Brain noradrenergic systems as a prerequisite for developing tolerance to barbiturates.

Mice treated with 6-hydroxydopamine before they were chronically fed phenobarbital did not develop functional barbiturate tolerance, measured by duration of the loss of righting reflex and hypothermia. Injection of 6-hydroxydopamine caused significant depletion of brain norepinephrine, while brain dopamine levels were not significantly depleted. Intact brain noradrenergic systems seem to be necessary for developing tolerance to the hypnotic and hypothermic effects of the barbiturates.

A behavioral animal model of posttraumatic stress disorder featuring repeated exposure to situational reminders.

The purpose of this study was to evaluate an animal model of posttraumatic stress disorder (PTSD) in mice. The model featured repeated exposures to situational reminders of a traumatic stress, which consisted of a brief electric shock, and included assessment of two behavioral parameters and the startle reflex. The findings indicated an initial, but unsustained, increase in locomotor activity in a neutral environment due to traumatic stress. Exposure to situational reminders was associated with a persistent bidirectional abnormal behavioral pattern in a fear-provoking environment and a progressive increase over time in the magnitude of the startle reflex. Exposure to situational reminders also produced an increase in aggressive behavior. This animal model appears to produce behavioral changes analogous to those seen in patients with PTSD.

Cyclo(Leu-Gly) has opposite effects on D-2 dopamine receptors in different brain areas.

Cyclo(Leu-Gly) (cLG), a diketopiperazine analog of Pro-Leu-Gly-NH2 (MIF), affects a number of physiological and behavioral responses to the endogenous neurotransmitter, dopamine (DA). In the present series of experiments, the effect of in vivo administration of cLG (8 mg/kg) was investigated five days following subcutaneous administration. It was found that cLG administration of cLG (8 mg/kg) was investigated five days following subcutaneous administration. It was found that cLG administration caused a supersensitive behavioral response, measured by increased stereotypic sniffing, to the DA agonist, apomorphine (APO). At the same time, an increase was found in the affinity for dopamine (DA), as measured by dopamine inhibition of 3H-spiroperidol binding to D-2 DA receptors in striatum (nigro-striatal DA tract). In contrast, the same peptide treatment caused a subsensitive physiological response to APO-induced hypothermia, concomitant with a decrease in affinity for dopamine, as measured by DA inhibition of 3H-spiroperidol binding to D-2 DA receptors in hypothalamus (incerto-hypothalamic DA tract). These results suggest that a single neuromodulatory agent, the peptide cLG, can elicit diametrically opposite effects on D-2 DA receptors and on the corresponding physiological endpoints in two different brain areas.

The effects of cyclo(leucyl-glycyl) on nigrostriatal dopaminergic supersensitivity--inhibition of apomorphine-induced climbing.

In a previous study we showed that cyclo(leu-gly) (CLG) prevents the behavioural supersensitivity induced in the mesolimbic dopamine (DA) tract (in mice) by chronic haloperidol (HAL). In the current study, we evaluated the effects of CLG on supersensitivity to DA agonists in the nigrostriatal DA tract induced by chronic HAL (1.0 mg/kg, i.p. x 21 days--Experiment 1) or by acute injection of a high dose of apomorphine (APO) (Experiment 2). In Experiment 1 CLG was given at doses of either (a) 0 mg/kg/day (b) 1 mg/kg every third day (30 minutes prior to HAL), (c) 1 mg/kg every day, or (d) 8 mg/kg every third day. In Experiment 2 the dose of CLG was 8 mg/kg, s.c., given 24h after APO. Co-administration of CLG with HAL attenuated the development of HAL-induced supersensitivity in both paradigms (b) and (c) above, although the attenuation was significantly greater in (c) compared to (b). This biphasic dose response (D-R) curve for CLG in Experiment 1 indicates that a therapeutic window exists for CLG (bell-shaped D-R curve) and is similar to previous curves for CLG effects on the mesolimbic DA tract. In Experiment 2, CLG attenuated the DA receptor supersensitivity caused by acute high dose APO. The capacity of CLG to down-regulate DA receptors and attenuate dopaminergic supersensitivity in these experiments suggests a potential therapeutic use in the prevention of tardive and/or L-dopa-induced dyskinesias.

Neuropeptides differentially effect various forms of morphine tolerance.

Two different forms of tolerance to morphine were shown to develop. One form, environment dependent (ED), was associated with cues paired with the arrival of the drug. The second form of tolerance, environment independent (EI), was not dependent on any cues. ED tolerance was induced using a multiple injection model in which a cue (orange scent) preceeded each morphine injection (ip). After 12 days the animals were tolerant to the i.p. injection. However if morphine was injected i.c.v. tolerance was no longer evident. EI tolerance was induced by a pellet implant method. These animals were tolerant to morphine regardless of the route of administration. Peptides arginine vasopressin (AVP) and cyclo (Leu-Gly) (cLG) were tested for their ability to alter tolerance. AVP was found to facilitate the development of ED tolerance but had no effect on EI tolerance. On the other hand cLG blocked the development of EI tolerance without affecting ED tolerance. The determination of brain morphine levels indicated that the ED tolerance produced by this method is dispositional while EI tolerance is functional.

The effect of selective lesions of brain noradrenergic systems on the development of barbiturate tolerance in rats.

A method for chronic infusion of barbiturates into the central nervous system of rats was developed and used to study the importance of noradrenergic systems in the development of barbiturate tolerance. Destruction of noradrenergic neurons by intraventricular administration of 6-hydroxydopamine or by specific lesions of the dorsal or ventral noradrenergic bundles prevented the development of barbiturate tolerance without altering the animal's response to the acute administration of barbiturate.

Acute tolerance in inbred and selected lines of mice.

Mice of the C57Bl and C3H strains regained their righting reflex at higher brain ethanol levels than those at which they had lost their righting reflex, indicating that these animals developed acute tolerance. DBA mice did not develop acute tolerance. DBA mice "slept" significantly longer than C57Bl mice, but all mice lost their righting reflex at similar brain ethanol levels. Mice of SS and LS lines also showed no evidence for developing acute tolerance but differed significantly in brain ethanol levels upon loss of righting reflex. Both acute tolerance development and initial brain sensitivity to ethanol seem to determine duration of ethanol "sleep time" in mice.

In vivo down-regulation of rat striatal opioid receptors by chronic enkephalin.

Administration of methionine enkephalin (ICV) to rats for 5 days resulted in the development of physical dependence as exemplified by a hypothermic response which peaked 2-8 hours after initiation of withdrawal. Twenty-four hours post-withdrawal, opioid receptor binding was determined in the striatum using a selective delta receptor ligand. These studies revealed a decreased in the number of receptors. Bmax decreased from 193 +/- 20.4 fmoles/mg protein in controls to 136 +/- 9.7 fmoles/mg protein in enkephalin treated rats. This difference is significant at p less than 0.001. Existing evidence suggests that this decrease in binding is predominantly due to a decrease in delta receptors. Hence, the present findings indicate that delta receptor down-regulation in vivo may be an important mechanism in the adaptive response to chronic exposure to an endogenous opioid peptide.

Cyclo(Leu-Gly) attenuates the striatal dopaminergic supersensitivity induced by chronic morphine: agonist binding to D2 dopamine receptors correlates with stereotypic behavior.

We have previously shown that MIF and its structural analog, cyclo-(Leu-Gly), block analgesic tolerance and some signs of physical dependence following chronic opiate administration. The mechanism of action of these peptides has not been clearly elucidated. The data presented here suggests that chronic opiate administration causes a behavioral supersensitivity to dopamine (DA) agonists which is highly correlated with an increase in D2-Hi receptor affinity for DA agonists, but not antagonists. Both the behavioral and receptor changes are blocked by prior administration of cyclo(Leu-Gly). This suggests that the ability of cyclo(Leu-Gly) to block the development of opiate addictive states may involve DA synaptic elements.

Peptide inhibition of morphine-induced dopaminergic supersensitivity.

Injection of the peptide cyclo(Leu-Gly) into rats prior to chronic exposure to morphine, inhibits: 1) the development of analgesic tolerance; 2) some signs of physical dependence; and, 3) morphine-induced increases in behavioral responses to dopamine agonists. Although there was no change in the total number of high affinity striatal dopamine receptors, chronic morphine treatment did increase the affinity of the ligand at the receptor. The peptide blocked not only the affinity change, but the increased behavioral response to apomorphine as well. These behavioral changes correlate significantly with the neurochemical changes in dopamine receptors following chronic morphine treatment. Therefore, some of the pharmacological efforts of morphine may be mediated by changes in CNS dopamine receptors and that the peptides may act by inhibiting these neurochemical changes.

Differential down-regulation of delta opioid binding sites during physical dependence on methionine enkephalin in the rat.

Post-synaptic receptor modulation is thought to be one important mechanism involved in the adaptation of a neuronal system during chronic exposure to a drug. However, initial studies of opioid receptor regulation following chronic in vivo administration of narcotic agonists, such as morphine, reported no down-regulation in the number of opioid receptors in the brain. Subsequent studies, employing in vitro preparations, have reported evidence of opioid receptor down-regulation under specific conditions. It remains to be determined whether the in vitro phenomena of opioid receptor plasticity is relevant to the intact mammalian central nervous system. The data in this report shows that chronic in vivo administration the opioid peptide methionine enkephalin, results in a significant, regionally specific down-regulation of delta opioid receptors in rat brain: 30% decrease in receptor density in the striatum; no change in hypothalamus.

Opiate dependence following acute injections of morphine and naloxone: the assessment of various withdrawal signs.

The injection of high dose of naloxone 15 minutes after a single injection of morphine in mice was found to produce a jumping response which was behaviorly similar to the jumping response observed during the withdrawal from chronic morphine administration. In addition the jumping response following the acute administration of morphine-naloxone was increased by the injection of atropine and attenuated by oxotremorine. These data are consistent with the reports of effect of these cholinergic drugs on the jumping response which occurred during withdrawal after chronic morphine administration. However, other symptoms associated with opiate withdrawal (hypothermia, weight loss and diarrhea) were not produced by the acute injection of morphine-naloxone. It is therefore suggested that this single injection paradigm is particular to the jumping response rather than a demonstration of the rapid development of opiate dependence.

Pregnenolone and pregnenolone sulfate, alone and with ethanol, in mice on the plus-maze.

The neurosteroids pregnenolone and pregnenolone sulfate were tested for anxiogenic/anxiolytic effects in mice on the elevated plus-maze. Pregnenolone in a dose of 0.01 micrograms/kg increased motor activity and caused an anxiogenic response, i.e., a decreased number of entries onto the open arms of the plus-maze. Pregnenolone sulfate had no effect on motor activity in the doses tested but showed a biphasic response on the plus-maze: at 10.0 and 1.0 micrograms/kg pregnenolone sulfate caused an anxiogenic response but at 0.1 microgram/kg it produced an anxiolytic response. When administered with 1.5 g/kg ethanol, neither neurosteroid altered the depression in motor activity caused by ethanol. However, all doses of pregnenolone tested blocked the anxiolytic effect of ethanol on the plus-maze while one dose of pregnenolone sulfate, 1.0 microgram/kg, attenuated the response to ethanol. These results support the suggestion that these neurosteroids could play a role in the initial response to stress and indicate that further work needs to be done to determine the mechanism for the interaction with ethanol.

Dehydroepiandrosterone enhances the hypnotic and hypothermic effects of ethanol and pentobarbital.

Recent reports have indicated that the neurosteroid dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) interact with the GABAA receptor complex. Because many of the behavioral effects of ethanol and pentobarbital are due to activity at this complex, DHEA and DHEAS were tested for their ability to interact with the hypnotic and hypothermic effects of ethanol and pentobarbital. DHEA, but not DHEAS, causes a dose-dependent increase in the sleep time induced by either ethanol or pentobarbital. At 20 mg/kg, DHEA and DHEAS themselves cause a fall in body temperature. DHEA enhances the hypothermic effect of both ethanol and pentobarbital. DHEAS enhances the hypothermic effect of ethanol, but with pentobarbital it only delays the return of body temperature to baseline levels. Neither DHEA nor DHEAS affects the metabolism of ethanol.

Neurosteroids block the memory-impairing effects of ethanol in mice.

Using a win-shift foraging paradigm to assess working memory in C57BL/6 mice, the memory-enhancing effect of low doses of the neurosteroids 5-pregnen-3 beta-ol-20-one [pregnenolone (PE)], 5-pregnen-3 beta-ol-20-one sulfate [pregnenolone sulfate (PS)], 5-androsten-3 beta-ol-17-one [dehydroepiandrosterone (DHEA)], and 5-androsten-3 beta-ol-17-one sulfate [dehydroepiandrosterone sulfate (DHEAS)] were demonstrated. The neurosteroids 5 beta-pregnan-3 alpha-ol-20-one [pregnanolone (PA)] and 5 beta-pregnan-3 beta-ol-20-one [epipregnanolone (EPI)] disrupted memory in this paradigm. PE, PS, DHEA, DHEAS, and PA were also capable of blocking the memory-impairing effect of 0.5 g/kg ethanol. EPI prevented PA from blocking the effect of ethanol. The influence of these compounds on memory and their interactions on this behavior are consistent with their actions on the GABAA system.

Inhibition of neuroleptic-induced dopamine receptor supersensitivity by cyclo (Leu-Gly).

Behavioral supersensitivity of dopamine receptors was induced in mice by chronic administration of haloperidol (1 mg/kg/day for 21 days) and its subsequent withdrawal for 48 hr. This was evidenced by enhanced spontaneous locomotor activity and hypothermic responses to a dopamine agonist, apomorphine. Concurrent administration of cyclo (Leu-Gly), the enzymatically resistant diketopiperazine, an analog of melantropin release inhibiting factor, blocked haloperidol-induced dopamine receptor supersensitivity as evidenced by the blockade of apomorphine induced responses. Since many studies have linked the development of neuroleptic induced tardive dyskinesias with enhanced sensitivity of brain dopamine receptors, and the latter was blocker by cyclo (Leu-Gly), this agent may be of value in preventing the development of symptoms of neuroleptic-induced tardive dyskinesias.

An inhibitor of opioid peptide degradation produces analgesia in mice.

Bacitracin, a known inhibitor of brain peptidases which degrade the enkephalins and endorphins, produced dose-dependent analgesia in mice when injected into the lateral ventricle of the brain as determined by the tail-flick assay. The analgesic response peaked at 15 min post-injection but was reduced to control levels by 3.5 hours. Naloxone reversed the analgesic response to bacitracin, suggesting that opiate receptors may be involved. It is proposed that bacitracin produces analgesia by preventing the enzymatic destruction of endogenously released opioid peptides.

Dehydroepiandrosterone is an anxiolytic in mice on the plus maze.

Dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) are neurosteroids that have been shown to interact with the GABA system. The present study examined the effects of these compounds in mice on motor activity and behavior in the elevated plus maze. Doses of 0.5 mg/kg and above of DHEA reduced motor activity. This effect was blocked by diazepam, RO15-1788, pentylenetetrazole (PTZ), and ethanol. Both DHEA and DHEAS showed anxiolytic activity in the plus maze test, with DHEA being effective over a very wide range of doses (5 micrograms/kg to 1.0 mg/kg). Both RO15-1788 and PTZ blocked the anxiolytic effect of DHEA, there was no interaction with diazepam, and ethanol enhanced the anxiolytic effect of DHEA. At 1.0 mg/kg, DHEAS blocked the anxiolytic effect of ethanol. These results support the hypothesis that neurosteroids could be involved in the termination of a stress response.

Neurohypophyseal peptide influences on ethanol tolerance and acute effects of ethanol.

The neurohypophyseal hormone, arginine vasopressin (AVP), was previously shown to prolong the duration of ethanol tolerance in mice. Since drug tolerance and certain memory-related processes are examples of CNS adaptation, these phenomena have been proposed to share underlying mechanisms. We investigated the effects on ethanol tolerance of two other neurohypophyseal peptides, both of which modulate memory consolidation or retrieval of information. (Des-9-glycinamide, 8-lysine) vasopressin (DGLVP), like AVP, maintained ethanol tolerance in C57Bl mice, while cyclo(Leu-Gly) (cLG), at an equimolar dose, was ineffective. Thus, various neurohypophyseal peptides may differentially influence CNS adaptive phenomena. Direct peptide effects on ethanol-induced hypothermia and "sleep time," the parameters used to evaluate ethanol tolerance, were also determined. AVP per se caused hypothermia in mice, but neither AVP nor cLG affected ethanol-induced hypothermia. Both peptides, however, increased "sleep time" after acute ethanol administration. Although these direct peptide-ethanol interactions do not account for the observed peptide effects on tolerance, the findings emphasize the importance of using several parameters to assess ethanol tolerance.

Development of narcotic tolerance and physical dependence: effects of Pro-Leu-Gly-NH2 and cyclo (Leu-Gly).

Administration of Pro-Leu-Gly-NH2 (MIF) and cyclo (Leu-Gly) blocked the development of tolerance to and physical dependence on morphine, induced by the pellet implanation procedure in mice. Inhibition of tolerance development by peptides was evidenced by the presence of an analgesic response (increase in jump threshold) as determined by measuring the jump threshold to an increasing electric current, after a challenge dose of morphine (40 mg/kg). The same dose of morphine did not alter the jump threshold in morphine tolerant mice which were injected with saline prior to pellet implantation. The inhibition of the development of physical dependence on morphine by these peptides was evidenced by the antagonism of the hypothermic response which occurs during abrupt or naloxone-induced withdrawal. The naloxone-induced withdrawal jumping response was unaffected by these peptides. Dose-response experiments indicated that cyclo (leu-Gly) was much more potent than MIF in these tests. These peptides, when given after the development of tolerance and dependence, did not modify either the analgesic response to morphine or the symptoms of abrupt and naloxone-precipitated withdrawal. The inhibition of development of analgesic tolerance and physical dependence was not associated with changes in brain morphine concentration. The data indicate that these peptides do not interfere withe the morphine-morphine receptor complex formation but alter a subsequent step in the genesis of some aspects of tolerance and dependence processes.