ORE identifies extreme expression effects enriched for rare variants.

MOTIVATION: Non-coding rare variants (RVs) may contribute to Mendelian disorders but have been challenging to study due to small sample sizes, genetic heterogeneity and uncertainty about relevant non-coding features. Previous studies identified RVs associated with expression outliers, but varying outlier definitions were employed and no comprehensive open-source software was developed. RESULTS: We developed Outlier-RV Enrichment (ORE) to identify biologically-meaningful non-coding RVs. We implemented ORE combining whole-genome sequencing and cardiac RNAseq from congenital heart defect patients from the Pediatric Cardiac Genomics Consortium and deceased adults from Genotype-Tissue Expression. Use of rank-based outliers maximized sensitivity while a most extreme outlier approach maximized specificity. Rarer variants had stronger associations, suggesting they are under negative selective pressure and providing a basis for investigating their contribution to Mendelian disorders. AVAILABILITY AND IMPLEMENTATION: ORE, source code, and documentation are available at https://pypi.python.org/pypi/ore under the MIT license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Integrative transcriptomic analysis reveals key drivers of acute peanut allergic reactions.

Mechanisms driving acute food allergic reactions have not been fully characterized. We profile the dynamic transcriptome of acute peanut allergic reactions using serial peripheral blood samples obtained from 19 children before, during, and after randomized, double-blind, placebo-controlled oral challenges to peanut. We identify genes with changes in expression triggered by peanut, but not placebo, during acute peanut allergic reactions. Network analysis reveals that these genes comprise coexpression networks for acute-phase response and pro-inflammatory processes. Key driver analysis identifies six genes (LTB4R, PADI4, IL1R2, PPP1R3D, KLHL2, and ECHDC3) predicted to causally modulate the state of coregulated networks in response to peanut. Leukocyte deconvolution analysis identifies changes in neutrophil, naive CD4+ T cell, and macrophage populations during peanut challenge. Analyses in 21 additional peanut allergic subjects replicate major findings. These results highlight key genes, biological processes, and cell types that can be targeted for mechanistic study and therapeutic targeting of peanut allergy.

Development and Characterization of Novel Rat Anti-mERß Sera.

Estrogens regulate normal sexual and reproductive development in females. Their actions are mediated mainly by estrogen receptor (ER)α and ERß. Understanding the function of ERs necessitates knowing their cellular location and protein partners, which, in turn, requires reliable and specific antibodies. Several antibodies are available for ERα; however, discrepancies in immunoreactivity have been reported for ERß. Here, we have developed antisera for mouse ERß (mERß) using a specific C-terminal 18-amino acid peptide conjugated to mariculture keyhole limpet hemocyanin. Sprague Dawley rats were immunized, and the resulting antisera were characterized by Western blot analysis of nuclear extracts from tissues of wild-type (WT) mice, and mice genetically modified to lack either ERα (CERαKO) or ERß (CERßKO). An approximately 56-kDa protein was detected in the hypothalamus, uterus, ovary, mammary gland, testes, and epididymis of WT mice, consistent with the predicted molecular size of ERß. In addition, the same protein band was identified in in vitro synthesized mERß protein and in the mammary glands of CERαKO mice. The approximately 56-kDa protein was not observed in in vitro synthesized mERα protein or in any tissue examined in the CERßKO mice. Immunohistochemistry using the antisera revealed ERß staining in the granulosa cells of WT ovaries and in the mediobasal hypothalamus, paraventricular nucleus, and cerebral cortex in the WT adult mouse brain. These data suggest that the novel rat anti-mERß sera are specific to ERß to allow investigators to explore to cellular and physiological role of ERß in the brain and other mouse tissues.

Kisspeptin and the preovulatory gonadotrophin-releasing hormone/luteinising hormone surge in the ewe: basic aspects and potential applications in the control of ovulation.

The identification of the neural mechanisms controlling ovulation in mammals has long been a 'holy grail' over recent decades, although the recent discovery of the kisspeptin systems has totally changed our views on this subject. Kisspeptin cells are the major link between gonadal steroids and gonadotrophin-releasing hormone (GnRH) neurones. In the female rodent, kisspeptin cells of the preoptic area are involved in the positive-feedback action of oestrogen on GnRH secretion, although the picture appears more complicated in the ewe. As in rodents, activation of preoptic kisspeptin neurones accompanies the GnRH surge in the ewe but an active role for arcuate kisspeptin neurones has also been proposed. Experimentally, kisspeptin is able to restore reproductive function when the hypothalamic-hypophyseal ovarian axis is quiescent. For example, i.v. infusion of a low dose of peptide in anoestrous ewes induces an immediate and sustained release of gonadotrophin, which subsides and then provokes a luteinising hormone (LH) surge a few hours later. This pharmacological intervention induces the same hormonal changes normally observed during the follicular phase of the oestrous cycle, including the secretion of oestrogen and its negative- and positive-feedback actions on the secretion of LH and follicle-stimulating hormone. Accordingly, a high percentage of kisspeptin-infused animals ovulated. Although the multiple facets of how the kisspeptin systems modulate GnRH secretion are not totally understood, the demonstration that exogenous kisspeptin administration can induce ovulation in anovulatory animals paves the way for future therapeutic applications aiming to control reproduction.

Temporal and spatial regulation of CRE recombinase expression in gonadotrophin-releasing hormone neurones in the mouse.

Gonadotrophin-releasing hormone (GnRH) neurones located within the brain are the final neuroendocrine output regulating the reproductive hormone axis. Their small number and scattered distribution in the hypothalamus make them particularly difficult to study in vivo. The Cre/loxP system is a valuable tool to delete genes in specific cells and tissues. We report the production of two mouse lines that express the CRE bacteriophage recombinase in a GnRH-specific manner. The first line, the GnRH-CRE mouse, contains a transgene in which CRE is under the control of the murine GnRH promoter and targets CRE expression specifically to GnRH neurones in the hypothalamus. The second line, the GnRH-CRETeR mouse, uses the same murine GnRH promoter to target CRE expression to GnRH neurones, but is modified to be constitutively repressed by a tetracycline repressor (TetR) expressed from a downstream tetracycline repressor gene engineered within the transgene. GnRH neurone-specific CRE expression can therefore be induced by treatment with doxycycline which relieves repression by TetR. These GnRH-CRE and GnRH-CRETeR mice can be used to study the function of genes expressed specifically in GnRH neurones. The GnRH-CRETeR mouse can be used to study genes that may have distinct roles in reproductive physiology during the various developmental stages.

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 and progesterone do not activate Fos in AVPV or LHRH neurons in male rats.

In rodents, females but not males, in response to escalating levels of estrogen, express a luteinizing hormone (LH) surge that is prompted by a surge in luteinizing hormone-releasing hormone (LHRH). It cannot take place if estrogen-sensitive afferents located in the anteroventral periventricular nucleus (AVPV) are either absent or disabled. Males appear to lack the ability to exhibit an LH surge, but it is unclear what level of the CNS contributes to this dimorphic response. This study was conducted to determine whether estrogen followed by progesterone treatment (E + P) of gonadectomized males evokes Fos activation in LHRH and AVPV neurons as it does in females. The results indicated that, consistent with the males' inability to express an LH surge in response to E + P treatment, LHRH and AVPV neurons in males failed to show increased Fos activation. Examination of neuron nuclear antigen (NeuN, a neuron-specific marker), estrogen receptor (ERalpha) and progesterone receptor (PR) neurons in AVPV neurons indicated that, while essentially all the neurons of the caudal AVPV in males and females are steroid responsive, the male possessed half the number of steroid responsive neurons within the caudal AVPV (where activation of Fos is maximal in females) compared to the female. Together, these data indicate that the male lacks a substantial population of steroid receptive AVPV neurons and is unable to respond to the presence of E and P and activate either AVPV or LHRH neurons.

Neonatal manipulations of oxytocin alter expression of oxytocin and vasopressin immunoreactive cells in the paraventricular nucleus of the hypothalamus in a gender-specific manner.

Early postnatal manipulations of oxytocin have long-term behavioral and physiological consequences; the present study examined the hypothesis that oxytocin or its absence influences the subsequent expression of either oxytocin or arginine vasopressin in the CNS. On postnatal day 1 female and male prairie voles (Microtus ochrogaster) received a single i.p. injection of oxytocin (3 microg), oxytocin antagonist (0.3 microg), or 50 microl of isotonic saline or were only handled. On postnatal days 1, 8 and 21, brains were fixed, sectioned and stained for oxytocin or vasopressin immunoreactivity and analyzed as a function of age, treatment and sex. Both oxytocin and vasopressin immunoreactivity were observed on day 1 in the supraoptic and paraventricular nuclei (PVN) of the hypothalamus. Numbers of oxytocin and vasopressin neurons increased with age in both nuclei. Females treated on postnatal day 1 with oxytocin or oxytocin antagonist displayed a significant increase in oxytocin immunoreactivity on day 21 in the PVN. In contrast, males treated with antagonist tended to have decreased vasopressin immunoreactivity in the same region. These results revealed that the effects of neonatal manipulation of oxytocin are age-dependent, site-specific and sexually dimorphic. The long-lasting effects of neonatal exposure to exogenous oxytocin and oxytocin antagonist indicate a role for oxytocin in the development of the CNS during the neonatal period, affecting the development of the oxytocinergic system in females and the vasopressinergic system in males. The developmental effects observed suggest one possible mechanism by which neonatal exposure to oxytocin or neonatal inhibition of endogenous oxytocin produces long-lasting behavioral and physiological alterations and could play a role in the development of male- and female-typical behavior.

Ovarian steroid modulation of seizure severity and hippocampal cell death after kainic acid treatment.

To determine whether maintained estrogen or progesterone levels affect kainic acid (KA) seizure patterns or the susceptibility of hippocampal neurons to death from seizures, ovariectomized Sprague-Dawley rats were implanted with estrogen pellets, 0.1 or 0.5 mg, that generated serum levels of 42.4 +/- 6.6 (mean +/- SEM) and 242.4 +/- 32.6 pg/ml or one to six capsules of progesterone that generated serum levels of 11.00 +/-.72 to 48.62 +/- 9.4 ng/ml. Seven days later, the rats were administered KA (8.5mg/kg, ip) and scored for seizure activity; 96 h later, the rats were killed and their brains processed for localization of neuron nuclear antigen (NeuN), a general neuronal marker. The hippocampus was scored for spread (the number of separate regions showing cell loss), and the area within the CA fields occupied by NeuN immunoreactivity was measured (indicating surviving neurons). Administration of estrogen or progesterone (independent of dose) significantly reduced mortality from KA seizures. Progesterone reduced seizure severity in animals that received one to four implants; compared with controls, no difference in seizure severity was noted for animals with six progesterone implants. The reduced seizures in progesterone-treated animals were accompanied by a reduction in the spread of hippocampal damage (r(2) = 0.87; P < 0.05). Likewise, in progesterone-treated rats, neuron survival and reduction in seizure scores were correlated (r(2) = 0.76; P < 0.0001). Estrogen had no effect on seizure severity (P > 0.05), but reduced both the spread (P < 0.05) and degree of neuronal loss (P < 0.05). Indeed, in the estrogen-treated rats, neuronal death was significantly lower than that observed in progesterone-treated animals with equally severe seizures (P < 0.05). These data are consistent with the hypothesis that progesterone produces its effects by reducing seizures, whereas estrogen has little beneficial effect on seizure behavior but protects the hippocampus from the damage seizures produce.

Anatomical markers of activity in neuroendocrine systems: are we all 'fos-ed out'?

It has now been nearly 15 years since the immediate early gene, c-fos, and its protein product, Fos, were introduced as tools for determining activity changes within neurones of the nervous system. In the ensuing years, this approach was applied to neuroendocrine study with success. With it have come advances in our understanding of which neuroendocrine neurones respond to various stimuli and how other central nervous system components interact with neuroendocrine neurones. Use of combined tract-tracing approaches, as well as double-labelling for Fos and transmitter markers, have added to characterization of neuroendocrine circuits. The delineation of the signal transduction cascades that induce Fos expression has led to establishment of the relationship between neurone firing and Fos expression. Importantly, we can now appreciate that Fos expression is often, but not always, associated with increased neuronal firing and vice versa. There are remaining gaps in our understanding of Fos in the nervous system. To date, knowledge of what Fos does after it is expressed is still limited. The transience of Fos expression after stimulation (especially if the stimulus is persistent) complicates design of experiments to assess the function of Fos and makes Fos of little value as a marker for long-term changes in neurone activity. In this regard, alternative approaches must be sought. Useful alternative approaches employed to date to monitor neuronal changes in activity include examination of (i) signal transduction intermediates (e.g. phosphorylated CREB); (ii) transcriptional/translational intermediates (e.g. heteronuclear RNA, messenger RNA (mRNA), prohormones); and (iii) receptor translocation. Another capitalizes on the fact that many neuroendocrine systems show striking stimulus-transcription coupling in the regulation of their transmitter or its synthetic enzymes. Together, as we move into the 21st Century, the use of multiple approach to study activity within neuroendocrine systems will further our understanding of these important systems.

Parallel declines in Fos activation of the medial anteroventral periventricular nucleus and LHRH neurons in middle-aged rats.

The middle-age decline in reproductive function is manifested by reduced LHRH release, resulting in a decreased magnitude and delay of onset of the LH surge. Earlier studies suggested that the reductions in LHRH neural activation in middle-aged rats resulted from deficits in the afferent drive to the LHRH neurons. One critical afferent to the LHRH neurons lies in the anteroventral periventricular preoptic area (AVPv) nucleus. The neurons of the medial AVPv are synchronously activated to express Fos with LHRH neurons at the time of an LH surge in young adult animals. The present study examined whether, in middle age, reductions in the activation of AVPv neurons accompany the reduction in Fos activation in LHRH neurons. Young (3- to 4-month-old) and middle-aged (10- to 12-month-old) spontaneously cycling and ovariectomized steroid-replaced rats were killed during peak and early descending phase of the LH surge, and their brains were examined for Fos in LHRH and AVPv neurons. Young animals had a characteristic increase in Fos expression in both LHRH and AVPv neurons. In middle-aged rats, the proportion of LHRH neurons expressing Fos at the time of an LH surge was reduced by approximately 50%, irrespective of whether surges were spontaneous or induced by exogenous steroids. A similar reduction in the number of Fos+ cells (by approximately 50%) was noted in the medial AVPv. Linear regression analysis of the relationship between the extent of Fos activation in LHRH and AVPv neurons revealed a strong positive correlation (r(2) = 0.66; P < 0.01), suggesting that changes in the AVPv's drive to LHRH neurons underlie the decrease in LHRH activity in middle age. A second series of experiments examined whether decreased input from the AVPv could account for reduced Fos activation in LHRH neurons seen in middle-aged animals. When the medial AVPv was lesioned, LHRH neurons failed to express Fos on the side ipsilateral to the lesion. Animals with lesioned medial AVPv also had significantly lower LH values than animals with an intact medial AVPv. Taken together, these data suggest that a principal deficit in middle-aged rats is the ability of the medial AVPv to stimulate LHRH neurons.

Divergent effects of ovarian steroids on neuronal survival during experimental allergic encephalitis in Lewis rats.

Experimental allergic encephalitis, (EAE) a Th1-cell-dependent autoimmune disease of the central nervous system (CNS) used to study immune responses relevant to multiple sclerosis (MS) displays gender susceptibility. The underlying basis of the sexual dimorphism may reflect multiple factors including gender-specific hormones. To study the relationship between ovarian hormones and CNS inflammation, we induced EAE in susceptible female Lewis rats ovariectomized (OVX) 7 days earlier and implanted with blank capsules or capsules containing estradiol (E), progesterone (P), or both (EP). Rats were immunized with complete Freunds' adjuvant alone or combined with guinea pig myelin basic protein. Motor function was scored 0-5 on standard criteria (days 7-11 postimmunization). On day 11, the rats were euthanized and the lumbar spinal cord was analyzed for Nissl, neuron nuclear antigen, and DNA fragmentation with a TUNEL assay. Inflammation was judged qualitatively on a scale of 0-4. Our immunization protocol induced limited sensorimotor deficits in OVX rats (2.3 +/- 0.6, mean +/- SEM) with moderate inflammation (2.5 +/- 0.4). E limited both behavioral impairments (1.0 +/- 0.4) and inflammation (0.5 +/- 0.2). P-treated rats had more severe sensorimotor deficits (3.1 +/- 0.5) with increased inflammatory infiltrates (3.6 +/- 0.4) and markedly increased numbers of TUNEL(+) neurons. Neuron counts of the outer two Rexed lamina (L3-L5) showed a 20% neuron loss (P < 0.02) in P-treated rats with EAE in comparison to other groups. Coadministration of E with P prevented the consequences of P, including neuronal apoptosis (behavioral score, 0.6 +/- 0.6; inflammation, 1.4 +/- 0.5). Our results suggest a potential and novel function of P that increases the vulnerability of neurons to apoptotic injury in EAE and may have pathophysiologic implications in the progression of disability in women with MS.

Suckling stimulus suppresses messenger RNA for tyrosine hydroxylase in arcuate neurons during lactation.

Tyrosine hydroxylase (TH) mRNA in tuberoinfundibular dopamine (TIDA) neurons is suppressed during lactation but rebounds upon pup removal. A time course of TH mRNA changes after pup removal revealed three phases: (1) a nuclear phase (evident 1.5 hours after pup removal, maximal at 3 hours) with TH mRNA appearing in 1 or 2 nuclear loci with little or no change in cytoplasmic mRNA; (2) a cytoplasmic phase (noted 6 hours after pup removal, peaked 12-24 hours) with a significant increase in total TH mRNA levels mainly in the cytoplasm; and (3) a stabilization phase (24-48 hours after pup removal) when nuclear signals were low and cytoplasmic RNA showed a slight decline with extension of RNA clusters into the cell dendrites. In rats whose pups could suckle only on one side, TH was up-regulated only on the side contralateral to nipple blockade. These data indicate that after suckling terminates, TH up-regulation is evident at 1.5 hours, but 6 hours is needed before the cells transport sufficient mRNA into the cytoplasm. The rapid signaling of TH up-regulation stems from the fact that the TIDA neurons respond to neural signals from termination of suckling.

Distribution of gonadal steroid receptor-containing neurons in the preoptic-periaqueductal gray-brainstem pathway: a potential circuit for the initiation of male sexual behavior.

The present study used anterograde and retrograde tract tracing techniques to examine the organization of the medial preoptic-periaqueductal gray-nucleus paragigantocellularis pathway in the male rat. The location of neurons containing estrogen (alpha subtype; ER alpha) and androgen receptors (AR) were also examined. We report here that injection of the anterograde tracer biotinylated dextran amine (BDA) into the medial preoptic (MPO) produced dense labeling within the periaqueductal gray (PAG); anterogradely labeled fibers terminated in close juxtaposition to neurons retrogradely labeled from the nucleus paragigantocellularis (nPGi). Dual immunostaining for Fluoro-Gold (FG) and ER alpha or FG and AR showed that over one-third of MPO efferents to the PAG contain receptors for either estrogen or androgen. In addition, approximately 50% of PAG neurons retrogradely labeled from the nPGi were immunoreactive for either ER alpha or AR. These results are the first to establish an MPO-->PAG-->nPGi circuit and further indicate that gonadal steroids can influence neuronal synaptic activity within these sites. We reported previously that nPGi reticulospinal neurons terminate preferentially within the motoneuronal pools of the lumbosacral spinal cord that innervate the pelvic viscera. Together, we propose that the MPO-->PAG-->nPGi circuit forms the final common pathway whereby MPO neural output results in the initiation and maintenance of male copulatory reflexes.

Brainstem catecholaminergic neurons activated by hypoxemia express GR and are coordinately activated with fetal sheep hypothalamic paraventricular CRH neurons.

In late gestation, challenges to fetal homeostasis are accompanied by increases in adrenocorticotropin (ACTH) concentrations in fetal peripheral plasma and Fos (c-fos protein) activation in corticotropin-releasing hormone (CRH) neurons of the fetal hypothalamic paraventricular nucleus (PVN). In adults, ventrolateral brainstem catecholaminergic (CA) neurons (A1/C1, A2/C2) project to the parvocellular neurons of the PVN, possess glucocorticoid receptors (GR) and are Fos activated in parallel with CRH neurons of the PVN during hypoxia. Such observations suggest a role for the aforementioned medullary neurons in the function of the hypothalamo-pituitary-adrenal axis. The present study utilized late gestation fetal sheep, stereotaxic methodology and retrograde axon tracing and immunocytochemical techniques to investigate the relationship between activation of fetal brainstem CA neurons and activation of fetal PVN CRH immunopositive neurons in response to hypoxemia. Results indicated that: (1) the largest brainstem CA projection to PVN CRH neurons is from A1/C1 neurons, (2) brainstem neurons exhibit GR immunostaining and (3) brainstem CA neurons show a strong correlation (A1/C1 - r(2)=0.894, P<0.005; A2/C2 - r(2)=0. 848; P<0.002) of Fos activation with Fos activation in PVN CRH cells. We conclude that in late gestation the brainstem A1/C1 and A2/C2 areas are in position to influence the function of the hypothalamo-pituitary-adrenal axis during hypoxemic challenges to homeostasis in a fashion similar to that which has been demonstrated in the adult rat.

Progesterone attenuates persistent inflammatory hyperalgesia in female rats: involvement of spinal NMDA receptor mechanisms.

The relationship between endogenous gonadal steroid levels and persistent or chronic pain is poorly understood. These studies used an inflammation model to examine the role of the gonadal steroid, progesterone, in the development of persistent pain and hyperalgesia in lactating ovary-intact and ovariectomized rats. The results indicate that constant high plasma levels of progesterone attenuate inflammatory hyperalgesia by a mechanism involving inhibition of N-methyl-D-aspartate receptor activation at the spinal cord level. Since the pattern of high progesterone in lactating rats mimics the progesterone component of the luteal phase of the human menstrual cycle, these findings have significance in persistent or chronic pain conditions that are most prevalent in females.

Androgen and estrogen (alpha) receptor distribution in the periaqueductal gray of the male Rat.

The midbrain periaqueductal gray (PAG) has been strongly implicated in numerous behaviors heavily influenced by the gonadal steroids estrogen and testosterone, including reproductive behavior, autonomic regulation, and antinociception. However, the location of receptors for these steroids within the PAG has not been carefully characterized. Immunocytochemical techniques were used to map the distribution of neurons immunoreactive for the androgen (AR) and estrogen receptor (alpha subtype; ERalpha) along the rostrocaudal axis of the PAG in the male rat. The results show that the PAG contains a large population of both androgen and estrogen receptor containing neurons. Neurons immunoreactive for either receptor were concentrated within the caudal two-thirds of the PAG. At midlevels of the PAG, ERalpha and AR immunoreactive neurons were located primarily within the dorsomedial and lateral PAG. In the caudal third of the PAG, immunoreactive cells were distributed primarily within the dorsal half. The distributions of ERalpha and AR were remarkably similar, and it is likely that some PAG neurons contain receptors for both gonadal steroids, similar to what has been previously reported for the male rat hypothalamus. The results of this study suggest that the PAG may provide the anatomical substrate for steroid mediated changes in nociceptive thresholds and reproductive behavior.

Periventricular preoptic area neurons coactivated with luteinizing hormone (LH)-releasing hormone (LHRH) neurons at the time of the LH surge are LHRH afferents.

Earlier studies demonstrated coactivation of the periventricular preoptic area (pePOA) with LHRH neurons at the time of an induced or spontaneous LH surge, suggesting that the pePOA might regulate LHRH neurons. To investigate this hypothesis, studies were conducted to determine the temporal pattern of pePOA Fos activation during the rat estrous cycle and establish the connections of the pePOA neurons with LHRH neurons. Fos activation within LHRH and pePOA neurons showed the same temporal pattern. Both were absent during diestrous I, diestrous II, and the morning of proestrus. Fos was induced in the pePOA and LHRH neurons beginning on the afternoon of proestrus (4 h before lights off), with a decline 8 h later on proestrous evening. Tract-tracing studies then established the relationship between LHRH and pePOA neurons. Retrograde labeling with fluorogold determined that a portion of the Fos-positive pePOA neurons present at the time of the LH surge sent a projection to regions that contain LHRH cells. Anterograde tracer (neurobiotin) injections established that the pePOA neurons sent axons to the LHRH cells. Taken together, these data indicate that the pePOA provides direct input to LHRH neurons that is likely to stimulate LHRH neurons at the time of the LH surge.

Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area.

Recent studies have identified several neuropeptide systems in the hypothalamus that are critical in the regulation of body weight. The lateral hypothalamic area (LHA) has long been considered essential in regulating food intake and body weight. Two neuropeptides, melanin-concentrating hormone (MCH) and the orexins (ORX), are localized in the LHA and provide diffuse innervation of the neuraxis, including monosynaptic projections to the cerebral cortex and autonomic preganglionic neurons. Therefore, MCH and ORX neurons may regulate both cognitive and autonomic aspects of food intake and body weight regulation. The arcuate nucleus also is critical in the regulation of body weight, because it contains neurons that express leptin receptors, neuropeptide Y (NPY), alpha-melanin-stimulating hormone (alpha-MSH), and agouti-related peptide (AgRP). In this study, we examined the relationships of these peptidergic systems by using dual-label immunohistochemistry or in situ hybridization in rat, mouse, and human brains. In the normal rat, mouse, and human brain, ORX and MCH neurons make up segregated populations. In addition, we found that AgRP- and NPY-immunoreactive neurons are present in the medial division of the human arcuate nucleus, whereas alpha-MSH-immunoreactive neurons are found in the lateral arcuate nucleus. In humans, AgRP projections were widespread in the hypothalamus, but they were especially dense in the paraventricular nucleus and the perifornical area. Moreover, in both rat and human, MCH and ORX neurons receive innervation from NPY-, AgRP-, and alpha-MSH-immunoreactive fibers. Projections from populations of leptin-responsive neurons in the mediobasal hypothalamus to MCH and ORX cells in the LHA may link peripheral metabolic cues with the cortical mantle and may play a critical role in the regulation of feeding behavior and body weight.

Medullary c-Fos activation in rats after ingestion of a satiating meal.

The distribution and chemical phenotypes of hindbrain neurons that are activated in rats after food ingestion were examined. Rats were anesthetized and perfused with fixative 30 min after the end of 1-h meals of an unrestricted or rationed amount of food, or after no meal. Brain sections were processed for localization of the immediate-early gene product c-Fos, a marker of stimulus-induced neural activation. Hindbrain c-Fos expression was low in rats that ate a rationed meal or no meal. Conversely, c-Fos was prominent in the medial nucleus of the solitary tract (NST) and area postrema in rats that ate to satiety. There was a significant positive correlation between postmortem weight of gastric contents and the proportion of NST catecholaminergic neurons expressing c-Fos. Cells in the ventrolateral medulla (VLM) were not activated in rats after food ingestion, in contrast with previous findings that stimulation of gastric vagal afferents with anorexigenic doses of cholecystokinin activates c-Fos expression in both NST and VLM catecholaminergic cells. These findings indicate that anatomically distinct subsets of hindbrain catecholaminergic neurons are activated in rats after food ingestion and that activation of these cells is quantitatively related to the magnitude of feeding-induced gastric distension.