component of the nerve appears at stage 21. Axons originating from epithelium, extend to the border of the CNS as confirmed by DiI labeling at stage 21. Small fascicles have entered the CNS at this stage. As previously reported, GnRH neurons begin their migration between stages 20-21 and have also arrived at the border of the brain at stage 21. Despite the penetration of neurites from the olfactory nerve into the CNS, GnRH neurons pause at the nerve-brain junction until stage 29 (2 1/2 days later) before entering the brain. Subsequent studies will examine the nature of the impediment to continued GnRH neuronal migration.

Embryonic development of the gonadotropin-releasing hormone (GnRH) system in the chick: a spatio-temporal analysis of GnRH neuronal generation, site of origin, and migration.

We present a quantitative immunocytochemical study of GnRH migration by developmental stage. GnRH peptide was detected in cells of the olfactory epithelium at stage 19. Migration was initiated a few hours later at stage 20. Of interest is the observation that GnRH neurons paused at the central nervous system border for 3 days, entering the brain at stage 29. The major expansions of the GnRH population occurred at two points; stages 26 and 42. In one animal a third population expansion occurred after hatching, with the number of GnRH cells reaching 6600. To determine the site of origin of GnRH cells, embryos were exposed to tritiated thymidine and killed 5 h later. Most GnRH cells incorporated label in the olfactory epithelium; however, some autoradiographically labeled GnRH cells, possessing a neuronal morphology, were found in the olfactory nerve and the forebrain, suggesting that some GnRH neurons divide as they migrate. A cumulative labeling method employing tritiated thymidine was used to examine the birth date of GnRH neurons. Postmitotic GnRH cells were first detected at stages 19-21. At stage 24, a peak in GnRH neurogenesis preceded the increase in GnRH neurons expressing their peptide at stage 26. After stage 24, there was a gradual addition of postmitotic cells to the population through stage 35. A pulse-chase paradigm indicated that birth date did not influence the final GnRH cell distribution. Injections at stage 29, when 10% of the GnRH neurons are born, generated double labeled cells in all locations where placode-derived GnRH neurons reside.

Brain mast cells lack the c-kit receptor: immunocytochemical evidence.

Mast cells are reported to differ from other cells of the hematopoietic lineage in that as mature cells, they retain the c-kit receptor, and are thus capable of responding to the stem cell factor (SCF) ligand. SCF is important for development and survival of mast cells. In this study, c-kit expression was examined immunocytochemically in the brains of mice, rats and doves. The results indicate that brain mast cells lack the c-kit receptor; those of the leptomeninges and other connective tissues are a mixed population of c-kit positive and negative cells. The mechanisms whereby brain mast cells might survive in the absence of SCF-c-kit signaling are discussed.

Preoptic area grafts implanted in mammillary bodies of hypogonadal mice: patterns of GnRH neuronal projections.

Gonadotropin-releasing hormone (GnRH) axons project to the median eminence, where the peptide is released to stimulate pituitary gonadotrophs. Hypogonadal mice (hpg) do not synthesize GnRH due to a deletion in the gene. When neonatal preoptic area (POA) tissue from normal mice containing GnRH neurons is transplanted into the third ventricle of hpg mice, GnRH axons exit the graft and specifically project to the median eminence, where the release of GnRH in the portal circulation induces the stimulation of the pituitary-gonadal axis. To test the hypothesis that the median eminence region is critical to targeting, we placed POA grafts in the region of the mammillary bodies, which never contains GnRH cell bodies, but is nevertheless close to the median eminence. Control mice received bilateral grafts into the anterior hypothalamus. GnRH axons innervated the median eminence in animals with grafts in the mammillary bodies and posterior hypothalamus. Mice with such grafts for 4-5 months had gonadal development, while those with grafts for shorter periods did not. Anterior hypothalamic grafts merged into the third ventricle and, consistent with previous studies, this resulted in GnRH innervation of the median eminence and gonadal development. However, when grafts were located within dorsal regions such as the thalamus, no median eminence innervation was seen. In these cases, GnRH axons borrowed other bundles of fibers to travel within the host brain. The pattern of innervation from grafts within ventro-caudal regions of the hypothalamus vs. that from dorsal regions supported the hypothesis that the median eminence releases diffusible substances directing GnRH outgrowth.

Evidence for estrogen receptor in cell nuclei and axon terminals within the lateral habenula of the rat: regulation during pregnancy.

The habenular complex is involved in several estrogen-dependent reproductive behaviors in female rats, namely, sexual behavior, maternal behavior, and postpartum sexual behavior. Although it is known that estrogen acts in other brain regions to mediate these behaviors, it is not known whether estrogen may also act directly on the habenular complex. To address this possibility, we examined this region for the presence of estrogen receptor (ER). This analysis was carried out in separate experiments by using in situ hybridization, immunocytochemistry at the light and electron microscopic levels, and steroid autoradiography. Neurons within the lateral habenula (LHb), but not the medial habenula, express ER mRNA, contain ER immunoreactivity (ER-ir) in their nuclei, and concentrate radiolabelled estradiol, providing strong evidence for the presence of functional ER in the lateral habenula. There were also ER-ir containing punctate fibers within the LHb, which, at the electron microscopic level, in part, proved to be axons and presynaptic axonal terminals. Both the level of ER-ir in cell nuclei and the density of ER-ir fibers within the LHb were regulated during the course of pregnancy and the postpartum period, suggesting that the sensitivity of the LHb to estrogen may be altered during this time. Taken together, these results demonstrate that the LHb is likely a more estrogen-sensitive region than was previously considered, and they suggest alternative mechanisms of action for ER. ER within the LHb may play a critical role in the involvement of the LHb in estrogen-dependent female reproductive behaviors.

New observations on the development of the gonadotropin-releasing hormone system in the mouse.

In ongoing efforts to study the ontogeny of gonadotropin-releasing hormone (GnRH) neurons, we serendipitously observed that increasing times of incubation in antibodies enhanced signal detection. Here, we describe significant differences in the early migration pattern, population dynamics, and growth cone morphology from published reports. The first immunoreactive GnRH cells were detected in the mouse at E10.75 (7.6 +/- 2.8 cells; morning after mating = E0.5), prior to the closure of the olfactory placode. Although half of these cells were in the medial wall of the olfactory pit, the other half had already initiated their migration, and approximately one quarter had reached the telencephalic vesicle. Although the migratory pattern of the GnRH cells after E11.00 was identical to that described previously, these earliest migrating cells traveled singly rather than in cords, with some reaching the presumptive preoptic area (posterior to the ganglionic eminence) by E11.75. The number of GnRH cells increased significantly (p < 0.05) to 777 +/- 183 at E11.75 and peaked at 1949.6 +/- 161.6 (p < 0.05) at E12.75. The adult population was approximately 800 cells distributed between the central nervous system (CNS) and the nasal region. Hence, the population of GnRH neurons during early development is much larger than previously appreciated; mechanisms for its decline are discussed. Neuritic extensions on the earliest GnRH neurons are short (30-50 microm) and blunt and may represent the leading edge of the moving cell. By E12.75, GnRH axons in the CNS had a ribboned or beaded morphology and increasingly more complex growth cones were noted from this time until the day of birth. The most complex growth cones were associated with apparent choice points along the axons' trajectory. By E13.75, GnRH axons were seen at the presumptive median eminence in all animals, and it was at this stage that the axons began to branch profusely. Branching, as well as the presence of growth cones, continued post-natally. These results provide further insights into the pathfinding mechanisms of GnRH cells and axons.

Mast cell number and maturation in the central nervous system: influence of tissue type, location and exposure to steroid hormones.

While it is well established that brain mast cells are usually associated with the cerebral vasculature, in ring doves mast cells lie directly in the neuropil of the medial habenula. During normal development mast cells enter the habenula and complete their differentiation in situ. In the present study, we asked what characteristics of the medial habenula contribute to mast cell entry and differentiation. Grafts of embryonic habenula or control optic tectal grafts were placed in the lateral ventricle or anterior chamber of the eye. Transplantation alters the location of the habenula as well as its neural and vascular connections. Three groups of hosts were used for the ventricular grafts: four-month-old and killed three months after transplantation; four-month-old and killed seven months later, and two- to three-year-old gonadectomized males killed three months later. Hosts for the intraocular grafts were four months of age and killed three months later. Mast cells were present in the habenular grafts but not in the control tissue. Mast cells in three- and seven-month-old grafts were phenotypically immature when compared to those of hosts. They contained fewer metachromatic granules, fewer granules immunoreactive to an antiserum against gonadotropin-releasing hormone, and no highly-sulphated proteoglycans. As previously described, gonadectomized adults had fewer mast cells in their medial habenula than did intact animals, but there was no change in mast cell number in habenular grafts. The current experiments indicate that the occurrence and survival of mast cells can occur within the microenvironment of the medial habenula, but that maturation of these cells requires the normal connections of this nucleus. Furthermore, gonadectomy appears to alter mast cell number in the medial habenula by generating a secondary signal which the transplanted tissue is incapable of receiving or processing.

Gonadotropin-releasing hormone axons target the median eminence: in vitro evidence for diffusible chemoattractive signals from the mediobasal hypothalamus.

The projection of GnRH neurons to the median eminence of the medial basal hypothalamus (MBH) is established early in development and is also seen when preoptic area-derived GnRH cell-containing grafts are placed in the third ventricle of hypogonadal mice. To further study the factors directing GnRH axonal targeting, we cultivated embryonic or postnatal day 1 preoptic area with a coexplant on collagen- and laminin-coated membranes in insert chambers. After 7 days of culture, GnRH-immunoreactive fibers extended significantly farther and in greater number onto the sector of membrane facing a MBH coexplant than in the opposite sector, but not toward coexplants of control tissue. Moreover, such effects were specific, as outgrowth of a general axonal population, immunoreactive for growth-associated protein 43 was not influenced by the presence of the MBH. Preferential GnRH outgrowth toward the MBH was established early and was maintained during 10 days of culture. The importance of substrate-derived guidance was also assessed with confocal microscopy. GnRH axons consistently traveled in the company of growth-associated protein 43-labeled axons, but only erratic associations were seen between GnRH and glial processes extending on the membrane. We suggest that although employing an axonal substrate, GnRH axons follow a diffusible chemoattractive signal(s) secreted by the MBH.

What nature's knockout teaches us about GnRH activity: hypogonadal mice and neuronal grafts.

The hypogonadal mouse is one of "nature's knockouts," bearing a specific deletion in the gene for gonadotropin-releasing hormone (GnRH), with the result that no GnRH peptide is detectable in the brain. The lack of reproductive development after birth provides an animal model that has proved fruitful in clarifying the role of GnRH in reproductive behavior and physiology. Behavioral studies with hypogonadal mice convincingly demonstrate that although GnRH may facilitate the appearance of sexual behavior, this peptide is not essential for either male or female sexual behavior in the mouse. Administration of GnRH to hypogonadal mice with regimens mimicking GnRH pulsatility initiates reproductive development. Surprisingly, continuous exposure to GnRH stimulates remarkable ovarian and uterine growth and increased FSH release, although pituitary content of LH and FSH remains unchanged. In contrast, when brain grafts of normal fetal preoptic area (POA), containing GnRH cells, are implanted in the third ventricle of adult hypogonadal mice, both pituitary and plasma gonadotropin levels increase. Grafted GnRH neurons innervate the median eminence of the host and support pulsatile LH secretion in the majority of animals with graft-associated gonadal development. Studies of hypogonadal mice with POA grafts demonstrate that distinct components of reproductive function are dissociable: hosts may demonstrate reflex but not spontaneous ovulation; others may show positive but not negative feedback. Activation of grafted GnRH cells in response to sensory input to the host, as revealed in Fos expression studies, is an example of the integration of the graft with the host brain that underlies such capabilities. A goal of these studies is to elucidate the specific connectivity underlying discrete aspects of reproductive function.

Brain mast cell degranulation regulates blood-brain barrier.

Mast cells synthesize vasoactive agents and a number of neurotransmitters. They are particularly numerous in the medial habenular region of the epithalamus, the attachment site of the choroid plexus. The present study examined whether degranulation of brain mast cells alters the permeability of the blood-brain barrier (BBB). To this end, doves were injected intramuscularly with the mast cell degranulator, compound 48/80 (C40/80), followed by i.v. injection of Evans blue. The distribution of the dye in the parenchyma was examined using digital imaging. Three brain areas were analyzed: the medial habenula (which also contains mast cells), the paraventricular nucleus (PVN, which abuts the third ventricle, but has no mast cells), and the lateral septal organ (LSO, a circumventricular organ with fenestrated capillaries). Significantly more Evans blue tracer and fewer toluidine blue-positive mast cells were detected in the medial habenula of subjects treated with C48/80 compared to saline controls. Evans blue did not enter the PVN in either the experimental or control group, while it entered the LSO equally in both. Degranulation of mast cells after C48/80 treatment was confirmed histochemically and ultrastructurally. The results support the hypothesis that brain mast cell degranulation locally alters BBB permeability. Activation of brain mast cells may provide a mechanism for regulated opening of the BBB.

Joint migration of gonadotropin-releasing hormone (GnRH) and neuropeptide Y (NPY) neurons from olfactory placode to central nervous system.

The olfactory epithelium in vertebrates generates the olfactory sensory neurons and several migratory cell types. Prominent among the latter are the gonadotropin-releasing hormone (GnRH) neurons that differentiate within the olfactory epithelium during embryogenesis and migrate along the olfactory nerve to the central nervous system. We initiated studies to characterize additional neuronal phenotypes of olfactory epithelial derivation. Neuropeptide Y (NPY) neurons are functionally related to the reproductive axis, modulating the release of GnRH and directly enhancing GnRH-induced luteinizing hormone (LH) secretion from gonadotrophs. We demonstrate that a population of migratory NPY neurons originates within the olfactory epithelium of the chick. At stage 25, NPY-positive fibers, but not cells, were detected in the epithelium and the nerve. By stages 28-34, NPY neurons and processes were present in the olfactory epithelium, olfactory nerve, and at the junction of the olfactory nerve and forebrain. In these regions the number of NPY neurons increased until stage 30 and then declined as development progressed. Electron microscopic immunocytochemistry confirmed the neuronal phenotype of the NPY-positive cells. The origin and migratory nature of some of these NPY cells was confirmed by double-label immunocytochemical detection of NPY and GnRH. A large percentage of the NPY-cells coexpressed the GnRH peptide. Between stages 28 and 34 single- and double-labeled NPY and GnRH neurons were found side by side along the GnRH migratory route emanating from the nasal epithelium, along the olfactory nerve, and into the ventral forebrain. These data suggest that an NPY population originates in the olfactory epithelium and migrates into the central nervous system during embryogenesis. By stage 42, no NPY/GnRH double-labeled cells were detected.

FOS expression in grafted gonadotropin-releasing hormone neurons in hypogonadal mouse: mating and steroid induction.

We used FOS expression, widely accepted as a marker for neuronal activation, to evaluate physiologically induced activation of gonadotropin-releasing hormone (GnRH) neurons within intraventricular preoptic area grafts in hypogonadal (hpg) female mice. Hpg mice lack endogenous GnRH due to a mutated gene, but can respond to grafted GnRH neurons with reproductive development. The purpose of this study was to determine the degree to which the host brain regulates grafted GnRH neurons. FOS expression in grafted GnRH neurons was induced in progesterone-primed female mice paired with sexually active males. The degree of sexual activity did not affect the outcome, with 40.9 +/- 12.2% of the grafted GnRH cells expressing FOS when male partners performed intromissions, and 47.5 +/- 10.2% when they also ejaculated. There was little or no FOS expression in the grafts of unprimed hpg mice paired with sexually active males, in unpaired mice primed with progesterone or sequential estradiol benzoate and progesterone, or in controls. The pattern of FOS expression in the brains of the female hpg mice engaged in mating behavior was similar to that reported in other species, with moderate to high expression in the medial preoptic area, ventromedial nucleus, and medial amygdala in females paired with males that ejaculated. The present results support the hypothesis that host-derived activation of grafted GnRH neurons underlies aspects of reproductive responses seen in hpg mice with grafts, and further, that at least a portion of the host-graft connectivity is steroid sensitive.