Inhibition of the preoptic area and anterior hypothalamus by tetrodotoxin alters thermoregulatory functions in exercising rats.

We have previously demonstrated a functional role of the preoptic area and anterior hypothalamus (PO/AH) in thermoregulation in freely moving rats at various temperature conditions by using microdialysis and biotelemetry methods. In the present study, we perfused tetrodotoxin (TTX) solution into the PO/AH to investigate whether this manipulation can modify thermoregulation in exercising rats. Male Wistar rats were trained for 3 wk by treadmill running. Body core temperature (Tb), heart rate (HR), and tail skin temperature (Ttail) were measured. Rats ran for 120 min at speed of 10 m/min, with TTX (5 microM) perfused into the left PO/AH during the last 60 min of exercise through a microdialysis probe (control, n=12; TTX, n=12). Tb, HR, and Ttail increased during the first 20 min of exercise. Thereafter, Tb, HR, and Ttail were stable in both groups. Perfusion of TTX into the PO/AH evoked an additional rise in Tb (control: 38.2 +/- 0.1 degrees C, TTX: 39.3 +/- 0.2 degrees C; P <0.001) with a significant decrease in Ttail (control: 31.2 +/- 0.5 degrees C, TTX: 28.3 +/- 0.7 degrees C; P <0.01) and a significant increase in HR (control: 425.2 +/- 12 beats/min, TTX: 502.1 +/- 13 beats/min; P <0.01). These results suggest that the TTX-induced hyperthermia was the result of both an impairment of heat loss and an elevation of heat production during exercise. We therefore propose the PO/AH as an important thermoregulatory site in the brain during exercise.     

Sex differences in cell migration in the preoptic area/anterior hypothalamus of mice

The preoptic area/anterior hypothalamus (POA/AH) sits as a boundary region rostral to the classical diencephalic hypothalamus and ventral to the telencephalic septal region. Numerous studies have pointed to the region's importance for sex‐dependent functions. Previous studies suggested that migratory guidance cues within this region might be particularly unique in their diversity. To better understand the early development and differentiation of the POA/AH, cytoarchitectural, birthdate, immunocytochemical, and cell migration studies were conducted in vivo and in vitro using embryonic C57BL/6J mice. A medial preoptic nucleus became discernible using Nissl stain in males and females between embryonic days (E) E15 and E17. Cells containing immunoreactive estrogen receptor‐α were detected in the POA/AH by E13, and increased in number with age in both sexes. From E15 to E17, examination of the radial glial fiber pattern by immunocytochemistry confirmed the presence of dual orientations for migratory guidance ventral to the anterior commissure (medial‐lateral and dorsal‐ventral) and uniform orientation more caudally (medial‐lateral). Video microscopy studies followed the migration of DiI‐labeled cells in coronal 250‐μm brain slices from E15 mice maintained in serum‐free media for 1–3 days. Analyses showed significant migration along a dorsal‐ventral orientation in addition to medial‐lateral. The video analyses showed significantly more medial‐lateral migration in males than females in the caudal POA/AH. In vivo, changes in the distribution of cells labeled by the mitotic indicator bromodeoxyuridine (BrdU) suggested their progressive migration through the POA/AH. BrdU analyses also indicated significant movement from dorsal to ventral regions ventral to the anterior commissure. The significant dorsal‐ventral migration of cells in the POA/AH provides additional support for the notion that the region integrates developmental information from both telencephalic and diencephalic compartments. The sex difference in the orientation of migration of cells in the caudal POA/AH suggests one locus for the influence of gonadal steroids in the embryonic mouse forebrain. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 252–266, 1999     

Sexually dimorphic estrogen receptor α mRNA expression in the preoptic area and ventromedial hypothalamus of green anole lizards

Estradiol (E2) is important in activation of male reproductive behaviors, and masculinizes morphology of associated brain regions in a number of mammalian and avian species. In contrast, it is testosterone, rather than its metabolites, that is the most potent activator of male sexual behavior in green anole lizards. As in other vertebrate groups, however, E2 is critical for receptivity in females of this species. Aromatase, the enzyme which converts testosterone to E2, is more active in the male than female green anole brain, and appears to be actively regulated on a seasonal basis, suggesting some role for E2 in males. This study was designed to enhance our understanding of potential E2 actions by localizing and quantifying relative levels of estrogen receptor-alpha (ERα) mRNA in forebrain regions involved in masculine and feminine behaviors in anoles. These areas include the preoptic area (POA), ventromedial amygdala (AMY) and ventromedial hypothalamus (VMH). In situ hybridization was conducted in adult males and females collected during both breeding and non-breeding seasons. ERα mRNA was expressed in each brain region across sexes and seasons. However, expression was up to 3 times greater in the VMH compared to the POA and AMY. In the POA and VMH, expression was higher in females compared to males, independent of season. The increased receptor expression in females is consistent with E2 playing a larger role in female than male reproductive behaviors.     

Sex differences in cell migration in the preoptic area/anterior hypothalamus of mice.

The preoptic area/anterior hypothalamus (POA/AH) sits as a boundary region rostral to the classical diencephalic hypothalamus and ventral to the telencephalic septal region. Numerous studies have pointed to the region's importance for sex-dependent functions. Previous studies suggested that migratory guidance cues within this region might be particularly unique in their diversity. To better understand the early development and differentiation of the POA/AH, cytoarchitectural, birthdate, immunocytochemical, and cell migration studies were conducted in vivo and in vitro using embryonic C57BL/6J mice. A medial preoptic nucleus became discernible using Nissl stain in males and females between embryonic days (E) E15 and E17. Cells containing immunoreactive estrogen receptor-alpha were detected in the POA/AH by E13, and increased in number with age in both sexes. From E15 to E17, examination of the radial glial fiber pattern by immunocytochemistry confirmed the presence of dual orientations for migratory guidance ventral to the anterior commissure (medial-lateral and dorsal-ventral) and uniform orientation more caudally (medial-lateral). Video microscopy studies followed the migration of DiI-labeled cells in coronal 250-microm brain slices from E15 mice maintained in serum-free media for 1-3 days. Analyses showed significant migration along a dorsal-ventral orientation in addition to medial-lateral. The video analyses showed significantly more medial-lateral migration in males than females in the caudal POA/AH. In vivo, changes in the distribution of cells labeled by the mitotic indicator bromodeoxyuridine (BrdU) suggested their progressive migration through the POA/AH. BrdU analyses also indicated significant movement from dorsal to ventral regions ventral to the anterior commissure. The significant dorsal-ventral migration of cells in the POA/AH provides additional support for the notion that the region integrates developmental information from both telencephalic and diencephalic compartments. The sex difference in the orientation of migration of cells in the caudal POA/AH suggests one locus for the influence of gonadal steroids in the embryonic mouse forebrain.     

Telencephalic and diencephalic origin of radial glial processes in the developing preoptic area/anterior hypothalamus

Neuronal birth-dating sudies using [³H]thymidine have indicated that neurons in the preoptic area/anterior hypothalamus (POA/AH) are derived primarily from progenitors in proliferative zones surrounding the third ventricle. Radial glial processes are potential guides for neuronal migration, and their presence and orientation during development may provide further information about the origin of cells in the POA/AH. In addition to determining the orientation of radial glial fibers, we examined the relationship of neurons with identified birth dates to radial glial processes in the developing POA/AH of ferrets. Neuronal birth dates were determined by injecting ferret fetuses with bromodeoxyuridine (BrdU) at several different gestational ages; brains were taken from ferret kits at subsequent prenatal ages. Sections were processed for immunocytochemistry to reveal vimentin or glial fibrillary acidic protein in radial glia, or BrdU-labeled cell nuclei. Numerous radial glial processes extended from the lateral ventricles through ventral portions of the septal region to the pial surface of the POA/AH. These fibers both encapsulated and coursed ventrally through and around the anterior commissure of ferret, rat, and mouse fetuses. These ventrally directed fibers were less evident at older ages. In double-labeled sections from ferrets, BrdU-labeled cells in the dorsal POA/AH were often aligned in the same dorsal-ventral orientation as adjacent radial glial fibers. We suggest that a subset of neurons, originating in telencephalic proliferative zones, migrates ventrally along radial glial guides into the dorsal POA/AH. © 1995 John Wiley & Sons, Inc.     

Sleep-related c-Fos protein expression in the preoptic hypothalamus: effects of ambient warming

Preoptic area (POA) neuronal activity promotes sleep, but the localization of critical sleep-active neurons is not completely known. Thermal stimulation of the POA also facilitates sleep. This study used the c-Fos protein immunostaining method to localize POA sleep-active neurons at control (22 degrees C) and mildly elevated (31.5 degrees C) ambient temperatures. At 22 degrees C, after sleep, but not after waking, we found increased numbers of c-Fos immunoreactive neurons (IRNs) in both rostral and caudal parts of the median preoptic nucleus (MnPN) and in the ventrolateral preoptic area (VLPO). In animals sleeping at 31.5 degrees C, significantly more Fos IRNs were found in the rostral MnPN compared with animals sleeping at 22 degrees C. In VLPO, Fos IRN counts were no longer increased over waking levels after sleep at the elevated ambient temperature. Sleep-associated Fos IRNs were also found diffusely in the POA, but counts were lower than those made after waking. This study supports a hypothesis that the MnPN, as well as the VLPO, is part of the POA sleep-facilitating system and that the rostral MnPN may facilitate sleep, particularly at elevated ambient temperatures.     

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. © 1996 John Wiley & Sons, Inc.     

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. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 242–252, 1998     

Changes of body temperature and extracellular serotonin level in the preoptic area and anterior hypothalamus after thermal or serotonergic pharmacological stimulation of freely moving rats

Although many studies has been shown that serotonin (5-HT) in the preoptic area and anterior hypothalamus (PO/AH) is important for regulating body temperature (Tb), the exact role is not established yet due to conflicting results probably related to experimental techniques or conditions such as the use of anesthesia. The purpose of present study was to clarify the role of 5-HT in the PO/AH using the combined methods of telemetry, microdialysis and high performance liquid chromatography (HPLC), with a special emphasis on the regulation of Tb in freely moving rats. Firstly, we measured changes in Tb and levels of extracellular 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the PO/AH during cold (5 degrees C) and heat (35 degrees C) exposure. We also perfused fluoxetine (5-HT re-uptake inhibitor) and 8-hydroxy-2-(Di-n-propylamino)tetralin (8-OH-DPAT: 5-HT1A agonist) into the PO/AH. During both exposures, although Tb changed significantly, no significant changes were noted in extracellular levels of 5-HT and 5-HIAA in the PO/AH. In addition, although perfusion of fluoxetine or 8-OH-DPAT into the PO/AH increased or decreased extracellular 5-HT and 5-HIAA levels in the PO/AH respectively, but Tb did not change at all. Our results suggest that 5-HT in the PO/AH may not mediate acute changes in thermoregulation.     

Changes in body temperature and metabolic rate following microinjection of Met-enkephalinamide in the preoptic/anterior hypothalamus of rats

The effects of Met-enkephalinamide (MET-ENKamide) on brain temperature (Tb) and metabolic rate (MR) were assessed following direct administration into the preoptic/anterior hypothalamus (PO/AH) of freely moving rats. Bilateral microinjections of saline or MET-ENKamide (1-25 micrograms/microliter) were delivered through cannula guide tubes previously implanted in nine animals. Thiorphan, an enkephalinase inhibitor, was microinjected into the PO/AH of two of the animals. All injections were made remotely at an ambient temperature of 22 +/- 1 degree C in a volume of 1 microliter. Measurements of Tb (via a brain-dwelling thermistor) and MR were recorded continuously. The ability of naloxone to antagonize the effects of MET-ENKamide was investigated by fashioning a double-barreled injection cannula to fit within each guide tube; 1 microliter of saline or naloxone (1-10 micrograms) was delivered bilaterally into the PO/AH followed by 1 microliter of MET-ENKamide (25 micrograms) 5-10 min later. PO/AH administration of MET-ENKamide (1-25 micrograms) produced dose-dependent increases in Tb preceded by dose-dependent increases in MR, with a characteristic time course of approximately 30 min. Naloxone antagonized the rise in Tb and MR, either partially or completely, depending on dose. When administered alone, naloxone had no effect on Tb or MR. Microinjection of thiorphan (10 micrograms) into the PO/AH evoked increases in Tb and MR that were similar to those responses induced by MET-ENKamide. These results support a role for endogenous Met-enkephalin in the regulation of Tb in the rat.     

Sim2 contributes to neuroendocrine hormone gene expression in the anterior hypothalamus

Paraventricular (PVN) and supraoptic nuclei of the hypothalamus maintain homeostasis by modulating pituitary hormonal output. PVN and supraoptic nuclei contain five major cell types: oxytocin-, vasopressin-, CRH-, somatostatin-, and TRH-secreting neurons. Sim1, Arnt2, and Otp genes are essential for terminal differentiation of these neurons. One of their common downstream genes, Brn2, is necessary for oxytocin, vasopressin, and CRH cell differentiation. Here we show that Sim2, a paralog of Sim1, contributes to the expression of Trh and Ss genes in the dorsal preoptic area, anterior-periventricular nucleus, and PVN. Sim2 expression overlaps with Trh- and Ss-expressing cells, and Sim2 mutants contain reduced numbers of Trh and Ss cells. Genetically, Sim1 acts upstream of Sim2 and partially compensates for the loss of Sim2. Comparative expression studies at the anterior hypothalamus at early stages reveal that there are separate pools of Trh cells with distinctive molecular codes defined by Sim1 and Sim2 expression. Together with previous reports, our results demonstrate that Sim1 and Otp utilize two common downstream genes, Brn2 and Sim2, to mediate distinctive sets of neuroendocrine hormone gene expression.     

Effect of testosterone and season on proenkephalin messenger RNA expression in the preoptic area of the hypothalamus in the ram

Enkephalin appears to exert an inhibitory action on LH secretion, but whether testosterone regulates enkephalin gene expression is unknown. This study tested the hypothesis that testosterone and/or season modulate preproenkephalin mRNA expression in specific areas of the hypothalamus. Romney Marsh rams were castrated (wethers) either during the breeding season or nonbreeding season and received intramuscular injections of either oil or testosterone propionate (five/group). Blood samples were taken for the assay of plasma LH and testosterone. Preproenkephalin mRNA expression was quantified in hypothalamic sections by in situ hybridization. Mean plasma LH concentrations were reduced and the interpulse interval for LH pulses was greater in testosterone propionate-treated wethers compared with oil-treated wethers, with no change in LH pulse amplitude. Testosterone propionate treatment reduced proenkephalin expression in the diagonal band of Broca, the caudal preoptic area, and the bed nucleus of the stria terminalis. Seasonal differences in proenkephalin expression were observed in the bed nucleus of the stria terminalis, lateral septum, periventricular nucleus, and paraventricular nucleus. No differences were observed between treatments in seven other regions examined. We conclude that testosterone and season regulate proenkephalin mRNA levels in the preoptic area/hypothalamus in the ram in a region-specific manner.     

Stimulation of single unit activity in the preoptic area and anterior hypothalamus, and secretion of luteinizing hormone, by ovarian steroids in ovariectomized rats with diencephalic islands

The spontaneous activity of 454 single hypothalamic neurons was recorded in 42 chronically ovariectomized rats after severance of all neural connections with the diencephalon. In 15 of these diencephalic island preparations progesterone was administered immediately before the recording session (and just after deafferentation of the diencephalon) and oestrogen 72 h beforehand. Thirteen rats were given two injections of oestrogen at these times and the remaining 14 rats were similarly treated with equal volumes of oil. Blood samples were obtained from all rats just before each hormone or oil injection, and 4, 5, 6 and 7 h after the second one, for subsequent measurement of plasma luteinizing hormone (LH) concentration. Only the group of rats given progesterone at the time of the second injection showed a significant increase in plasma LH concentration during the recording period. There was however some individual variation and the greatest LH surge was obtained from a rat given two injections of oestrogen. For steroid-treated rats the size of the LH surge was significantly correlated (P less than 0.01) with the mean firing rate of the neurons recorded in the preoptic and anterior hypothalamic areas (p.o.--a.h.). No similar correlation could be established for p.o.--a.h. cells recorded in oil-treated rats or for cells recorded in other parts of the hypothalamus in steroid-treated rats. The mean firing rate of all p.o.--a.h. cells recorded from rats treated with oestrogen followed by progesterone was significantly higher (P less than 0.05) than in either of the other two groups of animals. The oestrogen--progesterone treatment also significantly changed the regularity of discharge of the slow firing (less than 2 Hz) p.o.--a.h. cells, but this phenomenon could not be related to any alteration in plasma LH concentration. The experiments have demonstrated for the first time that the magnitude of the steroid-stimulated LH surge in ovariectomized rats is significantly correlated with the increase in the electrical activity of p.o.--a.h. neurons.     

Immunohistochemical localization of cholecystokinin in the medial preoptic area and anterior hypothalamus of the Brazilian gray short-tailed opossum: a sex difference

We have studied the anatomical localization of cholecystokinin-like immunoreactivity (CCK IR) in somata and fibers in the medial preoptic area (MPA) and anterior hypothalamus (AH) of the Brazilian gray short-tailed opossum, (Monodelphis domestica). With the aid of an avidin-biotin, nickel-enhanced, immunohistochemical technique, CCK IR neuronal elements were found within the MPA and AH. A large number of CCK IR cell bodies were located in the MPA of colchicine-treated opossums. The MPA also contained a CCK IR fiber plexus. Quantitative image analysis revealed that the periventricular preoptic area of noncolchicine-treated male opossums had a significantly higher percent of blocked light measurements than that of the noncolchicine-treated females, indicating a higher density of CCK IR neuronal elements in the males. Neuronal fibers and somata containing CCK IR were also found within the periventricular hypothalamic nucleus (Pe), and the suprachiasmatic nucleus (SCh). These results show that CCK IR neuronal elements are found within the MPA and AH of the Brazilian short-tailed opossum. Furthermore, there is a sexually dimorphic distribution of CCK IR elements within the MPA of this small marsupial.     

Cytosol and nuclear estrogen receptor binding in the preoptic area and hypothalamus of female rats during pregnancy and ovariectomized, nulliparous rats after steroid priming: correlation with maternal behavior

In a previous study, high nuclear estrogen receptor concentrations in the preoptic area (POA) were found on Day 16 of pregnancy to prime females to respond to a subsequent low dose of estradiol benzoate (EB) after hysterectomy-ovariectomy by exhibiting maternal behavior in 48 hr. Receptor concentrations in the POA were found to be higher than those in the hypothalamus (HYP). The present study investigated when nuclear estrogen receptors increase during pregnancy in POA and when the difference in receptor concentrations between POA and HYP occurs. An attempt was made to reproduce these pregnancy changes with a 16-day treatment of estrogen and progesterone in ovariectomized (OVX), nulliparous rats. In Experiment 1, we measured cytosol and nuclear estrogen receptor concentrations in the POA and HYP of female rats during pregnancy. Nuclear receptor concentrations in the POA increased beginning on Day 10, increased again on Day 16, and continued at this high level for the remainder of pregnancy. Nuclear estrogen receptor concentrations in the HYP remained at a lower level throughout most of pregnancy until Day 22 when they increased significantly. In Experiment 2, we tested the maternal behavior and measured estrogen receptor concentrations in OVX, steroid-primed, nulliparous rats after hysterectomy (H) and EB treatment. While 90% of estradiol (E) + progesterone (P)-primed females displayed short-latency maternal behavior 48 hr after H and EB treatment, 46% of E + vehicle (V)-treated controls were maternal. At 0 hr (prior to H and EB treatment), there was a significantly larger nuclear receptor accumulation in the POA but significantly attenuated receptor binding in the HYP. P treatment significantly affected cytosol and nuclear estrogen receptor dynamics. Differences in nuclear estrogen receptor concentrations were shown to be based on the number of available binding sites and not to changes in receptor affinity for estradiol.     

Progesterone receptor isoforms differentially regulate the expression of tryptophan and tyrosine hydroxylase and glutamic acid decarboxylase in the rat hypothalamus

Progesterone exerts a variety of actions in the brain through the interaction with its receptors (PR) which have two isoforms with different function and regulation: PR-A and PR-B. Progesterone may modulate neurotransmission by regulating the expression of neurotransmitters synthesizing enzymes or their receptors in several brain regions. The role of PR isoforms in this modulation is unknown. We explored the role of PR isoforms in the regulation of tryptophan (TPH) and tyrosine (TH) hydroxylase, and glutamic acid decarboxylase (GAD) expression in the hypothalamus of ovariectomized rats. Two weeks after ovariectomy, animals were subcutaneously injected with 5 μg of estradiol benzoate (EB), and 40 h later, progesterone (P) was intracerebroventricularly (ICV) injected. Each animal received two ICV injections of 1 μg/μl (4 nmol) of PR-B and total PR (PR-A + PR-B) sense or antisense (As) oligonucleotides (ODNs). First injection was made immediately before sc EB injection, and 24 h later animals received the second one. Twenty-four hours after P administration, rats were euthanized and brains removed to measure the expression of PR-A and PR-B, TPH, TH and GAD by Western blot. We observed that sense ODNs modified neither PR isoforms nor enzymes expression in the hypothalamus, whereas PR A + B antisense (PR A + B As) clearly decreased the expression of both PR isoforms in this region. ICV administration of PR-B As only decreased PR-B isoform expression with no significant effects on PR-A expression. A differential protein expression of TPH, TH and GAD was observed after PR isoforms antisense administration. PR-B As administration decreased the expression of TPH (65% with respect to control). In contrast, PR A + B As and PR-B As administration increased (51.6% and 34.4%, respectively) TH expression. The administration of PR A + B As and PR-B As diminished GAD expression (33.4% and 41.6%, respectively). Our findings indicate that PR isoforms play a differential role in the regulation of the content of TPH, TH and GAD in the rat hypothalamus.