Corticotropin-releasing factor and neuropeptide Y: role in emotional integration.

The amygdala complex integrates stressful stimuli and is critical in transducing their aversive value into autonomic, endocrine and behavioural responses. Stimulation within the amygdala complex produces signs of fear without a relevant external object, while lesions in this region abolish normal fear responses. In a manner characteristic of phylogenetically old limbic brain areas, the complex neurochemical anatomy of the amygdala involves a large number of phylogenetically old peptide mediators. The distribution and connectivity of these peptide systems have been extensively studied, but less is known about their functional role. Recent evidence suggests that two neuropeptides, corticotropin-releasing factor (CRF) and neuropeptide Y (NPY) exert a reciprocal regulation of responsiveness to stressful stimuli, possibly via an interaction of these two systems in the amygdala.

Supersensitive endocrine response to physostigmine in dopamine-depleted rats: a model of depression?

Depressed patients exhibit an abnormal "supersensitive" increase in the plasma concentration of several pituitary hormones following intravenous injection of the acetyl cholinesterase inhibitor physostigmine (PHY). In the present study, we examined the effects of PHY treatments on the plasma concentrations of prolactin (PRL) and adrenocorticotrophic hormone (ACTH) in the rat. Physostigmine (0-0.6 mg/kg, s.c.) produced a dose-dependent increase in PRL and ACTH immunoreactivity in unoperated animals. Neurotoxin-induced depletion of brain dopamine (DA) or norepinephrine (NE) did not significantly alter baseline plasma PRL or ACTH values. Following depletion of brain DA, but not NE, animals exhibited a "supersensitive" increase in plasma ACTH values, which was evidenced by a sixfold left shift in the dose-response properties of PHY. These results suggest that there are intriguing parallels between the abnormal endocrine response to PHY demonstrated by depressed patients and that demonstrated by rats following depletion of central nervous system (CNS) DA levels.

Potentiation of acoustic startle by corticotropin-releasing factor (CRF) and by fear are both reversed by alpha-helical CRF (9-41).

A series of experiments were performed to investigate the effects of alpha-helical CRF [9-41] (AHCRF), a structural analogue and functional antagonist of corticotropin-releasing factor (CRF), on CRF- and fear-potentiated acoustic startle amplitude. Intracerebroventricular (ICV) administration of the CRF antagonist AHCRF reversed the potentiation of startle amplitude that was produced by ICV administration of CRF (1.0 micrograms). Doses of AHCRF that antagonized CRF-potentiated startle amplitude also reversed the potentiation of startle produced by conditioned "fear" but failed to lower startle baseline or antagonize strychnine-potentiated acoustic startle. These results suggest that CRF and "fear" may potentiate acoustic startle through overlapping neural substrates.

Endocrine, immune, and neurochemical changes in rats during withdrawal from chronic amphetamine intoxication.

In humans who chronically abuse amphetamine (AMPH), sudden abstinence often precipitates an organic mood disorder that mimics many symptoms of major depression. We report that AMPH exposure and withdrawal in rats modifies hypothalamic-pituitary-adrenal axis endocrine responses, peripheral immune functions, and regional brain catecholamine levels. Compared to vehicle-treated animals, rats treated with AMPH for 10 days exhibit significantly decreased physostigmine-induced release of adrenocorticotropin hormone (ACTH). During AMPH withdrawal, these animals show a loss of the normal correlation between levels of plasma ACTH and corticosterone. Chronic AMPH treatment in rats causes a significant increase in natural killer cell activity. Brain dopamine levels in these animals are decreased in the caudate nucleus but are increased in the nucleus accumbens. AMPH withdrawal in rats may be a useful model for studying the physiologic and neural substrates of human AMPH withdrawal states.

Anti-stress action of a corticotropin-releasing factor antagonist on behavioral reactivity to stressors of varying type and intensity.

Central administration of a Corticotropin-Releasing Factor (CRF) antagonist is well documented to attenuate a variety of behavioral responses to several distinct stressors; however, it is not yet clear whether the activation of CRF neurons is dependent on the type or intensity of the experimental stressor, or rather on the particular behavioral response to stress under study. To test the generality of the stress-protective effect of the CRF antagonist, alpha-helical CRF9-41, (1, 5 or 25 micrograms intracerebroventricularly), the present experiments employed a sensitive index of anxiogenic-like behavior by measuring suppression in exploration on the elevated plus-maze following exposure to social, swim, or restraint stressors. A 1 but not 5 or 25 micrograms dose of the CRF antagonist administered just prior to social, swim, or restraint stress reversed the stress-induced inhibition of exploratory behavior. Chlordiazepoxide and the steroid anesthetic, alphaxalone, also attenuated the anxiogenic-like effect of restraint stress and elevated the baseline exploratory behavior of nonstressed control groups. Although the stressors produced a graded secretion of adrenocorticotropin (ACTH) with the ranking restraint > swim > social, the relative amplitude of behavioral reactivity to social, swim, and restraint stress was comparable. The relative efficacy of the CRF antagonist to reverse the stressor effects was also comparable. These results suggest that antagonism of activated brain CRF systems attenuates the behavioral response to stress regardless of the type or intensity of the stressor as measured by ACTH secretion.

Anxiolytic-like action of neuropeptide Y: mediation by Y1 receptors in amygdala, and dissociation from food intake effects.

Evidence from animal and human studies suggests that neuropeptide Y (NPY) may be a potent endogenous anxiolytic. The anatomic structures mediating this action of the peptide remain unknown. Furthermore, in addition to its anxiolytic-like effects, intracerebroventricular administration of NPY induces food intake through hypothalamic mechanisms, making the anxiolytic-like action of the peptide more difficult to interpret. The purpose of this study was to examine the anatomic substrate for the effects of NPY on anxiety, and to characterize the NPY receptors mediating these effects. Intracerebroventricular injection of NPY produced increased food intake in free-feeding animals, and dose-dependent anticonflict/anxiolytic-like effects in an established animal model of anxiety, the Geller-Seifter punished responding test. In contrast, microinjection of NPY into the central nucleus of the amygdala did not increase food intake in free-feeding animals, did not affect unpunished lever pressing for food, but did reproduce the anticonflict/anxiolytic-like effect with high potency. The selective NPY-Y1 agonist, p[Leu31,Pro34]NPY was approximately equipotent with native NPY in the conflict paradigm, and markedly more potent than the Y2 agonist, NPY13-36. Intrastriatal injections had no effect on conflict behavior. Thus, activation of Y1 receptors in the central nucleus of the amygdala produces effects similar to established anxiolytics without affecting food intake, suggesting that Y1-receptors in the amygdala may be a substrate for anxiolytic actions of NPY.

Identification of high-potency neuropeptide Y analogues through systematic lactamization.

In the pursuit of potent analogues of neuropeptide Y (NPY) that are selective for the Y1 receptor subtype, two lactam bridge scans of a centrally truncated parent compound were synthesized. A single lactam bridge (gamma-carboxyl of Glu to epsilon-amino of Lys) extending from residues i to i + 3 or i to i + 4 of the proposed alpha-helical region (residues 25-31 of NPY) was introduced in des-AA7-24[Gly6]NPY. Cyclogues (contraction of cyclic analogues), which were approximately one-half the size of native NPY, were initially screened for binding affinity at two discrete NPY receptor types using human neuroblastoma cell membranes, SK-N-MC and SK-N-BE2. Exploitation of the subtle differences present on each receptor type allowed for the identification of cyclogues which bound specifically to Y1 receptors with increased affinity when compared to the corresponding linear parent analogue, while one short Y1 specific cyclogue, des-AA2,3,5,7-24cyclo-(26/29)[Gly6,Glu26,Lys2 9,Pro34]NPY, bound with Ki = 16 nM. Other cyclogues showed distinct preference for Y2 receptors and bound in the low-nanomolar range. Functionally, the compounds inhibited the norepinephrine-stimulated accumulation of cAMP indicating that all acted as agonists with varying potencies.

Alphaxalone, a steroid anesthetic, inhibits the startle-enhancing effects of corticotropin releasing factor, but not strychnine.

Corticotropin releasing factor (CRF) is a 41 amino acid peptide implicated in the expression of stress- and fear-enhanced behaviors. CRF potentiates the amplitude of the startle reflex, and this effect is reversed by benzodiazepines (BDZ), suggesting that the startle-enhancing effects of CRF are modulated by changes in the GABA/BDZ receptor complex. In the present study, CRF-potentiated startle is inhibited by alphaxalone, a pregnane steroid anesthetic that is thought to act via the GABA/BDZ receptor complex. Alphaxalone (ALX) does not reduce CRF-potentiated startle by producing a generalized reduction in reactivity, since blockade of CRF-stimulated startle was not accompanied by an ALX-induced reduction in baseline startle amplitude and ALX does not reduce strychnine-potentiated startle. The effects of alphaxalone on CRF-potentiated startle may not be generalized to all CRF-stimulated behaviours, since alphaxalone failed to disrupt CRF-stimulated locomotor activity. CRF-potentiated startle is a useful assay for studying the effects of novel anxiolytic agents, and alphaxalone appears to be a steroid anesthetic with anxiolytic properties in this assay.

Corticotropin releasing factor and amphetamine exaggerate partial agonist properties of benzodiazepine antagonist Ro 15-1788 in the conflict test.

The central nervous system stimulants corticotropin releasing factor (CRF) and amphetamine were administered in combination with the benzodiazepine ligands Ro 15-1788 and FG 7142 in order to assess their benzodiazepine agonist and antagonist receptor properties in an operant conflict test in rats. Ro 15-1788, which was without behavioral activity in this test when given alone, reversed the suppression of punished responding produced by CRF and amphetamine in a dose-dependent manner. Chlordiazepoxide, which produced a release of punished responding by itself, also reversed the suppression of punished responding produced by CRF but not that of amphetamine. The benzodiazepine inverse agonist FG 7142, in contrast, enhanced the rate suppressing actions of both CRF and amphetamine. In a locomotor activity test, Ro 15-1788 failed to block the locomotor activation observed with CRF and amphetamine. The results suggest that "anxiety" or stress-enhancing compounds may enhance the partial agonist properties of Ro 15-1788 in certain test situations.

CRF antagonist reverses the "anxiogenic" response to ethanol withdrawal in the rat.

The role of the neuropeptide corticotropin-releasing factor (CRF) in mediating the behavioral effects of ethanol withdrawal in the rat was examined using the elevated plus-maze test. In Experiment 1, CRF (0.5 microgram ICV) reduced the percentage of time spent on the open arms of the elevated plus-maze, consistent with an "anxiogenic-like" effect. CRF also reduced the total number of arm entries, indicating a reduction in general activity. Low doses (5 and 25 micrograms ICV) of the CRF antagonist, alpha-helical CRF produced no behavioral effects in the elevated plus-maze, while a higher dose (50 micrograms ICV) elicited CRF-like activity. In experiment 2, rats were maintained for 2-3 weeks on a liquid diet containing ethanol (8.5-11.5% v/v) or sucrose. Eight hours after withdrawal from the ethanol diet rats displayed "anxiogenic-like" responses as well as a reduction in general activity in the elevated plus-maze compared with rats withdrawn from control diet. Alpha-helical CRF significantly antagonized the "anxiogenic-like" effects of ethanol withdrawal in the plus-maze. General activity and physical signs of ethanol withdrawal such as tail stiffness, body tremor and ventromedial distal flexion were unaffected by alpha-helical CRF. Blood Alcohol Levels (BALs) determined immediately after removal of the ethanol diet showed no group differences in ethanol consumption. These results suggest that increased activity of central CRF systems may mediate the anxiogenic effects of ethanol withdrawal.

Anxiolytic activity of NPY receptor agonists in the conflict test.

This investigation examined receptor subtype specificity and possible modulation by GABAa receptor ligands of NPY-induced behavioral responses to stressful stimuli. First, a series of NPY receptor agonists were examined for their potential effects on punished responding in a conflict test modified for incremental shock. NPY, peptide YY (PYY) and NPY Y1 receptor agonists [Leu31,Pro34]-NPY and [Gly6, Glu26,Lys26,Pro34]-NPY produced increases in punished responding in the conflict test. No significant effects on unpunished responding were noted. The pattern of responding was similar to that observed with the benzodiazepine agonist chlordiazepoxide. Neither pancreatic peptide (PP) nor the Y2 agonists NPY13-36 or [Glu2,32,Ala6,Dpr27,Lys28]-NPY significantly altered punished or unpunished responding. Of significance, the atypical Y1 agonist [Cys7,21,Pro34]-NPY produced negligible effects on punished responding, consistent with the presence of a subclass of Y1 receptors. Second, the anxiolytic effects of NPY were subjected to treatments that block actions at the GABAa receptor complex. The increase in punished responding produced by NPY was not altered by administration of the benzodiazepine antagonist flumazenil and only partially blocked by the picrotoxinin receptor ligand isopropylbicyclophosphate (10 and 15 microg/kg). These findings further support the hypothesis that the pharmacologic substrates for the anxiolytic-like actions of NPY may be mediated by the Y1 receptor subtype and suggest that these actions are independent of either the benzodiazepine or picrotoxinin binding sites of the GABA/benzodiazepine receptor complex.

Chlordiazepoxide attenuates response suppression induced by corticotropin-releasing factor in the conflict test.

The role of corticotropin-releasing factor (CRF) in mediating the stress response was studied using a behavioral test in which anxiety or conflict influence performance. Rats implanted with intraventricular cannulae were tested in a Geller-Seifter conflict test modified for incremental shock. CRF produced a dose-dependent attenuation of punished and nonpunished responding in the conflict test. Chlordiazepoxide increased punished, but not unpunished, responding and produced a dose-dependent reversal of CRF-induced response suppression. CRF had no effect on tail flick or hot-plate analgesia tests. The results support the hypothesis that CRF produces behavioral effects consistent with "anxiety" or an increased responsiveness to stress.

Time-dependent induction of anxiogenic-like effects after central infusion of urocortin or corticotropin-releasing factor in the rat.

RATIONALE: Corticotropin-releasing factor (CRF) and urocortin (Ucn) belong to the CRF-related family, share a high degree of structural homology and bind to CRF receptors. However, compared with CRF, Ucn was shown to display either weaker or similar anxiogenic-like effects in vivo. OBJECTIVE: To compare the anxiogenic-like responses of rats injected intracerebroventricularly (ICV) with different doses of either rat/human CRF (r/hCRF) or rat Ucn (rUcn) at different intervals after injection. METHODS: Rats were tested on three validated paradigms of emotional behavior [i.e. elevated plus-maze (EPM), defensive withdrawal (DW) and conflict test (CT)] 5 and 30 min after treatment. RESULTS: In the EPM test only r/hCRF, but not rUcn, produced anxiogenic-like effects at the dose of 1.0 microg, when the peptides were injected 5 min before testing. At 30 min after injection, both peptides caused a significant reduction of open arms exploration, rUcn being effective at 0.01 microg. In the DW test both peptides were equally potent in decreasing the exploratory behavior and increasing the time spent in the chamber at the dose of 1.0 microg when tested 30 min after injection. In the CT both rUcn (0.25-1.0 microg) and r/hCRF (0.75-1.0 microg) decreased significantly the responding in the punished component. However, rUcn reduced food responding also in the unpunished component possibly due to its powerful anorectic activity. CONCLUSIONS: Comparison of anxiogenic-like activities of r/hCRF and rUcn at doses up to 1.0 microg revealed striking differential effects that depended on the time of testing after ICV peptide injection, and on the paradigm of anxiety used. These results suggest that the onset of r/hCRF and rUcn actions related to behavioral responses to anxiety is likely to depend on brain peptide-specific mechanisms including binding properties to CRF-receptors, differential distribution to specific functional brain sites and the distribution and effectiveness of binding-protein interactions.

The role of CRF in behavioral aspects of stress.

CRF in the central nervous system appears to hve activating properties on behavior and to coordinate behavioral responses to stressors. These behavioral effects of CRF appear to be independent of the pituitary-adrenal axis and can be reversed by CRF antagonists. CRF antagonist administration reverses not only decreases in behavior associated with stress, but also increases in behavior associated with stress, thus suggesting that the role of CRF is stress dependent and not intrinsic to a given behavioral response. Further, microinjection of alpha-helical CRF 9-41 and immunotargeting of CRF neurons in separate brain compartments reveal a link between the anatomical sites that contain CRF and the nature of the behavioral response to stressors that can be modified by suppression of endogenous CRF activity therein. These actions of CRF in coordinating coping responses to stress at several bodily levels are consistent with a role for CRF similar to the dual role of other hypothalamic releasing factors in integrating hormonal and neural mechanisms by acting both as secretagogues for anterior pituitary hormones and as extrapituitary peptide neurotransmitters. Moreover, dysfunction in such a fundamental homeostatic system may be the key to a variety of pathophysiological conditions including mental disorders.

Naloxone blocks 'anxiolytic' effects of neuropeptide Y.

Intracerebroventricular injection of neuropeptide Y (NPY) produces potent 'anxiolytic' effects in animal models of anxiety. Administration of opioid receptor antagonists suppresses NPY-induced food intake and thermogenesis. The present study examined whether the opiate antagonist naloxone would also suppress the 'anxiolytic' effects of neuropeptide Y. Following training and stabilization of responding in an operant conflict model of anxiety, rats were injected with either NPY or diazepam. Both NPY (veh., 2, 4, 6 microg, i.c.v.) and chlordiazepoxide (veh., 2, 4, 6 mg/kg, i.p.) produced a dose-dependent increase in punished responding in the conflict test. The 'anxiolytic' effects of NPY were not blocked by the administration of flumazenil (3, 6, 12 mg/kg, i.p.). The administration of naloxone (0.25-2.0 mg/kg, s.c.) antagonized the effects of NPY. Central administration of the selective mu opiate antagonist CTAP (1 microg, i.c.v.) partially blocked NPY-induced conflict responding. These results support the hypothesis that NPY may play an important role in experimental anxiety independent of the benzodiazepine receptor and further implicate the opioid system in the behavioral expression of anxiety.

Cardiovascular actions of neuropeptide Y and social stress.

The role of central neuropeptide Y (NPY) in the cardiovascular response to social stress was evaluated in freely moving rats using telemetry. In unstressed rats, intracerebroventricular (ICV) administration of NPY and the selective Y1 receptor agonist [Leu31, Pro34]-NPY decreased blood pressure and heart rate, while the selective Y2 agonist NPY13-36 transiently raised blood pressure. NPY and [Leu31, Pro34]-NPY blunted elevations in blood pressure and pulse rate following exposure to the resident-intruder procedure, an established social stress paradigm. In contrast, the Y2 agonist significantly augmented stress-induced pressor effects. These observations indicate that the hypotensive effects of ICV NPY appear to be mediated by the Y1 receptor subtype and the NPY receptor subtypes may mediate opposing cardiovascular actions in response to stressful stimuli.

Neuropeptide Y blocks anxiogenic-like behavioral action of corticotropin-releasing factor in an operant conflict test and elevated plus maze.

Central administration of neuropeptide Y (NPY) produces anxiolytic-like behavioral effects in rat models of anxiety. Because previous evidence has suggested a relationship between NPY and corticotropin-releasing factor (CRF) in the brain, we have focused on the interaction of these neuropeptide systems in emotional responsiveness to stressful stimuli. Intracerebroventricular administration of CRF produced a marked response suppression in an operant incremental shock conflict paradigm. NPY [(1 microg, intracerebroventricularly (i.c.v.)] significantly antagonized the response-suppressing effects of CRF (0.75 microg, i.c.v.) on punished responding in the conflict test at doses that produced little or no behavioral effect when administered alone. Central administration of the CRF antagonist [D-Phe(12), Nle(21,38),C(alpha) MeLeu(37)]CRF (D-Phe CRF(12-41)) alone did not alter punished or unpunished responding in the conflict test. However, pretreatment with the CRF antagonist before a subthreshold dose of NPY (1 microg, i.c.v.) produced a significant potentiation of the release of punished responding relative to NPY alone and untreated controls. NPY also antagonized the "anxiogenic-like" behavioral effects of CRF in the elevated plus maze. These findings support the hypothesis that NPY and CRF may reciprocally modulate an animal's behavioral response to stressful stimuli.

Anxiolytic-like effect of neuropeptide Y (NPY), but not other peptides in an operant conflict test.

The peptide messengers neuropeptide Y (NPY), growth hormone-releasing hormone (GHRH), atrial natriuretic peptide (ANP) and beta-endorphin (BEND) were tested in an animal model of anxiety, the Geller-Seifter conflict test. Rats were subjected to a multiple schedule consisting of three components: in the first component, lever-pressing produced food-reward ('unpunished responding'). The second component was a time-out period, during which lever-pressing had no consequences. During the third component, lever-pressing produced food-reward, but was also punished by an incremental foot-shock ('punished responding'). After establishing a stable baseline of both unpunished and punished responding, animals were injected with various doses of NPY, GHRH, ANP, BEND, or with saline into the lateral cerebral ventricle, and testing was repeated. While changes in unpunished responding can reflect alterations in performance factors or motivational strength, increases in punished responding have previously been shown to be highly specific for anxiety-reducing drugs, such as the benzodiazepines. NPY markedly and dose-dependently increased punished responding. A smaller increase of unpunished responding was also seen. These results add further support to the hypothesis that NPY may be an endogenous anxiolytic. GHRH, ANP and END did not affect punished responding.

The amygdala mediates the anxiolytic-like effect of the neurosteroid allopregnanolone in rat.

The neurosteroid allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one) possesses clear anxiolytic-like effects. Other neurosteroids namely pregnenolone sulfate (PREG-S) and dehydroepiandrosterone sulfate (DHEA-S) influence anxiety-related behavior differently. In the present study, the implication of the amygdala, a key structure in mechanisms of fear and anxiety, was investigated as a potential neural substrate for the effects of neurosteroids on anxiety-like behavior in rat. Animals implanted with bilateral cannulae aimed at the central nucleus of the amygdala (CeA) and infused with neurosteroids, were tested in two animal models of anxiety. Allopregnanolone (8 microg/side) produced a significant increase in responding suppressed by punishment in the conflict test. In the elevated plus maze, allopregnanolone (8 microg/side) induced a significant increase in the time spent and the number of entries in open arms compared with the vehicle-infused controls. No significant changes in punished and unpunished responding of the conflict test were observed with PREG-S (0.001-8 microg/side) and DHEA-S (2-8 microg/side) administered into the CeA or into the lateral ventricle (1-20 microg). The results reveal the lack of activity of PREG-S and DHEA-S in the operant conflict test, but suggest that the central nucleus of the amygdala is a key region involved in the mechanisms underlying the anxiolytic-like action of allopregnanolone.