Richard M. Eisenberg

Effect of Amphetamine on Plasma Corticosterone in the Conscious Rat

The effect of amphetamine (AMPH) on plasma corticosterone was studied in the conscious, unstressed rat. AMPH (0.5-5.0 mg/kg) produced a dose-dependent increase in plasma corticosterone. This rise in plasma corticosterone was not altered by the adrenergic blocking agents phenoxybenzamine or propranolol. In contrast, the serotonergic depleting agent p-chloroamphetamine significantly inhibited the AMPH-induced rise in corticosterone. In addition, the serotonergic blocking agent methysergide, but not cyproheptadine, inhibited the corticosterone increase induced by AMPH.

Further studies on the acute dependence produced by morphine in opiate naive rats

Morphine appears to be capable of initiating the opiate dependence process with the first exposure. This can be demonstrated within 3 hrs by the administration of a low dose of naloxone which results in a significant elevation in plasma corticosterone. The response was still evident if the interval between morphine-priming and naloxone was extended to 6, 12, or 24 hours. The magnitude of the hormone elevation varied with the priming dose of morphine or with the dose of naloxone used to precipitate the response. Results are presented suggesting that the stress/withdrawal hormone response may be evident as early as 30 min after morphine-priming. Rats pretreated for eight days with either diazepam, phenobarbital, or amphetamine showed similarities in hormone responses after morphine-priming and naloxone administration when compared to saline-pretreated controls. The exception being the phenobarbital-pretreated group, where the response was attenuated and not observed at the 24 hr interval. These results emphasize the parallels between acute dependence and chronic dependence, suggesting that the same mechanism is involved.

Effects of naloxone on plasma corticosterone in the opiate-naive rat

Abstract Naloxone HCl (NX) has long been considered to be a pure narcotic antagonist, having an effect only subsequent to pretreatment with a narcotic. Characteristically, low doses of NX have been used to antagonize the effects of analgesic doses of narcotics and to precipitate withdrawal in chronically treated animals. In this study, the effects of high doses of NX (2.0–20.0 mg/kg) on changes in plasma corticosterone were examined in the opiate-naive animal. Using male rats with chronic intravenous catheters and one-way vision boxes, injections were made and serial blood samples were obtained in the conscious, unrestrained animal. The acute administration of NX to the opiate-naive animal produced a dose-related increase in plasma corticosterone with respect to both amplitude and duration. NX (10.0 mg/kg i.v.) produced a significant elevation in hormone level at 15 and 30 minutes. With NX (20.0 mg/kg i.v.) the duration of the response was extended to 60 minutes. To examine whether short-term tolerance to this effect could be produced, animals were given a single pretreatment with either NX (10.0 mg/kg) or saline i.v. Two hours later NX produced a similar elevation in hormone level in both groups. The effect of chronic injection of NX was also studied. Animals pretreated with either NX (10.0 mg/kg) or saline s.c. once daily for 7 days did not show a significant difference following the subsequent administration of NX. In both cases, a significant elevation of plasma corticosterone resulted. The results suggest that NX may have a direct effect on opiate receptors resulting in an elevation of plasma hormone levels or NX may be disrupting an endogenous opiate-receptor interaction producing a stress response.

Effects of naltrexone on plasma corticosterone in opiate-naive rats: A central action

We have previously reported the elevation of plasma corticosterone by i.v. naloxone HCl (NX). This work has been extended, with the current study, showing a similar effect with naltrexone HCl (NTX) and that this effect is due to a central action of the drug. Using opiate-naive male rats with chronic i.v. catheters, stereotaxically placed intracerebroventricular (ICV) cannula guides where necessary, and sound-attenuated one-way vision boxes, serial blood samples were obtained from conscious unrestrained animals. NTX (5.0, 10.0, or 20.0 mg/kg i.v.) resulted in a significant increase in plasma corticosterone 15 min following injection. I.V. administration of the methylbromide salts of each drug, which do not cross the blood-brain barrier, did not produce this same elevation in hormone level at several doses (0.4, 1.0, 2.0, 10.0 or 20.0 mg/kg). In contrast, ICV injection of either of the quaternary salts (50 micrograms/10 microliter/animal) resulted in an immediate and sustained rise in plasma corticosterone. Results obtained demonstrate that NTX has a similar effect on plasma corticosterone as NX at the appropriate doses and that the effect is a central rather than a peripheral one.

Further Evidence of a Central Alpha-Adrenergic Inhibitory Influence on the Hypothalamo-Pituitary-Adrenal Axis in the Rat

It has previously been shown that stimulation of an adrenergic system in the central nervous system (CNS) will depress the increase in plasma corticosterone (B) following surgical stress. Two aspects of hypothalamo-pituitary-adrenal function, namely adrenal compensatory hypertrophy (ACH) and plasma B response to ether stress, were studied to determine the influence of this adrenergic component. Phenoxybenzamine, an gamma-adrenergic blocking agent, chronically implanted into the lateral ventricle produced an augmented response to ether stress, whereas norepinephrine (NE), dopamine (DA), and propranolol did not. The augmented response was similar to that observed following the daily administration of reserpine (0.25 mg/kg, s.c.). The response to this dose of reserpine was blocked by the simultaneous administration of a low dose of dexamethasone (14 mu-g/kg, s.c.). Augmentation of the stress response was not produced by larger doses of reserpine but did occur with lower doses. The effects of the treatments on ACH were not consistent with those observed on the stress response, suggesting that different mechanisms are involved. The data support the concept of an gamma-adrenergic component in the CNS which serves to inhibit the plasma B stress response.

Diazepam withdrawal as demonstrated by changes in plasma corticosterone: A role for the hippocampus

Elevations in plasma corticosterone were shown to be a reliable indication of antagonist-precipitated withdrawal from diazepam in the rat. Dependence to the benzodiazepine was produced by a single daily injection for eight days at which time CGS-8216 was injected i.v. via a chronic indwelling catheter. This injection and subsequent serial blood samples were withdrawn from conscious, unrestrained animals that were placed previously in sound-attenuated one-way vision boxes. The magnitude of the hormone change was correlated with either the chronic dose of diazepam or the dose of the antagonist used to precipitate withdrawal. When CGS-8216 was administered chronically with the diazepam, antagonist-precipitated abstinence did not occur. Additional results showed that dependence could be produced by bilateral intracerebral placement of micropellets of diazepam into the dorsal and ventral hippocampus. These data show that dependence to diazepam can be demonstrated in a relatively short time using modest doses of drug, and, further, that exposure of the hippocampus, an area with a high concentration of benzodiazepine receptors, to diazepam will initiate dependence.

Effects of chronic treatment with diazepam, phenobarbital, or amphetamine on naloxone-precipitated morphine withdrawal

The present study examines the severity of naloxone-precipitated opiate withdrawal in morphine-pelleted rats and mice. Animals were chronically pretreated with either saline, diazepam, phenobarbital or amphetamine for 8 days prior to withdrawal precipitation. Indicators of withdrawal were changes in plasma corticosterone in rats and jumping behavior in mice. The use of chronic intravenous catheters in rats and one-way vision boxes allowed for serial blood sampling and sequential hormone determinations. Opiate dependence was established by the subcutaneous implantation of a 75-mg morphine pellet 72 h prior to withdrawal precipitation. All pretreated groups of rats with morphine pellets showed a substantial elevation of plasma corticosterone following the injection of naloxone (0.4 mg/kg, i.v.). Those with lactose pellets showed little change. The group pretreated with phenobarbital showed a lower, more attenuated withdrawal response as compared to saline or the other pretreated groups. The responsiveness of the hypothalamo-pituitary-adrenal axis to ether stress was evaluated. This was found to be unaffected by chronic phenobarbital suggesting that the drug may have a more specific effect on opiate dependence/withdrawal mechanisms. Similar studies in mice showed no differences in ED50 for naloxone-induced jumping behavior in any of the pretreatment groups.

Sound vibration, a non-invasive stress: antagonism by diazepam

Rats, subjected to sound-vibration stress, showed an abrupt increase in plasma corticosterone (CS). This stimulation was reliably produced using a Burgess brand “vibro-graver,” a standard tool used for engraving. With the tool set at “8” or coarse, the barrel of the tool was placed on the animals flank and the point held against the side of the metal cage for 15 s. Plasma CS increased to 29.3±4.7 µg/dl at 15 min and 15.7±1.8 µg/dl at 30 min. These levels were significantly higher than animals pretreated with diazepam, 5 mg/kg IV, 2 h prior to stimulation (9.2±2.0 and 7.4±1.5 µg/dl, respectively). Animals which were pretreated with CGS-8216 (a mixed agonist/antagonist at the benzodiazepine receptor), 2 mg/kg IV, 30 min prior to diazepam had the protective effects of diazepam abolished. Sound/vibration produced a significant elevation in plasma CS in animals given CGS-8216 alone; but, this elevation was significantly lower than in vehicle-treated controls. This comparatively lower plasma CS level suggests a partial-agonist, diazepam-like effect by CGS-8216. Experiments were done in conscious unrestrained male Sprague-Dawley rats with chronic IV catheters. Except for 15 s stimulation exposure, all animals remained isolated in sound-attenuated one-way vision boxes for the duration of the serial blood sampling. Control stimulation exposure involved similar handling without turning on the engraving tool. These results demonstrate: 1) the usefulness of this tool to provide a repeatable stress stimulus; 2) the ability of diazepam to abolish the stress response; 3) that CGS-8216 can antagonize the action of diazepam; and 4) a demonstration of the partial agonist effects of CGS-8216.

Influence of clonidine on the acute dependence response elicited in naive rats by naloxone

Clonidine has been used successfully in the treatment of opiate dependence. The discomforting effects of withdrawal are attenuated by the drug. The question of whether the more central process of dependence is affected by clonidine was tested in the present study. Change in plasma corticosterone was used as the indication of the stress of acute withdrawal from morphine. Conscious, unrestrained male rats showed a dose-related, though somewhat delayed, increase in plasma corticosterone after clonidine (0.01-0.1 mg/kg). The suggested mechanism for this effect involves presynaptic inhibition of noradrenergic neurons inhibiting CRF (corticotropin-releasing factor) release. Similar animals showed an elevation of plasma corticosterone after naloxone (0.4 mg/kg) was administered 3 hrs following a single morphine-priming (10 mg/kg). The naloxone-precipitated response was unaffected by clonidine (0.04 mg/kg). This dose of clonidine did not substitute for morphine-priming to produce the naloxone-precipitated response. The data suggests that clonidine elevated plasma corticosterone by an indirect mechanism. Further, the stress associated with acute withdrawal is unaffected by clonidine suggesting that the drug does not alter dependence development.

Short-term tolerance to morphine: Effects of indomethacin

Short-term tolerance to opiates has been demonstrated in as little as three hours after priming with a single dose of morphine in naive animals. Tail-flick latency in mice and changes in plasma corticosterone in rats were the indicators tested in these experiments. Rats primed wiht either saline or morphine, 10 mg/kg, were injected 3 hrs. subsequently with morphine, 5 mg/kg. Those primed with saline showed the characteristic plasma corticosterone elevation following morphine, when serial blood samples were examined, whereas those previously treated with morphine did not. Mice were primed with saline or either of two doses of morphine, 30 or 100 mg/kg, 3.5 hrs. prior to estimation of tail-flick latency and ED50 determinations. Mice primed with either dose of morphine had significantly higher ED50s than those primed with saline. The effects of indomethacin, 5 or 10 mg/kg, were examined on both systems. Rats and mice were pretreated with indomethacin at 2.25 or 3 hrs., respectively, before morphine-priming. In all cases, indomethacin did not produce alterations in responses previously observed in correspondently treated controls.