Effects of chronic weight perturbation on energy homeostasis and brain structure in mice.

Maintenance of reduced body weight in lean and obese human subjects results in the persistent decrease in energy expenditure below what can be accounted for by changes in body mass and composition. Genetic and developmental factors may determine a central nervous system (CNS)-mediated minimum threshold of somatic energy stores below which behavioral and metabolic compensations for weight loss are invoked. A critical question is whether this threshold can be altered by environmental influences and by what mechanisms such alterations might be achieved. We examined the bioenergetic, behavioral, and CNS structural responses to weight reduction of diet-induced obese (DIO) and never-obese (CON) C57BL/6J male mice. We found that weight-reduced (WR) DIO-WR and CON-WR animals showed reductions in energy expenditure, adjusted for body mass and composition, comparable (-10-15%) to those seen in human subjects. The proportion of excitatory synapses on arcuate nucleus proopiomelanocortin neurons was decreased by ∼50% in both DIO-WR and CON-WR mice. These data suggest that prolonged maintenance of an elevated body weight (fat) alters energy homeostatic systems to defend a higher level of body fat. The synaptic changes could provide a neural substrate for the disproportionate decline in energy expenditure in weight-reduced individuals. This response to chronic weight elevation may also occur in humans. The mouse model described here could help to identify the molecular/cellular mechanisms underlying both the defense mechanisms against sustained weight loss and the upward resetting of those mechanisms following sustained weight gain.

Enhanced anorexigenic signaling in lean obesity resistant syndecan-3 null mice.

Obesity is associated with increased risk of diabetes, cardiovascular disease and several types of cancers. The hypothalamus is a region of the brain critical in the regulation of body weight. One of the critical and best studied hypothalamic circuits is comprised of the melanocortinergic orexigenic agouti-related protein (AgRP) and anorexigenic α-melanocyte stimulating hormone (α-MSH) neurons. These neurons project axons to the same hypothalamic target neurons and balance each other's activity leading to body weight regulation. We previously showed that the brain proteoglycan syndecan-3 regulates feeding behavior and body weight, and syndecan-3 null (SDC-3(-/-)) mice are lean and obesity resistant. Here we show that the melanocortin agonist Melanotan II (MTII) potently suppresses food intake and activates the hypothalamic paraventricular nuclei (PVN) in SDC-3(-/-) mice based on c-fos immunoreactivity. Interestingly, we determined that the AgRP neuropeptide is reduced in the PVN of SDC-3(-/-) mice compared to wild type mice. In contrast, neuropeptide Y, coexpressed in the AgRP neuron, is not differentially expressed nor is the counteracting neuropeptide α-MSH. These findings are unprecedented and indicate that AgRP protein localization can be selectively regulated within the hypothalamus resulting in altered neuropeptide response and tone.

New approaches to the understanding of tamoxifen action and resistance.

Tamoxifen (TAM) provides an effective agent for treatment of hormone-dependent breast cancer but resistance uniformly ensues upon continued use. Additional studies are required to define more precisely the mechanisms involved in development of resistance. We conducted systematic experimental and clinical studies based on the hypothesis that tumors exposed to TAM long-term may develop resistance by becoming hypersensitive to its estrogenic effects. These investigations uncovered new features of the TAM resistance (TR) phenomenon and identified possible means for its prevention and/or elimination. Initially we confirmed that TR may be divided into two subtypes, primary and acquired resistance, and that these differ by certain important characteristics including the level of the possible involvement of adaptive and genetic components. Then we distinguished at least three consequent stages of this phenomenon: stage I when TAM behaves as an antiestrogen, stage II with development of increased sensitivity to the agonistic (pro-estrogenic) properties of TAM and stage III with an adaptive increase in sensitivity to estradiol (E(2)). During this evolutionary process, as shown in vitro, MAP kinase (MAPK) and aromatase activities increase. The time frame of the increase in MAPK activity as a rule outpaces the increase in aromatase activity during the course of the development of TR. This may occur as a response to estrogen deprivation or interruption of the process of estrogen signaling and can be one of the promoting factors of increased aromatase activation. On the other hand, the chronology of these events indicates that changes in the MAPK cascade can be more important for the early steps of the development and maintenance of the TR state. Changes in local estrogen production/sensitivity to E(2) are perhaps essential for the later steps of this phenomenon. We have explored the use of a growth factor-blocking agent to abrogate the adaptive changes in sensitivity. Farnesylthiosalicylic acid (FTS), an inhibitor of GTP-Ras binding to its membrane acceptor site, reduces the increase in the number of MCF-7 cells induced by long-term TAM treatment. It also decreases MAPK activity in TAM-treated MCF-7 cells and in established TR cell lines. Alone or in combination with letrozole (presumably, through the influence on MAPK pathway) FTS exerts moderate inhibitory effects on aromatase activity in estrogen-deprived or estrogen-exposed MCF-7 cells. Taken together, our observations suggest that FTS is a 'candidate drug' for the treatment of TR. Both the adaptive and genetic types of resistance may be amenable to this approach. Our studies underline the possible importance of starting the treatment/prevention of TR early on. From our clinical studies using immunohistochemistry, there is a rather strong rationale to include as a predisposing factor in the development of TR the increase in MAPK and aromatase activities in human primary breast tumors. In summary, data obtained during the course of this project may be considered as evidence supporting the principle that processes resulting in responses to TAM as an agonist and the development of estrogen hypersensitivity of breast cancer cells could potentially be mechanistically linked.

Estrogen, synaptic plasticity and hypothalamic reproductive aging.

Unlike primates who undergo ovarian failure and loss of sex steroids at the end of reproduction, aging rodents undergo constant vaginal estrus followed by constant diestrus and finally anestrus, which indicates the absence of responsive ovarian follicles. The latter state is analogous to menopause in women. The timing of the appearance of constant estrus is determined by many factors including estrogen exposure in the brain during development and the number of times that the animal gets pregnant. The chief site of this reproductive aging in rat brains is the arcuate nucleus of the hypothalamus. The transition from normal cycles to constant estrus parallels the females' gradually decreased ability to respond to administered estradiol with a cycle of inhibition followed by disinhibition of gonadotrophin-releasing hormone. Evidence has accumulated indicating this to be due to a loss of the rat's ability to respond to markedly elevated estradiol with the usual arcuate nucleus neuro-glial plasticity that supports the estrogen-induced gonadotrophin surge (EIGS). Just as male rats are not capable of an EIGS, aged females loose this ability through repeated EIGS. Experiments indicate that in male rats the hypothalamic synaptology that develops as a result of exposure to testicular androgens in the perinatal period (brain sexual differentiation) is a result of conversion of testosterone from the testes to estrogen in the brain and is therefore due to early estrogen exposure. Aging females appear to reach a synaptology similar to males and constant estrus as a result of repeated exposure to ovarian estrogens during their reproductive careers. The relative role of aging and hormonal factors remains unclear. Morphological evidence is presented that indicates the above effects of estrogen involve changes in hypothalamic arcuate nucleus neurons and glia, including changes in the organization of perikaryal membranes as well as arcuate nucleus synaptology and the load of peroxidase in the astroglia. A possible role for free radicals (reactive oxygen species) in hypothalamic reproductive aging has been proposed. Such a mechanism is supported by evidence that the anti-oxidant vitamin E delays the onset of constant estrus and the accumulation of glial peroxidase in aging female rats. However, since the synaptology and peroxidase load in constant estrus females is independent of the age at which the constant estrus occurs, it appears that the role of (repeated) estradiol exposure is more deterministic of hypothalamic failure than is aging, per se.

Supramammillary area mediates subcortical estrogenic action on hippocampal synaptic plasticity.

It is well established that systemically administered estrogen to ovariectomized rats positively affects the density of pyramidal cell spines in the hippocampal CA1 subfield and intact subcortical connections of the hippocampus are essential in this hormonal action. This study explored whether local estrogen administration into the supramammillary area influences the density of CA1 pyramidal cell spine synapses in ovariectomized rats. The first group of experiments using a combination of retrograde tracer technique and immunostaining for estrogen receptor-alpha demonstrated that a large population of supramammillary area estrogen receptor-alpha-containing neurons projects to the hippocampus. Animals belonging to the second experimental group were ovariectomized and received cannulae filled with 0.4% 17 beta-estradiol placed unilaterally into the supramammillary area. Control animals received a cholesterol-containing cannula into the supramammillary area or an estrogen-filled cannula implanted into the head of the caudate nucleus. One week later, rats were killed and CA1 pyramidal cell spine synapse density was determined using electron microscopic unbiased stereological procedures. Animals that received an estrogen-filled cannula into the supramammillary area exhibited a significantly higher (37%) density of CA1 pyramidal cell spine synapses than both other control groups. These observations indicate that the supramammillary area is involved in mediating synaptoplastic, estrogenic effects to the hippocampus.

Hormonal regulation of hippocampal spine synapse density involves subcortical mediation.

It is well established that estrogen has positive effects on the density of pyramidal cell spines in the hippocampal CA1 subfield. This study explored whether afferent connections of the hippocampus that come from estrogen-sensitive subcortical structures, including the septal complex, median raphe and supramammillary area, play a role in this estrogen-induced hippocampal synaptic plasticity. These particular subcortical structures have major influences on hippocampal activity, including theta rhythm and long-term potentiation. The latter also promotes the formation of new synapses. All of the rats were ovariectomized; the fimbria/fornix, which contains the majority of subcortical efferents to the hippocampus, was transected unilaterally in each, and half of the animals received estrogen replacement. Using unbiased electron microscopic stereological methods, the CA1 pyramidal cell spine synapse density was calculated. In the estrogen-treated rats, contralateral to the fimbria/fornix transection, the spine density of CA1 pyramidal cells increased dramatically, compared to the spine density values of both the ipsilateral and contralateral hippocampi of non-estrogen-treated animals and to that of the ipsilateral hippocampus of the estrogen replaced rats. These observations indicate that fimbria/fornix transection itself does not considerably influence CA1 area pyramidal cell spine density and, most importantly, that the estrogenic effect on hippocampal morphology, in addition to directly affecting the hippocampus, involves subcortical mediation.

Muscarinic tone sustains impulse flow in the septohippocampal GABA but not cholinergic pathway: implications for learning and memory.

Systemic infusions of the muscarinic cholinergic receptor antagonists atropine and scopolamine (atr/scop) produce an amnesic syndrome in humans, subhuman primates, and rodents. In humans, this syndrome may resemble early symptoms of Alzheimer's disease. Behavioral studies in rats have demonstrated that the medial septum/diagonal band of Broca (MSDB), which sends cholinergic and GABAergic projections to the hippocampus, is a critical locus in mediating the amnesic effects of atr/scop. The amnesic effects of atr/scop in the MSDB have been presumed but not proven to be caused by a decrease in hippocampal acetylcholine (ACh) release after blockade of a muscarinic tone in the MSDB. Using electrophysiological recordings and fluorescent-labeling techniques to identify living septohippocampal neurons in rat brain slices, we now report that, contrary to current belief, a blockade of the muscarinic tone in the MSDB does not decrease impulse flow in the septohippocampal cholinergic pathway; instead, it decreases impulse flow in the septohippocampal GABAergic pathway via M(3) muscarinic receptors. We also report that the muscarinic tone in the MSDB is maintained by ACh that is released locally, presumably via axon collaterals of septohippocampal cholinergic neurons. As such, cognitive deficits that occur in various neurodegenerative disorders that are associated with a loss or atrophy of septohippocampal cholinergic neurons cannot be attributed solely to a decrease in hippocampal acetylcholine release. An additional, possibly more important mechanism may be the concomitant decrease in septohippocampal GABA release and a subsequent disruption in disinhibitory mechanisms in the hippocampus. Restoration of impulse flow in the septohippocampal GABA pathway, possibly via M(3) receptor agonists, may, therefore, be critical for successful treatment of cognitive deficits associated with neurodegenerative disorders such as Alzheimer's and Parkinson's disease.

The supramammillo-hippocampal and supramammillo-septal glutamatergic/aspartatergic projections in the rat: a combined [3H]D-aspartate autoradiographic and immunohistochemical study.

It is well established that the supramammillary nucleus plays a critical role in hippocampal theta rhythm generation/regulation by its direct and indirect (via the septal complex) connections to the hippocampus. Previous morphological and electrophysiological studies indicate that both the supramammillo-hippocampal and supramammillo-septal efferents contain excitatory transmitter. To test the validity of this assumption, transmitter specific retrograde tracer experiments were performed. [3H]D-aspartate was injected into different locations of the hippocampus (granular and supragranular layers of the dentate gyrus and CA2 and CA3a areas of the Ammon's horn) and septal complex (medial septum and the area between the medial and lateral septum) that are known targets of the supramammillary projection. Consecutive vibratome sections prepared from the entire length of the posterior hypothalamus, including the supramammillary area, were immunostained for calretinin, tyrosine hydroxylase, or calbindin, and further processed for autoradiography. Radiolabeled, radiolabeled plus calretinin-containing, and calretinin-immunoreactive neurons were plotted at six different oro-caudal levels of the supramammillary area. The results demonstrated that following both hippocampal and septal injection of the tracer, the majority of the retrogradely radiolabeled (glutamatergic/aspartatergic) cells are immunoreactive for calretinin. However, non-radiolabeled calretinin-containing neurons and radiolabeled calretinin-immunonegative cells were also seen, albeit at a much lower density. These observations clearly indicate the presence of glutamatergic/aspartatergic projections to both the hippocampus and septal complex. It may be assumed that this transmitter could play a role in hippocampal theta rhythm generation/regulation.

Cholinergic excitation of septohippocampal GABA but not cholinergic neurons: implications for learning and memory.

The medial septum/diagonal band (MSDB), which gives rise to the septohippocampal pathway, is a critical locus for the mnemonic effects of muscarinic drugs. Infusion of muscarinic cholinergic agonists into the MSDB enhance learning and memory processes both in young and aged rats and produce a continuous theta rhythm in the hippocampus. Intraseptal muscarinic agonists also alleviate the amnesic syndrome produced by systemic administration of muscarinic receptor antagonists. It has been presumed, but not proven, that the cellular mechanisms underlying the effects of muscarinic agonists in the MSDB involve an excitation of septohippocampal cholinergic neurons and a subsequent increase in acetylcholine (ACh) release in the hippocampus. Using a novel fluorescent labeling technique to selectively visualize live septohippocampal cholinergic neurons in rat brain slices, we have found that muscarinic agonists do not excite septohippocampal cholinergic neurons, instead they inhibit a subpopulation of cholinergic neurons. In contrast, unlabeled neurons, confirmed to be noncholinergic, septohippocampal GABA-type neurons using retrograde marking and double-labeling techniques, are profoundly excited by muscarine. Thus, the cognition-enhancing effects of muscarinic drugs in the MSDB cannot be attributed to an increase in hippocampal ACh release. Instead, disinhibitory mechanisms, caused by increased impulse flow in the septohippocampal GABAergic pathway, may underlie the cognition-enhancing effects of muscarinic agonists.

Opioids suppress IPSCs in neurons of the rat medial septum/diagonal band of Broca: involvement of mu-opioid receptors and septohippocampal GABAergic neurons.

The medial septum/diagonal band region (MSDB), which provides a major cholinergic and GABAergic input to the hippocampus, expresses a high density of opioid receptors. Behaviorally, intraseptal injections of opioids produce deficits in spatial memory, however, little is known about the electrophysiological effects of opioids on MSDB neurons. Therefore, we investigated the electrophysiological effects of opioids on neurons of the MSDB using rat brain slices. In voltage-clamp recordings with patch electrodes, bath-applied met-enkephalin, a nonselective opioid receptor agonist, decreased the number of tetrodotoxin and bicuculline-sensitive inhibitory synaptic currents in cholinergic- and GABA-type MSDB neurons. A similar effect occurred in brain slices containing only the MSDB, suggesting that opioids decrease GABA release primarily by inhibiting spontaneously firing GABAergic neurons located within the MSDB. Accordingly, in extracellular recordings, opioid-sensitive, spontaneously firing neurons could be found within the MSDB. Additionally, in intracellular recordings a subpopulation of GABA-type neurons were directly inhibited by opioids. All effects of met-enkephalin were mimicked by a mu receptor agonist, but not by delta or kappa agonists. In antidromic activation studies, mu-opioids inhibited a subpopulation of septohippocampal neurons with high conduction velocity fibers, suggestive of thickly myelinated GABAergic fibers. Consistent with the electrophysiological findings, in double-immunolabeling studies, 20% of parvalbumin-containing septohippocampal GABA neurons colocalized the mu receptor, which at the ultrastructural level, was found to be associated with the neuronal cell membrane. Thus, opioids, via mu receptors, inhibit a subpopulation of MSDB GABAergic neurons that not only make local connections with both cholinergic and noncholinergic-type MSDB neurons, but also project to the hippocampus.

Estrogen receptor-alpha in the raphe serotonergic and supramammillary area calretinin-containing neurons of the female rat.

It is well established that estrogen affects hippocampal long-term potentiation and hippocampus-related memory processes. Furthermore, theta rhythm, in conjunction with long-term potentiation, influences memory and is regulated by subcortical structures, including the median raphe and supramammillary area. To test the validity of the hypothesis that the effects of estrogen on the hippocampus are mediated, at least partly, via these subcortical structures, it must first be determined whether the neurons of the median raphe and supramammillary area contain estrogen receptors. Light and electron microscopic double immunostaining for estrogen receptor-alpha plus serotonin and estrogen receptor-alpha plus calretinin on vibratome sections of the median raphe and supramammillary area, respectively, demonstrated that large populations of the median raphe serotonin and supramammillary area calretinin neurons exhibit estrogen receptor-immunoreactive nuclei. These observations indicate that circulating gonadal hormones can affect hippocampal electric activity indirectly, via those subcortical structures that are involved in theta rhythm regulation.

A novel method for concurrent visualization of immunostain under light and electron microscopy in pancreatic islets.

We developed a simple method employing the use of flat-embedding techniques on thick frozen sections which allows correlation of light and electron microscopic immunohistochemistry. This method has been particularly useful in visualization of pancreas sections, an adaptation especially important because this tissue is not amenable to conventional vibratome sectioning. In this study we demonstrate the use of this technique to examine the same tissue section at the light and the electron microscopic level while maintaining morphology.

Extrinsic and intrinsic substance P innervation of the rat lateral septal area calbindin cells.

The electrophysiological observations that substance P administration to the lateral septal area elicits both excitatory and inhibitory responses, together with earlier reports on the multiple sources of substance P innervation of the septum, implies that these axons with distinct origins have different functions. This prompted us to examine the origin and neurochemical character of substance P afferents to the lateral septal area. Chronic surgical isolation of the septum from its ventral afferents and retrograde tracer experiments using wheat germ agglutinin-conjugated horseradish peroxidase, both followed by an immunostaining for substance P, were employed to elucidate the origin of these axon terminals. In order to assess the possible co-existence of substance P with other neurotransmitter substances in the parent cells of the septopetal projections, co-localization studies for substance P and choline acetyltransferase, as well as substance P and GABA, were performed. The comparative distribution of substance P fibers and septal calbindin-containing neurons was also investigated using correlated light and electron microscopic double immunostaining. The results are summarized as follows: (i) the substance P innervation of the lateral septal area derives from several hypothalamic nuclei (including the lateral and lateroanterior hypothalamic area, tuber cinereum and ventromedial hypothalamic nucleus) and tegmental nuclei (the majority of fibers from the laterodorsal and a few from the pedunculopontine tegmental nucleus), as well as intrinsic septal cells; (ii) the septopetal substance P fibers of tegmental origin are cholinergic; intraseptal substance P neurons located in the dorsolateral part of the lateral septum also contain GABA, while substance P neurons seen on the border between the medial and lateral septal area and septopetal hypothalamic substance P cells do not contain GABA or acetylcholine; (iii) substance P fibers from pericellular baskets around calbindin-containing lateral septal neurons with a high degree of selectivity; (iv) approximately 90% of the entire calbindin cell population are postsynaptic targets of substance P axons; (v) their terminals contact the soma and the dendrites of these cells, among them the somatospiny neurons; and (vi) the extrinsic substance P boutons establish asymmetric, while the intrinsic substance P axon terminals form symmetric membrane specializations. Because neurons in the lateral septal area receive hippocampal input and project massively to hypothalamic areas, the different types of substance P input on these neurons can modify the information flow arriving from the hippocampus to diencephalic brain structures at the level of the lateral septal area.

Hypothalamic Leu-enkephalin-immunoreactive fibers terminate on calbindin-containing somatospiny cells in the lateral septal area of the rat.

Correlated light and electron microscopic double-immunostaining experiments for Leu-enkephalin and calbindin were employed to determine the postsynaptic targets in the septal complex of Leu-enkephalin fibers. Chronic surgical isolation of the septal complex from its hypothalamic afferents and retrograde tracer studies using wheat germ agglutinin-conjugated horseradish peroxidase, both followed by an immunostaining for Leu-enkephalin, were performed to elucidate the location of the origin of these axon terminals. Furthermore, a colocalization study for glutamic acid decarboxylase and Leu-enkephalin was carried out on hypothalamic sections to determine their possible coexistence in cells projecting to the lateral septum. These studies revealed that 1) Leu-enkephalin-immunoreactive axons form pericellular baskets around a population of lateral septal area neurons; 2) they establish exclusively asymmetric synaptic contacts on their soma and initial dendritic segments; 3) 10% of the lateral septal area calbindin-containing cells, which are all of the gamma-aminobutyric acid (GABA)-ergic somatospiny type, are innervated by Leu-enkephalin-immunoreactive baskets; 4) only 40% of the Leu-enkephalin target neurons are calbindin immunopositive; 5) the septopetal Leu-enkephalin fibers derive from neurons located in the ipsilateral perifornical area and anterior hypothalamus; and 6) none of their cells of origin cocontains the inhibitory transmitter GABA. These observations indicate that hypothalamic Leu-enkephalin-containing neurons are non-GABAergic excitatory cells. Hence, they can effectively stimulate a population of lateral septal area neurons, including the somatospiny cells, which are all GABAergic. Therefore, after stimulatory Leu-enkephalin action, these neurons can inhibit their postsynaptic targets, including other projective lateral septal neurons.

Neuropeptide-Y innervation of estrogen-induced progesterone receptor-containing dopamine cells in the monkey hypothalamus: a triple labeling light and electron microscopic study.

Light and electron microscopic triple immunostaining was performed on coronal vibratome sections prepared from the hypothalamus of ovariectomized (OVX) and OVX plus estrogen-treated African green monkeys (Cercopithecus aethiops). Immunoreactivity for progesterone receptors (PRs) and neuropeptide-Y (NPY) was visualized by a dark blue to black nickel diaminobenzidine reaction, while the tyrosine hydroxylase-containing perikarya were labeled with a light brown diaminobenzidine reaction. In the OVX plus estrogen-treated material, 30% of the tyrosine hydroxylase-immunoreactive neurons contained PR-immunopositive nuclei. The majority of these cells were found in the central portion of the periventricular area, and a few could be observed in the anterior hypothalamus and the arcuate and dorsomedial hypothalamic nuclei. These tyrosine hydroxylase-immunoreactive PR-containing cells were surrounded with NPY-immunoreactive axon terminals. A correlated electron microscopic analysis of the same sections revealed synaptic contacts between these NPY-immunoreactive boutons and the PR-containing tyrosine hydroxylase-immunoreactive neurons. In contrast, in the OVX animals, no PR-containing tyrosine hydroxylase-immunoreactive neurons could be detected. In these monkeys, the frequency of synaptic contacts between the NPY-immunoreactive axon terminals and tyrosine hydroxylase-immunopositive cells was similar to that in the OVX plus estrogen-treated monkeys. These observations indicate that in a population of hypothalamic dopamine cells, the presence of nuclear PRs is estrogen dependent, show that these cells are innervated by NPY axons, and suggest that these estrogen-induced PR-containing dopamine neurons are involved in mediation of the effect of NPY on hypophyseal hormone secretion, including ovarian steroid hormone-dependent LH and PRL release.

Estrogen induces ultrastructural changes in progesterone receptor-containing GABA neurons of the primate hypothalamus.

Estrogen affects gonadotrophin levels and sex behavior in monkeys. This action could be via inhibitory GABA-ergic neurons in the hypothalamus. We tested for direct estrogen actions on such neurons. Seven days after ovariectomy (OVX) or OVX + estrogen treatment (10 mg estradiol valerate in 1 ml sesame oil s.c. on the day of OVX), light- and electron-microscopic double immunostaining procedures were used for simultaneous visualization of immunoreactivity for progesterone receptors (PR) and glutamic acid decarboxylase (GAD), and to detect ultrastructural changes in PR-containing neurons in the arcuate and ventromedial hypothalamic nuclei of colchicine- and noncolchicine-treated African green monkeys (Cercopithecus aethiops). Immunoreactivity for PR was found only in cell nuclei, and estrogen treatment enhanced the intensity of the immunostaining: in estrogen-treated monkeys in the arcuate nucleus 62%, while in the ventromedial nucleus 42% of the neurons contained PR-immunoreactive nuclei. All of the PR-containing neurons were immunopositive for GAD in colchicine-pretreated monkeys. OVX induced whorl body formation, while estrogen treatment of OVX animals resulted in a large number of nematosomes. While all of the whorl bodies and the majority of nematosomes were observed in PR-immunopositive GAD neurons, nematosomes were also found in non-PR-containing GAD-immunoreactive cells.

Comparison of age- and sex-related changes in cell nuclear estrogen-binding capacity and progestin receptor induction in the rat brain.

Previous studies have suggested parallels between the mechanisms underlying sexual differentiation and age-related loss of reproductive cyclicity in the female rat. Both appear to involve the actions of estrogen on the brain and are associated with a reduction in hypothalamic estrogen sensitivity. In this study the effects of sex and aging on cell nuclear estrogen receptor-binding capacity and cytosol progestin receptor induction in the pituitary gland, periventricular preoptic area (PVP), medial preoptic area (mPO), bed nucleus of the stria terminalis, arcuate median eminence region (ARC), ventromedial nucleus (VMN), and corticomedial amygdala were directly compared. Young (2.5 months old) and middle-aged (8-10 months old) male and female and old (19 months old) female rats were gonadectomized 14 days before adrenalectomy (ADX). For comparison of cell nuclear estrogen receptor-binding capacity, animals received a saturating dose of estradiol (36.0 micrograms/kg BW) 3 days after ADX and 1 h before death. Cell nuclear estrogen binding was measured by an in vitro exchange assay. For comparison of estrogen-induced progestin receptors, animals received a sc placed Silastic capsule containing 10% 17 beta-estradiol at the time of ADX and were killed 3 days later. Cytosol progestin binding was measured by an in vitro binding assay. Cell nuclear estrogen-binding capacities and cytosol progestin receptor induction were lower in the PVP, mPO, and VMN of the young male than in the young female. In the female, the level of progestin receptor induction in the pituitary and brain was unaffected by age; however, cell nuclear estrogen-binding capacity in the mPO, VMN, ARC, and pituitary gland was lower in old than in middle-aged females. These results demonstrate that the effects of sexual differentiation and aging on the hypothalamus involve similar, but not identical, region-specific reductions in cell nuclear estrogen receptor-binding capacity. The consequences of these reduced estrogen receptor binding levels in terms of the induction of progestin receptor in response to estrogen exposure are, however, very different in the male compared to those in old female rats.

Progestin receptor-containing cells in guinea pig hypothalamus: Afferent connections, morphological characteristics, and neurotransmitter content.

Progestin receptor-containing cells in the guinea pig hypothalamus were examined by immunocytochemical methods to characterize their morphological properties, afferent connections, and neurotransmitter content. Through the use of a monoclonal mouse antibody raised against the rabbit uterine progestin receptor, progrestin receptor-containing cells were identified after estrogen treatment in the retrochiasmatic area, medial and periventricular preoptic area, arcuate nucleus, and lateral hypothalamic area including the ventrolateral aspect of the ventromedial nucleus. Ultrastructural analysis revealed a heterogeneously distributed immunoreaction product restricted to the cell nucleus. A similar pattern of nuclear progestin receptor immunostaining was observed in the uterine endometrium. Progesterone administration intensified progestin receptor immunostaining, without appearing to alter the distribution of immunoreaction product within the cell nucleus. No immunostaining was observed in sections from ovariectomized (OVX) or OVX progesterone-treated animals. Hypothalamic sections from estrogen/progesterone-treated guinea pigs were double-immunostained for progestin receptor and neurotransmitter- or neuropeptide-related antigens. Synaptic contacts of tyrosine hydroxylase-, glutamic acid decarboxylase (GAD)-, serotonin-, adrenocorticotropic hormone-, and cholecystokinin-immunoreactive axon terminals were found on progestin receptor-immunopositive neurons. In addition, nonconventional junctional complexes were observed between TH-immunoreactive soma and the soma of progestin receptor- immunoreactive neurons. No evidence of immunostaining for neurotransmitters or neuropeptides other than GAD was observed in progestin receptor- immunoreactive neurons. In addition to ultrastructural characterization of progestin receptor immunoreactivity in the guinea pig hypothalamus, these studies demonstrate, for the first time, direct connections of neurotransmitter/neuropeptide systems involved in neuroendocrine regulation with steroid receptor-containing neurons in the rodent hypothalamus. Estrogen/progesterone-sensitive cells in the hypothalamus may act as centers for the integration of hormonal and neural influences on the control of reproductive function.

Catecholaminergic innervation of luteinizing hormone-releasing hormone and glutamic acid decarboxylase immunopositive neurons in the rat medial preoptic area. An electron-microscopic double immunostaining and degeneration study.

Catecholaminergic innervation of luteinizing hormone-releasing hormone (LHRH) and glutamic acid decarboxylase (GAD) immunoreactive neurons in the rat medial preoptic area (MPO) was studied using electron-microscopic (EM) double-label immunostaining and combinations of single- and double-label immunostaining with acute axonal degeneration. The EM double-immunostaining experiments included double staining for either tyrosine hydroxylase (TH) and LHRH, or TH and GAD. Analysis of TH and LHRH double-immunostained material revealed synaptic connections between TH immunoreactive axons and LHRH immunopositive neurons. The TH and GAD double-staining experiments also demonstrated synaptic connections between axons immunoreactive for TH and GAD immunopositive neurons. Two days following unilateral surgical transection of the ventral and dorsal noradrenergic bundles, synaptic connections were found between degenerated boutons and GAD immunoreactive neurons in the ipsilateral MPO. However, no synapses could be observed in the same area between degenerated axons and the LHRH immunopositive neurons. Following the same operation and immunostaining for TH, a moderate number of degenerating TH axons as well as a large number of nondegenerated TH immunoreactive boutons were observed. Double immunostaining for TH and GAD in MPO sections ipsilateral to the operation revealed synaptic connections between the degenerating TH immunopositive axons and GAD immunoreactive neurons. These results suggest that there are direct synaptic connections between catecholaminergic axons and GAD and LHRH immunoreactive neurons in the medial preoptic area of the rat. Some of the connections between TH immunopositive afferents and GAD immunoreactive neurons may represent connections from noradrenergic neurons in the brain stem, while the majority of TH-GAD and TH-LHRH connections may represent innervation of GABA and LHRH neurons from local dopamine-containing cells.

Immunohistochemical evidence for synaptic connections between pro-opiomelanocortin-immunoreactive axons and LH-RH neurons in the preoptic area of the rat.

Connections between adrenocorticotropic hormone (ACTH)-immunoreactive neurons in the arcuate nucleus and the preoptic area were studied in the female rat. ACTH-immunopositive terminals were observed in the medial preoptic area in contact with dendritic shafts, while in the ventrolateral preoptic area the majority of ACTH-immunoreactive synapses were found on dendritic spines. Double-label electron microscopic immunocytochemistry using peroxidase and avidin-ferritin as contrasting electron-dense markers revealed numerous synaptic contacts between ACTH-immunopositive boutons and luteinizing hormone-releasing hormone (LH-RH)-immunoreactive dendritic shafts in the medial preoptic area. Following injection of horseradish peroxidase (HRP) into the medial preoptic area, retrogradely HRP-labeled perikarya were observed throughout the arcuate nucleus. Double-staining experiments revealed that a proportion of these retrogradely labeled cells, in the ventromedial arcuate nucleus, are also immunoreactive for ACTH. These results suggest that pro-opiomelanocortin peptide-producing neurons in the ventromedial arcuate nucleus project to the medial preoptic area. Some of these neurons establish direct synaptic contacts with LH-RH-immunoreactive cells.