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.     

Activating and 'anxiogenic' effects of corticotropin releasing factor are not inhibited by blockade of the pituitary-adrenal system with dexamethasone

Central administration of corticotropin releasing factor (CRF) in rats produces pituitary-adrenal activation and a variety of "anxiogenic-like" effects. The present study was designed to explore the contribution of the peripheral pituitary-adrenocortical axis in mediating these CRF responses. Intraventricularly administered CRF produced suppression of responding in the conflict test and a marked locomotor activation. Neither behavioral effect was altered by the prior administration of dexamethasone in a dose that blocked pituitary-adrenal activation to CRF. These results support the hypothesis that behavioral effects of CRF are mediated by its action at central sites and not via an action on the pituitary-adrenocortical system.     

Selective effect on punished versus unpunished responding in a conflict test as the criterion for anxiogenic activity

The punished drinking test has been used successfully for identifying and studying anxiolytic agents. By reducing the level of punishment (i.e., decreasing the intensity of shock), it has also been used as a method for measuring anxiogenic activity. Because anxiogenic behavior is a novel and important concept that is not yet fully established, we have reinvestigated the effects of two putative inverse benzodiazepine agonists and pentylenetetrazol in this conflict test. In a series of experiments, using both our version of the procedure and a replication of a previously published method, we were unable to demonstrate a selective reduction in punished responding over unpunished responding caused by CGS 8216 (3 to 40 mg/kg), FG 7142 (2 to 6 mg/kg), and pentylenetetrazol (10 to 20 mg/kg) as reported previously. A careful comparison of the details of our method and the published procedure failed to reveal the source of this discrepancy. If anxiogenic behavior is to be defined as a selective effect of a drug on punished response, the value of this test will depend on identification of its critical variables.     

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.     

Opposite effects of two ligands for peripheral type benzodiazepine binding sites, PK 11195 and RO5-4864, in a conflict situation in the rat

The effects of two drugs acting at the peripheral type benzodiazepine binding sites, PK 11195 and RO5-4864, were examined in shock-induced suppression of drinking in rats. These two compounds have opposite effects : RO5-4864 (3.1-1205 mg/kg i.p.) enhanced whereas PK 11195 (25-50 mg/kg i.p.) decreased the punished responding, and PK 11195 (6.25 mg/kg, a dose which did not alter the punished responding) blocked the proconflict action of RO5-4864 (6.25 and 12.5 mg/kg). The effects of RO5-4864 and PK 11195 were not antagonized by RO15-1788, a selective antagonist of the central benzodiazepine site. In addition, PK 11195 (6.25 mg/kg) did not reverse the proconflict effect of two beta-carbolines : beta-CEE and FG 7142. AS picrotoxin did not change the punished responding, these data imply that the effects of RO5-4864 and PK 11195 on the one hand and those of chlordiazepoxide and beta-carbolines on the other hand are differentially mediated and suggest that the peripheral type benzodiazepine binding sites are involved in this conflict model.     

Anxiolytic versus sedative properties in the benzodiazepines series: differencies in structure-activity relationships.

The effects of three benzodiazepines (Ro 5-3448, Ro 5-3367 and Ro 5-3438) upon conflict behavior and motor activity were investigated. In addition data related to nine other compounds (already published elsewere) were considered in order to study the relationships between anxiolytic and sedative properties in the benzodiazepine series. As regard conflict behavior, Ro 5-3448, Ro 5-3367 and Ro 5-3438 attenuated, in a dose related manner, the suppression of behavior during the punished schedule and, beyond certain doses, they also depressed responding in the nonpunished schedule. Motor activity was constantly depressed by suitable doses of the three drugs.Analysis of variance of data related to all the twelve benzodiazepines showed that the optimal requirements for antianxiety and sedative effects are quite different; furthermore a correlation coefficient of 0.40 between punished and nonpunished behaviors and of 0.88 between nonpunished behavior and motor activity was found, taking in account only the eight very potent alogenated compounds. It was concluded that, in benzodiazepine series, the similarity of effects upon learned and unlearned behaviors is not complete and, more important, that a substantial separation between anxiolytic and sedative activities can be obtained.     

Mechanisms involved in the control of punished responding in mother rats

In a previous study we found that mother rats show more drinking responses than virgins in the punished drinking paradigm, an animal model for anxiety. The present investigation was carried out to determine the possible mechanisms underlying this naturally occurring anticonflict effect. In Experiment 1, we investigated whether the induction of maternal behavior in virgin females (by long-term estrogen and progestin treatment in combination with pup exposure) enhances punished drinking. However, no release from shock-induced (0.25 mA) suppression of drinking was observed in maternally responsive virgins deprived of water for 24 hr. Unlike natural mothers, then, no anticonflict effect is seen in maternal virgins. A considerable body of evidence suggests that facilitation of gamma-aminobutyric acid (GABA) activity in the brain increases punished responding in rats. In the second experiment, therefore, lactating females were injected with pentylenetetrazol, a GABA antagonist, before being monitored for punished drinking. The drug attenuated the enhanced acceptance of shock in mothers, the effect being observed at a dose level that did not reliably affect unpunished responding. Experiment 3 addressed the possible influence of ovarian and adrenal hormones on punished drinking in lactating females. No significant behavioral effects were observed in mothers subjected to adrenalectomy or ovariectomy 4 days before testing.     

Do benzodiazepine receptors mediate the anticonflict action of pentobarbital?

The effects of the benzodiazepine antagonist CGS 8216 (2-phenylpyrazolo[4,3-c]quinoline-3(5H)-one) were examined in a thirsty rat conflict test in the presence and absence of pentobarbital. CGS 8216 (2.5-10 mg/kg i.p.) did not affect nonpunished responding, but doses of 5 and 10 mg/kg significantly reduced the rate of punished responding (i.e., the number of 3 second drinking episodes in a "shock" contingency). However, a dose of CGS 8216 which did not significantly alter punished responding (2.5 mg/kg) antagonized the anticonflict actions of pentobarbital. These observations suggest that while high doses of CGS 8216 may elicit an "anxiogenic" response in rodents, lower doses of CGS 8216 antagonize the anticonflict actions of a compound which has been shown to enhance benzodiazepine affinity in vitro. These data imply that the anticonflict actions of pentobarbital may be mediated through benzodiazepine receptors.     

Multiple benzodiazepine receptors: evidence of dissociation between anticonflict and anticonvulsant properties by PK 8165 and PK 9084 (two quinoline derivatives)

PK 8165 and PK 9084, two quinoline derivatives, displace [³H]-diazepam and its binding sites. These drugs appeared to be more potent in the presence of halides suggesting that they are acting on benzodiazepine receptors associated to a chloride ionophore. Like the benzodiazepines PK 8165 and PK 9084 increase punished responding in the rat conflict procedure but do not produce ataxia or sedation even at doses 5 to 20 times higher than those which are effective in the conflict test. Moreover they do not possess anticonvulsant properties. PK 8165 and PK 9084 do not change cGMP content of the cerebellar cortex and do not antagonize the increase in cGMP induced by the GABA antagonists isoniazid and picrotozin. Thus PK 8165 and PK 9084 “pure anticonflict drugs” might act on GABA-independent benzodiazepine receptors associated to a chloride ionophore.     

Effects of ethanol on punished and nonpunished responding under conditions of equated reinforcement rates and similar response rates

Lever pressing by pairs of rats was maintained under random-ratio (first subject) and yoked-interval (second subject) schedules of food presentation. The inter-reinforcement intervals generated under the ratio schedule comprised the interval values for the second (yoked) subject. This arrangement yielded nearly equivalent rates of food presentation for each subject pair. For the first rat of each pair a random-ratio schedule of shock presentation was added to the ratio schedule of food presentation. This manipulation resulted in similar rates of punished (first rat) and nonpunished (second rat) responding within subject pairs. Ethanol administration (0.25–1.5 g/kg) generally resulted in dose-related decreases in both punished and nonpunished responding. In general, punishment-specific effects were not obtained. These results suggest that ethanol may not be as effective as chlordiazepoxide or pentobarbital in increasing punished responding even when the effects of baseline response and reinforcement rates are controlled.     

GABA-B receptor activation and conflict behaviour

Baclofen and oxazepam enhance extinction of conflict behaviour in the Geller-Seifter test while baclofen and diazepam release punished behaviour in Vogel's conflict test. In order to investigate the possibility that the effect of the selective GABA-B receptor agonist baclofen is mediated indirectly via the GABA-A/benzodiazepine receptor complex, the effect of pretreatment of rats with baclofen on [3H]-diazepam binding to washed and unwashed cortical and cerebellar membranes of rats has been studied. Baclofen pretreatment increased Bmax in washed cerebellar membranes when bicuculline was present in the incubation mixture. No effect was seen in cortical membranes. The present results render it unlikely that the effect of baclofen on extinction of conflict behaviour and punished drinking is mediated via the GABA-A/benzodiazepine receptor complex.     

Determinants of drug effects on punished responding.

The effects of drugs on punished responding depend on interactions among a large number of experimental variables. Among these variables are the drug history of the animal, the dose of the drug administered, the type of stimulus used to punish responding, the intensity and duration of the punishing stimulus, the schedule of presentation of the punishing stimulus, the control rate and pattern of punished responding, the schedule of positive reinforcement maintaining the punished responding, the species of animal, the deprivation state of the animal, the behavioral history of the animal, and the nature of the required response. Although it is not known how all of these variables interact to determine the effect of drugs on punished responding, there is evidence that many of these variables are important as determinants of drug effects. The task facing behavioral pharmacologists studying drug effects on punished responding is to determine under what conditions drugs produce their characteristic effects on punished responding.     

Effects of corticotropin-releasing factor (CRF), tuftsin and dermorphin on behavior of squirrel monkeys maintained by different events

The effects of intracerebroventricular (ICV) administration of ovine CRF (0.1–30.0 μg/kg), dermorphin (0.3–30.0 μg/kg) and tuftsin (10–3000 μg/kg) were examined in squirrel monkeys trained to respond under a multiple 3-min fixed-interval schedule of food presentation and either shock presentation or stimulus-shock termination. Initial administration of the 41-amino acid polypeptide CRF increased food-maintained responding by 150–200% in 2 of 3 subjects. However, no other doses tested affected response rates, a result that may have been due to the rapid development of tolerance. The tetrapeptide tuftsin selectively increased responding maintained by food presentation at doses that decreased shock-maintained responding. The heptapeptide dermorphin selectively increased food-maintained responding when responding in the other component of the multiple schedule was maintained by shock presentation. When responding was maintained by a multiple food, stimulus-shock termination schedule, dermorphin decreased response rates in both components. Dermorphin's rate increases were blocked by the opiate antagonist naloxone, indicating that dermorphin's actions were mediated through the opiate receptor. These results indicate that the behavioral effects of tuftsin, dermorphin, and perhaps CRF, depend on the manner in which responding is controlled by its consequences. While the actions of tuftsin and dermorphin are believed to be mediated through the opiate system, the behavioral effects observed in primates appear different from the effects of morphine under similar schedule conditions.     

Corticotropin-releasing factor: an inhibitor of vascular leakage in rat skeletal muscle and brain cortex after injury

Corticotropin-releasing factor (CRF) and other peptides of the corticoliberin superfamily inhibit development of edema in skin and mucosa after noxious stimuli. Here, the breadth of CRFs protective activity on small blood vessels was examined after injury to skeletal muscle or to brain cortex. Male rats (243 +/- 15 g) were anesthetized with sodium pentobarbital 60 mg/kg i.p. and Monastral blue 60 mg/kg i.v. was injected 3 min before mechanical injury to muscle produced by a 4 cm midline surgical incision in the rectus abdominis or before freeze injury to the cortex produced by applying a cold probe (-50 degrees C) to the skull for 4 min. Vascular leakage, measured as area of dye staining multiplied by its light intensity, was quantified with an image-analysis system. CRF, having the human/rat sequence, 30 micrograms/kg s.c., injected once (30 min) or twice (30 min and 10 min) before injury to muscle or to brain, inhibited the lesion size by 58% and 55%, respectively (tissues taken at 0.5 and 1 h). Microscopy showed that CRF inhibited Monastral blue labeling of small blood vessels. The ED50 (95% C.L.) of CRF for reducing vascular leakage in muscle after celiotomy was 24 (9 to 64) micrograms/kg s.c. h/rCRF injected 30 micrograms/kg s.c. 2 h before celiotomy inhibited vascular leakage after celiotomy in adrenalectomized rats and this effect was not obtained with dexamethasone phosphate, 1 mg/kg s.c. alpha-Helical CRF (9-41), a CRF receptor antagonist, attenuated the actions of CRF on celiotomy. Laser-Doppler flowmeter measurements of skeletal muscle showed that the anti-inflammatory effects of CRF occurred when there were no significant concurrent changes in blood flow. From these results, we surmise that CRF has a versatile protective effect on small blood vessels when it inhibits leakage within different vascular beds.     

Effects of psychotropic drugs of different classes injected in super small doses

Effects of benzodiazepine tranquillizers (phenazepam, flunitrazepam), antidepressants (amitriptiline, imipramine) and nootropic piracetam injected in supersmall dozes were studied in outbred albino rats. It was found that in supersmall doses (10(-12)-10(-14) mol/kg) all these substances exert characteristic for each of these classes specific effects revealed by means of adequate pharmacological techniques. Benzodiazepine tranquillizers increased the number of punished water lickings in the conflict situation test. Antidepressants increased the number of wheel rotations in the test of the forced swimming in a tank with freely rotating wheels and enhanced a correlation between the number of wheel turns during the first and the second five minutes of the experiment. Nootrop piracetam increased the rate of acquisition of the active avoidance reflex in a shuttle box. The effects of all investigated drugs injected in supersmall doses were not accompanied by side effects, characteristic for them at administration in usual dosages. The conclusion is made, that the action of the drugs injected in supersmall doses is an universal property of psychotropic drugs. When administered in supersmall doses the pharmacological substances still exert their specific activity, but are devoid of side effects.     

Effects of cocaine on conflict behavior in the rat

The present studies examined the effects of acute cocaine administration, chronic cocaine administration and cocaine withdrawal on behavior in the Conditioned Suppression of Drinking (CSD) conflict paradigm, an animal model for the study of anxiety. In daily 10-minute sessions, water deprived rats were trained to drink from a tube that was occasionally electrified (0.25 mA), electrification being signalled by a tone. Within 3-4 weeks, control (i.e., non-drug) CSD behavior stabilized (30-50 shocks and 10-12 ml/session) and drug studies were initiated. Acute administration of cocaine (30-min pretreatment) produced a selective pro-conflict effect only at a dose of 10 mg/kg cocaine, with lower doses (2.5, 5 mg/kg) exerting no effect on CSD behavior and a higher dose (20 mg/kg) depressing both punished and unpunished responding. In a second experiment, cocaine (10 mg/kg, IP, 2/day) or saline was administered to separate groups of subjects for 7 weeks. In this chronic treatment study, CSD testing was conducted 12 hours after each evening cocaine administration. Although it had no effect on CSD behavior during the first week of treatment, this chronic cocaine administration produced a significant and selective pro-conflict effect which was stable during the period from Weeks 2-7. In a final experiment, a high dose of cocaine (20 mg/kg, 3/day) or saline was given to separate groups of subjects for 2 weeks and the behavioral effects of these treatments and their subsequent termination were examined. In this study, CSD testing was conducted 8 hours after each evening cocaine treatment. During the first week of high dose cocaine treatment, a decrease in punished responding was observed; this parameter returned to baseline levels by Week 2. Discontinuation of this high dose chronic cocaine treatment resulted in a selective decrease in punished responding. This pro-conflict effect was greatest at 3 days, and lasted for 6 days after the last cocaine dose. These data are consistent with clinical findings demonstrating the anxiogenic effects of both acute and chronic cocaine treatment as well as cocaine withdrawal and suggest that conflict paradigms such as the CSD may be useful for the study of cocaine-induced anxiety states.     

Effect of Corticotropin Releasing Factor (CRF) Injected Into the Median Eminence on LH Secretion in Male Rats

We determined the dose-response relationship and examined the time-related effect of CRF (corticotropin releasing factor) injected directly into the Median Eminence (ME) on LH secretion in conscious intact and castrated male rats. Doses of 0.25, 0.75, 1, and 1.5 nmol CRF dissolved in 1 l of saline (or saline only in the controls) were injected into the ME and blood samples collected 30, 60, 90, and 120 min postinjection to determine by RIA serum LH. CRF at doses of 0.75, 1 and 1.5 nmol significantly decreased serum LH in castrated and intact animals. The lower dose of CRF did not decrease LH in the two groups studied. The results suggest that in males as in females, CRF inhibits by itself LH secretion, at least in part, by a central action in the ME; the inhibitory effect of CRF on LH is similar in castrated and intact males; the dose of 0.25 nmoles of CRF was ineffective in decreasing LH and finally that CRF at ME levels may participate in a variety of stress-related responses, including reproduction inhibition, through LH suppression.     

Effects of corticotropin releasing factor on locomotor activity in hypophysectomized rats

Corticotropin releasing factor (CRF) injected intracerebroventricularly to hypophysectomized and sham hypophysectomized rats produced a dose dependent increase in locomotor activity, but in untreated hypophysectomized rats 10× more CRF was needed to produce a significant increase in activity. Concomitant daily supplements of rat growth hormone, thyroxine, and corticosterone to the hypophysectomized rats eliminated locomotor activity differences between the two groups. There was no statistically significant difference in locomotor response to either saline, 0.1 μg CRF, 1.0 μg CRF or 10.0 μg CRF in the group of animals receiving hormonal supplements. These results demonstrate that CRF can produce behavioral activation in rats independently of its effects on releasing hormones from the pituitary gland.     

Potentiation of the behavioral effects of pentobarbital, chlordiazepoxide and ethanol by thyrotropin-releasing hormone

Effects of pentobarbital, chlordiazepoxide and ethanol were studied alone and in combination with thyrotropin-releasing hormone (TRH), IM, on punished behavior. Key-peck responses of pigeons were maintained by food presentation under a fixed-interval 3-min schedule in which every 30th response produced shock. Moderate doses of pentobarbital, chlordiazepoxide and ethanol increased punished responding to 150-200% of control values while the higher doses of these drugs almost completely eliminated responding. TRH (0.01-1 mg/kg) had little effect on punished responding and 3 mg/kg produced 50% decreases. Although the lower doses of TRH were without effect when given alone, doses of 0.03 mg/kg and greater markedly potentiated the rate-increasing effects of pentobarbital, chlordiazepoxide and ethanol. Increases in punished responding of 350% were obtained with combinations of TRH and these drugs. The rate-decreasing effects of the sedative-hypnotic and anxiolytic compounds were not reversed by TRH. Potentiation of the behavioral effects of sedative-hypnotic and anxiolytic drugs by TRH suggests that TRH may play an important role in modulating the behavioral effects of these compounds and that combinations of neuroactive peptides with certain psychotherapeutic agents may be of some therapeutic value.