Arousal-related reticular neurons during reduced oxygen tension: resilience and recovery of electrical activity.

Whole-cell patch clamp recordings of the electrical activity of large medullary reticular formation neurons, in nucleus gigantocellularis, were performed under control conditions and under conditions of hypoxia or anoxia. Neurons were discovered whose activity was remarkably resilient during and after the reduction or loss of oxygen. Such cells may relate to the ability of the newborn brain to survive hypoxia/anoxia, and also may demonstrate the preservation of neurons involved in generalized CNS arousal, as would be appropriate for activating behavioral responses to the reduction or loss of oxygen.

Thyroid hormone action: nongenomic modulation of neuronal excitability in the hippocampus.

Years of effort have failed to establish a generally-accepted mechanism of thyroid hormone (TH) action in the mature brain. Recently, both morphological and pharmacological evidence have supported a direct neuroactive role for the hormone and its triiodinated metabolites. However, no direct physiological validation has been available. We now describe electrophysiological studies in vivo in which we observed that local thyroxine (T4) administration promptly inhibited field excitatory postsynaptic potentials recorded in the dentate gyrus (DG) with stimulation of the medial perforant pathway, a result that was found to be especially pronounced in hypothyroid rats. In separate in vitro experiments, we observed more subtle but statistically significant responses of hippocampal slices to treatment with the hormone. The results demonstrate that baseline firing rates of CA1 pyramidal cells were modestly reduced by pulse-perfusion with T4. By contrast, administration of triiodothyronine (T3) was often noted to have modest enhancing effects on CA1 cell firing rates in hippocampal slices from euthyroid animals. Moreover, and more reliably, robust firing rate increases induced by norepinephrine were amplified when preceded by treatment with T3, whereas they were diminished by pretreatment with T4. These studies provide the first direct evidence for functional, nongenomic actions of TH leading to rapid changes in neuronal excitability in adult rat DG studied in vivo and highlight the opposing effects of T4 and T3 on norepinephrine-induced responses of CA1 cells studied in vitro.

Reverse engineering the lordosis behavior circuit.

Reverse engineering takes the facts we know about a device or a process and reasons backwards to infer the principles underlying the structure-function relations. The goal of this review is to apply this approach to a well-studied hormone-controlled behavior, namely the reproductive stance of female rodents, lordosis. We first provide a brief overview on the considerable amount of progress in the analysis of female reproductive behavior. Then, we propose an analysis of the mechanisms of this behavior from a reverse-engineering perspective with the goal of generating novel hypotheses about the properties of the circuitry elements. In particular, the previously proposed neuronal circuit modules, feedback signals, and genomic mechanisms are considered to make predictions in this manner. The lordosis behavior itself appears to proceed ballistically once initiated, but negative and positive hormonal feedback relations are evident in its endocrine controls. Both rapid membrane-initiated and slow genomic hormone effects contribute to the behavior's control. We propose that the value of the reverse-engineering approach is based on its ability to provide testable, mechanistic hypotheses that do not emerge from either traditional evolutionary or simple reductionistic perspectives, and several are proposed in this review. These novel hypotheses may generalize to brain functions beyond female reproductive behavior. In this way, the reverse-engineering perspective can further develop our conceptual frameworks for behavioral and systems neuroscience.

Presynaptic actions of opioid receptor agonists in ventromedial hypothalamic neurons in estrogen- and oil-treated female mice.

Estrogens act upon ventromedial hypothalamic (VMH) neurons, and their effects on female arousal and sexual behaviors mediated by VMH neurons involve several neurotransmitters and neuromodulators. Among these are opioid peptides which might be predicted to oppose estrogenic action on VMH because they tend to decrease CNS arousal. Spontaneous excitatory postsynaptic currents were recorded from VMH neurons from 17beta-estradiol- (E, 10 mug/0.1 ml) or oil-treated control ovariectomized (OVX) mice using whole-cell patch-clamp techniques. To examine the impact of opioidergic inputs, recordings of neurons from both treatment groups were obtained in the presence of the general opioid receptor agonist methionine enkephalin-Arg-Phe (MERF, 3 muM), or mu-receptor specific agonist [d-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO, 1 muM). Compared with oil, E treatment for 48 h significantly increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) without affecting their amplitude. MERF and DAMGO each abolished this E effect, causing significant reductions in sEPSCs. The effect of MERF was abolished by naltrexone (general opioid receptor antagonist, 3 muM) and the effect of DAMGO by d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP) (mu-opioid receptor selective antagonist, 1 muM); in contrast, kappa- and delta-opioid receptor agonists, U69593 (300 nM) and [d-Pen(2),d-Pen(5)]-enkephalin (DPDPE, 1 muM) respectively, had little effect on the sEPSCs compared with DAMGO. To consider presynaptic vs. postsynaptic effects of opioids, miniature excitatory postsynaptic currents (mEPSCs) were investigated in E- and oil-treated VMH neurons and opioid receptor antagonist effects on mEPSCs were observed. Both MERF and DAMGO reduced the frequency of mEPSCs, but had no effect on their amplitude. Our findings indicate that opioids suppress excitatory synaptic transmissions in VMH neurons primarily through mu-receptors and could thereby decrease sexual arousal in mice.

Voltage-dependent calcium channels in ventromedial hypothalamic neurones of postnatal rats: modulation by oestradiol and phenylephrine.

Oestradiol actions in the hypothalamus play an important role in reproductive behaviour. Oestradiol treatment in vivo induces alpha(1b)-adrenoceptor mRNA and increases the density of alpha(1B)-adrenoceptor binding in the hypothalamus. Oestradiol is also known to modulate neuronal excitability, in some cases by modulating calcium channels. We assessed the effects of phenylephrine, an alpha(1)-adrenergic agonist, on low-voltage-activated (LVA) and high-voltage-activated (HVA) calcium channels in ventromedial hypothalamic (VMN) neurones from vehicle- and oestradiol-treated female rats. Whole-cell and gramicidin perforated-patch recordings were obtained, with barium as the charge carrier. In the absence of phenylephrine, oestradiol treatment increased the magnitude of LVA currents compared to controls, but had no effect on HVA currents. Phenylephrine enhanced HVA currents in a significantly greater proportion of neurones from oestradiol-treated rats (76%) than from vehicle-treated (41%) rats. The L-channel blocker nifedipine abolished this oestradiol effect on phenylephrine-enhanced HVA currents. Preincubating slices with the N-type channel blocker omega-conotoxin GVIA completely blocked the phenylephrine response, suggesting that the N-type channel is essential. Phenylephrine also stimulated LVA currents in approximately two-thirds of neurones in slices from both vehicle- and oestradiol-treated rats. Our data show that oestradiol increases LVA currents in the VMN. Oestradiol also amplifies alpha(1)-adrenergic signalling by increasing the proportion of neurones showing phenylephrine-stimulated HVA currents mediated by N- and L-type calcium channels. In this way, oestradiol may increase excitatory responses to arousing adrenergic inputs to VMN neurones governing oestradiol-dependent reproductive behaviour.

Hormonal induction of lordosis and ear wiggling in rat pups: gender and age differences.

To assess how early can estrogens induce female mating behaviors, rat pups 8-29 days old (D8-D29, respectively) were injected twice daily with estradiol benzoate (E) or oil (O) followed by progesterone (P) or oil, and then observed for the estrogen-dependent ear wiggling (EW) and lordosis in response to natural stimulation from male rats. In female pups treated with E + E + P, the incidence of EW appeared as early as D13 and increased gradually to reach maximum at D18, when all pups tested showed EW. EW also occurred in E + E + O females, but never in O + O + P females or in any E + E + P male. Lordosis in E + E + P, as well as E + E + O, female pups occurred later, starting at D15. O + O + P females or E + E + P males never display lordosis. To explore the possibilities that the age and gender differences are due to distribution and/or function of estrogen receptor-alpha (ERalpha) or progesterone receptor (PR), separate pups were used for immunocytochemical (ICC) staining of these receptors in the hypothalamic ventromedial nucleus (VMN). There was no age difference in female pups in the density of ERalpha or the induction of PR between D11/D12, when no sexual behavior was observed, and D19/D20, when almost all pups tested performed the behaviors. There were gender differences: male pups had less ERalpha than females at D19/D20, though not at D11/D12, and did not respond to E in the induction of PR in the VMN. These results show that ERs and their signaling systems in the VMN of rat pups are functional at least after D11 but only in females, and that the gender differences appeared to be due to differences in the molecular biology of ERalpha.

Acute estradiol application increases inward and decreases outward whole-cell currents of neurons in rat hypothalamic ventromedial nucleus.

Acute estradiol (E2) can potentiate the excitatory responses of hypothalamic ventromedial nucleus (VMN) neurons to neurotransmitters. To investigate the mechanism(s) underlying the potentiation, the whole-cell patch voltage clamp technique was used to study VMN neurons in hypothalamic slices prepared from female juvenile (3-5 weeks) rats. A voltage step and/or ramp was applied every 5 min to evoke whole-cell currents before, during and after a treatment with E2 (10 nM), corticosterone (10 nM) or vehicle for up to 20 min. Acute E2 increased inward currents in 38% of neurons tested. Their average peak inward current amplitudes started to increase within 5 min and reached the maximum of 163% of pretreatment level (Pre) at 20 min of treatment before recovering toward Pre. These increases are significantly greater than the Pre and corresponding vehicle controls and non-responsive neurons. Outward currents were decreased significantly by E2 in 27% of E2-treated cells, down to 60% of Pre levels. E2 also appeared to affect the kinetics of the inward and outward currents of estrogen-responsive neurons. Whenever observed, the effects of acute E2 were reversible after a 5- to 10-min washing. Probability analysis indicates that E2 affected the inward and the outward currents independently. The E2 effects are specific in that they were not produced by similar treatment with vehicle or corticosterone. Pharmacological characterizations using ion replacement and channel blockers showed that the inward currents were mediated practically all by Na(+) and the outward currents mainly by K(+). Thus, acute E2 can enhance inward Na(+) and attenuate outward K(+) currents. Since both effects will lead to an increase in neuronal excitability, they may explain our previous observation that E2 potentiates the excitation of VMN neurons.

Sex and estrogenic effects on coexpression of mRNAs in single ventromedial hypothalamic neurons.

Regulated gene expression in single neurons can be linked to biophysical events and behavior in the case of estrogen-regulated gene expression in neurons in the ventrolateral portion of the ventromedial nucleus (VMN) of the hypothalamus. These cells are essential for lordosis behavior. What genes are coexpressed in neurons that have high levels of mRNAs for estrogen receptors (ERs)? We have been able to isolate and measure certain mRNAs from individual VMN neurons collected from rat hypothalamus. Large numbers of neurons express mRNA for ERalpha, but these neurons are not identical with the population of VMN neurons expressing the likely gene duplication product, ERbeta. An extremely high proportion of neurons expressing either ER also coexpress mRNA for the oxytocin receptor (OTR). This fact matches the known participation of oxytocin binding and signaling in sexual and affiliative behaviors. In view of data that ER and OTR can signal through PKCs, we looked at coexpression of selected PKCs in the same individual neurons. The most discriminating analysis was for triple coexpression of ERs, OTR, and each selected PKC isoform. These patterns of triple coexpression were significantly different for male vs. female VMN neurons. Further, individual neurons expressing ERalpha could distribute their signaling across the various PKC isoforms differently in different cells, whereas the reverse was not true. These findings and this methodology establish the basis for systematic linkage of the brain's hormone-sensitive signaling pathways to biophysical and behavioral mechanisms in a well studied mammalian system.

Female oxytocin gene-knockout mice, in a semi-natural environment, display exaggerated aggressive behavior.

Compared to results from a generation of neuropharmacological work, the phenotype of mice lacking the oxytocin (OT) peptide gene was remarkably normal. An important component of the current experiments was to assay OT-knockout (OTKO) and wild-type (WT) littermate control mice living under controlled stressful conditions designed to mimic more closely the environment for which the mouse genome evolved. Furthermore, our experimental group was comprised of an all-female population, in contrast to previous studies which have focused on all-male populations. Our data indicated that aggressive behaviors initiated by OTKO during a food deprivation feeding challenge were considerably more intense and diverse than aggressive behaviors initiated by WT. From the measures of continuous social interaction in the intruder paradigm, it emerged that OTKO mice were more offensively aggressive (attacking rumps and tails) than WT. In a test of parental behaviors, OTKO mice were 100% infanticidal while WT were 16% infanticidal and 50% maternal. Finally, 'alpha females' (always OTKO) were identified in each experiment. They were the most aggressive, the first to feed and the most dominant at nesting behaviors. Semi-natural environments are excellent testing environments for elucidating behavioral differences between transgenic mice and their WT littermates which may not be ordinarily discernible. Future studies of mouse group behavior should include examining female groupings in addition to the more usual all-male groups.

Acute estrogen potentiates excitatory responses of neurons in rat hypothalamic ventromedial nucleus.

In a previous behavioral study, brief application of a membrane-limited estrogen to neurons in rat hypothalamic ventromedial nucleus (VMN) facilitated lordosis behavior-inducing genomic actions of estrogen. Here, electrophysiological recordings from single neurons were employed to characterize these membrane-initiated actions. From rat hypothalamic slices, electrical activity was recorded from neurons in the ventrolateral VMN, the cell group crucial for estrogen induction of lordosis. In addition to the resting activity, neuronal responses to histamine (HA) and N-methyl-d-aspartate (NMDA) were also recorded before, during, and after a brief (10-15 min) application of estradiol (E, 10 nM). These two transmitters were chosen because their actions are mediated by different mechanisms: HA through G protein-coupled receptors and NMDA by ligand-activated ion channels. Vehicle applications did not affect either resting activity or neuronal responses. In contrast, acute E exposure modulated neuronal responses to transmitters, with no significant effect on the resting activity. It potentiated excitatory responses to HAs (20 out of 48 cells tested) and to NMDA (10 out of 19 cells), but attenuated inhibitory responses to HA (3 out of 6 units). Both of these hormonal actions would increase VMN neuronal excitation. In separate experiments, neuronal excitation was found to be suppressed by anesthetics, which would block E's induction of lordosis when administered at the time of estrogen application. These data are consistent with the notion that increasing electrical excitation of VMN neurons can be a mechanism by which acute E exposure facilitates the lordosis-inducing genomic actions of estrogens.

Potentiation of the excitatory action of NMDA in ventrolateral periaqueductal gray by the mu-opioid receptor agonist, DAMGO.

Several lines of evidence have suggested that mu-opioids, generally regarded as inhibitory, also have effects that stimulate neural activity. To look for possible excitatory opioid action in the rat periaqueductal gray (PAG), we first re-examined data from a previous study and found that met-enkephalin could evoke a delayed, sluggish excitation, suggestive of modulation by the opioid on the action of certain excitants. This observation, coupled with other studies that show mu-opioids can modulate NMDA receptor activation, prompted us to perform extracellular recording of the responses of single ventrolateral PAG (vlPAG) neurons in brain slices to DAMGO, a mu-opioid, and to NMDA. When applied alone, DAMGO at nM concentrations, like met-enkephalin, often evoked the delayed excitation and occasionally an inhibition. When applied after a brief exposure to NMDA, DAMGO at doses as low as 0.1 nM potentiated the excitation produced by a subsequent pulse of NMDA. This occurred, depending on cell type, in 23-100% of vlPAG neurons. The potentiating action of DAMGO was blocked by naloxone, suggesting it was mediated by mu-opioid receptors. Characterization of these mu-opioid actions revealed that the potentiation and the delayed excitation, unlike the inhibition, was not blocked by another opioid antagonist, nalmefene, nor by an inhibitor of the G protein of the G(i) class, N-ethylmaleimide. Moreover, the potentiating action was distinct from the inhibition in that it was: (a) enhanced by repeated opioid applications, (b) exhibited low effective doses, (c) had a long time course (minutes to develop and last tens of minutes) and (d) was present in distinct though overlapping cell populations. These data reveal an unconventional action of opioids in PAG neurons, that is, a potentiation of excitation produced by NMDA. This effect appeared mechanistically distinct from opioid inhibition or disinhibition and may be related to established examples of direct opioid excitation. These observations may help understanding behaviorally important mechanisms linked to acute and chronic opioid functions in the vlPAG.

Brain glucose-sensing mechanisms: ubiquitous silencing by aglycemia vs. hypothalamic neuroendocrine responses.

Interest in brain glucose-sensing mechanisms is motivated by two distinct neuronal responses to changes in glucose concentrations. One mechanism is global and ubiquitous in response to profound hypoglycemia, whereas the other mechanism is largely confined to specific hypothalamic neurons that respond to changes in glucose concentrations in the physiological range. Although both mechanisms use intracellular metabolism as an indicator of extracellular glucose concentration, the two mechanisms differ in key respects. Global hyperpolarization (inhibition) in response to 0 mM glucose can be reversed by pyruvate, implying that the reduction in ATP levels acting through ATP-dependent potassium (K-ATP) channels is the key metabolic signal for the global silencing in response to 0 mM glucose. In contrast, neuroendocrine hypothalamic responses in glucoresponsive and glucose-sensitive neurons (either excitation or inhibition, respectively) to physiological changes in glucose concentration appear to depend on glucokinase; neuroendocrine responses also depend on K-ATP channels, although the role of ATP itself is less clear. Lactate can substitute for glucose to produce these neuroendocrine effects, but pyruvate cannot, implying that NADH (possibly leading to anaplerotic production of malonyl-CoA) is a key metabolic signal for effects of glucose on glucoresponsive and glucose-sensitive hypothalamic neurons.

Early membrane estrogenic effects required for full expression of slower genomic actions in a nerve cell line.

Interpretations of steroid hormone actions as slow, nuclear, transcriptional events have frequently been seen as competing against inferences of rapid membrane actions. We have discovered conditions where membrane-limited effects potentiate later transcriptional actions in a nerve cell line. Making use of a two-pulse hormonal schedule in a transfection system, early and brief administration of conjugated, membrane-limited estradiol was necessary but not sufficient for full transcriptional potency of the second estrogen pulse. Efficacy of the first pulse depended on intact signal transduction pathways. Surprisingly, the actions of both pulses were blocked by a classical estrogen receptor (ER) antagonist. Thus, two different modes of steroid hormone action can synergize.

Presynaptic and postsynaptic relations of mu-opioid receptors to gamma-aminobutyric acid-immunoreactive and medullary-projecting periaqueductal gray neurons.

The ventrolateral portion of the periaqueductal gray (PAG) is one brain region in which ligands of the mu-opioid receptor (MOR) produce analgesia. In the PAG, MOR ligands are thought to act primarily on inhibitory [e.g., gamma-aminobutyric acidergic (GABAergic)] neurons to disinhibit PAG output rather than directly on medullary-projecting PAG neurons. In this study, the ultrastructural localization of MOR immunolabeling was examined with respect to either GABAergic PAG neurons or PAG projection neurons that were labeled retrogradely from the rostral ventromedial medulla. Immunoreactivity for MOR and GABA often coexisted within dendrites. Dual-labeled profiles accounted for subpopulations of dendrites containing immunoreactivity for either MOR (65 of 145 dendrites; 45%) or GABA (65 of 183 dendrites; 35%). In addition, nearly half of PAG neuronal profiles (148 of 344 profiles) that were labeled retrogradely from the ventromedial medulla contained MOR immunoreactivity. MOR was distributed equally among retrogradely labeled neuronal profiles in the lateral and ventrolateral columns of the caudal PAG. With respect to the presynaptic distribution of MOR, approximately half of MOR-immunolabeled axon terminals (35 of 69 terminals) also contained GABA. Some MOR and GABA dual-immunolabeled axon terminals contacted unlabeled dendrites (11 of 35 terminals), whereas others contacted GABA-immunoreactive dendrites (15 of 35 terminals). Furthermore, axon terminals synapsing on medullary-projecting PAG neurons sometimes contained immunoreactivity for MOR. These data support the model that MOR ligands can act by inhibiting GABAergic neurons, but they also provide evidence that MOR ligands may act directly on PAG output neurons. In addition, MOR at presynaptic sites could affect both GABAergic neurons and output neurons. Thus, the disinhibitory model represents only partially the potential mechanisms by which MOR ligands can modulate output of the PAG.

Hypothalamic glucose sensor: similarities to and differences from pancreatic beta-cell mechanisms.

Glucose-responsive neurons in the ventromedial hypothalamus (VMH) are stimulated when glucose increases from 5 to 20 mmol/l and are thought to play an essential role in regulating metabolism. The present studies examined the role of glucose metabolism in the mechanism by which glucose-responsive neurons sense glucose. The pancreatic, but not hepatic, form of glucokinase was expressed in the VMH, but not cerebral cortex, of adult rats. In brain slice preparations, the transition from 5 to 20 mmol/l glucose stimulated approximately 17% of the neurons (as determined by single-cell extracellular recording) from VMH but none in cortex. In contrast, most cells in both VMH and cortex were silent below 1 mmol/l and active at 5 mmol/l glucose. Glucosamine, 2-deoxyglucose, phloridzin, and iodoacetic acid blocked the activation of glucose-responsive neurons by the transition from 5 to 20 mmol/l glucose. Adding 15 mmol/l mannose, galactose, glyceraldehyde, glycerol, and lactate to 5 mmol/l glucose stimulated glucose-responsive neurons. In contrast, adding 15 mmol/l pyruvate to 5 mmol/l glucose failed to activate glucose-responsive neurons, although pyruvate added to 0 mmol/l glucose permitted neurons to maintain activity. Tolbutamide activated glucose-responsive neurons; however, diazoxide only blocked the effect of glucose in a minority of neurons. These data suggest that glucose-responsive neurons sense glucose through glycolysis using a mechanism similar to the mechanism of pancreatic beta-cells, except that glucose-responsive neurons are stimulated by glycerol and lactate, and diazoxide does not generally block the effect of glucose.

Neural oxytocinergic systems as genomic targets for hormones and as modulators of hormone-dependent behaviors.

At the molecular level, estradiol turns on the gene for oxytocin in a subset of paraventricular hypothalamic neurons and turns on the gene for the oxytocin receptor in other limbic and hypothalamic cell groups. As a result, oxytocin deposition, whose signal is transduced both through G alpha (q/11) and Gi to stimulate phosphatidylinositol turnover, facilitates electrical activity in certain hypothalamic neurons. Consequently, affiliative behaviors including those closely associated with reproduction--mating behaviors and parental behaviors--are promoted. One important aspect of this effect is the preservation of instinctive behaviors associated with reproduction, in the face of disturbances due to mild stress.

In the ventromedial nucleus of the rat hypothalamus, GABA-immunolabeled neurons are abundant and are innervated by both enkephalin- and GABA-immunolabeled axon terminals.

Immunohistochemical-labeling for the neurochemicals gamma-aminobutyric acid (GABA) and enkephalin are abundant in the ventromedial nucleus of the hypothalamus (VMN). In VMN, both GABA and enkephalin may function to regulate feeding behavior, as well as other hormone-controlled behaviors. Importantly, in several brain areas, enkephalin is often thought to modulate GABAergic neurotransmission. Therefore, we used dual-labeling immunohistochemistry with electron microscopic analysis to study the circuitry of neurons containing GABA- and/or enkephalin-labeling within the VMN. Somato-dendritic profiles containing GABA-labeling were three fold more abundant than GABA-labeled axon terminals (117 soma or dendrites vs. 34 axons). In addition, axon terminals containing GABA-labeling sometimes synapsed onto GABA-labeled somata or dendrites (25% or 9/34). In contrast, under these conditions labeling for enkephalin was primarily restricted to axon terminals, which were very abundant throughout VMN. Enkephalin-containing terminals accounted for a large fraction (25% 23/92) of the axons in contact with GABA-labeled dendrites, although they also contacted unlabeled dendrites. These observations suggest that a population of VMN neurons are GABAergic. These may be either local circuit 'interneurons' or projection neurons. In addition, GABA-labeled VMN neurons may be regulated by either enkephalin or GABA. These morphologic observations provide the basis for disinhibitory mechanisms to function within the VMN.

Mapping of neural and signal transduction pathways for lordosis in the search for estrogen actions on the central nervous system.

Estrogen can act on the brain to regulate various biological functions and behavior. In attempts to elucidate the estrogen action, the rodent female reproductive behavior, lordosis, was used as a model. Lordosis is an estrogen-dependent reflexive behavior and, hence, is mediated by discrete neural pathways that are modulated by estrogen. Therefore, a strategy of mapping the pathways, both neural and biochemical, and examining them for estrogen effect was used to localize and subsequently analyze the central action of estrogen. Using various experimental approaches, an 'inverted Y-shaped' neural pathway both sufficient and essential for mediating lordosis was defined. The top portion is a descending pathway conveying the permissive estrogen influence which originated from hypothalamic ventromedial nucleus relayed via midbrain periaqueductal grey down to medullary reticular formation, the top of the spino-bulbo-spinal reflex arc at the bottom. This estrogen influence alters the input-output relationship, shifting the output toward more excitation. With this shift in output, estrogen can enable the otherwise ineffective lordosis-triggering sensory stimuli to elicit lordosis. In the ventromedial nucleus, the origin of the estrogen influence, a multidisciplinary approach was used to map intracellular signaling pathways. A phosphoinositide pathway involving a specific G protein and the activation of protein kinase C was found to be involved in the mediation of lordosis as well as a probable target of the permissive estrogen action. The action of estrogen on this signal transduction pathway, a potentiation, is consistent with and, hence, may be an underlying mechanism for the estrogen influenced shift toward excitation. Thus, further investigation on this specific signal transduction pathway should be helpful in elucidating the action of estrogen on the brain.

Sexual reinforcement in the female rat.

Sexual reinforcement in the female rat was studied in a preparation that allowed continuous operant responding for access to a male rat leading to intromission. Experiment 1 used a high operant level nose-poke response to test the possible reinforcing effects of some components of access to a male. A simple tone stimulus used as a conditioned reinforcer and two odor stimuli, target male bedding and emulsified preputial gland, were tested. None of these contingent events altered responding above or below operant level. Access to the male, which was always accompanied by intromission, immediately increased response rate when it was made contingent upon the nose-poke response. Performance on fixed-ratio schedules was erratic, and response rate was low in comparison to typical food-reinforced responding. An interresponse-time analysis indicated, however, that some effect of the ratio contingency may have been present. In Experiment 2, several modifications of the procedure were tested with the objective of creating a more tractable preparation for behavior analysis. Response type and the hormone delivery method were changed, and 2 target males were used instead of 1. The latter tripled the average number of reinforcers earned in a single session. Differences between sexual and other reinforcers are discussed in terms of procedural, quantitative, and motivational aspects of the sexual reinforcement procedure.

Estradiol regulation of nitric oxide synthase mRNAs in rat hypothalamus.

Expression and estrogen regulation of the genes for nitric-oxide (NO)-synthesizing enzymes (NO synthase, NOS) were investigated by in situ hybridization. This study focused on regions of the hypothalamus that contain estrogen receptors and regulate specific neuroendocrine functions related to female sexual behavior and food intake, among others. Ovariectomized (OVX) rats were treated with vehicle or 3 micrograms/100 g estradiol benzoate (EB) for 7 days. Brains were sectioned and hybridized with antisense riboprobes for neuronal NOS, macrophage NOS and endothelial NOS. In the hypothalamus, mRNA was clearly detectable only for the neuronal NOS with the probes used. A strong hybridization signal was observed in the supraoptic paraventricular and ventromedial nuclei (SON, PVN and VMN, respectively). Quantitative analysis showed an increase in neuronal NOS mRNA in the VMN of the OVX rats treated with EB. The increase was mainly in the ventrolateral aspect of the VMN. No significant changes were observed in the hypothalamic SON and PVN. The data suggest that the expression of neuronal NOS mRNA in VMN can be regulated by estrogen.