Ventromedial Preoptic Area Research Articles

(291) The Effect of Administration and Discontinuation of Finasteride in Male Rats

Abstract Introduction It has been suggested that 5AR (5-alpha reductase) inhibitors may have negative effects on psychosexual activity, but there is still debate. Objective We aimed to observe the effect of 5AR type 2 inhibition on brain tissue by using finasteride. Methods 14 weeks old, eight male rats were assigned to each group (group 1 as the control group, group 2 as the finasteride group, and group 3 as the finasteride withdrawal group). Each rat was isolated and acclimatized in a single cage, then mated for 2 days. Rats in group 2 and 3 were administrated with finasteride for 4 weeks, then rats in group 2 were sacrificed whereas rats in group 3 were sacrificed 4 weeks thereafter. Before sacrifice, they were exposed to female bedding at least for 2 days. RT-PCR, western blot, and immunohistochemistry were performed for brain tissue evaluation where the target genes/proteins were 5AR (type 2) and c-Fos. Results Dihydrotestosterone (ng/dl) and dihydrotestosterone to testosterone ratio (%) plunged after 1 month administration of finasteride (group 2), and they were ameliorated after discontinuation of the drug (group 3) (Figure 1). These results were parallel to those from the western blot and immunohistochemistry (Figure 2 and 3). On the contraty, RT-PCR showed elevation of each gene (5AR and c-Fos) expression (Figure 2). Conclusions Finasteride exerted an influence on brain tissue including hippocampus and ventromedial preoptic area via 5AR-2 inhibition, resulting in decrease of c-Fos protein activation. However, the negative impact of finasteride on rat brain in regard with c-Fos activation may fall away in a month cessation of the drug. Disclosure No.

Circadian Oscillations in the Murine Preoptic Area Are Reset by Temperature, but Not Light.

Mammals maintain their internal body temperature within a physiologically optimal range. This involves the regulation of core body temperature in response to changing environmental temperatures and a natural circadian oscillation of internal temperatures. The preoptic area (POA) of the hypothalamus coordinates body temperature by responding to both external temperature cues and internal brain temperature. Here we describe an autonomous circadian clock system in the murine ventromedial POA (VMPO) in close proximity to cells which express the atypical violet-light sensitive opsin, Opn5. We analyzed the light-sensitivity and thermal-sensitivity of the VMPO circadian clocks ex vivo. The phase of the VMPO circadian oscillations was not influenced by light. However, the VMPO clocks were reset by temperature changes within the physiological internal temperature range. This thermal-sensitivity of the VMPO circadian clock did not require functional Opn5 expression or a functional circadian clock within the Opn5-expressing cells. The presence of temperature-sensitive circadian clocks in the VMPO provides an advancement in the understanding of mechanisms involved in the dynamic regulation of core body temperature.

The EP3 and EP4 Receptor Subtypes both Mediate the Fever-producing Effects of Prostaglandin E2 in the Rostral Ventromedial Preoptic Area of the Hypothalamus in Rats

This study aimed to re-examine the receptor subtype that mediates the fever-producing effects of prostaglandin E2 (PGE2) in the rostral ventromedial preoptic area (rvmPOA) of the hypothalamus. Among the four subtypes of PGE2 receptors (EP1, EP2, EP3, and EP4), EP3 receptor is crucially involved in the febrile effects of PGE2. However, it is possible for other subtypes of PGE2 receptor to contribute in the central mechanism of fever generation. Accordingly, effects of microinjection of PGE2 receptor subtype-specific agonists or antagonists were examined at the locus where a microinjection of a small amount (420 fmol) of PGE2 elicited prompt increases in the O2 consumption rate (VO2), heart rate, and colonic temperature (Tc) in the rvmPOA of urethane–chloralose-anesthetized rats. The EP3 agonist sulprostone mimicked, whereas its antagonist L-798,106 reduced, the febrile effects of PGE2 microinjected into the same site. Similarly, the EP4 agonist rivenprost mimicked, whereas its antagonist ONO-AE3-208 reduced, the effects of PGE2 microinjected into the same site. In contrast, microinjection of the EP1 agonist iloprost induced a very small increase in VO2 but did not have significant influences on the heart rate and Tc, whereas its antagonist, AH6809, did not affect the PGE2-induced responses. Microinjection of the EP2 agonist butaprost had no effects on the VO2, heart rate, and Tc. The results suggest that the EP3 and EP4 receptor subtypes are both involved in the fever generated by PGE2 in the rvmPOA.

0163 Investigating the Role of Vasoactive Intestinal Peptide-Containing Neurons of the Ventromedal Preoptic Area in Sleep-Wake Control

Abstract Introduction A role for vasoactive intestinal peptide (VIP) in promoting rapid eye movement (REM) sleep has been suggested, but the anatomical location of the neurons that release VIP to promote REM sleep has not been identified. Here, we investigated the role of VIP-containing cell groups in the ventromedial preoptic area (VMPOVIP) in sleep-wake regulation. The VMPO has also previously been implicated in thermoregulation and the febrile response. Methods We first investigated the native firing activity of VMPOVIP neurons, over repeated sleep-wake cycles, using in vivo fiber photometry in VIP-ires-Cre mice. We next examined the afferent and efferent profile of this cell group using conditional retrograde (pseudotyped modified rabies) and anterograde (adeno-associated viral vector-based) tracers. We finally utilized a chemogenetic strategy to selectively activate VMPOVIP neurons cells while monitoring electroencephalogram/electromyogram activity and core body temperature, in order to determine their role in sleep-wake and thermoregulatory control. Results We found that VMPOVIP cells were predominantly and strikingly REM-active, that they received many synaptic inputs from surrounding hypothalamic regions (including the ventromedial hypothalamus, dorsomedial hypothalamus and the arcuate nucleus), and that they targeted established sleep-wake nodes, such as the ventrolateral preoptic nucleus, tuberomammillary nucleus, lateral hypothalamus and ventrolateral periaqueductal gray area. To our surprise, chemogenetic activation of the VMPOVIP cell population had little effect upon all measures of sleep-wake analysed and no effect upon core body temperature. Conclusion We conclude that VMPOVIP neurons do not promote REM sleep per se. However, their REM-active profile and anatomical connectivity suggest that these neurons may play a functional role in generating certain cardinal features of REM sleep, which is an active focus of on-going research in our laboratory. Support SRSF CDA #016-JP-17 to A.V. and NS073613, NS092652 and NS103161 to P.M.

Luteinizing hormone-releasing hormone distribution in the anterior hypothalamus of the female rats.

Luteinizing hormone-releasing hormone (LHRH) neurons and fibers are located in the anteroventral hypothalamus, specifically in the preoptic medial area and the organum vasculosum of the lamina terminalis. Most luteinizing hormone-releasing hormone neurons project to the median eminence where they are secreted in the pituitary portal system in order to control the release of gonadotropin. The aim of this study is to provide, using immunohistochemistry and female brain rats, a new description of the luteinizing hormone-releasing hormone fibers and neuron localization in the anterior hypothalamus. The greatest amount of the LHRH immunoreactive material was found in the organum vasculosum of the lamina terminalis that is located around the anterior region of the third ventricle. The intensity of the reaction of LHRH immunoreactive material decreases from cephalic to caudal localization; therefore, the greatest immunoreaction is in the organum vasculosum of the lamina terminalis, followed by the dorsomedial preoptic area, the ventromedial preoptic area, and finally the ventrolateral medial preoptic area, and in fibers surrounding the suprachiasmatic nucleus and subependymal layer on the floor of the third ventricle where the least amount immunoreactive material is found.

Neural Systems Mediating Seasonal Breeding in the Ewe

Seasonal reproduction in ewes is caused by a dramatic increase in response to oestradiol (E(2)) negative feedback during the nonbreeding (anoestrous) season. Considerable evidence supports the hypothesis that A15 dopaminergic neurones in the retrochiasmatic area (RCh) play a key role in these seasonal changes. These A15 neurones are stimulated by E(2) and inhibit gonadotrophin-releasing hormone (GnRH) secretion in anoestrus, but not the breeding season. Because A15 neurones do not contain oestrogen receptors-alpha (ER alpha), it is likely that E(2)-responsive afferents stimulate their activity when circulating E(2) levels increase during anoestrus. Retrograde tract tracing studies identified a limited set of ER alpha-containing afferents primarily found in four areas [ventromedial preoptic area, RCh, ventromedial and arcuate (ARC) nuclei]. Pharmacological and anatomical data are consistent with GABA- and glutamate-containing afferents controlling A15 activity in anoestrus, with E(2) inhibiting GABA and stimulating glutamate release at this time of year. Tract tracing demonstrated that A15 efferents project posteriorly to the median eminence and the ARC, suggesting possible direct actions on GnRH terminals or indirect actions via kisspeptin neurones in the ARC to inhibit GnRH in anoestrus. Identification of this neural circuitry sets the stage for the development of specific hypotheses for morphological or transmitter/receptor expression changes that would account for seasonal breeding in ewes.

Prostaglandin E2 Attenuates Preoptic Expression of GABAA Receptors via EP3 Receptors

Prostaglandin E(2) (PGE(2)) has been shown to produce fever by acting on EP3 receptors within the preoptic area of the brain. However, there is little information about the molecular events downstream of EP3 activation in preoptic neurons. As a first step toward this issue, we examined PGE(2)-induced gene expression changes at single-cell resolution in preoptic neurons expressing EP3. Brain sections of the preoptic area from PGE(2)- or saline-injected rats were stained with an anti-EP3 antibody, and the cell bodies of EP3-positive neurons were dissected and subjected to RNA amplification procedures. Microarray analysis of the amplified products demonstrated the possibility that gene expression of gamma-aminobutyric acid type A (GABA(A)) receptor subunits is decreased upon PGE(2) injection. Indeed, we found that most EP3-positive neurons in the mouse preoptic area are positive for the alpha2 or gamma2 GABA(A) receptor subunit. Moreover, PGE(2) decreased the preoptic gene expression of these GABA(A) subunits via an EP3-dependent and pertussis toxin-sensitive pathway. PGE(2) also attenuated the preoptic protein expression of the alpha2 subunit in wild-type but not in EP3-deficient mice. These results indicate that PGE(2)-EP3 signaling elicits G(i/o) activation in preoptic thermocenter neurons, and we propose the possibility that a rapid decrease in preoptic GABA(A) expression may be involved in PGE(2)-induced fever.

Endothelial-specific knockdown of interleukin-1 (IL-1) type 1 receptor differentially alters CNS responses to IL-1 depending on its route of administration.

Interleukin-1 (IL-1) has been implicated as a critical mediator of neuroimmune communication. In the brain, the functional receptor for IL-1, type 1 IL-1 receptor (IL-1R1), is localized primarily to the endothelial cells. In this study, we created an endothelial-specific IL-1R1 knockdown model to test the role of endothelial IL-1R1 in mediating the effects of IL-1. Neuronal activation in the hypothalamus was measured by c-fos expression in the paraventricular nucleus and the ventromedial preoptic area. In addition, two specific sickness symptoms, febrile response and reduction of locomotor activity, were studied. Intracerebroventricular injection of IL-1 induced leukocyte infiltration into the CNS, activation of hypothalamic neurons, fever, and reduced locomotor activity in normal mice. Endothelial-specific knockdown of IL-1R1 abrogated all these responses. Intraperitoneal injection of IL-1 also induced neuronal activation in the hypothalamus, fever, and reduced locomotor activity, without inducing leukocyte infiltration into the brain. Endothelial-specific knockdown of IL-1R1 suppressed intraperitoneal IL-1-induced fever, but not the induction of c-fos in hypothalamus. When IL-1 was given intravenously, endothelial knockdown of IL-1R1 abolished intravenous IL-1-induced CNS activation and the two monitored sickness symptoms. In addition, endothelial-specific knockdown of IL-1R1 blocked the induction of cyclooxygenase-2 expression induced by all three routes of IL-1 administration. These results show that the effects of intravenous and intracerebroventricular IL-1 are mediated by endothelial IL-1R1, whereas the effects of intraperitoneal IL-1 are partially dependent on endothelial IL-1R1.

C-Fos immunoreactivity induced by intraperitoneal LPS administration is reduced in the brain of mice lacking the microsomal prostaglandin E synthase-1 (mPGES-1)

The aim of the present study was to investigate the impact of the deletion of the microsomal prostaglandin E synthase-1 (mPGES-1) gene on lipopolysaccharide (LPS)-induced neuronal activation in central nervous structures. The mPGES-1 catalyses the conversion of COX-derived PGH 2 to PGE 2 and has been described as a regulated enzyme whose expression is stimulated by proinflammatory agents. Using the immediate-early gene c-fos as a marker of neuronal activation, we determined whether deletion of the mPGES-1 gene altered the neuronal activation induced by LPS in structures classically recognized as immunosensitive regions. No significant difference in the c-Fos immunostaining was observed in the brain of saline-treated mPGES-1+/+, mPGES-1+/− and mPGES-1−/− mice. However, we observed that LPS-induced neuronal activation was reduced in most of the centres known as immunosensitive nuclei in mPGES-1−/− mice compared with heterozygous and wild-type mice. The decrease in the number of c-Fos positive nuclei occurred particularly in the caudal ventrolateral medulla, the medial, intermediate and central parts of the nucleus tractus solitarius, area postrema, parabrachial nucleus, locus coeruleus, paraventricular nucleus of the hypothalamus, ventromedial preoptic area, central amygdala, bed nucleus of the stria terminalis and to a lesser extent in the ventrolateral part of the nucleus tractus solitarius and rostral ventrolateral medulla. These results suggest that the mPGES-1 enzyme is strongly needed to provide sufficient PGE 2 production required to stimulate immunosensitive brain regions and they are discussed with regard to the recent works reporting impaired sickness behavior in mPGES-1−/− mice.

Does nitric oxide act in the ventromedial preoptic area to mediate oestrogen negative feedback in the seasonally anoestrous ewe?

Seasonal anoestrus in the ewe results from enhanced oestrogen negative feedback. Recent data have implicated the ventromedial preoptic area (vmPOA) as an important site of oestrogen action. This study addressed whether NO acts within the vmPOA to inhibit LH during seasonal anoestrus. In Experiment 1, microimplants containing Nomega-nitro-l-arginine methyl ester (l-NAME, NOS inhibitor), S-methyl thiocitrulline (SMTC, neural NOS (nNOS) inhibitor) or empty implants (control) were administered during mid-anoestrus to the vmPOA. l-NAME, but not SMTC, significantly increased LH pulse frequency. For Experiment 2, ewes in late anoestrus were administered 7-nitroindazole (7NI; nNOS inhibitor), l-NAME, SMTC, or empty implants. 7NI, but not l-NAME or SMTC, increased LH pulse frequency. In Experiment 3, the effects of microimplants and microinjections of l-NAME were compared in mid-anoestrus. Microinjections of l-NAME (300 nl at 10 microg/microl) increased LH pulse frequency, but microimplants did not. In late anoestrus, similar microinjections were ineffective. Taken together, the results of Experiments 1-3 suggested that NO inhibition may be stronger during the middle than at the end of seasonal anoestrus. To test this hypothesis, ewes in Experiment 4 received microinjection of l-NAME or vehicle thrice during the non-breeding season; none of the treatments increased LH pulse frequency. These results indicate that NO plays a role in the vmPOA in suppressing LH secretion during seasonal anoestrus because NOS inhibitors were consistently stimulatory when LH pulse frequency was low. However, the inconsistent and modest effects of these inhibitors suggest that NO actions in this area cannot completely account for the effects of inhibitory photoperiod.

Effects of prostaglandin E2 on the electrical properties of thermally classified neurons in the ventromedial preoptic area of the rat hypothalamus

BackgroundPhysiological and morphological evidence suggests that activation of the ventromedial preoptic area of the hypothalamus (VMPO) is an essential component of an intravenous LPS-dependent fever. In response to the endogenous pyrogen prostaglandin E2 (PGE2), the majority of temperature insensitive neurons in the VMPO show an increase in firing rate, while warm sensitive neurons are inhibited. We have hypothesized that these PGE2 dependent effects on firing rate are due to changes in the inherent electrical properties of VMPO neurons, which are regulated by the activity of specific ionic currents.ResultsTo characterize the electrical properties of VMPO neurons, whole-cell recordings were made in tissue slices from male Sprague-Dawley rats. Our results indicate that PGE2 dependent firing rate responses were not the result of changes in resting membrane potential, action potential amplitude and duration, or local synaptic input. However, PGE2 reduced the input resistance of all VMPO neurons, while increasing the excitability of temperature insensitive neurons and decreasing the excitability of warm sensitive neurons. In addition, the majority of temperature insensitive neurons responded to PGE2 with an increase in the rate of rise of the depolarizing prepotential that precedes each action potential. This response to PGE2 was reversed for warm sensitive neurons, in which the prepotential rate of rise decreased.ConclusionWe would therefore suggest that PGE2 is having an effect on the ionic currents that regulate firing rate by controlling how fast membrane potential rises to threshold during the prepotential phase of the action potential.

Evidence that thyroid hormones act in the ventromedial preoptic area and the premammillary region of the brain to allow the termination of the breeding season in the ewe.

Thyroid hormones are permissive for various species to enter seasonal anestrus. In the ewe they act centrally to permit the onset of potent estradiol-negative feedback responsible for anestrus, but the specific sites of action are unknown. Therefore, we tested whether T(4) replacement via chronic microimplants in any of five brain areas could reverse the reproductive effects of thyroidectomy. Diffusion of (125)I-T(4) from the microimplant was largely (>98%) limited to a 1.2-mm radius. A marked decline in LH concentration in ovariectomized, estradiol-treated ewes was used as an index for anestrus. In experiment 1, all thyroidectomized (THX) ewes with microimplants in the medial preoptic area, A15 area, and medial basal hypothalamus failed to enter anestrus; instead, LH levels remained elevated, similar to those in untreated THX controls. In ventromedial preoptic area (vmPOA)-microimplanted ewes, only the two animals with the most caudal microimplants entered anestrus, as did thyroid-intact controls and THX ewes receiving icv or sc T(4) replacement. In experiment 2, all vmPOA-treated ewes with similar placements to those effective in experiment 1 along with all ewes microimplanted in the premammillary region entered neuroendocrine anestrus. Thus, the premammillary region and vmPOA are brain sites in which thyroid hormones act to permit the onset of seasonal anestrus.

The effects of prostaglandin E 2 on the firing rate activity of thermosensitive and temperature insensitive neurons in the ventromedial preoptic area of the rat hypothalamus

In response to an immune system challenge with lipopolysaccharide (LPS), recent work has shown that Fos immunoreactivity is displayed by neurons in the ventromedial preoptic area of the hypothalamus (VMPO). In addition, neurons in this region show distinct axonal projections to the anterior perifornical area (APFx) and the paraventricular nucleus (PVN). It has been hypothesized that neurons within the VMPO integrate their local responses to temperature with changes in firing activity that result from LPS induced production of prostaglandin E 2 (PGE 2). This may be an important mechanism by which the set-point regulation of thermoeffector neurons in the APFx and PVN is altered, resulting in hyperthermia. To characterize the firing rate activity of VMPO neurons, single-unit recordings were made of neuronal extracellular activity in rat hypothalamic tissue slices. Based on the slope of firing rate as a function of tissue temperature, neurons were classified as either warm sensitive or temperature insensitive. Neurons were then treated with PGE 2 (200 nM) while tissue temperature was held at a constant level (∼36 °C). The majority of temperature insensitive neurons responded to PGE 2 with an increase in firing rate activity, while warm sensitive neurons showed a reduction in firing rate. This suggests that both warm sensitive and temperature insensitive neurons in the VMPO may play critical and contrasting roles in the production of a fever during an acute phase response to infection.

Oestradiol microimplants in the ventromedial preoptic area inhibit secretion of luteinizing hormone via dopamine neurones in anoestrous ewes.

Oestradiol exerts a season-specific negative feedback effect on the GnRH/LH neurosecretory system of the Suffolk ewe. This neuroendocrine suppression is mediated in part by dopamine A15 neurones, but these neurones do not possess the oestrogen receptor. Based on indirect evidence, we hypothesized that oestrogen receptor-containing neurones in the ventromedial preoptic area (vmPOA) may be the initial step in a neuronal system whereby oestradiol suppresses GnRH secretion during the non-breeding season. To test this, three experiments were conducted using ovariectomized ewes receiving either empty or oestradiol-containing bilateral microimplants directed at the vmPOA or s.c. subcutaneous oestradiol-containing implants. In the first experiment, LH pulse frequency was measured on days 0, 1, 7 and 14 of treatment during seasonal anoestrus. In vmPOA oestradiol and s.c. oestradiol groups only, LH pulse frequency was suppressed on days 7 and 14, with maximal suppression evident by day 7. In the second experiment, this protocol was repeated during the breeding season, with LH pulses examined on days 0 and 7; LH pulse frequency did not change in any group. The third experiment tested if the effect of vmPOA oestradiol during anoestrus could be overcome by an injection of the dopamine-D2 receptor antagonist (-)-sulpiride. The vmPOA microimplants and s.c. oestradiol implants again suppressed LH pulse frequency and this was reversed by sulpiride in vmPOA oestradiol ewes. We conclude that oestradiol acts on cells in the vmPOA to stimulate a system involving dopamine neurones that inhibits GnRH/LH pulsatility in the anoestrous ewe.

The central pyrogenic action of interleukin-6 is related to nuclear translocation of STAT3 in the anteroventral preoptic area of the rat brain

(1) In animals treated with i.c.v. pyrogen free saline constitutive STAT3-expression was low and predominantly detected within the cytoplasm of large cells in the medio-caudal hypothalamus. (2) Wistar rats treated with i.c.v. IL-6 (100–200 ng) developed a clear febrile response 90–120 min post treatment and showed a strong STAT3-upregulation and -translocation into cell nuclei at various fore- and hindbrain sites that was already observed 15–30 min after the treatment. (3) The most intense IL-6-induced STAT3-activation pattern was found within the vascular organ of the lamina terminalis and the adjacent ventromedial preoptic area. (4) STAT3-immunohistochemistry therefore represents a novel tool to functionally map IL-6 actions on the brain. The results further support the importance of the anteroventral preoptic area as main central IL-6 target involved in the mediation of fever responses.

The vagus nerve mediates behavioural depression, but not fever, in response to peripheral immune signals; a functional anatomical analysis.

Cytokines act on the brain to induce fever and behavioural depression after infection. Although several mechanisms of cytokine-to-brain communication have been proposed, their physiological significance is unclear. We propose that behavioural depression is mediated by the vagus nerve activating limbic structures, while fever would primarily be due to humoral mechanisms affecting the preoptic area, including interleukin-6 (IL-6) action on the organum vasculosum of the laminae terminalis (OVLT) and induction of prostaglandins. This study assessed the effects of subdiaphragmatic vagotomy in rats on fever, behavioural depression, as measured by the social interaction test, and Fos expression in the brain. These responses were compared with induction of the prostaglandin-producing enzyme cyclooxygenase-2 and the transcription factor Stat3 that translocates after binding of IL-6. Vagotomy blocked behavioural depression after intraperitoneal injection of recombinant rat IL-1beta (25 microg/kg) or lipopolysaccharide (250 microg/kg; LPS) and prevented Fos expression in limbic structures and ventromedial preoptic area, but not in the OVLT. Fever was not affected by vagotomy, but associated with translocation of Stat3 in the OVLT and cyclooxygenase-2 induction around blood vessels. These results indicate that the recently proposed vagal link between the immune system and the brain activates limbic structures to induce behavioural depression after abdominal inflammation. Although the vagus might play a role in fever in response to low doses of LPS by activating the ventromedial preoptic area, it is likely to be overridden during more severe infection by action of circulating IL-6 on the OVLT or prostaglandins induced along blood vessels of the preoptic area.

Electrophysiology and pharmacology of projections from the suprachiasmatic nucleus to the ventromedial preoptic area in rat

Extracellular and whole-cell patch-clamp recordings were made from neurons in the ventromedial preoptic area in rat horizontal brain slices. Responses to single-pulse electrical stimulation of the ipsilateral suprachiasmatic nucleus were characterized using peristimulus time histograms or postsynaptic current recordings, and bath application of neurotransmitter receptor antagonists. Extracellular recordings showed that suprachiasmatic nucleus stimulation (50–150 μA) elicited a short-latency suppression in 35 of 64 neurons (55%), with the majority (29/35, 83%) showing a biphasic response consisting of a short-latency suppression followed by a long-duration activation. In addition, 14 cells (22%) showed activation only, while 15 (23%) were unresponsive. The GABA A receptor antagonist bicuculline (5–10 μM) reversibly blocked suppressions evoked by suprachiasmatic nucleus stimulation (20/20 cells). In the majority of these neurons (13/20), bicuculline also unmasked an activation in response to stimulation. Activations elicited by suprachiasmatic nucleus stimulation, either in the presence or absence of bicuculline, were blocked by the non- N-methyl- d-aspartate and N-methyl- d-aspartate glutamate receptor antagonists 6,7-dinitroquinoxaline-2,3-dione and (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (10/10 cells). 6,7-Dinitroquinoxaline-2,3-dione (10 μM) selectively and reversibly blocked the initial, short-duration (<50 ms) activation, but had no effect on the longer-duration activation. In contrast, (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (10 μM) appeared to inhibit the long-duration activation selectively without affecting the initial rapid activation. Combined applications of the two ionotropic glutamate receptor antagonists blocked stimulation-induced activations completely. All the pharmacological effects were concentration dependent. Whole-cell patch-clamp recordings showed that suprachiasmatic nucleus stimulation elicited inhibitory postsynaptic currents or a combination of inhibitory and excitatory postsynaptic currents in 25 of 33 neurons tested. The inhibitory postsynaptic currents had short onset latencies (4.9±0.3 ms) and a reversal potential of −56.0±3.8 mV ( n=5), and were reversibly blocked by bicuculline (5–10 μM, 4/4 cells). In the presence of bicuculline (5–10 μM), excitatory postsynaptic currents had short onset latencies (4.7±0.5 ms), and had a fast and a slow component. (±) 3-(2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid blocked the slow, but not the fast, component, whereas 6,7-dinitroquinoxaline-2,3-dione blocked the fast, but not the slow, component ( n=7). These results suggest that the projection from the suprachiasmatic nucleus conveys both inhibitory (GABA) and excitatory (glutamate) inputs to the ventromedial preoptic area. GABA A receptor and both non- N-methyl- d-aspartate and N-methyl- d-aspartate glutamate receptors mediate these influences. These inputs may be responsible for conveying information related to circadian phase from the suprachiasmatic nucleus to regions of the preoptic area known to be involved in regulation of sleep/waking and other physiological functions.

Localization of immunoreactive lamprey gonadotropin-releasing hormone in the rat brain☆

A highly specific antiserum against lamprey gonadotropin-releasing hormone (GnRH) was used to localize l-GnRH in areas of the rat brain associated with reproductive function. Immunoreactive l-GnRH-like neurons were observed in the ventromedial preoptic area (POA), the region of the diagonal band of Broca and the organum vasculosum lamina terminalis, with fiber projections to the rostral wall of the third ventricle and the organum vasculosum lamina terminalis. Another population of l-GnRH-like neurons was localized in the dorsomedial and lateral POA, with nerve fibers projecting caudally and ventrally to terminate in the external layer of the median eminence. Other fibers apparently projected caudally and circumventrically to terminate around the cerebral aqueduct in the mid-brain central gray. By using a highly specific antiserum directed against mammalian luteinizing hormone-releasing hormone (m-LHRH), the localization of the LHRH neuronal system was compared to that of the l-GnRH system. There were no LHRH neurons in the dorsomedial or the lateral region of the POA that contained the l-GnRH neurons. As expected, there was a large population of LHRH neurons in the ventromedial POA associated with the diagonal band of Broca and organum vasculosum lamina terminalis. In both of these regions, there were many more LHRH neurons than l-GnRH neurons and the LHRH neurons extended more dorsally and laterally than the l-GnRH neurons. The LHRH neurons seemed to project to the median eminence in the same areas as those that were innervated by the l-GnRH neurons. Absorption studies indicated that l-GnRH cell bodies were eliminated by adding 1 μg of either l-GnRH-I or 1-GnRH-III, but not m-LHRH to the antiserum before use. Fibers were largely eliminated by the addition of 1 μg l-GnRH-III to the antiserum. No chicken GnRH-II neurons or nerve fibers could be visualized by immunostaining. Because the antiserum recognized GnRH-I and GnRH-III equally, we have visualized an l-GnRH system in rat brain. The results are consistent with the presence of either one or both of these peptides within the rat hypothalamus. Because l-GnRH-I has only weak nonselective gonadotropin-releasing activity, whereas l-GnRH-III is a highly selective releaser of follicle-stimulating hormone, and because l-GnRH neurons are located in areas known to control follicle-stimulating hormone release selectively, our results support the hypothesis that l-GnRH-III, or a closely related peptide, may be mammalian follicle-stimulating hormone-releasing factor.

Temporal and spatial relationships between lipopolysaccharide-induced expression of fos, interleukin-1 β and inducible nitric oxide synthase in rat brain

Interleukin-1 β plays an important role in mediating central components of the host response to peripheral infection such as fever and neuroendocrine activation by acting in the brain. The present study assessed whether interleukin-1(3 produced in the brain is relevant to neuronal activation and the fever response induced by intraperitoneal injection of bacterial lipopolysaccharide. The distributions of Fos protein, interleukin-1β protein and inducible nitric oxide synthase messenger RNA, used as an anatomical indicator of interleukin-1 β bioactivity, were compared in brains of animals killed 2, 4 or 8 b after lipopolysaccharide (250 μg/kg) or saline injection. Saline did not induce interleukin-1β or Fos immunoreactivity in the brain. Interleukin-1β positive cells were found 2h after lipopolysaccharide injection in circumventricular organs. Fos immunoreactivity at this time-point was not found in circumventricular organs, but in parenchymal structures such as the nucleus of the solitary tract, paraventricular hypothalamus and ventromedial preoptic area. Fos expression did occur in circumventricular organs only 8 h after lipopolysaccharide injection. This late pattern of Fos expression coincided with the rise in body temperature and the induction of inducible nitric oxide synthase messenger RNA. These data show that after peripheral lipopolysaccharide administration interleukin-1β is synthesized and bioactive in circumventricular organs. Interleukin-1 β may activate local neurons that induce fever and neuroendocrine activation via projections to the ventromedial preoptic area and the nucleus of the solitary tract.

Neurobiological basis of fever.

Fever is a coordinated endocrine, autonomic, and behavioral response organized by the brain in response to inflammatory stimuli. We examined the mechanism of the febrile response to intravenous lipopolysaccharide (LPS) in the rat. LPS caused activation of microglia and tissue macrophages in the meninges and along penetrating blood vessels. The microglia produce cytokines, such as tumor necrosis factor-alpha, and cyclooxygenase type 2. The latter produces prostaglandins, which may cross the blood-brain barrier. We found that inhibition of COX at the preoptic area prevented fever, whereas injection of PGE2 at this site produced fever. Either i.v. LPS or PGE2 into the preoptic area activated a specific set of pathways, including the ventromedial preoptic area, which we believe to be a key regulatory site, and the paraventricular nucleus, which we believe produces autonomic and endocrine responses that cause elevation of body temperature. We hypothesize that the pathway connecting these two sites involves a double inhibitory relay through temperature-sensitive GABAergic neurons in the hypothalamus. This pathway would essentially "turn up the thermostat" during a fever, causing an increase in body temperature via normal thermoregulatory pathways.