Effect of prenatal exposure to diazepam on brain GABA(A) receptor mRNA levels in rats examined at late fetal or adult ages.

This study tested the hypotheses that in utero exposure to diazepam (DZ): (1) exerts long-lasting effects on GABA(A) receptor function by altering GABA(A) receptor subunit mRNA levels in specific brain regions of adult animals and/or (2) alters GABA(A) subunit mRNA expression in exposed fetuses. We assayed levels of mRNAs encoding several of the most predominant GABA(A) receptor subunits as well as cyclophilin mRNA. Analysis of mRNA levels in the cortex in adults showed that only gamma2S mRNA levels varied significantly with prenatal drug exposure, an effect unrelated to DZ action to the GABA(A) receptor. Analysis in fetuses indicated that mRNA levels varied as a function of both fetal sex and fetal drug environment. Irrespective of sex, DZ exposure increased both alpha1 and cyclophilin mRNAs in fetal brainstem whereas the mRNA levels of gamma2S were increased and decreased, respectively, in the telencephalon and hypothalamus of DZ-exposed fetuses.

Sex-specific effects of in utero manipulation of GABA(A) receptors on pre- and postnatal expression of BDNF in rats.

Exposure to diazepam (DZ) during the last week of in utero development in rats induces neurobehavioral effects that do not become apparent in exposed animals until young adult ages. Some of the effects are sex specific. This study evaluated the hypothesis that late gestational exposure to DZ, a positive modulator of GABA(A) receptors, affects the developmental appearance of brain-derived neurotrophic factor (BDNF), an effect that could be linked to the later consequences of the exposure. Pregnant Long-Evans rats were injected with DZ (2.5 mg/kg) over gestation days 14-20, and their male and female offspring were evaluated for levels of BDNF mRNA and protein in the cerebral cortex and hypothalamus at fetal day 20 and at postnatal ages spanning birth to young adulthood. The effects of the exposure were sex and region specific. At fetal day 20 the expression of BDNF was reduced by about 20% in the hypothalamus of males only. The early exposure affected postnatal expression of BDNF in the hypothalamus only modestly, influencing the age-related profile in both sexes. Postnatal development of BDNF in the cerebral cortex was significantly affected by the in utero exposure in males only with mRNA levels lower in the exposed group and protein levels higher during juvenile ages. At adulthood, both levels were lower in DZ-exposed males. GABA serves a role as a trophic factor during early development, and these results suggest that manipulation of GABA(A) receptors during early development could interact with the developmental action of other trophic factors thereby leading to altered neural organization and later neurobehavioral dysfunction.

Synchronous postnatal increase in alpha1 and gamma2L GABA(A) receptor mRNAs and high affinity zolpidem binding across three regions of rat brain.

The objective of this study was to correlate postnatal changes in levels of mRNAs encoding predominant GABA(A) receptor subunits with a functional index of receptor development. This study is the first to quantify the temporal relationship between postnatal changes in predominant GABA(A) receptor mRNAs and zolpidem-sensitive GABA(A) receptor subtypes. In Experiment 1, we measured zolpidem displacement of 3H-flunitrazepam from rat cerebral cortex, hippocampus, and cerebellum at 0, 6, 14, 21, 29, and 90 postnatal days. Three independent 3H-flunitrazepam sites with high (K(i)=2. 7+/-0.6 nM), low (K(i)=67+/-4.8 nM), and very low (K(i)=4.1+/-0.9 mM) affinities for zolpidem varied in regional and developmental expression. In Experiment 2, we used RNAse protection assays to quantify levels of alpha1, alpha2, beta1, beta2, gamma2S and gamma2L mRNAs in the above regions at the same postnatal ages. Although there was a high degree of regional variation in the developmental expression of zolpidem-sensitive GABA(A) receptors and subunit mRNAs, a dramatic increase in high affinity zolpidem binding sites and alpha1 mRNA levels occurred within all three regions during the second postnatal week. Furthermore, a temporal overlap was observed between the rise in alpha1 mRNA and high affinity zolpidem binding and a more prolonged increase in gamma2L in each region. These results point to the inclusion of the alpha1 and gamma2L subunits in a GABA(A) receptor subtype with a high zolpidem affinity and suggest that a global signal may influence the emergence of this subtype in early postnatal life.

Sex differences in long-term consequences of prenatal diazepam exposure: possible underlying mechanisms.

Prenatal exposure to diazepam, a benzodiazepine (BZD) compound, leads to pronounced effects on responses to stressors in exposed animals when they reach adulthood. Many of the responses are sex specific. The mechanisms mediating the effects of the exposure on the organism have not been elucidated; however, the time course for the appearance of altered function following in utero drug exposure indicates that the exposure interfered with neural organization of mechanisms mediating responses to stressors. The article discusses possible mechanisms that relate to sites of action of the drug in the developing brain: the GABA(A) receptor, and the mitochondrial BZD receptor. The mechanisms mediating the sex-specific impact of diazepam on the developing brain appear to be complex and interactive.

Reduced progesterone metabolites are not critical for plus-maze performance of lactating female rats.

Lactation has been associated with anxiolysis in several tests of anxiety. These observations, considered together with observations that progesterone and its 5alpha-reduced metabolites are anxiolytic in cycling, nonlactating females, raised the question of whether the changes in anxiety-related behaviors that accompany lactation are driven by reduced progesterone metabolites. Lactating female rats were tested on the plus-maze on postpartum days 2 or 7, and demonstrated enhanced open-arm performance relative to cycling, nonlactating females. Hormonal analysis indicated that while serum levels of both progesterone and its 3alpha,5alpha-reduced metabolite were increased in lactating females, the turnover of progesterone to the metabolite was markedly reduced during lactation. Furthermore, treatment with a 5alpha-reductase inhibitor for 3 days prior to testing potentiated the open-arm performance in lactating females, implying that enhanced open-arm performance was not mediated by the reduction of progesterone or other steroids. Additionally, analysis of GABA(A) receptor function indicated that parturition and lactation did not alter the sensitivity of the receptor to GABA or to modulation by reduced steroids. The mechanisms driving enhanced plus-maze behavior in lactating females appear to differ from mechanisms identified in nonlactating females.

Endogenous levels of 5 alpha-reduced progestins and androgens in fetal vs. adult rat brains.

5 Alpha-reduced metabolites of certain steroids have been shown to have important functions in adult brains and may play a role in brain development. To assess which 5 alpha-reduced steroid metabolites may have an impact during development, endogenous levels of 5 alpha-reduced androgens and progestins and their parent hormones were measured in male and female fetal brains over the last 5 days of gestation. These levels were compared to levels measured in adult male and female brains (evaluated at different stages of the estrous cycle). Neither the brain levels of parent hormones nor of their 5 alpha-reduced metabolites varied as a function of fetal sex or of gestational age. Therefore, the data from the two sexes were combined. In fetal brains, the levels of the progesterone reduced metabolites were 20-fold higher than levels of progesterone itself whereas levels of testosterone reduced metabolites were 10-fold lower than testosterone levels. In contrast to fetal brain, conversion of progesterone to reduced metabolites was much lower in adult brain, but the level of 5 alpha-reduced androgens was 3-10-fold higher than the level of testosterone in all adult tissue, indicating more conversion of androgen to 5 alpha-reduced metabolites in adult than in fetal brains. These results imply that the reduction of progesterone to reduced metabolites may play a critical role in brain development.

Brain androgen-inducible aromatase is critical for adolescent organization of environment-specific social interaction in male rats.

Previous observations have indicated that specific behavioral responses to anxiogenic stimuli emerge over adolescent development in male rats and that gonadal androgens during puberty are essential for this emergence. The objective of the current study was to evaluate mechanisms via which androgens might be organizing the brain during adolescence for appropriate mature adaptive responses. Male rats were exposed to fadrozole (an aromatase inhibitor, 5 mg/kg), flutamide (an androgen receptor antagonist, 10 mg/kg), or MK-434 (a 5 alpha-reductase inhibitor, 10 mg/kg) from day 29 to 60 and tested for environment-specific social interaction (SI) at 60 days of age. The emergence of adult-typical SI was impaired by exposure to the aromatase inhibitor and to the antiandrogen, whereas exposure to the 5 alpha-reductase inhibitor was without effect. Peripheral indices of drug effects indicated that the respective mechanisms had been altered by the different compounds. These results suggest that testosterone induction of aromatase is critical for the organization of mature SI behavior in male rats over adolescent development.

Early developmental modulation of GABAA receptor function. Influence on adaptive responses.

If GABA is serving a trophic role during early brain development, before taking on its function as a neurotransmitter, interference with the function of GABA during this period should have a profound influence on neural organization. We have addressed this hypothesis by evaluating the effects of exposing rat fetuses to diazepam (DZ), a positive modulator of GABA at the GABAA receptor, over gestation days 14 to 20. Studies have shown that adult rats exposed in utero to DZ over this developmental period make inappropriate behavioral responses and have altered neural and hormonal responses to environmental stimuli that threaten the organism's stability and homeostasis. Thus, the early exposure led to altered adaptive responses. These effects of the early exposure did not become apparent until late in adolescent development. Furthermore, specific behavioral and neural responses to environmental challenges normally emerge over adolescent development. Other studies have shown that the GABAA receptor in adult brains is responsive to environmental challenges. Thus, we hypothesize that early modulation of the action of GABA mediated via the GABAA receptor interfered with the neural organization of adaptive responses.

Neurosteroid action at the GABAA receptor in fetal rat forebrain.

In utero exposure to diazepam (DZ), a positive modulator of the GABAA (gamma-aminobutyric acid type A) receptor exerts profound effects on the offspring that become most apparent after the maturation of the brain during puberty and that are often sex specific, suggesting that the early exposure might have interfered with organizing actions of sex steroids. In addition to genomic actions, many reduced steroids interact directly with membrane receptors, including the GABAA receptor. In the present study, the effect of in vitro exposure to neurosteroids on GABA-stimulated 36chloride uptake in synaptoneurosomes from adult cerebral cortex or fetal forebrain (gestation day 20) was examined. The initial study examined the effects of incubation with DZ (10 microM) and the neuroactive steroid, 3 alpha,5 beta-THP (500 nM), alone and in combination. In adult tissue, the presence of either drug alone decreased the EC50 for GABA stimulation, and incubation with both drugs had an additive effect. In fetal tissue, while both compounds decreased the EC50, an additive effect was apparent only when comparing the combined exposure to 3 alpha,5 beta-THP alone. DZ alone reduced the EC50 as much as both drugs together. In the second study, the effect of in vitro exposure to androsterone (2.5 microM) was evaluated in male and female fetal tissue separately as well as in the adult. Androsterone enhanced the sensitivity to GABA in all groups but also reduced the efficacy of GABA in fetal tissue, irrespective of gender. While neurosteroids and DZ elicited similar responses in fetal and adult tissue, the study identified a greater vulnerability of fetal GABAA receptors to modulatory compounds.

Adolescent development alters stressor-induced Fos immunoreactivity in rat brain.

Adult-typical behavioural responses to environmental challenges as well as the stressor responsiveness of several neural systems emerge over adolescent development. The present study was undertaken to determine whether stressors might activate different neural populations in adult vs juvenile male rats. Fos-immunoreactivity was determined in various forebrain nuclei following 15 min or 2 h of restraint in 28- and 60-day-old male rats (representing late juvenile and young adult ages, respectively) and compared to non-restrained control animals at each age. Few Fos-positive cells were identified in unrestrained controls at either age. Restraint, however, induced the production of Fos in several areas. Fos immunoreactivity was marked in parvocellular regions of the paraventricular nucleus of the hypothalamus following both restraint periods and at both ages, an observation consistent with previous observations that restraint increases plasma corticosterone at both ages. And at both ages, Fos immunoreactivity was evident in magnocellular regions of the hypothalamus only following the longer restraint period. Fos immunoreactivity, however, clearly varied as a function of adolescent age in several regions. Moderate to intense Fos immunoreactivity was observed in adults in all divisions of the anterior olfactory nucleus, cortical and medial amygdaloid nuclei, pyriform cortex and tenia tecta. In contrast to the adult, only a few Fos positive cells were observed in any of these regions in juveniles. Exposure to the same stressor induced Fos in a broader spectrum of neurons in young adult than in juvenile male rats. The lack of Fos-positive cells in specific areas of juveniles may relate to maturation in specific amygdaloid nuclei, which project to many of the other regions that showed age-related differences in Fos production. The emergence over adolescence of Fos-positive cells in specific areas in response to stressors may underlie the emergence of adult-typical behavioural and neural stressor-responsiveness.

Control of endogenous norepinephrine release in the hypothalamus of male rats changes over adolescent development.

In order to evaluate mechanisms that could contribute to the effect of adolescent development on the in vivo utilization of norepinephrine (NE) in the hypothalamus, the depolarized release of endogenous norepinephrine (using 50 mM potassium) was measured in vitro in hypothalamic explants from male rats over late juvenile (28 days) to young adult (70 days) ages. Depolarized release, expressed as a percent of the total endogenous pool, was significantly greater in juveniles than in either adolescents (42 days) or young adults. Incubation in the presence of idazoxan, an alpha 2-adrenoceptor antagonists, increased the depolarized fractional NE release in adolescent and young adult rats; however, the same drug decreased depolarized release in juveniles. Inhibition of norepinephrine reuptake by incubation in the presence of nisoxetine (1 microM) significantly increased depolarized release (fractional and absolute) in young adults only. A higher concentration of nisoxetine (5 microM) significantly increased depolarized release in juveniles, but significantly reduced release in adults. Nisoxetine did not influence release in adolescents at either concentration. The possibilities that adolescents development brings about a change in alpha 2-adrenoceptor subtype and that juveniles may have a greater NE reuptake capacity than adults are discussed. Hypothalamic NE projections are important to several regulatory functions, and changes that take place in this system over adolescence may be important for the emergence of adult-typical responses as well as render adolescents vulnerable to specific dysfunctions.

Neonatal exposure to cocaine enhances the reward-potentiating properties of the drug in young adult animals.

Either cocaine (20 mg/kg) or saline vehicle was administered to rat pups once daily on postnatal days 1-8. The enhancement of brain stimulation reward (BSR) by acute administration of cocaine (2.5, 5, and 10 mg/kg i.p.) was assessed in adult offspring (70-90 days of age) using a rate-frequency curve-shift paradigm. Acute administration of cocaine produced orderly dose-related shifts of the rate-frequency function toward lower frequencies in all groups indicating a reward-enhancing effect of the drug on BSR. However, offspring neonatally exposed to cocaine displayed a greater drug-induced potentiation of BSR. Of particular note, the small but significant enhancement of the reward-potentiating properties of cocaine was more pronounced in female offspring neonatally exposed to the drug. These findings indicate that the rewarding properties of cocaine were altered by neonatal exposure to the drug in a sexually dimorphic fashion.

Adolescent development influences functional responsiveness of noradrenergic projections to the hypothalamus in male rats.

Hypothalamic noradrenergic utilization in vivo and the in vitro depolarized release of norepinephrine (NE) were measured at 28, 42, and 70 days of age in male rats to determine the impact of adolescent development on the functional responsiveness of this transmitter system. At each age, function was determined in control rats and rats challenged by restraint. NE utilization in vivo was estimated by measuring the decrease in NE levels following administration of a synthesis inhibitor, alpha-methyl-p-tyrosine methyl ester (alpha-MT, 250 mg/kg). The half-life of approach to a new steady-state was determined. To measure depolarized release of NE, the hypothalamus was incubated in vitro in a high potassium (50 mM) medium and the percent of endogenous NE released into the medium was determined. The in vivo results indicated that hypothalamic NE utilization in control animals decreases as animals mature. Additionally, the in vitro results indicated that the percent NE released upon depolarization also decreased with maturation in control animals. Restraint shifted the NE decay curve measured in vivo to the right at all ages. Overall, however, restraint tended to increase NE utilization at 70 days, have little effect at 42 days, and retard utilization at 28 days. Furthermore, restraint markedly reduced the depolarized release of NE at 28 days, had no effect at 42 days and slightly, but significantly, increased release at 70 days. Restraint significantly increased plasma corticosterone at all ages. Hypothalamic NE projections are important to an organism's regulatory responses, and changes that take place over adolescence in this system may be important for the emergence of adult-typical responses as well as render adolescents vulnerable to specific dysfunctions.

Hypothalamic GABAA receptor blockade modulates cerebral cortical systems sensitive to acute stressors.

Pharmacologic blockade of GABA binding sites in the hypothalamus elicits a pattern of physiological and behavioral arousal. The latter outcome implicates a perturbation in the neural functioning of higher brain centers. The effect that hypothalamic GABAA receptor modulation has on the function of cerebral cortical neural substrates linked with responses to stressors was assessed using microinfusion of bicuculline methiodide (BMI) into the medial hypothalamus of freely moving, handling habituated rats. BMI led to rapid increases in frontal cortical dopamine (DA) utilization (calculated from the sum of the levels of the DA metabolites, homovanilic and dihydroxyphenylacetic acids, divided by DA levels) resembling that identified following restraint-induced stress. Also, cortical GABAA receptor function [using chloride (Cl-) enhancement of 3H-flunitrazepam (Flu) binding as an index] was disrupted; i.e. there was a loss of typical Cl- enhancement of 3H-Flu binding in animals after BMI infusions. However, placing animals in restraint after BMI infusion reversed the effects of BMI, with both DA utilization and Cl- facilitated 3H-Flu binding similar to control basal values. Muscimol infusions in separately prepared animals did not alter either frontal cortical DA utilization or GABAA receptor function. The present results implicate GABA in the hypothalamus as "gating" activity of cortical systems involved in sensation of and/or responses to stressors. These findings may have important implications for effects of autonomic arousal on neural substrates involved in mediating stress responses.

Immunohistochemical and neurochemical evidence for GABAA receptor heterogeneity between the hypothalamus and cortex.

This study examined both the function of the GABAA receptor complex and the expression of its alpha 1, alpha 2 and alpha 3 subunits within the hypothalamus as compared to that of the cerebral cortex. A large number of different GABAA receptor subunit combinations potentially exist in various brain regions which, presumably, would intimate differing receptor structure and function. Here, we present evidence that the average functional characteristics of GABAA receptors within the rat hypothalamus are considerably different from those of the cerebral cortex. We assessed two neurochemical measures of GABAA receptor function: namely, chloride-facilitation of [3H]flunitrazepam binding and GABA-mediated 36chloride uptake. [3H]Flunitrazepam binding in the rat cortex was facilitated by increasing concentrations (12.5-500 mM) of chloride, and this facilitation was responsive to 15 min restraint. Yet, hypothalamic [3H]flunitrazepam binding was not responsive to increasing chloride-concentration in either the basal or restraint conditions. Also, maximal facilitation of GABA-mediated 36chloride uptake was significantly blunted in the hypothalamus relative to cortex (7.4 +/- 0.9 versus 35.8 +/- 1.5 nmoles/mg protein, respectively). While in vitro addition of 10 microM diazepam shifted GABA-mediated 36chloride uptake curves of the cortex to the left, diazepam addition appeared to be without effect in the hypothalamus. However, the blunted maximal facilitation of GABA on hypothalamic 36chloride uptake made accurate determination of the EC50 for the diazepam-potentiation difficult. In addition to these functional disparities between the regions, differences in subunit expression were also apparent. Distributions of alpha 1, alpha 2 and alpha 3 subunit immunoreactivities within cingulate, parietal and temporal cortices and 8 major hypothalamic regions were assessed. Staining of the alpha 1 subunit was prevalent throughout the hypothalamus and cortex, and dense in both regions. However, the alpha 2 and alpha 3 subunits, while of intermediate density in cortex, were of low density or absent (alpha 3) in the hypothalamus. The alpha 2-immunoreactivity was restricted to cell bodies of the arcuate nucleus, dorsomedial nucleus and overlying dorsal area and to neuropil staining of the median eminence. Thus, functional responsiveness of the GABAA receptor differs in the hypothalamus relative to the cortex and this would seem related to the presence of different receptor alpha subunits in homogenate preparations of the two regions.

Alterations in behavioral responses to stressors following excitotoxin lesions of dorsomedial hypothalamic regions.

The dorsomedial hypothalamus is important for regulation of cardiovascular responses associated with emotional arousal. This region has also been identified as a component of neural circuitry involved in fear/anxiety, yet clear evidence as to the effects of lesioning on stress-related behaviors is missing. In this study, we lesioned the dorsomedial hypothalamic region with the neurotoxin, ibotenic acid (IBO; 2.0 micrograms in 0.2 microliter), and studied the impact on spontaneous and unlearned behavioral responses to stressors. In the open field test, we observed non-generalized increases in motility parameters in the IBO rats with the differences occurring in the latter two-thirds of the test. In the elevated plus-maze, the IBO rats displayed a classic anxiolytic response with a greater proportion of entries into (and greater time spent in) the open arms of the maze. In the environment-specific social interaction (SI) test, the IBO rats showed a normal familiar/unfamiliar environment discrimination with respect to Total SI; however, the composition of the behaviors ('curiosity' vs. physical contact) by the IBO rats was markedly altered, with there being a 2-fold increase in non-violent physical interactions. Additionally, the differences in these traditional indices of anxiety were associated with lesioned animals exhibiting greater acoustic startle responsiveness than controls as a function of prepulse intensity. Overall, the results following IBO lesions indicate an altered responsiveness to sudden stressors, particularly as relates to novelty or exploration-oriented behaviors. The hypothalamic lesion may, therefore, have resulted in a disinhibition of normally suppressed responding to innate fear or challenging stimuli. This study contributes to those that have begun to define neural interactions that are essential for integrated stress responses.

Treatment with an anabolic-androgenic steroid affects anxiety-related behavior and alters the sensitivity of cortical GABAA receptors in the rat.

The putative psychotropic effect of the anabolic-androgenic steroid, testosterone propionate (TP), was determined in intact adult male rats after 1 or 2 weeks of continued exposure via subcutaneously implanted capsules. Behavior was assessed in a novel open-field arena and in the elevated plus-maze. In addition, gamma-aminobutyric acid (GABA)-stimulated 36chloride (Cl-) influx was determined in cerebral cortical synaptoneurosomes as a function of TP exposure. The weight of the prostate gland was taken and blood serum level of total testosterone (T) was assayed. One week of TP exposure (approximately 3.5-5.0 mg/kg per day) resulted in anxiolytic behavior, as evidenced by an increase in the exploration of the open arms of the elevated plus-maze. The behavioral effect in the elevated plus-maze was not observed in animals exposed to TP for a 2-week period. Ambulation scores in the novel open field did not change as a function of TP exposure. Blood T levels were increased 7-fold by 1 week of exposure, and increased 10-fold in animals with implants for a 2-week period. After 1 week of TP exposure, the concentration of GABA that elicited 50% of the maximal Cl- influx in cortical synaptoneurosomes (i.e., EC50) was significantly decreased; this effect was not seen in animals exposed to TP for 2 weeks. The maximal efficacy of the GABAA receptor-gated Cl- influx was not affected after 1 or 2 weeks of TP treatment. Thus, 1 week of treatment with TP resulted in anxiolytic behavior that was accompanied by an increase in the sensitivity of cortical GABAA receptors. However, the behavioral and neurochemical changes were no longer present after 2 weeks of TP exposure. These results are discussed in terms of the agonist effects of reduced androgen metabolites at the GABAA receptor and the possible development of tolerance to these effects.

Anxiolytic effect of progesterone is associated with increases in cortical allopregnanolone and GABAA receptor function.

The effects of a SC injection of progesterone (0, 1, or 4 mg) on locomotor behavior and exploration of an elevated plus-maze were examined in ovariectomized rats. At the completion of the behavioral tests, blood serum and cerebral cortical level of the 3 alpha-hydroxy ring-A metabolite of progesterone, 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone), was also assessed. GABA-stimulated 36Cl- influx was studied in cortical synaptoneurosomes from a subgroup of ovariectomized females treated with vehicle or 4 mg progesterone. Whereas progesterone treatment did not affect ambulation in a novel arena, significant anxiolytic behavior was detected in the plus-maze 4 h after administration of 1 or 4 mg progesterone. A dose-dependent increase in allopregnanolone level was found in serum and cortical homogenates. Studies of GABA-stimulated Cl- influx demonstrated that progesterone treatment increased the sensitivity of cortical synaptoneurosomes to GABA (i.e., decreased the EC50) and increased the maximal efficacy with which GABA stimulated Cl- transport (i.e., increased the Emax). Together, these data support the hypothesis that the psychotropic effects observed after progesterone administration are due to the bioconversion of progesterone to allopregnanolone, which subsequently augments GABAA receptor-mediated function.

Importance of hypothalamic function to stressor-induced responsiveness of the GABAA receptor in the cerebral cortex: a non-corticosterone influence.

Catecholamine terminals in the paraventricular nucleus (PVN) of the hypothalamus of 60-day-old rats were destroyed by the stereotaxic injection of 6-hydroxydopamine into the PVN (6-OHDA; 9 micrograms/1.5 microliters bilaterally), and the rats were tested 2 weeks later. Lesions led to a 70% reduction of norepinephrine in the hypothalamus and a loss of dopamine-beta-hydroxylase immunoreactivity in the PVN. Furthermore, 6-OHDA lesions in the hypothalamus disrupted stressor-induced (15 min of restraint) changes in GABAA receptor function in the cerebral cortex (assessed by measuring chloride-facilitated benzodiazepine binding) but did not alter stressor-induced increases in plasma corticosterone levels. Additionally, the lesion did not change the responsiveness of the GABAA receptor to the corticosterone metabolite, allotetrahydrodeoxycorticosterone. These results indicate that stressor-induced changes in cortical GABAA receptor function are not driven by the stressor-induced release of corticosterone. A separate group of animals were tested for behavioral responses to challenge, and while 6-OHDA-induced lesions did not alter total scores in the test of environment-specific social interaction, the lesions did induce a change in composition of the behavior. Lesioned animals demonstrated increased physical (vigorous contact) interactions, similar to behavior previously observed in younger rats. The results of the behavioral study support a role for the GABAA receptor in the cerebral cortex in mediating appropriate behavioral responses to challenge in the adult rat. Thus, a hypothalamic lesion that prevented challenge-induced changes in GABAA receptor function in the cortex (with no change in the corticosterone response to the stressor) also led to altered behavioral responses to challenge.