Juvenile rats show reduced c-fos activity in neural sites associated with aversion to pups and inhibition of maternal behavior.

Juvenile rats (18-23 days old) interact avidly with pups as novel stimuli and show maternal behavior after only 1-3 days of pup exposure; adults initially avoid pups and require 3-9 days of pup exposure. Upon exposure to pups as novel stimuli, adults had more c-Fos-immunoreactive neurons in the hypothalamus and amygdala--regions associated with aversion to pups--than adults exposed to familiar pup stimuli (maternal) or not exposed to pups (p < .05). In juvenile rats exposed to pups as novel stimuli, only the medial amygdala had a small significant increase of c-Fos neurons. In juveniles, this blunted engagement of c-Fos neurons may reflect the diminished activation of inhibitory neurons, facilitating the interaction of juveniles with pups as novel stimuli and onset of maternal behavior.

Region-specific effects of ovarian hormones on estrogen receptor immunoreactivity.

The cell groups in which nuclear estrogen receptor (ER) expressing neurons are found have unique, often coordinated, functions. Regulation of ER content may be one mechanism through which feedback responses can be adjusted to match the function of a specific brain region and physiological circumstance. In these immunocytochemical experiments, estrogen decreased staining intensity for ER in the ventrolateral hypothalamus and bed nucleus of the stria terminalis, but not in the periventricular preoptic area. ER staining intensity was further decreased by progesterone, following estrogen, but not in all brain regions. These results suggest that ER is regulated by estrogen in a region-specific manner. Furthermore, inhibition of responses to estrogen by progesterone may involve progesterone-induced down-regulation of ERs.

Neuroanatomical tract tracing provides histological verification of neuron loss following cytotoxic lesions.

In the present study we report a neuroanatomical procedure that provides direct histological verification of the extent of neuron loss following cytotoxic lesions of the lateral subdivision of the habenular complex (Lhb). Following kainic acid-induced lesions of Lhb neurons, the fluorescent retrograde tracer Fluoro-Gold was injected into the ventral midbrain where many medial habenula (Mhb) and Lhb neurons project. The absence of retrogradely labeled neurons in the Lhb indicated the extent of neuron loss there, and the presence of Fluoro-Gold-labeled neurons in the Mhb indicated that its neurons were intact. The extent of neuron loss in the Lhb was significantly correlated with behavioral data. Retrograde tract tracing can be used as an effective histological tool to verify the extent of neuron loss following a lesion procedure.

Sex differences in the distribution and projections of testosterone target neurons in the medial preoptic area and the bed nucleus of the stria terminalis of rats.

Retrograde tracing was combined with steroid hormone autoradiography to investigate the projections of testosterone-target neurons in preoptic and limbic regions to the midbrain in male and female rats. Autoradiograms were prepared from the brains of male and female rats that had received an injection of a fluorescent retrograde tracer into the midbrain, and an intravenous injection of [3H]testosterone. Testosterone target neurons that project to the midbrain were abundant in the medial preoptic nucleus (MPN) and bed nucleus of the stria terminalis (BST) and were also observed in the ventromedial nucleus of the hypothalamus. Testosterone target neurons and testosterone target neurons that project to the midbrain were more abundant in the caudal half of the MPN compared to the rostral half. Moreover, male rats had more testosterone target neurons in the caudal MPN than female rats, and the number of testosterone target neurons in the MPN that project to the midbrain was higher in male than in female rats. Male rats also had more testosterone target neurons than females throughout the encapsulated subdivision of the BST. We hypothesize that sex differences in the neuronal connectivity of testosterone target neurons may underly sex specific behavioral responsiveness to androgens.

Hormonal priming and triggering of maternal behavior in the rat with special reference to the relations between estrogen receptor binding and ER mRNA in specific brain regions.

Estrogen stimulation of maternal behavior during pregnancy in the rat has been studied at several levels of analysis. These include (a) changes in maternal responsiveness during pregnancy; (b) hormonal stimulation of maternal behavior; and (c) correlation between nuclear binding of estradiol in the medial preoptic area and the stimulation of maternal behavior (i.e., in pregnancy-terminated, ovariectomized females treated with estradiol benzoate). These studies have given rise to the concepts of hormonal priming and triggering of maternal behavior during pregnancy and at parturition. More recently, using in situ hybridization, ER mRNA was measured during pregnancy (also diestrus and postpartum) in brain regions in which binding previously had been studied, to investigate further the regulation of hormonal priming. Steady state levels of ER mRNA per cell and cell densities of ER mRNA produced a measure of total ER mRNA per brain region which was then compared to nuclear estrogen receptor binding. The relation between binding and ER mRNA is presented for one of the brain regions, the rostral medial preoptic nucleus. The results indicate that ER transcription is regulated during pregnancy, but regulation is specific to each brain region and there is no simple relation between ER mRNA and nuclear estrogen receptor binding.

Gonadotropin-releasing hormone mRNA in the rat: distribution and neuronal content over the estrous cycle and after castration of males.

The decapeptide gonadotropin-releasing hormone (GnRH) stimulates release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary. In the present study we used a 51-base oligonucleotide probe and in situ hybridization to study the neuronal content of GnRH mRNA at several time points in the estrous cycle and 7 days after castration of male rats. GnRH mRNA containing cells were found in the medial septum (SEPT), the vertical and horizontal limbs of the diagonal band of Broca (DBB), and throughout the preoptic area (POA) from the organum vasculosum of the lamina terminalis (OVLT) to its caudal merger with the anterior hypothalamus. The number of neurons producing detectable quantities of GnRH mRNA was not different either among females killed at 0700 h proestrus, 1000 h estrus, or 1900 h of diestrus 1 or between intact male rats and male rats killed 1 week after castration. We did, however, detect a significant difference in the number of GnRH mRNA producing neurons between males and females (P less than 0.05), where females had 20% more labeled cells. We detected no significant difference in the relative copy number of GnRH mRNA molecules (grains per labeled cell) either over the estrous cycle or between intact and castrate males. However, females overall had 24% more grains per labeled cell than males (P less than 0.05). These results suggest that gonadal steroid regulation of GnRH both over the estrous cycle and after short-term castration of males is mediated primarily by cellular processes subsequent to GnRH gene regulation. Furthermore, these results suggest that biosynthetic activity of GnRH is higher in females than in males.

Distribution of estrogen receptor-immunoreactive cells in the forebrain of the female guinea pig.

We mapped the distribution of estrogen receptor-containing cells in the forebrain of the adult female guinea pig. Cellular estrogen receptor content was detected using monoclonal antibody H222, directed against the estrogen receptor, and the avidin-biotin method with nickel-intensified diaminobenzidine as the chromagen. A complete set of deletion, titration, and adsorption controls established the specificity of the staining. The most dense collections of estrogen receptor-immunoreactive cells were found in medial preoptic, medial hypothalamic, and limbic nuclei (amygdala, bed nucleus of the stria terminalis, lateral septum). Numerous estrogen receptor-immunoreactive cells were also found in additional, specific subregions of the remainder of the preoptic area, hypothalamus, and limbic system, and also in the midbrain (central gray). Elsewhere, estrogen receptor-immunoreactive cells were present in smaller numbers or were absent. This map confirms and extends previous maps based on estrogen binding. The majority of estrogen receptor-immunoreactive cells are found in areas known to be involved in some aspect of reproduction. In addition, many estrogen receptor-immunoreactive cells are found in areas not typically considered to have a primary role in reproductive behavior or neuroendocrine function.

A subset of progesterone target neurons have axonal projections to the midbrain.

Progestin-concentrating neurons in the preoptic area and hypothalamus that project to the midbrain in the female rat were identified using the combined steroid hormone autoradiography-retrograde axonal tracing technique. Progesterone target neurons were most abundant in the periventricular preoptic area and the medial preoptic nucleus, and in the ventromedial and arcuate nuclei of the hypothalamus. In the medial preoptic area as a whole, about 14% of the progestin-concentrating cells were afferent to the midbrain. More specifically, 23% of medial preoptic nucleus progesterone target neurons communicated directly with midbrain cell groups, whereas a much smaller percentage (2%) of periventricular preoptic target neurons projected to the midbrain. In the medial basal hypothalamus as a whole, 11% of the progestin-concentrating cells detected sent axons to the midbrain. This proportion was slightly higher in the ventromedial nucleus (15%), and much lower in the arcuate nucleus (3%). In the dorsal and lateral hypothalamic areas, close to 30% of the progesterone target neurons sent axons to the midbrain, although the total number and density of target cells in the two latter areas was low. These data support the idea that transduction by forebrain target neurons of the progesterone signal into altered synaptic transmission in the midbrain is one route through which progesterone can influence a variety of behaviors.

Estradiol-concentrating forebrain and midbrain neurons project directly to the medulla.

The location and number of estradiol (E2)-concentrating neurons afferent to the dorsal medulla were determined by combining retrograde fluorescent tract tracing with steroid hormone autoradiography. Injections of Fluro-Gold were made into the medulla of 80 day old, ovariectomized, and adrenalectomized female rats. After 7 days survival to allow for retrograde transport, females were injected with [3H]estradiol; they were then perfused and their brains processed for steroid hormone autoradiography. Following a 4-12 month exposure period, autoradiograms were developed and microscopically analyzed for the presence of E2-concentrating neurons that project to the medulla. Numerous E2-concentrating neurons were identified that send axons directly to the medulla; the majority were found in the bed nucleus of the stria terminalis, paraventricular nucleus of the hypothalamus, central nucleus of the amygdala, and the central gray. Of the E2-concentrating neurons in the bed nucleus of the stria terminalis, 12.7% also projected to the medulla. E2-concentrating neurons that sent axons to the medulla were also identified in and ventromedial to the lateral parvicellular subdivision in the caudal half of the paraventricular nucleus of the hypothalamus (69.4%). Over one-third of the E2-concentrating neurons found in the central nucleus of the amygdala coursed to the medulla. The central gray was the only mesencephalic brain region that contained E2-concentrating neurons that projected to the medulla (41.9%). The medulla-bound E2-concentrating forebrain and midbrain neurons identified in the present study may influence autonomic tone via direct projections.

Estrogen plus progesterone increases progestin receptor immunoreactivity in the brain of ovariectomized guinea pigs.

The goal of these experiments was to determine the number and distribution of brain cells that contain progestin receptors (PR) and to determine the effect of estrogen and estrogen plus progesterone on PR content of those cells. Ovariectomized adult female guinea pigs were treated with oil (control), or estrogen followed by oil, or estrogen followed by progesterone. As expected, only those animals treated with estrogen plus progesterone became sexually receptive. The cellular content of PR was determined using a monoclonal antibody to the receptor, and standard immunocytochemical techniques. Analysis of the PR-immunoreactive (PR-IR) cells consisted of: (1) mapping the anatomical distribution of PR-IR cells; (2) analyzing the effect of steroid hormones on PR-IR cell number, and (3) determining the effect of steroid hormones on PR immunoreactivity per cell. PR immunoreactivity was located exclusively in the nuclei of cells in the preoptic area and hypothalamus. The most dense collections of PR-IR cells were found in the preoptic area, ventrolateral nucleus of the hypothalamus, and infundibular nucleus. Estrogen caused a dramatic increase in the number of PR-IR cells in these cell groups. Sequential treatment with estrogen plus progesterone further increased PR-IR cell number, in the preoptic area by 65%, in the ventrolateral nucleus by 38%, and in the infundibular nucleus by 49%. A cell-by-cell rating of the PR immunoreactivity was carried out in these three cell groups. We found that the staining intensity across the populations of PR-IR cells was increased by estrogen and further increased by sequential estrogen plus progesterone. Alterations in cellular PR content may contribute importantly to the ability of progesterone target cell groups to perform their specialized roles in steroid-regulated activity.

Distribution of luteinizing hormone-releasing hormone in the nervus terminalis and brain of the mouse detected by immunocytochemistry.

Immunoreactive luteinizing hormone-releasing hormone (LHRH) was localized in a relatively large number of ganglion cells and fibers of the nervus terminalis of neonatal and adult mice, indicating that this nerve is a substantial source of LHRH in the mouse brain. Whole-head specimens of neonatal mice, prior to calcification of the cranium, revealed an extensive distribution of LHRH neurons and fine fibers throughout the peripheral, intracranial, and central parts of the nervus terminalis. The most striking difference between the neonatal and adult animals, in the nervus terminalis, was the increase in immunoreactive axons that made up the fiber bundles of this nerve. In the adult mouse, the intracranial and central projections were composed of thick fascicles of immunoreactive axons, ensheathed by glial cells and accompanied by ganglia that contained both LHRH-reactive and nonimmunoreactive neurons. LHRH-immunoreactive cells and axons were seen in a branch of the nervus terminalis that coursed along the medial, posterodorsal aspect of the olfactory bulb and in branches of this nerve that accompany the vomeronasal nerves to the accessory olfactory bulb. A few LHRH neurons and many immunoreactive processes were seen in the accessory and main olfactory bulbs. LHRH-reactive neurons were seen in the hypothalamus and extrahypothalamic structures. Examination of adult mouse brains revealed a pattern of distribution and number of immunoreactive neurons similar to that seen in the neonate. However, many more LHRH-reactive axons were seen in all areas of the brain of the mature animal.

Quantitative autoradiographic analysis of estradiol retention by cells in the preoptic area, hypothalamus and amygdala.

These experiments were done to compare quantitatively, on a cell-by-cell basis, estradiol retention by cells in the medial preoptic area, arcuate nucleus, ventrolateral subdivision of the ventromedial nucleus, and the caudal half of the medial nucleus of the amygdala. The steroid autoradiograms were prepared from 2 mu sections of brains from ovariectomized, adrenalectomized adult female rats that had been infused intravenously with [3H] estradiol (E2) in a regimen which kept circulating hormone concentration at or above proestrus levels for 3-4 h. Even in these brain regions, containing the most dense collections of E2-concentrating cells, a maximum of only 27-61% of the cells concentrated E2. Therefore, in these regions only a particular subset of the cells retain hormone; other cells in the region do not retain hormone. Frequency distribution histograms of the number of grains per cell versus the number of cells in each region showed a wide range in the amount of E2 retained per cell, and no modes among E2-retaining cells. The data followed a distribution markedly different from that predicted by a simple Poisson distribution, confirming that E2-retention does not result from a random, passive process such as diffusion. The overall quantitative characteristics of the frequency distribution histograms were similar across the four brain areas. Therefore, we propose that the different E2-sensitive functions of these brain areas must depend on differences in the neural connectivity or differences in hormone regulated peptide content of the areas.

Brattleboro rat hypothalamic neurons transcribe vasopressin gene: evidence from in situ hybridization.

In situ hybridization has been used to identify specific hypothalamic magnocellular neurons, in normal and Brattleboro rats, that contain vasopressin (VP) or oxytocin (OT) mRNA. The subnuclear distribution of identified neurons in hypothalamic magnocellular nuclei after hybridization with several probes specific for OT or VP mRNA was in direct agreement with immunocytochemical descriptions of the distribution of cells containing VP or OT hormone. The number of grains per cell suggested that Brattleboro rats contained greater levels of OT mRNA, while hybridization with VP probes produced fewer grains in tissue from Brattleboro rats compared to normal rats. This paper provides the first cell-by-cell description of VP gene expression in the Brattleboro rat and demonstrates that VP gene transcription is confined precisely to the magnocellular neurons that normally synthesize VP hormone in normal rats.

Localization of forebrain neurons which project directly to the medulla and spinal cord of the rat by retrograde tracing with wheat germ agglutinin.

Wheat germ agglutinin (WGA) in a slow-release polyacrylamide gel pellet was implanted in the medulla or spinal cord of the rat. Large numbers of retrogradely labeled cells were visualized by immunocytochemical procedures in specific nuclei of the forebrain mainly ipsilateral to the implant site following implants as far caudal as the sacral segments of the spinal cord. Total average number of labeled forebrain cells (three brains per category; 100 micron per 150 micron of brain tissue were examined microscopically): medulla, 2,115; cervical, 1,878; lumbar, 1,017; sacral, 385. After WGA-gel implants in the medulla or cervical cord the majority of retrogradely labeled neurons were seen in the lateral hypothalamic area, the zona incerta, and in subdivisions of the paraventricular nucleus. A continuum of labeled cells extended from the caudal part of the paraventricular nucleus into the posterior hypothalamus and into the central gray of the midbrain. Labeled cells were also seen in the medial basal hypothalamus and the rostral part of the bed nucleus of the stria terminalis. A few labeled cells were observed in the medial and lateral preoptic areas, the rostral part of the paraventricular nucleus, and in the arcuate nucleus. Following WGA-gel implants in the lumbar or sacral cord many retrogradely labeled cells were observed mainly in the paraventricular nucleus, the lateral hypothalamus, zona incerta, medial basal hypothalamus, and posterior hypothalamic area. The continuum of labeled cells described above was also seen following these implants. Our data indicate that the lateral hypothalamus and zona incerta, as well as specific parts of the paraventricular nucleus, are major loci of neurons which project directly to the medulla and spinal cord of the rat. The consistency with which labeled cells were localized across all brains examined within categories of implant sites and the large numbers of labeled cells counted within these areas appeared to verify the sensitivity of our retrograde tracing method. Therefore, we interpret the paucity or absence of labeled cells in particular brain regions to indicate that cells of these regions do not project to the medulla or spinal cord.

Temporal pattern of HRP spread from an iontophoretic deposit site and description of a new HRP-gel implant method.

In the course of examining afferents to ventromedial hypothalamic (VMH) neurons using horseradish peroxidase (HRP), we needed to know how close to an iontophoretic deposit site neurons could be proved to be retrogradely labeled. In evaluating cells near but clearly outside HRP deposit sites visualized after a 24-hour survival period, for example, neurons which had been filled with HRP by somal or dendritic uptake could not be treated as retrogradely labeled and thus would not add to studies of intrahypothalamic connections. Rats were given standardized iontophoretic applications of HRP into VMH (continuous positive current 0.25 mu amp for 1 minute) and sacrificed after 5 or 15 minutes, 1, 4, 8, 12, or 24 hours in order to examine the pattern of HRP spread. The chromogen was tetramethylbenzidine. The volume of the application site visualized at 24 hours was less than 10% of maximum site size, which occurred at 1 hour. Since the cells located within the maximal spread boundary are candidates for nonretrograde labeling, HRP data on local connections obtained even from small iontophoretic deposits must be evaluated in the light of the demonstrated expansion and subsequent contraction of the application site. These results may also hold implications for the precision with which distant connections can be examined using the HRP retrograde method, as sites that appear discrete when visualized after 24-hour survival may have overlapped at shorter times post-iontophoresis. Incorporation of retrograde tracers into polyacrylamide gels provides an effective alternative to pressure injection or iontophoresis of aqueous tracer solutions. We describe a method for filling micropipettes with HRP-polyacrylamide gel. The pipettes are then implanted into brain sites to provide a confined pool of HRP. With postimplantation survival of 24 hour or longer, this method produces sites comparable in size to iontophoretic sites examined at 24 hours and results in improved retrograde labeling. Some results obtained with this method concerning the afferent connections of the dorsomedial hypothalamus are described.

Axonal projections and peptide content of steroid hormone concentrating neurons.

The axonal projections of cell groups containing the most dense collections of steroid hormone concentrating cells have been demonstrated with retrograde neuroanatomical tracing methods. Horseradish peroxidase revealed large numbers of neurons in ventrolateral ventromedial nucleus (VL-VM) which project to dorsal midbrain. Wheat germ agglutinin (immunocytochemical recognition method) revealed large numbers of neurons in medial basal hypothalamus (MBH) and particular subdivisions of paraventricular nucleus (PVN) that project to dorsal caudal medulla or spinal cord. Fluorescent dyes revealed that many preoptic area (POA), anterior hypothalamic (AHA), and bed nucleus of the stria terminalis (BNST) neurons project to ventral tegmental area of Tsai (VTA). Also many neurons in POA and BNST project to amygdala. A method which enabled simultaneous demonstration of the steroid binding capacity and axonal projections of neurons in the same tissue section revealed that 26-36% estradiol (E2) concentrating cells in VL-VM project to dorsal midbrain. E2 concentrating neurons in POA and BNST project to amygdala and E2 concentrating POA neurons project to VTA. These neurons, which send their axons to cell groups located in different brain regions, are probably under the genomic-regulatory influence of E2. Using a method which allows simultaneous demonstration of peptide content and steroid hormone concentrating capacity of cells, many oxytocin-neurophysin and vasopressin-neurophysin containing magnocellular neurons in the caudal PVN were found to concentrate E2. About 4% of the beta-endorphin and about 6% of the dynorphin containing neurons in the MBH concentrate E2. In contrast, virtually none (less than 0.2%) of the LHRH containing hypothalamic neurons concentrate E2.

Characterization of estrogen-concentrating hypothalamic neurons by their axonal projections.

A method combining steroid autoradiography and fluorescent dye retrograde neuroanatomical tracing has been devised. This method makes it possible to demonstrate that some estrogen-concentrating cells in the ventrolateral subdivision of the ventromedial nucleus of the rat hypothalamus are neurons that send axons to the dorsal midbrain. Other cells only concentrate estrogen or only project to the midbrain. Estrogen-concentrating neurons in the ventromedial hypothalamus that project to the dorsal midbrain are likely to transmit hormone-influenced signals that regulate circuits for reproductive or other behaviors or autonomic functions.

Autoradiographic identification of estradiol-concentrating cells in the spinal cord of the female rat.

The topography and number of estradiol (E)-concentrating cells in the lower lumbar and sacral segments of the spinal cord of the female rat have been examined by the steroid autoradiography method. A nuclear-saturating does of E was administered by intravenous infusion, which kept blood estrogen at or above proestrus levels for 3.5-4 h, much longer than usual for steroid receptor studies. The cord segments selected for examination are known to receive somatosensory information relevant for estrogen-dependent behavior, and to contain some of the motoneurons for epaxial muscles responsible for this behavior. Small numbers of E-concentrating cells were found in the dorsal portion of the gray matter of L4, L5, L6 and the sacral segments. These cells were found in lamina II, in the midline region which includes lamina X, and the medial portions of laminae III, IV and V when they cross in the midline. E-concentrating cells were also found in the lateral portions of laminae III, IV, and V, and in lamina VII. Virtually no E-concentrating cells were found in the ventral portion of the gray matter or in the white matter. The spinal cord had few E-concentrating cells compared to the hypothalamus.