Preoptic-anterior Hypothalamic Area Research Articles

Hypothalamic Neurons Preferentially Respond to Female Nest Coo Stimulation: Demonstration of Direct Acoustic Stimulation of Luteinizing Hormone Release

Avian vocalizations are generally understood to play a pivotal role in reproductive functions. The role of the hypothalamus in gonadotropin release in higher vertebrates including birds is well established. To date, however, a direct linkage between the neuronal processing of vocal input and the contingent luteinizing hormone (LH) response has not been demonstrated. In this study, using female ring doves, we recorded neuronal activity from hypothalamic nuclei that, as we have shown previously, receive acoustic inputs from the auditory thalamic relay. Concurrently with recording single-unit responses to stimulation with species-specific coo vocalizations, we sampled LH levels in blood from the pituitary veins. LH concentration in the plasma was significantly elevated in birds hearing species-typical coos but not in birds exposed to experimentally altered coos or white noise or in birds that received no vocal stimulation. We found two types of neurons in the preoptic and anterior hypothalamus that selectively responded to the female nest coo: excitatory units and inhibitory units. Among the excitatory neurons are units characterized by two bursts separated by a period of slow spiking or complete silence, in a pattern approximately corresponding temporally to the two-note coo. We designate them as female-nest-coo-specific units. Most neurons in the posterior hypothalamus were nonselective in their response. Female nest coo and male nest coo stimulation evoked an equal magnitude of discharge changes from responsive units in the preoptic-anterior hypothalamic area. We found, however, that the LH increment was three times greater for birds hearing female nest coos than for birds hearing male nest coos. These observations suggest that feature-detecting neurons such as the female-nest-coo-specific units are involved in gonadotropin-releasing hormone output. The present findings are consistent with the well established behavioral evidence that female nest coos mediate ovarian growth.

Injection of Oxytocin into the Medial Preoptic–Anterior Hypothalamus Increases Ultrasound Production by Female Hamsters

Recent studies of hamsters have documented the facilitation of lordosis and other sociosexual responses by injections of oxytocin (OXT) into the medial preoptic area-anterior hypothalamus (MPOA-AH). These data suggest the regulation of social interaction and bonds by OXT. In turn, this suggests that OXT could act in the MPOA-AH to control other behaviors involved in the initiation or maintenance of social contact, including the ultrasonic vocalizations that female hamsters use to alert and attract potential mates. To test this possibility, we compared the ultrasound rates of 11 naturally estrous hamsters before and after injections of OXT (200 ng/200 nl of saline) or saline (200 nl) into the MPOA-AH. The data revealed a clear facilitation of ultrasound rate 30 min after OXT treatment. This result suggests the modulation of ultrasound rate by endogenous OXT acting within the MPOA-AH. It extends the range of social behaviors sensitive to control by OXT and supports the possibility that OXT acts within the MPOA-AH to facilitate a variety of behaviors involved in the establishment or maintenance of the social interactions required for successful reproduction. At the same time, these data extend earlier observations linking ultrasound production to the MPOA-AH, and begin to describe the peptidergic mechanisms controlling this form of reproductive behavior.

Effects of kappa and delta opioid agonists on activity and thermosensitivity of rat hypothalamic neurons

Extracellular recordings were made from 161 warm-sensitive, six cold-sensitive and 153 temperature-insensitive neurons in slices of the preoptic area/anterior hypothalamus (PO/AH) of rats, to investigate the effects of the κ-receptor opioid agonist dynorphin A 1–17 and the δ-receptor opioid agonist DPDPE on neuronal response characteristics. While 61% of the neurons exhibited κ-receptors, δ-receptors were only present in 37% of the neurons. No co-localization was observed between κ- and δ-receptors, whereas μ-receptors could be co-localized with κ- as well as δ-receptors. Antagonistic effects on tonic activity were induced by different concentrations of the κ-agonist dynorphin A 1–17. At 0.5 nM, the excitatory effect was predominant, while 50% of the neurons were already inhibited at 5 nM and inhibition was the major effect at 100 nM. A significant increase in temperature sensitivity was observed in warm-sensitive neurons during administration of 0.5 nM dynorphin A 1–17; in contrast, the temperature sensitivity was significantly decreased at the high dose of 100 nM. In most of the neurons responding to the δ-receptor agonist DPDPE (0.5–100 nM) the firing rate was decreased. The temperature sensitivity was only affected in warm-sensitive neurons, and was increased in the majority of neurons at 0.5 and 5 nM, but predominantly decreased at higher concentrations. The effects of low concentrations of dynorphin A 1–17 and DPDPE were prevented by pre- and co-perfusion of the appropriate antagonists. The present results suggest that changes of the temperature sensitivity of warm-sensitive PO/AH neurons are an important mechanism for the effect of low doses of opioids on body temperature.

Cell death in the sexually dimorphic dorsal preoptic area/anterior hypothalamus of perinatal male and female ferrets.

A sexually dimorphic male nucleus (MN) is present in Nissl-stained sections through the dorsal (d) preoptic area/anterior hypothalamus (POA/AH) of male ferrets. The MN-POA/AH is composed of a cluster of large cells which is organized in males by the action of estradiol, formed via the neural aromatization of circulating testosterone (T), during the last quarter of a 41-day gestation. Several recent studies using rodent species have raised the possibility that the hormone-induced masculinization of POA/AH morphology is mediated at least in part by a perinatal modulation of cell death. We asked whether a perinatal reduction in cell death contributes to the differentiation of the MN-POA/AH in the male ferret, which is a carnivore species. The appearance of internucleosomal DNA fragmentation, detected by in situ end labeling (ISEL) using the ApopTag kit (Oncor Corp.) and of pyknotic cell nuclei in Nissl-stained sections were used to estimate the occurrence of cell death. Male and female ferret kits were killed at four different ages spanning the perinatal period during which the MN-POA/AH is organized and assumes an adult phenotype. A peak density of dying cells was present in both sexes at postnatal day (P) 2, which is nearly 1 week after the age, embryonic day (E) 37, when the MN-POA/AH is first visible in male ferrets using Nissl stains. The density of cells in the sexually dimorphic dPOA/AH which were either ISEL-positive or pyknotic was similar in males and females on E34, as well as on P2, 10, and 20. In the nondimorphic ventral POA/AH, the highest density of dying cells was present in both sexes at E34, and there were significantly more ISEL-positive cells present in males than females at this particular age. In contrast to previous studies using rodents, our results suggest that in fetal male ferrets a modulation of the incidence of cell death contributes little to estradiol's organizational action in the dPOA/AH.

Examination of guinea pig luteinizing hormone-releasing hormone gene reveals a unique decapeptide and existence of two transcripts in the brain.

We sequenced the complementary DNA (cDNA) encoding guinea pig LHRH from an expression library derived from the preoptic area-anterior hypothalamus. Data from in situ hybridization and RNase protection assays verified that the cloned cDNA was complementary to guinea pig LHRH messenger RNA. The architecture of the deduced precursor resembles that of LHRH precursors identified in other species. In contrast, the predicted sequence of the decapeptide differs from mammalian LHRH by two amino acid substitutions in positions 2 and 7. This is a novel finding, because the amino acid sequence that comprises LHRH decapeptide is identical in all mammals studied to date. Moreover, the predicted substitution in amino acid position 2 is unique among vertebrates. A second observation of potential significance is the existence of two subspecies of LHRH messenger RNA differing only in the length of their 3' untranslated regions. These two transcripts were verified by sequence analysis of positive clones from the cDNA library and by RNase protection analysis of preoptic area-anterior hypothalamus extracts, and their presence is consistent with the two polyadenylation signals identified in the untranslated regions of the LHRH gene. Future studies will examine LHRH gene expression in guinea pigs, which like primates but unlike rats, have a true luteal phase as a component of their reproductive cycle.

An Alternative Gonadotropin-releasing Hormone (GnRH) RNA Splicing Product Found in Cultured GnRH Neurons and Mouse Hypothalamus

Gonadotropin-releasing hormone (GnRH) is encoded by the proGnRH gene which contains four exons and three introns. In this study, two immortalized GnRH-expressing cell lines (Gn11 and NLT) were characterized. The NLT and Gn11 cells, derived from a same brain tumor in a transgenic mouse, display neuronal morphology and neuron-specific markers. However, NLT cells secrete much higher levels of GnRH than Gn11 cells. To delineate the mechanism underlying this difference, reverse transcriptase-polymerase chain reaction and RNase protection assays were performed to examine proGnRH gene expression. While the mature proGnRH mRNA was predominately expressed in NLT cells, Gn11 cells express an abundant short transcript. Sequence analysis revealed that this short transcript contains exons 1, 3, and 4, but not exon 2, which encodes the GnRH decapeptide. RNase protection assays demonstrated that NLT cells express much higher levels of mature proGnRH mRNA than Gn11 cells. The lower level of GnRH secreting capacity in Gn11 cells is due, in part, to decreased expression of mature proGnRH mRNA. When proGnRH gene expression in the mouse brain was examined, the same short splicing variant was observed in the olfactory area and preoptic area-anterior hypothalamus. But the prevalent transcript in these regions was the mature proGnRH mRNA. In contrast, only the mature proGnRH mRNA was found in the caudal hypothalamus. These results suggest that alternative splicing may be one of the mechanisms regulating proGnRH gene expression in the animal brain.

Antibodies to macrophage inflammatory protein-1 β in preoptic area of rats fail to suppress PGE 2 hyperthermia

This study determined whether macrophage inflammatory protein-1 β (MIP-1 β) plays a role in the hyperthermia caused by prostaglandin E 2 (PGE 2) given intracerebroventricularly (i.c.v.) in the rat. In these experiments, anti-murine MIP-1 β antibody (anti-MIP-1 β) was micro-injected in the anterior hypothalamic, preoptic area (AH/POA) just before i.c.v. PGE 2. The results showed that anti-MIP-1 β failed to alter the PGE 2 hyperthermia. However, immunocytochemical studies revealed MIP-1 β immunoreactivity detectable in both the organum vasculosum laminae terminalis (OVLT) and AH/POA in the febrile rat. These data thus demonstrate that MIP-1 β is sequestered in diencephalic structures underlying thermoregulation even though it is not involved in PGE 2 hyperthermia. This dissociation supports the viewpoint that at least two distinct systems exist in the brain which underlie a febrile response: MIP-1 β underlies one component whereas PGE 2 comprises the other.

Further studies on the suppression of luteinizing hormone release due to activation of medial preoptic-anterior hypothalamic area mu-opioid receptors.

Our laboratory has previously demonstrated that specific activation of mu-opioid receptors in the medial preoptic-anterior hypothalamic area with [D-Ala2, N Me-Phe4, Gly-ol5]-enkephalin (DAGO) suppresses luteinizing hormone secretion in the ovariectomized rat [29]. In the present study, three experiments were undertaken to ascertain whether changes in the activity of norepinephrine or dopamine neurons modulate the decrease in luteinizing hormone release in response to DAGO. The first experiment utilized push-pull perfusion in conjunction with HPLC to assess in vivo norepinephrine release in the medial preoptic-anterior hypothalamic area in response to perfusion of this site with DAGO (5 micrograms/h). DAGO significantly decreased luteinizing hormone release, but perfusate norepinephrine levels did not change. In the second experiment, push-pull perfusion in the medial preoptic-anterior hypothalamic area with cerebrospinal fluid (CSF) or CSF containing DAGO was done in rats pretreated with the norepinephrine synthesis inhibitor, FLA-63. This drug pretreatment had no effect on the DAGO-induced suppression of luteinizing hormone secretion. In experiment 3, push-pull perfusion in the medial preoptic-anterior hypothalamic area with CSF followed by CSF containing DAGO was done in rats pretreated with vehicle, or a dopamine receptor antagonist, either pimozide or d-butaclamol. Neither dopamine receptor antagonist had any effect on the DAGO-induced suppression of luteinizing hormone release. Thus, these studies do not support a role for alterations in the activity of norepinephrine or dopamine neurons in mediating the suppression of luteinizing hormone release in response to activation of mu-opioid receptors in the medial preoptic-anterior hypothalamic area in the ovariectomized rat.

Characterization of gonadotropin-releasing hormone gene transcripts in a mouse hypothalamic neuronal GT1 cell line

We have characterized the nuclear and cytoplasmic RNA transcripts derived from the gonadotropin releasing hormone (GnRH) gene in a mouse hypothalamic neuronal GT1 cell line. Analyses of nuclear GnRH RNA precursors present in the GT1 cells by RNase protection assay show that there is no particular order of intron excision, suggesting the existence of multiple processing pathways. A similar pattern is observed in mouse preoptic area–anterior hypothalamus (POA-AH). In GT1 cells, approximately 5% of the total GnRH RNA transcripts are found in the nucleus. In contrast, in the POA-AH of mice, nuclear transcripts comprise 40% of the total GnRH transcripts. Thus the GT1 cells, while similar in overall GnRH RNA processing to mouse hypothalamic GnRH neurons, do not exhibit the high abundance of nuclear GnRH RNA transcripts seen in the rodent GnRH neuron in vivo. Quantitative analysis of the nuclear RNA species shows that the GnRH primary transcript comprises more than 90% of the total nuclear GnRH mRNA precursors in both GT1 cells and mouse POA-AH and thus GnRH processing intermediates account for fewer than 10% of these precursors. Using these probes, we have examined changes in GnRH primary transcript expression in GT1-7 cells. In the presence of RNA synthesis inhibitors, the half-life of the GnRH primary transcript was found to be quite short, ∼ 18 min, suggesting that the level of primary transcript would reflect levels of GnRH gene transcription. When GT1-7 cells are treated with the phorbol ester PMA (phorbol, 12-myristate, 13-acetate) for 1 h, GnRH primary transcript levels decrease by ∼ 70%. Supporting the hypothesis that GnRH primary transcript is a good indicator of GnRH gene transcription is the finding that 1 h of PMA treatment results in a similar (∼ 50%) decrease in GnRH gene transcription, as assayed by nuclear run-on assay. Our observation that GT1 cells resemble mouse hypothalamic GnRH neurons in their pattern of intron excision and in the ratio of primary transcript to other nuclear transcripts emphasizes the utility of these cells for studying the regulation of GnRH gene expression in this immortalized hypothalamic cell line.

Intrahypothalamic Implantation of Progesterone in Castrated Male Whiptail Lizards (Cnemidophorus inornatus) Elicits Courtship and Copulatory Behavior and Affects Androgen Receptor- and Progesterone Receptor-mRNA Expression in the Brain

A primary tenet of behavioral neuroendocrinology is that gonadal steroid hormones act on limbic nuclei to activate mating behavior in vertebrates. Traditionally, research has focused on the regulation of male-typical sexual behavior by testicular androgens and female-typical sexual behavior by ovarian estrogen and progesterone. Indeed, progesterone generally is regarded as an antiandrogen, acting centrally to inhibit sexual behavior in males. However, experiments with lizards, and more recently with rats, have challenged this paradigm. For example, exogenous progesterone induces mating behavior in some, but not all, castrated male whiptail lizards. The present study determined that implantation of progesterone into the anterior hypothalamus preoptic area of castrated, progesterone-sensitive males completely restored sexual behavior but failed to elicit sexual activity in castrated, progesterone-insensitive males. Further, androgen receptor -and progesterone receptor-mRNA expression in specific brain regions was significantly different in progesterone-sensitive versus progesterone-insensitive animals. Progesterone-sensitive males showed significantly higher relative abundance of androgen receptor-mRNA in the preoptic area, amygdala, and lateral septum, as compared with progesterone-insensitive animals receiving the same treatment. In contrast, progesterone receptor-mRNA abundance was lower in preoptic area of progesterone-sensitive males than in progesterone-insensitive males. No differences were found in the baseline abundance of androgen receptor-or progesterone receptor-mRNA in these nuclei between control groups of progesterone-sensitive and progesterone-insensitive males who were castrated but not implanted. This suggests that progesterone differentially regulates its own receptor as well as androgen receptor in areas of the brain involved in the control of sexual behavior of males and that the nature of this regulation shows individual variability.

Neuronal basis for the hyperthermic effect of μ-opioid agonists in rats: decrease in temperature sensitivity of warm-sensitive hypothalamic neurons

The effect of the selective μ-opioid receptor agonist Tyr-Pro- N-MePhe- d-Pro-NH 2 (PL-017) on tonic activity and temperature sensitivity of neurons in the preoptic area/anterior hypothalamus (PO/AH) has been examined in rat brain slices using extracellular recordings. The tonic activity of both warm-sensitive and temperature-insensitive neurons was inhibited in a dose-dependent manner by superfusion with the μ-agonist (0.5–100 nM). The temperature sensitivity was selectively decreased in warm-sensitive neurons in concentrations up to 10 nM. Only in the high concentration of 100 nM did the μ-agonist reduce the temperature coefficient of both types of neurons. Pretreatment with equimolar concentrations of the μ-antagonist d-Phe-Cys-Tyr- d-Trp-Orn-Thr-Pen-Thr-NH 2 (CTOP) prevented the effects of the μ-agonist (0.5–10 nM) on tonic activity as well as temperature sensitivity. The results suggest that a specific change of the temperature sensitivity of PO/AH neurons is involved in the hyperthermia induced by μ-agonists in rats.

Melatonin Influences Gonadotropin II Secretion in the Atlantic Croaker (Micropogonias undulatus)

The role of melatonin in the control of gonadotropin II (GtH II) secretion was investigated in Atlantic croaker (Micropogonias undulatus) during different phases of the day–night cycle and seasonal reproductive cycle. Intraperitoneal injection of melatonin during the late-light phase of the day–night cycle elicited a significant elevation in plasma GtH II levels in croaker with fully developed gonads. The increase in GtH II levels was observed 30 min postinjection; the levels remained elevated for 1 hr and started to decline at the 2-hr sampling time. The stimulatory effect of melatonin was dose-dependent over the range 5 to 500 ng/g body weight. Circulating GtH II levels in fish with fully developed gonads showed significant diurnal variation, with elevated levels during the dark phase. The GtH II secretion in response to stimulation by a LHRH analog (LHRHa) also varied during the day–night cycle, with the maximum levels during the early-dark phase. Interestingly, melatonin stimulated GtH II release during the late-light and early- and mid-dark phases. The increased gonadotropic response to melatonin preceded (late-light phase) or corresponded to the period of elevated circulating GtH II levels and increased responsiveness to stimulation by LHRHa during the dark phase, suggesting that melatonin may be involved in the control of daily GtH II cycles in this species. Melatonin inhibited LHRHa-induced GtH II release during the mid-dark phase of the day–night cycle in a dose-dependent manner; the physiological significance of this inhibitory influence is not understood at present. No significant effect of melatonin on basal or LHRHa-induced GtH II release was observed in regressed croaker. Melatonin injection (1 or 10 ng/g body weight) into the third ventricle in the preoptic anterior hypothalamic area (POAH) of croaker with fully developed gonads resulted in an elevation in plasma GtH II concentrations. In addition, a low concentration (0.2 ng/ml) of melatonin stimulatedin vitroGtH II release from the pituitary fragments of fish with fully developed gonads. These results suggest that melatonin can influence GtH II secretion by acting at the level of POAH and also directly at the pituitary to stimulate GtH II release.

Dopaminergic A14/A15 neurons are activated during estradiol negative feedback in anestrous, but not breeding season, ewes

A major factor responsible for seasonal anestrus in sheep is a striking increase in the ability of estradiol (E) to inhibit pulsatile GnRH and LH secretion. Previous studies suggest that dopaminergic neurons in the A14 and A15 groups of the ovine hypothalamus play a key role in conveying the inhibitory effects of E in anestrous ewes. The present study tested the hypothesis that A14/A15 neurons in anestrous ewes are activated in response to E, and that this activation is specifically related to seasonal changes in E negative feedback. Expression of the immediate early gene products, Fos and the Fos-related antigens (FRAs), was used as a marker of neuronal activation. Ovariectomized anestrous ewes received either blank implants (no E) or 0.5-cm long E implants sc and were killed 6 h later (E+6h) or 7 days later (E+7d and no E groups). During the breeding season, two additional groups of ovariectomized ewes were perfused 7 days after insertion of either blank or E implants. During anestrus, E completely suppressed LH pulses in the E+7d group, but had no effect in the E+6h group. In the E+7d anestrous group, there was also a significant increase in the mean percentage of tyrosine hydroxylase (TH)-positive cells that expressed nuclear Fos/FRAs in A14 and A15 areas compared to that in either the no E or E+6h group. By contrast, during the breeding season, E had no effect on LH pulse frequency, and there were relatively few TH-positive cells in A14 and A15 that coexpressed Fos/FRAs in either the no E or E+7d group. No significant steroidal or seasonal differences in Fos/FRA expression were seen in other hypothalamic dopaminergic cell groups (A12 and A13) or in the preoptic area-anterior hypothalamus or suprachiasmatic nucleus. Furthermore, E did not alter the total number of TH-positive neurons in A14/A15 or other cell groups. There were seasonal differences in the number of TH-positive neurons, with a significantly greater number of cells in the A13 and A15 of breeding season animals compared to anestrous ewes. Thus, E increased Fos/FRA expression in A14/A15 neurons only during anestrus at a time when it also inhibited LH pulse frequency. These findings are consistent with the view that activation of dopaminergic cells in A14 and A15 is a critical link in the chain of events leading to seasonal shifts in sensitivity to E negative feedback in the ewe.

Dopaminergic A14/A15 neurons are activated during estradiol negative feedback in anestrous, but not breeding season, ewes.

A major factor responsible for seasonal anestrus in sheep is a striking increase in the ability of estradiol (E) to inhibit pulsatile GnRH and LH secretion. Previous studies suggest that dopaminergic neurons in the A14 and A15 groups of the ovine hypothalamus play a key role in conveying the inhibitory effects of E in anestrous ewes. The present study tested the hypothesis that A14/A15 neurons in anestrous ewes are activated in response to E, and that this activation is specifically related to seasonal changes in E negative feedback. Expression of the immediate early gene products, Fos and the Fos-related antigens (FRAs), was used as a marker of neuronal activation. Ovariectomized anestrous ewes received either blank implants (no E) or 0.5-cm long E implants sc and were killed 6 h later (E+6h) or 7 days later (E+7d and no E groups). During the breeding season, two additional groups of ovariectomized ewes were perfused 7 days after insertion of either blank or E implants. During anestrus, E completely suppressed LH pulses in the E+7d group, but had no effect in the E+6h group. In the E+7d anestrous group, there was also a significant increase in the mean percentage of tyrosine hydroxylase (TH)-positive cells that expressed nuclear Fos/FRAs in A14 and A15 areas compared to that in either the no E or E+6h group. By contrast, during the breeding season, E had no effect on LH pulse frequency, and there were relatively few TH-positive cells in A14 and A15 that coexpressed Fos/FRAs in either the no E or E+7d group. No significant steroidal or seasonal differences in Fos/FRA expression were seen in other hypothalamic dopaminergic cell groups (A12 and A13) or in the preoptic area-anterior hypothalamus or suprachiasmatic nucleus. Furthermore, E did not alter the total number of TH-positive neurons in A14/A15 or other cell groups. There were seasonal differences in the number of TH-positive neurons, with a significantly greater number of cells in the A13 and A15 of breeding season animals compared to anestrous ewes. Thus, E increased Fos/FRA expression in A14/A15 neurons only during anestrus at a time when it also inhibited LH pulse frequency. These findings are consistent with the view that activation of dopaminergic cells in A14 and A15 is a critical link in the chain of events leading to seasonal shifts in sensitivity to E negative feedback in the ewe.

Pharmacokinetics of extracellular melatonin in Siberian hamster forebrain

In vivo brain microdialysis was used to characterize the pharmacokinetics of subcutaneously injected melatonin in the anterior hypothalamic-preoptic area (AH-POA) of the male Siberian hamster. Animals with a microdialysis probe implanted in the AH-POA were treated with a subcutaneous melatonin injection at 0900 h (3 h after lights-on) or 2000 h (2 h prior to lights-off). Treatment with 2.5 or 0.25 mg/kg dosages of melatonin in saline vehicle induced peak concentrations of melatonin in AH-POA microdialysates within 20 min of injection (165 ± 34 and 18 ± 8 pg/20 min, respectively). For the 2.5 and 0.25 mg/kg dosages, the half-life of the absorbed melatonin ( t 1 2 elimination) was less than 20 min, and the concentrations fell to baseline within 60 min after injection. There were no significant time of day effects on the kinetic profile of extracellular melatonin associated with either of these dosages. These results confirm the rapid accumulation and clearance of extracellular melatonin in the vicinity of its putative target tissues.

Role of interleukin-1β, interleukin-6 and macrophage inflammatory protein-1β in prostaglandin-E 2-induced hyperthermia in rats

The purpose of this study was to investigate the role of pyrogenic cytokines, such as IL-1β, IL-6 and MIP-1β, in the mechanisms underlying the hyperthermic response of rats to central injection of PGE 2. Thus, specific murine neutralizing antibodies against these cytokines were microinjected directly into the anterior hypothalamic, preoptic area (AH/POA) of unrestrained rats just before intracerebroventricular injection of PGE 2. The significant hyperthermia induced by PGE 2 was markedly suppressed by micro-injection of anti-IL-6 and partially attenuated by anti-IL-1β. However, the micro-injection of anti-MIP-1β failed to alter the hyperthermic response. The results indicate that PGE 2-induced hyperthermia is presumably mediated through actions of IL-6 on the thermosensitive cells of the AH/POA and confirm that distinct and alternate pathways exist in the rat brain for the induction of fever.

Neurogenesis and cell migration into the sexually dimorphic preoptic area/anterior hypothalamus of the fetal ferret.

A sexually dimorphic male nucleus (MN) of the preoptic area/anterior hypothalamus (POA/AH), comprising large, estradiol-receptor containing neurons, is formed in male ferrets due to the action of estradiol, derived from the neural aromatization of circulating testosterone, during the last quarter of a 41-day gestation. Two experiments were conducted to compare the birthdates and the migration pattern of cells into the sexually dimorphic portion of the dorsomedial POA/AH as well as the nondimorphic ventral nucleus (VN) of the POA/AH of males and females. In experiment 1 the thymidine analog, bromodeoxyuridine (BrdU), was injected into the amniotic sacs of fetuses of different mothers between embryonic (E) days 18 and 30. Kits from all mothers were sacrificed on E38, and brains were processed to localize BrdU immunoreactivity (IR) for determining the birthdates of neurons in the POA/AH. Cells in the MN-POA/AH of males and in a comparable region of females were born between E22 and E28; cells in the nondimorphic VN-POA/AH of both sexes were born between these same ages. These results suggest that cells in the sexually dimorphic as well as the nondimorphic subdivision of the ferret POA/AH are born during the same embryonic period. This is well before the ages (E30-E41) when administering testosterone to females can stimulate, and blocking androgen aromatization in males can inhibit, MN-POA/AH differentiation. In experiment 2 BrdU was injected on E24, and kits from different litters were perfused on E30, E34, or E38. Brains were processed for BrdU-IR as well as glial fibrillary acidic protein (GFAP), which served as a marker for radial glial processes. The orientation of radial glial processes in fetal brains of both sexes suggested that cells migrate into the dorsomedial POA/AH from proliferative zones lining the lateral as well as the third ventricles. Quantitative, computer-assisted image analysis of BrdU-IR in groups of male and female brains supported this hypothesis. There were no significant sex differences in the distribution of BrdU-IR over the three ages studied, suggesting that formation of the MN-POA/AH in males cannot be attributed to an effect of estradiol on the migration of those cells born on E24 into this sexually dimorphic structure. Finally, total BrdU-IR did not change significantly in the POA/AH of male and female kits killed at E30, E34, or E38 while the area of the POA/AH increased more than 2.5-fold over this period, suggesting that few of the POA/AH cells born on E24 die during this period in either sex. In the absence of evidence that formation of the male ferret's MN-POA/AH depends on steroid-induced changes in neurogenesis, cell migration, or death, we suggest that the specification of a particular neuronal phenotype (e.g., large somal size; capacity to produce some undetermined neurotransmitter or neuropeptide) may be responsible.

Neuropeptide Y perfused in the preoptic area of rats shifts extracellular efflux of dopamine, norepinephrine, and serotonin during hypothermia and feeding.

This study examined the localized action of neuropeptide Y (NPY) on monoamine transmitter activity in the hypothalamus of the unrestrained rat as this peptide induced hypothermia, spontaneous feeding or both responses simultaneously. A guide tube was implanted in the anterior hypothalamic pre-optic area (AH/POA) of Sprague-Dawley rats. Then either control CSF vehicle or NPY in a dose of either 100 ng/microliter or 250 ng/microliter was perfused by push-pull cannulae in this structure in the fully sated, normothermic rat. Successive perfusions were carried out at a rate of 20 microliters/min for 6.0 min with an interval of 6.0 min elapsing between each. Samples of perfusate were assayed by HPLC for their levels of dopamine (DA), norepinephrine (NE), serotonin (5-HT) and their respective metabolites. Whereas control CSF was without effect on body temperature (Tb) or feeding, repeated perfusions of NPY over 3.0 hr caused dose-dependent eating from 4 to 39 g of food, hypothermia of 0.9 to 2.3 degrees C or both responses concurrently. As the rats consumed 11-39 g of food, the efflux of NE, MHPG, DOPAC and 5-HT was enhanced significantly, whereas during the fall in Tb the efflux of NE, DOPAC and 5-HIAA from the AH/POA increased. When the Tb of the rat declined simultaneously with eating behavior, the levels in perfusate of DOPAC and HVA increased significantly while MHPG declined. During perfusion of the AH/POA with NPY the turnover of NE declined while DA and 5-HT turnover increased during hypothermia alone or when accompanied by feeding. These results demonstrate that the sustained elevation in NPY within the AH/POA causes a selective alteration in the activity of the neurotransmitters implicated in thermoregulation, satiety and hunger. These findings suggest that both DA and NE comprise intermediary factors facilitating the action of NPY on neurons involved in thermoregulatory and ingestive processes. The local activity of NPY on hypothalamic neurons apparently shifts the functional balance of serotonergic and catecholaminergic neurons now thought to play a primary role in the control of energy metabolism and caloric intake.

Estrogenic control of preoptic area development in a carnivore, the ferret.

1. Evidence is reviewed which shows that a sexually dimorphic nucleus located in the dorsomedial portion of the male ferret's preoptic area/anterior hypothalamus (POA/AH), called the male nucleus of the POA/AH (Mn-POA/AH), develops during fetal life in response to the action of estradiol, which is formed directly in the nervous system from circulating testosterone over the final quarter of a 41-day gestation. 2. Results are summarized which establish that neurons which make up the Mn-POA/AH are born prior to the critical period of estradiol's action in the male brain. Other data show that some radial glial processes, visualized immunocytochemically using antibodies against GFAP, emanate from proliferative zones at the base of the lateral ventricles in a dorsal-ventral orientation, whereas other glial processes emanate laterally from proliferative zones lining the third ventricle. 3. We suggest that at least some neurons which constitute the dorsomedial POA/AH are born in proliferative zones surrounding the lateral ventricles, raising the question of whether estradiol acts in developing males to influence the migration of these neurons along radial glial guides into the Mn-POA/AH. 4. Finally, evidence is summarized showing that excitotoxic lesions of the dorsomedial POA/AH enhance males' preference to approach and interact with another sexually active male, as opposed to an estrous female, when adult subjects are castrated and treated with estradiol benzoate. These data suggest that the sexually dimorphic Mn-POA/AH is an essential part of a CNS circuit which determines heterosexual partner preference in the male ferret.

The capacity to accumulate cyclic AMP in the preoptic-anterior hypothalamic area of the rat is affected by the exposition to low ambient temperature and the subsequent recovery

The accumulation of adenosine 3':5'-cyclic monophosphate (cAMP) was measured in the preopticanterior hypothalamic area, the cerebral cortex, and the hippocampus of rats exposed to different ambient temperatures: (1) 23 +/- 0.5 degrees C, for 53 h +/- 20 min (control); (2) -10 +/- 1 degrees C, for 53 h +/- 20 min (exposure to low ambient temperature); (3) -10 degrees C for 48 h and 23 degrees C for the following 5 h +/- 20 min (recovery). The capacity to accumulate cAMP was tested by subjecting animals to acute hypoxia, a stimulus which is known to induce a large increase in brain cAMP concentration. In the control condition, hypoxic stimulation increases cAMP concentration in all the brain regions studied. In contrast, during the exposure to low ambient temperature, whilst both the cerebral cortex and the hippocampus show the same levels of accumulation found in the control condition, cAMP accumulation is reduced in the preoptic-anterior hypothalamic area. However, during the first few hours of the recovery period, the preoptic-anterior hypothalamic area is able to reattain the capacity for cAMP accumulation observed in the control condition.