TRIMU-5, a mu 2-opioid receptor agonist, stimulates the hypothalamo-pituitary-adrenal axis.

Previous work in our laboratory has shown that DAMGO (ICV) will cause an elevation in plasma corticosterone (CS). The effect was blocked by pretreatment with beta-FNA but not by naloxonazine, suggesting indirectly that DAMGO's effect was via a mu 2-opioid receptor. TRIMU-5, a mu 2 agonist/mu 1 antagonist, was tested in a similar series of experiments to show more directly that the effect of DAMGO to increase plasma CS was via the mu 2 receptor. Experiments were conducted on conscious, unrestrained, male Sprague-Dawley rats with chronic IV catheters and ICV cannula guides allowing for serial blood sampling and drug injection into the right lateral ventricle. During this process, animals remained isolated in sound-attenuated one-way vision boxes. TRIMU-5, 50 micrograms, produced a sustained increase in plasma CS for a 3-h period. The response peaked at 30 min, showing a plasma CS level of 19.7 +/- 1.4 micrograms/dl. A lower dose, 10 micrograms, did not produce a significant response. A higher dose, 100 micrograms, produced an elevated hormone response in a pilot study but was lethal in half the animals. The plasma CS increase was blocked by pretreatment with beta-FNA, 20 micrograms ICV, given 18 h before TRIMU-5, but was unaffected by naloxonazine pretreatment, 20 mg/kg i.v., also administered 18 h before TRIMU-5. These data confirm our earlier conclusion that the effect of DAMGO to elevate plasma CS was through a mu 2-opioid receptor.

DAMGO stimulates the hypothalamo-pituitary-adrenal axis through a mu-2 opioid receptor.

DAMGO, a highly selective mu opioid agonist, is capable of stimulating the hypothalamo-pituitary-adrenal (HPA) axis to produce a dose-related elevation in plasma corticosterone (CS). The purpose of this study was to confirm that this action was mu receptor selective and to determine which of the mu receptors was involved using naloxonazine, a mu-1 receptor-selective antagonist. Experiments were done in male rats with chronic i.v. catheters and i.c.v. cannula guides. This enabled the withdrawal of serial blood samples in conscious unrestrained animals that were isolated in sound-attenuated one-way vision boxes. DAMGO, 8 and 16 micrograms administered i.c.v. caused significant and prolonged elevation of plasma CS. beta-funaltrexamine (beta-FNA) in progressively increasing doses (i.c.v.), antagonized the effect of DAMGO. The hormone response to DAMGO was unaffected by pretreatment with norbinaltorphimine or naltrindole (both i.c.v.). Naloxonazine, 50 micrograms, administered i.c.v. 18 hr before DAMGO did not antagonize the response to DAMGO. The same dose of naloxonazine given 2 hr before did reduce the response to DAMGO. Naloxonazine, 20 mg/kg i.v., given 18 hr before did not alter DAMGO's effect on plasma CS; however, the analgesic response to DAMGO in the same animals 24 hr later was antagonized by naloxonazine pretreatment. Pretreatment with beta-FNA or naloxonazine i.c.v. did not alter the plasma CS increase after exposure to ether vapor.(ABSTRACT TRUNCATED AT 250 WORDS)

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 animal's flank and the point held against the side of the metal cage for 15 s. Plasma CS increased to 29.3 +/- 4.7 micrograms/dl at 15 min and 15.7 +/- 1.8 micrograms/dl at 30 min. These levels were significantly higher than animals pretreated with diazepam, 5 mg/kg i.v., 2 h prior to stimulation (9.2 +/- 2.0 and 7.4 +/- 1.5 micrograms/dl, respectively). Animals which were pretreated with CGS-8216 (a mixed agonist/antagonist at the benzodiazepine receptor), 2 mg/kg i.v., 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 i.v. 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.

Effects of beta-carboline-ethyl ester on plasma corticosterone--a parallel with antagonist-precipitated diazepam withdrawal.

Diazepam has been shown to produce physical dependence based on observations of behavioral stimulation or, in our laboratory, by increases in plasma corticosterone (CS) during antagonist-precipitated withdrawal. The behavioral excitation appears similar to that observed following the administration of beta-carboline esters--agents reported to interact with benzodiazepine receptors and termed "inverse agonists." The focus of the present study is to correlate the occurrence of changes in CS with behavioral excitation previously observed by others. Further, these studies are designed to show a parallel between the manifestations of benzodiazepine withdrawal and the pharmacologic effects of beta-carboline ethyl ester. Experiments were done in conscious unrestrained male Sprague-Dawley rats, with chronic i.v. catheters, using sound-attenuated one-way vision boxes. These studies compared the hormonal and behavioral changes induced by beta-carboline ethyl ester (beta CCE) with CGS-8216-precipitated withdrawal in rats treated with diazepam for 8 days. Rats treated chronically with diazepam (5 mg/kg/day), showed a significant increase in plasma (CS) following CGS-8216. Behavioral abstinence scores were also significantly elevated. beta CCE (0.5-5.0 mg/kg) showed a significant dose-related increase in plasma CS. Behavioral scores were also increased at doses of 0.5 and 2.0 mg/kg. beta CCE-induced plasma CS increases were antagonized by CGS-8216 at doses of 1.0 and 2.0 mg/kg but not by 0.5 mg/kg. In animals chronically treated with diazepam, beta CCE evoked a more prolonged plasma CS elevation than in vehicle-treated animals suggesting a dual agonist/antagonist effect. These data suggest a parallel between CS elevations and behavioral effects during withdrawal as well as similarities between the action of beta CCE and the manifestations of this withdrawal.

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.

The occurrence of cross-tolerance between morphine and ethyl-ketocyclazocine using the tail-flick test: lack of effect of diazepam, phenobarbital, or amphetamine.

Experiments were designed to test for short-term tolerance to morphine and ethyl-ketocyclazocine (EKC), mu and kappa agonists, respectively, and cross-tolerance between the two drugs. Mice were primed with one of the drugs, using doses that did not affect the tail-flick response when tested at a time 1 or 3 hours later, when the same or alternate test drug was administered. All animals were injected with the priming drug IP. In one series of experiments, the test drugs were given SC, and in the other, the test drugs were injected ICV under brief halothane anesthesia. Priming with morphine (30 or 100 mg/kg) significantly raised the ED50 for ICV morphine. Priming with EKC (2 or 6 mg/kg) similarly elevated the ED50's for SC and ICV EKC. Symmetrical cross--tolerance was produced in experiments where the test drugs were administered SC when tested at 3 hrs. The effects of priming with EKC on morphine analgesia was evident when the interval between priming and test drugs was 1 hour. When the test drugs were given ICV, cross-tolerance was also symmetrical: priming with EKC significantly raised the ED50 for morphine and priming with morphine raised the ED50 for EKC when tested at 3 hrs. These data suggest that both agonists act on a common site to produce analgesia as similar pA2 values for naloxone antagonism were determined. The occurrence of short-term tolerance and cross-tolerance to the opiates was unaltered by chronic pretreatment with diazepam, phenobarbital, or amphetamine.

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.

Plasma corticosterone changes in response to central or peripheral administration of kappa and sigma opiate agonists.

The acute administration of morphine produces a characteristic increase in plasma corticosterone. By using the male rat with a chronic i.v. catheter, a stereotaxically placed cannula in the lateral ventricle and a sound-attenuated one-way vision chamber, the effects of prototypic kappa and sigma opiate receptor agonists were studied. Both ethyl-ketocyclazocine (EKC) and N-allyl-normetazocine [SKF 10047] (SKF) also produced such a rise in hormone level after i.v. or i.c.v. administration. The former effect was blocked by concurrent treatment with naloxone (NX), 0.4 mg/kg. The latter effect was not blocked by i.c.v. pretreatment with beta-funaltrexamine, a long-acting mu receptor antagonist, whereas the response to i.c.v. morphine was attenuated significantly. The development of acute dependence and short-term tolerance to EKC and SKF was also studied. Priming with either drug (i.v.) did not result in a NX-precipitated plasma corticosterone withdrawal response 3 hr later. Similar studies priming with morphine (i.c.v.) did result in the plasma corticosterone-elevated response when NX was administered i.c.v. after 3 hr. When EKC or SKF was substituted for morphine, no NX-induced response was observed. Short-term tolerance to the effects of EKC and SKF on the hypothalamo-pituitary-adrenal axis did not appear to occur. These data support the notion that stimulation of several subclasses of opiate receptors will result in the activation of the hypothalamo-pituitary-adrenal axis. Furthermore, it appears that the mu opiate receptor is involved in the initiation of acute opiate dependence.

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.

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.

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.

Short-term tolerance to morphine: effects of diazepam, phenobarbital, and amphetamine.

Short-term tolerance to morphine, which can be demonstrated in as little as 3 hours after a single administration of the opiate, was examined in animals chronically pretreated with diazepam, phenobarbital, or amphetamine. Tail-flick latency in mice and changes in plasma corticosterone in rats were the parameters tested in these experiments. Rats primed with either saline or morphine, 10 mg/kg, were injected 3 hours 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 hours prior to estimation of tail-flick latency and ED50 determinations. Mice primed with either dose of morphine had significantly higher ED50's than those primed with saline. Chronic treatment with diazepam or amphetamine in either species did not significantly alter short-term tolerance development by either parameter. However, with phenobarbital pretreatment, the plasma corticosterone response was attenuated and short-term tolerance to morphine's analgesic effects did not occur. Further studies in morphine-pelleted mice showed that analgesic tolerance occurred similarly in all groups. This suggests that barbiturates may delay the process.

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 ED50's 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.

Changes in plasma corticosterone levels as a measure of acute dependence upon levorphanol in rats.

Changes in plasma corticosterone levels have been utilized as a sensitive and reliable indicator of opiate withdrawal. By using rats prepared with chronic indwelling i.v. catheters, drug injections and sequential blood sampling were accompanied in conscious undisturbed animals. Acute administration of levorphanol tartrate (LT) at 0.5, 1.0 or 2.0 mg/kg b.wt. caused an elevation in circulating corticosterone. With the lowest dose of LT, hormone levels returned to pretreatment values by 180 min. Naloxone hydrochloride (NX), 0.4 mg/kg, administered 3 hr after pretreatment with LT, 0.5 mg/kg, produced a significant elevation in plasma corticosterone. In contrast, animals pretreated with saline did not show the same increase in hormone levels after NX. NX, administered at several doses, along with LT, suppressed the plasma corticosterone increase which is normally observed when LT is given acutely. When NX is administered at sufficient dosage, along with LT pretreatment, the subsequent administration of the antagonist did not elicit the withdrawal response. These data indicate that a similar increase in plasma corticosterone, upon challenge with NX after a single dose of morphine, generalizes to other opiates. Blockade of the initial rise in plasma corticosterone and subsequent increase upon injection of the antagonist speak to the probability of the responses being related and opiate specific.

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.

The breeding system of Polysphondylium pallidum, a cellular slime mold.

A mating type analysis was performed on 231 isolates of the cellular slime mold, Polysphondylium pallidum found in 61 samples collected in eastern North America between northern Florida and sourthern Canada. Seventy-eight percent of the isolates belonged to one of 2 mating types; 18% were incapable of mating with any partner; 3% were homothallic; and 1%, consisting of 2 isolates from a Florida sample, belonged to a separate breeding group. It is suggested that the majority of isolates represent a species capable of local genetic adaptation to a niche, the parameters of which undergo considerable variation over space and time.

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.