Role of hypothalamic corticotropin-releasing factor in mediating alcohol-induced activation of the rat hypothalamic-pituitary-adrenal axis.

Alcohol stimulates the hypothalamic-pituitary-adrenal (HPA) axis through brain-based mechanisms in which endogenous corticotropin-releasing factor (CRF) plays a major role. This review first discusses the evidence for this role, as well as the possible importance of intermediates such as vasopressin, nitric oxide and catecholamines. We then illustrate the long-term influence exerted by alcohol on the HPA axis, such as the ability of a first exposure to this drug during adolescence, to permanently blunt neuroendocrine responses to subsequent exposure of the drug. In view of the role played by CRF in addiction, it is likely that a better understanding of the mechanisms through which this drug stimulates the HPA axis may lead to the development of new therapies used in the treatment of alcohol abuse, including clinically relevant CRF antagonists.

Corticotropin-releasing factor peptide antagonists: design, characterization and potential clinical relevance.

Elusive for more than half a century, corticotropin-releasing factor (CRF) was finally isolated and characterized in 1981 from ovine hypothalami and shortly thereafter, from rat brains. Thirty years later, much has been learned about the function and localization of CRF and related family members (Urocortins 1, 2 and 3) and their 2 receptors, CRF receptor type 1 (CRFR1) and CRF receptor type 2 (CRFR2). Here, we report the stepwise development of peptide CRF agonists and antagonists, which led to the CRFR1 agonist Stressin1; the long-acting antagonists Astressin2-B which is specific for CRFR2; and Astressin B, which binds to both CRFR1 and CRFR2.This analog has potential for the treatment of CRF-dependent diseases in the periphery, such as irritable bowel syndrome.

Roger Guillemin (1924-2024).

Father of neuroendocrinology.

Corticosteroid-dependent plasticity mediates compulsive alcohol drinking in rats.

Alcoholism is characterized by a compulsion to seek and ingest alcohol, loss of control over intake, and the emergence of a negative emotional state during abstinence. We hypothesized that sustained activation of neuroendocrine stress systems (e.g., corticosteroid release via the hypothalamic-pituitary-adrenal axis) by alcohol intoxication and withdrawal and consequent alterations in glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation drive compulsive alcohol drinking. Our results showed that rats exposed to alcohol vapor to the point of dependence displayed increased alcohol intake, compulsive drinking measured by progressive-ratio responding, and persistent alcohol consumption despite punishment, assessed by adding quinine to the alcohol solution, compared with control rats that were not exposed to alcohol vapor. No group differences were observed in the self-administration of saccharin-sweetened water. Acute alcohol withdrawal was accompanied by downregulated GR mRNA in various stress/reward-related brain regions [i.e., prefrontal cortex, nucleus accumbens (NAc), and bed nucleus of the stria terminalis (BNST)], whereas protracted alcohol abstinence was accompanied by upregulated GR mRNA in the NAc core, ventral BNST, and central nucleus of the amygdala. No significant alterations in MR mRNA levels were found. Chronic GR antagonism with mifepristone (RU38486) prevented the escalation of alcohol intake and compulsive responding induced by chronic, intermittent alcohol vapor exposure. Chronic treatment with mifepristone also blocked escalated alcohol drinking and compulsive responding during protracted abstinence. Thus, the GR system appears to be involved in the development of alcohol dependence and may represent a potential pharmacological target for the treatment of alcoholism.

Effects of two types of numerical problems on the emotions experienced in adults and in 9-year-old children.

It is widely acknowledged that emotions and cognition are closely related, and that negative emotions are detrimental on school achievement, especially on mathematical performance. On the other hand, positive emotions have a positive impact on motivation and cognitive abilities underlying the learning processes. Nevertheless, studies about the effects of experienced emotions on problem solving, a specific type of mathematical activity, are sparse. The present research focuses on experienced epistemic and achievement emotions after the resolution of two types of numerical word problems: the application problems, that requires the use of a specific and expected algorithm to be solved and are regularly proposed at school; and the non-application problems, which cannot be solved directly but using different solving strategies. This type of numerical word problems appears less frequently in French school curricula. In experiment 1, 105 adults (M = 24.4 years), of which the majority was university students, were involved in an online experiment with APs and NAPs problems and were asked to rate their experienced emotions after the resolution of the problems. In experiment 2, 65 children aged 9-year-old were asked to individually solve APs and NAPs problems with age-appropriate difficulty and then rate their associated emotions. The adults' sample reported higher epistemic and achievement positive emotions towards APs compared to NAPs. In both adults and children NAPs were more associated to surprise than APs. In children anxiety was more experienced after resolution of NAPs than APs. Results suggest the importance of varying the types of problems proposed in school curricula so that children become accustomed to using different solving strategies. This approach could be useful in decreasing negative emotions toward mathematics such as anxiety, which begins to settle as early as elementary school.

Immediate and prolonged effects of alcohol exposure on the activity of the hypothalamic-pituitary-adrenal axis in adult and adolescent rats.

Alcohol stimulates the hypothalamic-pituitary-adrenal (HPA) axis. Part of this influence is likely exerted directly at the level of the corticotropin-releasing factor (CRF) gene, but intermediates may also play a role. Here we review the effect of alcohol on this axis, provide new data on the effects of binge drinking during adolescence, and argue for a role of catecholaminergic circuits. Indeed, acute injection of this drug activates brain stem adrenergic and noradrenergic circuits, and their lesion, or blockade of α1 adrenergic receptors significantly blunts alcohol-induced ACTH release. As alcohol can influence the HPA axis even once discontinued, and alcohol consumption in young people is associated with increased adult drug abuse (a phenomenon possibly mediated by the HPA axis), we determined whether alcohol consumption during adolescence modified this axis. The number of CRF-immunoreactive (ir) cells/section was significantly decreased in the central nucleus of the amygdala of adolescent self-administering binge-drinking animals, compared to controls. When another group of adolescent binge-drinking rats was administered alcohol in adulthood, the number of colocalized c-fos-ir and PNMT-ir cells/brain stem section in the C3 area was significantly decreased, compared to controls. As the HPA axis response to alcohol is blunted in adult rats exposed to alcohol vapors during adolescence, a phenomenon which was not observed in our model of self-administration, it is possible that the blood alcohol levels achieved in various models play a role in the long-term consequences of exposure to alcohol early in life. Collectively, these results suggest an important role of brain catecholamines in modulating the short- and long-term consequences of alcohol administration.

Amyloid as a depot for the formulation of long-acting drugs.

Amyloids are highly organized protein aggregates that are associated with both neurodegenerative diseases such as Alzheimer disease and benign functions like skin pigmentation. Amyloids self-polymerize in a nucleation-dependent manner by recruiting their soluble protein/peptide counterpart and are stable against harsh physical, chemical, and biochemical conditions. These extraordinary properties make amyloids attractive for applications in nanotechnology. Here, we suggest the use of amyloids in the formulation of long-acting drugs. It is our rationale that amyloids have the properties required of a long-acting drug because they are stable depots that guarantee a controlled release of the active peptide drug from the amyloid termini. This concept is tested with a family of short- and long-acting analogs of gonadotropin-releasing hormone (GnRH), and it is shown that amyloids thereof can act as a source for the sustained release of biologically active peptides.

Urocortin 1 inhibits rat leydig cell function.

Corticotropin-releasing factor (CRF) has previously been reported in rat testes in which it inhibits Leydig cells activity. However, recent studies in our laboratory have suggested that some of the effects originally attributed to CRF were instead due to the related peptide Urocortin 1 (Ucn 1) and that this latter hormone, not CRF, was detectable in Leydig cells. We show here that Ucn 1 [a mixed CRF receptor (CRFR) type 1 and CRFR2 agonist] and the CRFR1-selective peptide Stressin 1, but not Ucn 2 or Ucn 3 (both considered selective CRFR2 ligands), significantly blunt the testosterone response to human chorionic gonadotropin. The effect of Ucn 1 is observed regardless of whether this peptide is injected iv or directly into the testes, and it is reversed by the mixed CRFR1/R2 antagonist Astressin B. Blockade of GnRH receptors with the antagonist Azalin B does not interfere with the influence of Ucn 1, thereby demonstrating that pituitary luteinizing hormone does not appear to be involved in this model. Collectively these results suggest that Ucn 1, not CRF, is present in the rat testes and interferes with Leydig cell activity. However, whereas we previously reported that alcohol up-regulated gonadal Ucn 1 gene expression, CRF receptor antagonists were unable to reverse the inhibitory effect exerted by alcohol on human chorionic gonadotropin-induced testosterone release. The functional role played by testicular Ucn 1 in stress models characterized by blunted androgen levels therefore needs to be further investigated.

Alcohol self-administration acutely stimulates the hypothalamic-pituitary-adrenal axis, but alcohol dependence leads to a dampened neuroendocrine state.

Clinical studies link disruption of the neuroendocrine stress system with alcoholism, but remaining unknown is whether functional differences in the hypothalamic-pituitary-adrenal (HPA) axis precede alcohol abuse and dependence or result from chronic exposure to this drug. Using an operant self-administration animal model of alcohol dependence and serial blood sampling, we show that longterm exposure to alcohol causes significant impairment of HPA function in adult male Wistar rats. Acute alcohol (voluntary self-administration or experimenter-administered) stimulated the release of corticosterone and its upstream regulator, adrenocorticotropic hormone, but chronic exposure sufficient to produce dependence led to a dampened neuroendocrine state. HPA responses to alcohol were most robust in 'low-responding' non-dependent animals (averaging < 0.2 mg/kg/session), intermediate in nondependent animals (averaging approximately 0.4 mg/kg/session), and most blunted in dependent animals (averaging approximately 1.0 mg/kg/session) following several weeks of daily 30-min self-administration sessions, suggesting that neuroendocrine tolerance can be initiated prior to dependence and relates to the amount of alcohol consumed. Decreased expression of corticotropin-releasing factor (CRF) mRNA expression in the paraventricular nucleus of the hypothalamus and reduced sensitivity of the pituitary to CRF may contribute to, but do not completely explain, neuroendocrine tolerance. The present results, combined with previous studies, suggest that multiple adaptations to stress regulatory systems may be brought about by excessive drinking, including a compromised hormonal response and a sensitized brain stress response that together contribute to dependence.

Stressin1-A, a potent corticotropin releasing factor receptor 1 (CRF1)-selective peptide agonist.

The potencies and selectivity of peptide CRF antagonists is increased through structural constraints, suggesting that the resulting ligands assume distinct conformations when interacting with CRF1 and CRF2 receptors. To develop selective CRF receptor agonists, we have scanned the sequence -Gln-Ala-His-Ser-Asn-Arg- (residues 30-35 of [DPhe12,Nle21,38]Ac-hCRF4-41) with an i-(i+3) bridge consisting of the Glui-Xaa-Xbb-Lysi+3 scaffold, where residues i=30, 31, and 32. When i=31, stressin1-A, a potent CRF1 receptor-selective agonist was generated. In vitro, stressin1-A was equipotent to h/rCRF to release ACTH. Astressin1-A showed a low nanomolar affinity for CRF1 receptor (Ki=1.7 nM) and greater than 100-fold selectivity versus CRF2 receptor (Ki=222 nM). Stressin1-A released slightly less ACTH than oCRF in adult adrenal-intact male rats, with increased duration of action. Stressin1-A, injected intraperitoneally in rats, induced fecal pellet output (a CRF1 receptor-mediated response) and did not influence gastric emptying and blood pressure (CRF2 receptor-mediated responses).

Activation of a neural brain-testicular pathway rapidly lowers Leydig cell levels of the steroidogenic acute regulatory protein and the peripheral-type benzodiazepine receptor while increasing levels of neuronal nitric oxide synthase.

Activation of a neural brain-testicular pathway by the intracerebroventricular injection of the beta-adrenergic agonist isoproterenol (ISO), the hypothalamic peptide corticotropin-releasing factor (CRF), or alcohol (EtOH) rapidly decreases the testosterone (T) response to human chorionic gonadotropin. To elucidate the intratesticular mechanisms responsible for this phenomenon, we investigated the influence of intracerebroventricular-injected ISO, CRF, or EtOH on levels of the steroidogenic acute regulatory (StAR) protein, the peripheral-type benzodiazepine receptor (PBR), and the cytochrome P450 side-chain cleavage enzyme in semipurified Leydig cells. ISO (10 microg), CRF (5 microg), or EtOH (5 microl of 200 proof, a dose that does not induce neuronal damage nor leaks to the periphery) rapidly decreased StAR and PBR but not cytochrome P450 side-chain cleavage enzyme protein levels. Levels of the variant of the neuronal nitric oxide synthase (nNOS) that is restricted to Leydig cells, TnNOS, significantly increased in response to ISO, CRF, and EtOH over the time course of altered StAR/PBR concentrations. However, pretreatment of the rats with N(w)nitro-arginine methylester, which blocked ISO-induced increases in TnNOS, neither restored the T response to human chorionic gonadotropin nor prevented the decreases in StAR and PBR. These results provide evidence of concomitant changes in Leydig cell StAR and PBR levels in live rats. They also indicate that activation of a neural brain-testicular pathway rapidly decreases concentrations of these steroidogenic proteins while up-regulating testicular NO production. However, additional studies are necessary to elucidate the functional role played by this gas in our model.

Role played by brainstem neurons in regulating testosterone secretion via a direct neural pathway between the hypothalamus and the testes.

We previously reported anatomical and functional evidence for a direct, inhibitory neural pathway that regulates testosterone (T) secretion independently of the pituitary. This pathway is activated by the intracerebroventricular (icv) administration of agents that stimulate stress responses, such as IL-1beta, corticotropin-releasing factor (CRF), and norepinephrine (NE), which results in a blunted T response to the administration of human chorionic gonadotropin (hCG). Blunting of the T response is mediated by central beta-adrenergic receptor stimulation. CRF, but not ethanol (EtOH) or IL-1beta, acts directly on the paraventricular nucleus of the hypothalamus to activate the pathway. Here we explored the role played by brain areas hypothesized to be part of this pathway, such as neurons in the dorsal pons [including the locus coeruleus (LC) of the brainstem], where NE is produced. Microinfusion of EtOH or IL-1beta, but not CRF, into these neurons activated the pathway. Electrolytic lesions of this region significantly reversed the inhibitory effect of icv-administered EtOH on hCG-induced T release, while having no effect on the ability of IL-1beta or CRF to do so. However, the icv administration of IL-1beta, EtOH, or CRF, in doses that rapidly inhibit the T response to hCG, all caused a significant depletion of NE from the LC. Collectively, these results indicate that in addition to the paraventricular nucleus, the brainstem area containing the LC is part of a neural pathway that connects the brain to the testes independently of the pituitary. We also speculate that EtOH may stimulate this pathway through NE-dependent activation of the dorsal pons.

Exposure to repetitive versus varied stress during prenatal development generates two distinct anxiogenic and neuroendocrine profiles in adulthood.

Early life experiences can shape brain function and behavior in adulthood. The present study sought to elucidate the effects of repetitive, predictable vs. varied, unpredictable prenatal stress on sexually dichotomous neuroendocrine and anxiety-related behavioral responses in adult offspring. Rat dams were exposed repeatedly during the last week of pregnancy to no stress, only restraint stress [prenatal stress (PS)-restraint], or a randomized sequence of varied stressors (PS-random), and several behavioral and endocrine measures were assessed in adult male and female offspring. Repeated exposure to the same stressor (restraint) generated the most robust changes, including increased anxiety-related behaviors (both passive, measured on the elevated plus maze, and active, measured using defensive burying tests), a delayed and prolonged hypothalamic-pituitary-adrenal (HPA) axis response to stress in female offspring. Conversely, PS-restraint males showed no changes in anxiety-like behavior and had elevated basal ACTH and a blunted HPA response to stress; consistent with attenuated HPA responsivity was an increase in glucocorticoid receptor immunoreactivity in the hippocampus, suggesting increased negative feedback on the HPA axis in these animals. Prenatal exposure to a varied, unpredictable pattern of stressors did not have as much effect on HPA function, with most neuroendocrine measures residing intermediate to PS-restraint and control animals within each sex. Gonadal steroids were altered independent of the type of prenatal stress, but changes were measurable only in males (lower testosterone). The present data exemplify the differential sensitivity of the developing nervous and endocrine systems to stress, depending on not only gender but also the nature of the stressful experience endured by the mother during pregnancy.

Novel analogues of degarelix incorporating hydroxy-, methoxy-, and pegylated-urea moieties at positions 3, 5, 6 and the N-terminus. Part III.

Novel degarelix (Fe200486) analogues were screened for antagonism of GnRH-induced response (IC(50)) in a reporter gene assay. Inhibition of luteinizing hormone release over time was measured in the castrated male rat. N(omega)-Hydroxy- and N(omega)-methoxy-carbamoylation of Dab and Dap at position 3 (3-6), and N(omega)-hydroxy-,N(omega)-methoxy-carbamoylation and pegylation of 4Aph at positions 5 and 6 (7-10, 15-17, 22-25) were carried out. Modulation of hydrophobicity was achieved using different acylating groups at the N-terminus (11-14, 18-21, 26-28). Analogues 8, 15-17, 22, and 23 were equipotent to acyline (IC(50) = 0.69 nM) and degarelix (IC(50) = 0.58 nM) in vitro. Analogues 7, 17, and 23 were shorter acting than acyline, when 9, 11, 13, 15, 16, and 22 were longer acting. Only 9 and 14 were inactive at releasing histamine. No analogue exhibited a duration of action comparable to that of degarelix. Analogues with shorter and longer retention times on HPLC (a measure of hydrophilicity) than degarelix were identified.

Characterization of Multisubstituted Corticotropin Releasing Factor (CRF) Peptide Antagonists (Astressins).

CRF mediates numerous stress-related endocrine, autonomic, metabolic, and behavioral responses. We present the synthesis and chemical and biological properties of astressin B analogues {cyclo(30-33)[D-Phe(12),Nle(21,38),C(α)MeLeu(27,40),Glu(30),Lys(33)]-acetyl-h/r-CRF(9-41)}. Out of 37 novel peptides, 17 (2, 4, 6-8, 10, 11, 16, 17, 27, 29, 30, 32-36) and 16 (3, 5, 9, 12-15, 18, 19, 22-26, 28, 31) had k(i) to CRF receptors in the high picomolar and low nanomole ranges, respectively. Peptides 1, 2, and 11 inhibited h/rCRF and urocortin 1-induced cAMP release from AtT20 and A7r5 cells. When Astressin C 2 was administered to adrenalectomized rats at 1.0 mg subcutaneously, it inhibited ACTH release for >7 d. Additional rat data based on the inhibitory effect of (2) on h/rCRF-induced stimulation of colonic secretory motor activity and urocortin 2-induced delayed gastric emptying also indicate a safe and long-lasting antagonistic effect. The overall properties of selected analogues may fulfill the criteria expected from clinical candidates.

Role played by hypothalamic nuclear factor-{kappa}B in alcohol-mediated activation of the rat hypothalamic-pituitary-adrenal axis.

The DNA binding protein nuclear factor-kappaB (NF-kappaB) is a transcription factor translocated from the cytosol to the nucleus in response to stressors. Here we determined whether the known ability of alcohol to activate the hypothalamic-pituitary axis was mediated by NF-kappaB, tested the hypothesis that this phenomenon was accompanied by increased hypothalamic NF-kappaB transcripts, and investigated some of the mechanisms involved in this response. We found that alcohol-induced increase in plasma ACTH was blunted by the intracerebroventricular (icv) injection of a cell-permeable peptide that inhibits NF-kappaB translocation. Alcohol also increased hypothalamic inhibitory factor kappaB (IkappaB) mRNA levels, a factor that regulates NF-kappaB protein activation and the activity of NF-kappaB DNA binding and whose expression is thought to reflect NF-kappaB activity. This response, which was not accompanied by detectable changes in brain levels of proinflammatory cytokines, was partially retained in adrenalectomized/corticosterone-replaced rats. The icv injection of corticotropin-releasing factor (CRF), a hypothalamic peptide that is released by alcohol and mediates its influence on ACTH secretion, also stimulated hypothalamic IkappaB transcription. We therefore determined whether brain CRF played a role in the influence of alcohol on NF-kappaB signaling pathways. Indeed, the icv injection of the CRF antagonist alpha-helCRF(9-41) decreased alcohol-induced hypothalamic IkappaB transcripts. Because this antagonist did not alter corticosterone levels, our data suggest that the role played by CRF was not modulated by this steroid. Collectively, our results provide evidence for a functional interaction between alcohol and NF-kappaB-dependent pathway in stimulating the rat hypothalamic-pituitary axis activity that involves independent roles of corticosterone and CRF.

Iterative approach to the discovery of novel degarelix analogues: substitutions at positions 3, 7, and 8. Part II.

Degarelix (FE200486, Ac-d-2Nal(1)-d-4Cpa(2)-d-3Pal(3)-Ser(4)-4Aph(l-Hor)(5)-d-4Aph(Cbm)(6)-Leu(7)-ILys(8)-Pro(9)-d-Ala(10)-NH(2)) is a potent and very long acting antagonist of gonadotropin-releasing hormone (GnRH) after subcutaneous administration in mammals including humans. Analogues of degarelix were synthesized, characterized, and screened for the antagonism of GnRH-induced response in a reporter gene assay in HEK-293 cells expressing the human GnRH receptor. The duration of action was also determined in the castrated male rat assay to measure the extent (efficacy and duration of action) of inhibition of luteinizing hormone (LH) release. Structurally, this series of analogues has novel substitutions at positions 3, 7, and 8 and N(alpha)-methylation at positions 6, 7, and 8 in the structure of degarelix. These substitutions were designed to probe the spatial limitations of the receptor's cavity and to map the steric and ionic boundaries. Some functional groups were introduced that were hypothesized to influence the phamacokinetic properties of the analogues such as bioavailability, solubility, intra- or intermolecular hydrogen bond forming capacity, and ability to bind carrier proteins. Substitutions at positions 3 ([N(beta)-(2-pyridyl-methyl)d-Dap(3)]degarelix, IC(50) = 2.71 nM) (5), 7 ([Pra(7)]degarelix, IC(50) = 2.11 nM) (16), and 8 ([N(delta)-(IGly)Orn(8)]degarelix, IC(50) = 1.38 nM) (20) and N-methylation ([N(alpha)-methyl-Leu(7)]degarelix, IC(50) = 1.47 nM) (32) yielded analogues that were equipotent to degarelix (2) in vitro (IC(50) = 1.64 nM) but shorter acting in vivo. Out of the 33 novel analogues tested for the duration of action in this series, two analogues ([N(epsilon)-cyclohexyl-Lys(8)]degarelix, IC(50) = 1.50 nM) (23) and ([N(beta)-(IbetaAla)Dap(8)]degarelix, IC(50) = 1.98 nM) (26) had antagonist potencies and duration of action similar to that of azaline B {inhibited LH (>80%) release for >72 h after sc injection to castrated male rats at a standard dose of 50 mug/rat in 5% mannitol}. Under similar conditions analogues ([N(gamma)-(IGly)Dab(8)]degarelix, IC(50) = 1.56 nM) (21) and ([IOrn(8)]degarelix, IC(50) = 1.72 nM) (18) had a longer duration of action {inhibited LH (>96 h) release} than azaline B; however they were shorter acting than degarelix. Hydrophilicity of these analogues, a potential measure of their ability to be formulated for sustained release, was determined using RP-HPLC at neutral pH yielding analogues with shorter as well as longer retention times. No correlation was found between retention times and antagonist potency or duration of action.

Importance of the paraventricular nucleus of the hypothalamus as a component of a neural pathway between the brain and the testes that modulates testosterone secretion independently of the pituitary.

We previously reported that in adult male rats, the intracerebroventricular (icv) injection of corticotropin-releasing factor (CRF) or the beta-adrenergic agonist isoproterenol (ISO) significantly inhibited the ability of human chorionic gonadotropin (hCG) to stimulate testosterone (T) secretion. The finding that this phenomenon also took place when LH release had been blocked with an LHRH antagonist suggested that icv CRF and ISO did not alter Leydig cell function by influencing the activity of pituitary gonadotrophs. We therefore proposed the existence of a neural pathway connecting the brain to the testes, whose activation by icv CRF or ISO interfered with T secretion. Based on the intratesticular injection of the transganglionic tracer pseudorabies virus, we recently identified the paraventricular nucleus (PVN) of the hypothalamus as a component of this neural link. The aim of the present work was to investigate the functional role of this brain area in mediating the ability of CRF and ISO to inhibit the ability of hCG to stimulate T secretion. We first demonstrated that local microinfusion of CRF or ISO directly into the PVN mimicked the effect of their icv injection, suggesting that the PVN does indeed represent a site of action of ISO and CRF in altering Leydig cell responsiveness to gonadotropin. In contrast, neither CRF nor ISO microinfusion into the central amygdala or the frontal cortex influenced hCG-stimulated T secretion. To further investigate the role of the PVN in ISO- and CRF-induced blunting of hCG stimulation of T, we determined the effect of icv CRF or ISO on testicular activity of rats with electrolytic lesions of the PVN. These lesions, which did not in themselves influence Leydig cell responsiveness to hCG, blocked the effect of both icv ISO and CRF on hCG-induced T release. Collectively, these results support the hypothesis that CRF- and ISO-induced activation of cells in the area of the PVN decreases the ability of gonadotropin to release T and suggests that this nucleus represents an important site of the proposed neural connection between the brain and the testes.