The effects of chronic haloperidol administration on GABA receptor binding

The effect of chronic administration of haloperidol for 30 days and its subsequent withdrawal for 0,4 and 8 days on the binding of [³H]-GABA and [³H]-muscimol to GABA binding sites in rat brain membranes obtained from six discrete regions of the brain and spinal cord was investigated. The chronic administration of haloperidol resulted in a marked increase in the number of GABA binding sites within the substantia nigra, but did not affect GABA binding sites in other regions of rat brain or spinal cord. The increase in GABA binding sites in the substantia nigra was evident for at least 4 days following termination of haloperidol treatment; however, 8 days following withdrawal from haloperidol, when dopaminergic supersensitivity had become maximally expressed, the number of GABA binding sites in the substantia nigra had declined and was the same as that observed in the control rats. These observations suggest that blockade of dopamine receptors in the striatum by haloperidol results in a compensatory decrease in the activity of the GABAergic strionigral system that leads to an increase in the number of GABA binding sites within the substantia nigra. This increase in the number of GABA binding sites is gradually reversed when dopamine is again allowed to interact with its receptors.     

Chronic morphine administration augments benzodiazepine binding and GABAA receptor function

Behavioral and neurochemical evidence indicates links between the opioid and GABA neurotransmitter systems. To assess effects of chronic opiates on the major site of postsynaptic GABAergic activity, the GABA_A receptor, we administered chronic morphine and naltrexone to mice and evaluated binding at the benzodiazepine andt-butylbicyclophosphorothionate (TBPS) sites and GABA-dependent chloride uptake. After morphine (3 days), benzodiazepine receptor binding in vivo but not in vitro was increased in cortex compared to placebo-treated mice. TBPS binding was unchanged in cortex, but muscimol-stimulated chloride uptake was increased at low doses of muscimol. Benzodiazepine and TBPS binding and muscimol-stimulated chloride uptake were unchanged in naltrexone-(8 days) compared to placebo-treated mice. When naltrexone was administered previously to block opiate sites, the increases in benzodiazepine binding and chloride uptake observed with chronic morphine were reversed. These results indicate that chronic morphine but not naltrexone enhances benzodiazepine binding and GABA_A receptor function, perhaps by an action at opioid receptors.Supported in part by Grants DA-05258 and MH-34223 from the US Public Health Service. Dr. Miller is the recipient of a Faculty Development Award in Clinical Pharmacology from the Pharmaceutical Manufacturers Association Foundation     

Sex differences in muscarinic receptor binding after chronic ethanol administration in the rat

Male and female rats were administered ethanol (5% v/v) in a liquid diet for 18 weeks. Pair-fed control animals were fed the same diet except that dextrose was substituted isocalorically for ethanol. Normal controls received a commercial laboratory chow for the same duration. Results showed that, in females, chronic ingestion of an ethanol liquid diet significantly increased the number of muscarinic receptor binding sites compared to both control groups. In contrast, for males, there was no significant difference in the mean number of binding sites among the treatment groups. Furthermore, the mean maximum number of binding sites for males and females varied across brain areas. Males had a significantly greater number of receptor binding sites than females in the striatum, while females had a greater number in the cortex. It was suggested that the geuder differences observed in the present study could be mediated by hormonal effects on central muscarinic functioning.     

Timolol pharmacokinetics and effects on heart rate and blood pressure after acute and chronic administration

Summary The pharmacokinetics and effects of various oral doses of timolol administered either acutely or after chronic medication for 7 days were studied in healthy volunteers. After acute administration of timolol maximum plasma concentrations were attained within 1–2 h and thereafter declined exponentially with time. The mean apparent half-life of elimination from plasma was 2.5 h and was independent of dose. Area under the plasma concentration-time curve (AUC) was proportional to the orally administered dose. Plasma concentrations, apparent elimination half-life and AUC were not altered after one week of chronic administration. The effect of timolol on heart rate and blood pressure response to three sequentially increasing steady state work loads were studied. After acute administration of timolol maximum reduction of systolic blood pressure, resting heart rate, and the different parameters of the work-heart rate (or blood pressure) relationships were produced by 5 mg timolol. Increasing the dose prolonged the duration over which these variables were reduced. The relationship between timolol plasma concentration and inhibition of different parameters of the exercise response was hyperbolic with half maximum inhibition at concentrations of about 3–4 ng/ml of timolol and maximum inhibition above 30 ng/ml. Maximum drug effects and duration of action of timolol on the different variables were similar after acute and chronic administration.     

Stimulation of GABA receptor binding by barbiturates

Barbiturates increase Na⁺-independent GABA binding to bovine cerebral cortex membranes. The relative activity of a series of compounds to enhance GABA binding correlates significantly with their anesthetic activity and with their ability to reverse bicuculline antagonism of GABA responses. There are regional differences in the maximal percent stimulation of GABA binding by pentobarbital; a low stimulation in cerebellum.     

The influence of morphine dependence on some specific effects mediated by monoaminergic and GABAergic systems

Physical dependence in rats was attained by increasing doses of morphine twice daily for 14 days. The effects of drugs modifying neurotransmitter functions were then monitored during withdrawal from the morphine treatment in comparison with controls withdrawn from saline at 40, 64, 112, and 184 h after the last injection. The stereotypy due to dopaminergic stimulation by apomorphine (1.5 and 2 mg/kg SC) was significantly potentiated in the rats withdrawn from morphine. Characteristic wet-dog shake behavior induced by elevating brain levels of serotonin (5-HT) with 5-hydroxy-DL-tryptophan (150 mg/kg SC) preceded by the decarboxylase inhibitor benserazide (50 mg/kg IP) was similar in animals withdrawn from either morphine or saline. The hypothermia and the general behavior following elevation of brain GABA levels with the GABA transaminase inhibitors D,L-4-amino-hex-5-ynoic acid (RMI 71645) (-acetylenic GABA) (100 mg/kg IP) or D,L-4-amino-hex-5-enoic acid (RMI 71754) (-vinyl GABA) (800 mg/kg IP) did not differ significantly between morphine-withdrawn and saline-treated rats. The central cholinergically mediated hypothermia and tremor induced by tremorine (15 mg/kg IP) was similar in both groups, but the peripheral chromodacryorrhea was blocked during morphine withdrawal. It is thus concluded that during morphine withdrawal in the rat under these conditions, the behavior mediated by dopaminergic system shows enhancement, but effects dependent upon central 5-HT, GABA, and cholinergic systems are not affected.A preliminary account of these findings was communicated to the Eleventh C.I.N.P. Meeting (Buckett, 1978)     

The effects of morphine treatment and morphine withdrawal on the dynorphin and enkephalin systems in sprague-dawley rats

The effect of morphine tolerance and withdrawal on prodynorphin peptides was studied in relevant brain areas and in the pituitary gland of male Sprague-Dawley rats, and compared with effects on the proenkephalin-derived peptide Met-enkephalin. After 8 days of morphine injections (twice daily), dynorphin A and B levels increased in the nucleus accumbens and dynorphin A levels increased also in the striatum. Morphine treatment increased striatal Met-enkephalin. Leu-enkephalinArg⁶ levels were reduced in the ventral tegmental area (VTA). Morphine-treated rats had very low Leu-enkephalinArg⁶ levels in the hippocampus as compared to saline control rats. Comparison of the relative amounts of dynorphin peptides and the shorter prodynorphin-derived peptides, Leu-enkephalinArg⁶ and Leu-enkephalin, revealed a relative increase in dynorphin peptides versus shorter fragments in the nucleus accumbens, VTA and hippocampus. Morphine-tolerant rats had lower levels of dynorphin A in both lobes of the pituitary gland, whereas hypothalamic dynorphin levels were unaffected by morphine. Leu-enkephalinArg⁶ levels were reduced in the hypothalamus, but not changed in the pituitary gland. Naloxone-precipitated withdrawal accentuated the increase in dynorphin A and B levels in the accumbens and dynorphin A levels in the striatum, while inducing an increase in enkephalin levels in the accumbens and Met-enkephalin in the VTA. In the hippocampus, Leu-enkephalinArg⁶ levels remained low in the withdrawal state. The low dynorphin levels in the anterior part of the pituitary gland were reversed by naloxone, whereas the low dynorphin A levels in the neurointer-mediate lobe were even lower in the withdrawal state. In conclusion, morphine tolerance and withdrawal affected prodynorphin-derived peptides in areas related to central reward mechanisms, and in the pituitary gland. The dynorphin peptides and the LeuenkephalinArg⁶ fragment were not affected similarly, indicating an effect also on metabolic interconversion.     

Behavioral effects of morphine and naloxone following chronic morphine administration

The effects of morphine, naloxone, and combinations of these drugs were examined in squirrel monkeys under shock-postponement schedules. In the absence of a lever press, shocks could be presented every 4s, and each response postponed shock for 20s. Acutely, morphine (0.10–3.00 mg/kg) produced not only overall response-rate decreases, but also increases in the number of shocks, whereas naloxone (0.10–30.00 mg/kg) had little effect on responding. When given in combination with morphine, several doses of naloxone antagonized the rate-reducing and shock-increasing effects of morphine. Daily administration of morphine resulted in a substantial decrease in the number of shocks received and a moderate attenuation of the rate-decreasing effects of morphine (tolerance). Lower doses substituted for the fixed daily dose resulted in a smaller effect on behavior than under acute administration. Naloxone given in combination with the daily morphine dose or substituted for the daily administration of morphine, produced effects similar to those seen prior to chronic drugging. Thus, behavioral effects of naloxone were not altered even though tolerance to morphine was observed. Larger doses of naloxone continued to antagonize the effects of morphine for at least 24h. No signs of physical dependence were noted when naloxone was administered or when administration of morphine ended.     

The effects of acute and chronic administration of morphine on the turnover of brain and adrenal catecholamines in rats

Brain and adrenal catecholamine turnover in adult female rats treated with morphine was investigated. A different time course response of brain and adrenal catecholamines to -methyl-p-tyrosine methylester (AMT) administration in normal rats was observed; the catecholamine turnover rate in adrenal glands appeared to be much slower than in the brain. Acute morphine increased the turnover of brain dopamine and noradrenaline as well as of adrenal catecholamines, whereas chronic morphine treatment induced a decrease in the turnover of brain noradrenaline. Withdrawal induced by nalorphine produced an increase in the utilization of brain noradrenaline and adrenal catecholamines; this effect could be related to the withdrawal stress situation induced by the opiate antagonist. Although the mechanism of morphine action may implicate other neurotransmitters besides catecholamines, our results contribute to evidence that brain and adrenal catecholamines could be involved in the mechanism of morphine tolerance and/or dependence.     

Changes in benzodiazepine/GABA receptor complex function in benzodiazepine-tolerant mice

Mice were given flurazepam, 40 mg k^–1, IP, once daily for 7 consecutive days. Twenty-four and forty-eight hours after the last injection measurements were made of the effects on convulsion threshold, body temperature and locomotor activity, of drugs acting on the GABA receptor complex. Significant decreases were seen in the hypothermic and hypomobility effects of progabide at 48 h, but no significant changes were seen in the effects of pentylenetetrazol or pentobarbitone. The actions of picrotoxin in all three types of test and the convulsant action of bicuculline (IP) were significantly decreased at 24 h but not at 48 h. The convulsive, but not the hypothermic, effects of picrotoxin were increased at the 48 h interval. These results may suggest that the chronic benzodiazepine treatment decreased some aspects of GABA receptor function at 48 h after the last dose; however, such an effect probably does not explain the previously reported increases in the effects of inverse agonists following chronic agonist treatment.     

Comparison of withdrawal precipitating properties of various morphine antagonists and partial agonists in relation to their stereospecific binding to brain homogenates

In morphine-dependent rats the withdrawal precipitating properties of various morphine antagonists and partial agonists were studied by quantitatively evaluating a variety of different withdrawal signs. A comparison of the dose response curves of the various substances obtained for the different signs revealed marked differences in respect to the lowest effective doses (EDs) necessary to precipitate the withdrawal signs as well as in the maximum frequencies of the signs induced. The pure antagonist, naloxone, which was judged very potent according to the ED, precipitated the lowest levels of jumping, whereas certain partial agonists of the benzomorphane type, which were less potent according to the ED, induced very high levels of this sign. These latter compounds, however, failed to precipitate complete withdrawal, as evidenced by the nearly complete absence of some of the withdrawal signs.The jumping precipitating potency of the antagonists as judged from the ED was found to be highly correlated to the stereospecific binding of these substances to rat brain homogenate. On the other hand, the ability of the substances to precipitate high levels of jumping was seen to increase, at least within a certain range, with increasing degree of agonistic properties, as indicated by the ratio of stereospecific binding in the presence and absence of sodium.     

The influence of single and chronic morphine administration on some central effects of amphetamine and apomorphine

In experiments on mice acute toxicity of morphine did not differ in aggregated and non-aggregated animals. Morphine in doses of 5, 10, 20, 40, 80 mg/kg s.c. did not significantly influence the amphetamine group toxicity in mice.Preliminary injection of morphine increased the occurance of amphetamine stereotypy and prolonged the duration of stereotypy caused by amphetamine and apomorphine in intact rats, but failed to prolong the duration of the stereotypy in rats tolerant to morphine.Some differences in the behavioural patterns after injection of amphetamine and apomorphine were observed. The development of tolerance to morphine affected differently the duration of the stereotypies produced by amphetamine and apomorphine.     

Influence of GABA receptor agonists and antagonists on the binding of 3H-diazepam to the benzodiazepine receptor

The GABA receptor agonists, GABA and muscimol, increased, while the GABA receptor antagonists, (+)-bicuculline, decreased tha affinity of the benzodiazepine receptor for ³H-diazepam. The effect was seen at both 0 and 25°C in spite of a large difference of affinity for ³H-diazepam at the two temperatures.     

Alteration in the effect of GTP on cerebral beta-adrenergic receptor binding during morphine withdrawal

The alteration in the effect of GTP on the binding of β-adrenergic agonists and antagonists in the rat cerebral cortex during morphine withdrawal was examined. Guanosine 5′-triphosphate (GTP), which is known to increase the coupling of β-receptors and adenylate cyclase, had no effect on the inhibition of cerebral [³H]dihydroalprenolol ([³H]DHA) binding by 1-isoproterenol (1-ISP) in animals rendered morphine-dependent. In morphine-abstinent rats, however, GTP increased the dissociation constant (K_D) of the high affinity sites of 1-ISP for the inhibition of [³H]DHA binding as well as the number of high affinity binding sites for 1-ISP. These GTP-induced changes in the binding sites in morphine-abstinent animals were not observed for pindolol, a β-adrenergic antagonist, but could be reproduced with 5′-guanylimidodiphosphate, a GTP analogue. On the other hand, increasing the temperature for the assay induced a similar effect on 1-ISP binding sites in normal synaptic membranes. However, at the maximal point of the temperature-induced effect, the addition of GTP further increased the K_D value of the high affinity binding site for 1-ISP in the cerebral cortex from morphine-abstinent animals. This suggests that the mechanisms underlying the alteration in the density and affinity of β-receptors following morphine abstinence may be different from the effects of temperature on cerebral β-adrenergic receptor sites. The present results suggest that in addition to the previously reported increase in β₁-receptors and activation of adenylate cyclase, the coupling between β-adrenergic receptor and adenylate cyclase in the brain may be facilitated during morphine withdrawal and that this change may also contribute to the occurrence of the morphine withdrawal syndrome.     

Impact of anti-cancer drugs and other determinants on serum protein binding of morphine 6-glucuronide

BACKGROUND AND THE PURPOSE OF THE STUDY: The aim of the present study was to examine factors that may influence the protein binding of morphine 6-glucuronide (M6G), the most active metabolite of morphine. METHODS: An enzyme-linked immunoabsorbent assay technique was used to measure the M6G concentration in serum of 18 healthy adults, 18 neonatal and 7 children with cancer. Total and free M6G concentrations were measured following equilibrium dialysis for 3 hrs and at physiological pH at 37°C. The influence of vincristine, methotrexate, 6-mercaptopurine, morphine, human albumin, alpha-1-acid glycoprotein, palmitic acid, oleic acid and pH on M6G protein binding was examined. RESULTS: M6G was 66.87±0.73 percent free in human serum at physiological pH and temperature. The percentage free (unbound) was increased significantly by vincristine (4.33%) and methotrexate (9.68%), but 6- mercaptopurine and morphine had no significant effect on it. Free percentages of M6G was reduced by decreasing serum albumin concentration but was unaffected by the presence of alpa-1-acid glycoprotein (AAG) or changes in serum pH. Similar results were obtained in human serum albumin (HAS) solutions. Addition of palmitic acid and oleic acid reduced protein binding significantly by 6.3% and 7.4%, respectively. MAJOR CONCLUSION: Although M6G in this study was not highly bounded, but because of its high analgesic potency, any change in its free concentration due to concurrent medication or disease caused significant changes in its effects. This dearth of evidence has been implicated in the reluctance of professionals to be cautious in prescribing them to children, particularly in the neonatal period.     

Tolerance to behavioral effects of clonidine after chronic administration of morphine

Male rats (Buffalo strain) were studied under a procedure in which each 30th lick of a drinking tube resulted in the delivery of 0.01 ml water. The effects of clonidine HC1 (0.003-0.3 mg/kg, IP) were determined before, during and after exposure to conditions in which a morphine sulfate solution (0.5 mg/ml in 0.4% saccharin) was the only source of fluid. After either 10 or 80 days exposure to the chronic morphine regimen, rats were maintained under a repetitive cycle in which the morphine was available for 3 days and then removed for 4 days. The subjects consumed an average of 100 mg/kg/day morphine during the times it was available. The effects of clonidine were redetermined once weekly, on the 4th day after removal of the morphine solution. The effects of clonidine were also determined after morphine was removed for more prolonged periods (18-67 days). Chronic exposure to the morphine solution resulted in a 4- to 5-fold shift to the right in the dose-effect curve for clonidine (decreased responding). ED50 values returned to pre-morphine levels when rats were tested at longer post-morphine times (e.g., 18 days). Under the conditions of this experiment, chronic exposure to morphine produced marked cross-tolerance to the behavioral effects of clonidine.     

Alterations in benzodiazepine and GABA receptor binding in rat brain following systemic injection of kainic acid

The binding of gamma-aminobutyric acid (GABA) and benzodiazepine to receptors was examined in regions of rat brain at various times after subcutaneous injection of kainic acid (KA, 15 mg/kg). The animals exhibited pronounced convulsions 90 min-4 hr after this treatment. During this period (2 hr after the injection of kainic acid) no alterations in the binding of [3H]-GABA or [3H]flunitrazepam to receptors were detected in the frontal cortex, the hippocampus or the amygdala-pyriform cortex. After recovery from the acute convulsive phase, the rats appeared to be hyperexcitable, hyperactive, and displayed marked aggression and occasional clonic convulsions one to 80 days later. During this period a marked increase (80-200%) in the number of binding sites for GABA in the amygdala-pyriform cortex occurred but this was associated with a slow decrease in the number of binding sites for [3H]flunitrazepam to 70% control value at 3 weeks. Binding of the "peripheral"-type of benzodiazepine ligand, [3H]-Ro5-4864, was increased to 450% of control 3 weeks after injection. In addition, the ability of GABA to stimulate the binding of [3H]flunitrazepam was reduced when measured 3 days after the injection of kainic acid. It is suggested that the long-term behavioural syndrome observed in kainic acid-treated rats, as well as the reduced effectiveness of diazepam in preventing seizures in animals treated with kainic acid, (Czuczwar, Turski, Turski and Kleinrock, 1981) may be explained in part by a reduction in the number of neuronal benzodiazepine receptors and a "desensitization" of the GABA receptors which are coupled to benzodiazepine receptors.     

Concurrent nimodipine attenuates the withdrawal signs and the increase of cerebral dihydropyridine binding after chronic morphine treatment in rats

Summary The effect of chronic administration of dihydropyridine calcium channel antagonist nimodipine (1 mg/kg/day) given concurrently with morphine on the signs of morphine withdrawal and on the [³H]nitren-dipine binding in the rat brain has been investigated. Chronic morphine administration in increasing daily doses from 20 mg/kg to 70 mg/kg for 24 days and consequent withdrawal for 24 h induced loss of body weight, wet dog shakes, episodes of writhing and yawning behaviour. The density of [³H]nitrendipine binding was elevated in the cortex and limbic structures but not in the striatum after chronic morphine treatment. Chronic concurrent administration of nimodipine prevented the loss of body weight and reduced the scores of wet dog shakes and writhing, but did not affect yawning behaviour at 24h after morphine withdrawal. The concurrent nimodipine treatment also prevented the rise in the density of central dihydropyridine binding sites which occurred upon chronic morphine treatment. These results suggest that chronic nimodipine treatment attenuates the development of the withdrawal signs which occur upon the termination of chronic morphine treatment by preventing the up-regulation of the central dihydropyridine-sensitive binding sites. Correspondence to: L. Ahtee at the above address     

Modulation of GABA release during morphine withdrawal in midbrain neurons in vitro

Chronic treatment with opioids induces adaptations in neurons leading to tolerance and dependence. Studies have implicated the midbrain periaqueductal gray (PAG) in the expression of many signs of withdrawal. Patch-clamp recording techniques were used to examine whether augmentation of adenylyl cyclase signalling produces hyperexcitation in GABAergic nerve terminals within the mouse PAG. Both the rate of mIPSCs and the amplitude of evoked IPSCs during naloxone-precipitated withdrawal was profoundly enhanced in chronically morphine treated mice, compared to vehicle treated controls, in the presence but not the absence an adenosine A(1) receptor antagonist DPCPX. Enhanced GABAergic transmission in the presence of DPCPX was abolished by blocking protein kinase A. Inhibitors of cAMP transport, phosphodiesterase and nucleotide transport mimicked the effect of DPCPX. Coupling efficacy of micro-receptors to presynaptic inhibition of GABA release was increased in dependent mice in the presence of DPCPX. The increased coupling efficacy was abolished by blocking protein kinase A, which unmasked an underlying micro-receptor tolerance. These findings indicate that enhanced adenylyl cyclase signalling following chronic morphine treatment produces (1) GABAergic terminal hyperexcitability during withdrawal that is retarded by a concomitant increase in endogenous adenosine, and (2) enhanced micro-receptor coupling to presynaptic inhibition that overcomes an underlying tolerance.     

Excitatory amino acids and morphine withdrawal: differential effects of central and peripheral kynurenic acid administration

The non-selective excitatory amino acid antagonist kynurenic acid, which does not readily cross the blood-brain barrier, dose-dependently attenuated the behavioral signs of naltrexone-precipitated withdrawal in morphine-dependent rats following both intraventricular and subcutaneous administration. However, intraventricular and subcutaneous administration of kynurenic acid and different effects on individual withdrawal behaviors. Moreover, single unit recordings in anesthetized animals showed that intraventricular, but not subcutaneous, kynurenic acid administration attenuated the withdrawal-induced increased firing of locus coeruleus neurons. These studies indicate that: (1) both central and peripheral excitatory amino acid receptors may play an important role in opiate withdrawal; and (2) excitatory amino acid antagonist treatments might be developed to reduce opiate abstinence symptoms in man.