Barbiturate Tolerance Research Articles

Changes of [3H]muscimol binding and GABA(A) receptor beta2-subunit mRNA level by tolerance to and withdrawal from pentobarbital in rats.

Effects of continuous pentobarbital administration on binding characteristics of [3H]muscimol were examined by autoradiography, and levels of GABA(A) receptor beta2-subunit mRNA were investigated by in situ hybridization histochemistry in the rat brain. In order to eliminate the induction of hepatic metabolism by systemic administration of pentobarbital, an i.c.v. infusion model of tolerance to and withdrawal from pentobarbital was used. An experimental model of barbiturate tolerance and withdrawal was developed using i.c.v. infusion of pentobarbital (300 microg/10 microl/hr for 7 days) by osmotic minipumps and abrupt withdrawal from pentobarbital. The levels of [3H]muscimol binding were elevated in cingulate of frontal cortex (46%) and granule layer of cerebellum (32%) of rats 24-hr after withdrawal from pentobarbital, while it was only elevated in cingulate (58%) of tolerant rats. The GABA(A) receptor beta2-subunit mRNA was increased in the withdrawal rats only: in the cortex (9-14%), hippocampus (15-21%), inferior colliculus (21%), and granule layer of cerebellum (24%). These results show the involvement of GABA(A) receptor and its beta2-subunit up-regulations in pentobarbital withdrawal rats, and suggest that the levels of [3H]muscimol binding and GABA(A) receptor beta2-subunit mRNA are altered in a region-specific manner during pentobarbital withdrawal.

Changes in [3H]forskolin binding to adenylate cyclase and [3H]phorbol dibutyrate binding to protein kinase C in pentobarbital tolerant/dependent rats.

These studies were designed to examine the effect of chronic administration of pentobarbital on activity of adenylate cyclase (AC) and protein kinase C (PKC) in the rat brain by autoradiography. Recently, it has been suggested that the phosphorylation of specific proteins may be involved in the development of physical dependence. An experimental model of barbiturate tolerance and dependence was developed using i.c.v. infusion of pentobarbital (300 microg/10 microl/hr for 7 days) by osmotic minipumps and abrupt withdrawal from pentobarbital. The levels of [3H]forskolin binding were elevated (28-67%) in cortex, thalamus, dentate gyrus, hippocampal CA3 and cerebellum of the pentobarbital withdrawal animals, while these changes were not observed in tolerant rats. The levels of [3H]phorbol dibutyrate binding were highly elevated (38-65%) in the region of cortex, caudate putamen, septum, thalamus, dentate gyrus, and cerebellum of rats withdrawal from pentobarbital. These results show that the levels of AC and PKC were significantly elevated in pentobarbital withdrawal rats, and suggest that the levels of AC and PKC are altered in a region-specific manner during pentobarbital withdrawal.

Effect of NMDA antagonists on development of rapid tolerance to various barbiturates

We recently reported that the noncompetitive antagonists, (+)-MK-801 and ketamine, block the development of rapid tolerance to ethanol. We now show that pretreatment with these NMDA antagonists also blocks rapid tolerance to the various barbiturates (pentobarbital, barbital, and phenobarbital) examined. Tolerance to pentobarbital occurred under three difference conditions: (a) in groups of rats that were tested at repeated times on day 1 (intoxicated practice or testing group), (b) in groups of rats that were not tested on the apparatus but handled at the same times on day 1 (dummy testing or associative learning group), and (c) in groups of rats that were not subjected to testing at all on day 1 (nontesting). However, NMDA antagonists blocked intoxicated practice and associative tolerance, but not tolerance produced in the nontesting group. In the last experiment NMDA antagonist failed to block tolerance (unlearned) when animals were treated in the animal quarters and tested in a different room (i.e., in the laboratory). These findings suggest that NMDA antagonists affect barbiturate tolerance in a manner similar to their effect on ethanol tolerance.

Dihydropyridine-sensitive calcium channels and barbiturate tolerance and withdrawal

We have shown previously that the dihydropyridine calcium channel antagonist nitrendipine, given chronically, prevents the development of ethanol tolerance and physical dependence. The present study examines the effects on barbiturate tolerance and physical dependence. Nitrendipine, given acutely during withdrawal, provided little protection against barbiturate withdrawal, as measured by convulsive behaviour on handling. When nitrendipine was given chronically concurrently with the barbiturate, a prolonged protection against the withdrawal syndrome was seen. Acute nitrendipine significantly increased the latency of seizures in response to the partial benzodiazepine inverse agonist FG7142 during barbiturate withdrawal, but there was no effect on the seizure incidence in response to bicuculline. Chronic treatment with nitrendipine did not alter the development of tolerance to the ataxic or general anaesthetic actions of barbiturates, but evidence was found of a possible interaction between nitrendipine and pentobarbitone, which may have been pharmacokinetic. The results suggest that neuronal calcium channels may be involved to some degree in the development of the changes responsible for barbiturate withdrawal, but to a less extent than found previously for ethanol dependence.

Barbiturate tolerance: effects on GABA-operated chloride channel function

Male ICR mice were fed powdered laboratory chow containing phenobarbital for 7 days to induce tolerance. Mice were sacrificed and brains assayed for changes in GABA-mediated chloride flux into membrane vesicles (microsacs). Concentration-dependent stimulation of chloride flux by GABA alone was not affected by the development of tolerance of phenobarbital. Phenobarbital potentiation of GABA-mediated chloride flux was significantly attenuated in the membranes prepared from phenobarbital-tolerant mice compared with those from pair-fed control mice. Similarly, stimulation of GABA-mediated flux by the benzodiazepine, flunitrazepam was also depressed in membranes from tolerant mice. However, the ability of ethanol and the benzodiazepine inverse agonist FG-7142 to modulate GABA-gated chloride flux was not affected by the development of phenobarbital tolerance. No significant changes in saturation [ 3H]diazepam binding parameters were observed. These findings suggest that there is a degree of cross-tolerance between phenobarbital and benzodiazepine agonist at the level of the GABA-operated chloride channel. Furthermore, although some reports have demonstrated behavioral cross-tolerance between ethanol and barbiturates, the present data suggest different mechanisms of tolerance development for these intoxicants at the level of the GABA A receptor chloride channel complex.

Effects of acute barbiturate administration, tolerance and dependence on brain GABA system: Comparison to alcohol and benzodiazepines

Central nervous system depressants, e.g., barbiturates, alcohol and benzodiazepines, have a wide spectrum of activity in humans and animals. Evidence accumulated suggests that some of the pharmacological actions exerted by these agents may be mediated through GABA system by mimicking GABAergic transmission. This review attempts to summarize the evidence available as to how the GABA system plays a part in the barbiturate actions and the development of tolerance to and physical dependence on barbiturates. The comparisons of the effects of alcohol, barbiturates and benzodiazepines at different steps of GABA synapse are also presented. Furthermore, the results which have been reported in the literature are inconsistent. This may be due to differences in: (a) animal models used; (b) brain regions used; (c) protocols (dose, duration, form and route of administration, etc.) used in treating animals and/or (d) techniques (pharmacological, biochemical, physiological, etc.) used.

Postmortem evaluation of barbiturate poisonings

The evaluation of barbiturate intoxication as the cause of death is often difficult when the concentration in body fluids and organs is not extremely high. The problem arises because of the great capacity of barbiturates to produce tolerance after chronic use, a property that is often unknown. Therefore, the most abused barbiturates were studied to assess whether chronic intake causes morphological liver changes or not. It was found that the chronic abuse of drugs containing seco-, cyclo-, brallo-, and/or pentobarbital produces hypertrophy of the smooth endoplasmic reticulum of hepatocytes corresponding to that of phenobarbital. Neither acute barbiturate overdose (without a history of abuse) nor chronic abuse of opiates causes similar liver changes. In conclusion, barbiturate tolerance can be evaluated postmortem by light microscopic examination of the liver.

The effect of age and pentobarbitone tolerance on pentobarbitone depression of calcium-45 uptake by mouse brain synaptosomes

Nerve terminals (synaptosomes) isolated from mouse brains accumulate 45Ca 2+ at a rate which can be stimulated to increase by immersion in a high K + saline. Low concentrations of pentobarbitone depress stimulated 45Ca 2+ accumulation in a dose-dependent manner. We investigated the effect of 1 mM pentobarbitone on calcium accumulation by synaptosomes isolated from the brains of young and old mice, some of which had been made tolerant to barbiturates, to see if age and tolerance to barbiturate would modify 45Ca 2+ accumulation. In the absence of barbiturate, neither advancing age nor barbiturate tolerance could be associated with any significant change in 45Ca 2+ uptake by potassium-stimulated or non-stimulated synaptosomes. However, both factors modified the depressant effect of barbiturate. Age and tolerance did not influence resting or potassium-stimulated 45Ca 2+ uptake, nor did they influence the suppression by pentobarbitone of resting 45Ca 2+ uptake. However, when correcting for suppression of resting uptake by barbiturate, age significantly reduced barbiturate suppression of potassium-stimulated 45Ca 2+ uptake. Tolerance significantly reduced this corrected effect of barbiturate in synaptosomes from the 3-month old but not the 24-month-old mice.

Barbiturate tolerance and hypersensitivity of arterial smooth muscle.

Experiments were undertaken on rats chronically treated with pentobarbital sodium, 50 mg/kg three times per day for a period of five days, to determine whether excised aortic or venous (portal vein) smooth muscle would demonstrate any change in reactivity to catecholamines or potassium chloride. Although sleeping time was reduced 50% (indicative of tolerance to the barbiturate), mean arterial blood pressure was not affected. Aortic strips, but not portal veins, excised from barbiturate-treated animals exhibited hypersensitivity to both contractile stimulants. Potassium-depolarized calcium-depleted aortic tissue, but not portal venous smooth muscle, from the barbiturate-treated animals contracted to lower concentrations of Ca2+ than did the arterial smooth muscle from the saline-treated control rats. The present observations thus indicate that chronic barbiturate treatment in rats can induce drug supersensitivity of excitable tissue other than one associated with the central nervous system. Our results on arterial smooth muscle could help to shed light on the unexplained rises in arterial blood pressure observed in humans undergoing barbiturate withdrawal.

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.

Barbiturate tolerance and dependence investigated in tissue culture

Barbiturate tolerance and dependence investigated in tissue culture

Development and mechanism of barbiturate tolerance in glial cell cultures

The effects of exposure of glial cells in primary culture and in continuous line (clone NN) to pentobarbital over various periods of time on cellular respiration and activities of enzymes involved in carbohydrate metabolism were studied. The results obtained in glial cells in primary culture were qualitatively identical to those obtained in glial cells in clonal line (NN). Both types of glial cells were shown to develop biochemical tolerance to pentobarbital as defined by an attenuated response to the depressant effects of a challenging dose of pentobarbital on cellular respiration in barbiturate-cultivated cells compared to those grown in drug-free medium. The biochemical tolerance was evident in the presence of glucose and succinate but not malate as substrate. This tolerance to pentobarbital was accompanied by increased activities of hexokinase, glucose-6-phosphate dehydrogenase, succinate dehydrogenase, and glutamate dehydrogenase and by a marked increase in the number of glial cell mitochondria as observed in electron micrographs. The results are interpreted to indicate a compensation of glial cells to the continuous presence of PB by an accelerated glucose uptake and metabolism, an accelerated metabolism of succinate, and an increased mitochondrial activity.

Effect of tolerance to morphine, ethanol and barbiturate on amphetamine circling in rats with a striatal dopamine lesion.

The (+)-amphetamine circling rate of rats with unilateral 6-OHDA lesions in the striatum was recorded. Morphine tablets were implanted subcutaneously for chronic treatment. In the morphine-dependent animal the circling rate to amphetamine given 4 days after morphine was first implanted was depressed but after withdrawal with naloxone a day later the rate increased, returning to normal after 21 days. Barbiturate physical dependence was induced by adding increasing amounts of barbitone to the drinking water of lesioned rats over four weeks after which the amphetamine circling response was depressed and remained so after the barbituate was withdrawn. Ethanol tolerance was induced by adding ethanol to the drinking water of lesioned rats for four weeks. Neither the induction of tolerance over this period nor ethanol withdrawal had any effect on the circling response to amphetamine. The change in the response of striatal dopamine neurons to amphetamine that occurs after chronic morphine treatment, cannot be produced by chronic treatment with either barbitone or ethanol. The neurochemical bases of barbiturate and ethanol tolerance are different from morphine tolerance.

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.

On the possible role of brain protein synthesis in functional barbiturate tolerance

Pentobarbital pellet implantation increased more than 200% the ED 50 dose of pentobarbital required to induce loss of the righting reflex within 2 min of i.p. injection and increased the onset of barbital-induced sleep. Both tests of functional barbiturate tolerance were blocked by the intraventricular injection of cycloheximide. The effects of acute (45 mg/kg i.p.) and chronic (pellet implantation) pentobarbital treatment on the incorporation of 3H-lysine into the protein of various subcellular fractions of the cortex and subcortex were studied. In the subcortex, chronic pentobarbital treatment significantly stimulated protein synthesis 40–50% in the microsomal, soluble and mitochondrial fractions. Both acute and chronic pentobarbital treatments significantly increased ( 3H-lys)-protein accumulation in a fraction of synaptic plasma membranes derived from a population of nerve ending particles (NEP) enriched in γ-aminobutyric acid (GABA). The possible significance of these data to pentobarbital tolerance and dependence development is discussed.

Vigilance and Arousal in Depressive States

An experiment was performed to investigate predictions of vigilance performance among depressive patients, based on the assumptions that vigilance would vary in a predictable manner with level of arousal, and that levels of arousal among diagnostic categories of depressive patients are well known. It was found that psychotic depressives, presumed to be hypo-aroused relative to normals, exhibited poor signal detection performances and committed few false positive errors relative to normals. This was consistent with predictions. Neurotic depressives, presumed to be hyper-aroused relative to normals, detected fewer signals than did normals, but also made more false positive errors than normals. Again this was consistent with predictions. A measure of arousal in experimental subjects, namely barbiturate tolerance, was found to directly relate to the false positive error rate in all subjects. The relationship between arousal and total signal detection rate was significantly curvilinear, and an 'inverted U' (quadratic) function provided the best fit. This justified the conclusion that vigilance performance is a function of at least that component of arousal measured by barbiturate tolerance.

Acute tolerance to barbiturate in the rat

3 experimental approaches to the quantitation of acute barbiturate tolerance have been compared in the rat. There was no difference between the brain hexobarbitone or barbitone concentration found at the time of loss of righting reflex compared with the concentration found on return of the righting reflex following the period of anaesthesia produced by a single i.p. injection of the drug. However, tolerance was induced by a 7 hr infusion of pentobarbitone which kept rats anaesthetized for approximately 8 hr. Such rats awakened with a significantly higher brain pentobarbitone concentration compared with rats awakening after a single i.p. injection. Repeated i.p. injections of pentobarbitone, sufficient to keep animals anaesthetized for 12 hr, also induced a tolerance to pentobarbitone, as indicated by a reduced sleeping time and higher brain barbiturate concentration on awakening following intracerebroventricularly administered pentobarbitone injected 12 hr after the last i.p. injection. The possible relationship between acute cellular tolerance and physical dependence is discussed.

A study on the development of barbiturate tolerance and dependence in hamster glial cells in culture

Hamster astroblast glial cells (clone NN) in cell culture were exposed to from 1 × 10 −5 to 3 × 10 −3 M pentobarbital-Na and from 1 × 10 −5 to 2·5 × 10 −4 M morphine hydrochloride for various periods of time. Profound morphological changes were induced in a dose/time-related fashion by pentobarbital only. These consisted of growing of cellular expansions, parallel cellular arrangement and increased amounts of intracellular material. Exposure of the cells to morphine up to 6 weeks resulted in a dose-correlated decreased rate of proliferation, but no specific morphological alteration could be observed. The morphological changes induced in cultured glial cells by pentobarbital were accompanied by an increase in oxygen consumption (35–45%) as well as an increase in glucose uptake (90–110%). These effects were compared to those obtained with bromodeoxyuridine which affected glucose metabolism similarly. Furthermore, glial cells that had been treated for 4 weeks with the barbiturate were less sensitive to the depression of oxygen consumption by a challenging dose of pentobarbital-Na signifying the development of cellular tolerance. After having cultured the cells in barbiturate for 4, 9 or 14 weeks, normal medium was substituted. This resulted in severe degenerative changes and cell deaths as well as altered growth characteristics in the surviving cells, demonstrating some degree of cellular dependence on the barbiturate. In addition, cross-tolerance with ethyl alcohol was established since it could be substituted for the barbiturate without the occurence of degenerative changes.

A model for the rapid development of dispositional and functional tolerance to barbiturates

The s.c. implantation of a 75 mg pentobarbital pellet in the back of a conscious mouse resulted in a much more rapid development of tolerance to barbiturates than that produced in mice receiving daily i.p. injections of 75 mg/kg sodium pentobarbital. Acceleration in tolerance development by pentobarbital pellet implantation was evidenced by a decrease in sleeping time after the challenge with either sodium pentobarbital or sodium barbital. The degree of hepatic microsomal drug enzyme induction after pentobarbital pellet implantation also was found to be significantly higher than that produced by the injection technique. Further studies demonstrated that the threshold for pentylenetetrazol-induced seizures was significantly reduced compared to that of the sodium pentobarbital daily injected and control groups. These studies provide an animal model for studying the mechanism of barbiturate tolerance and dependence.

Effects of Passive Immunization with Rabbit Anti-Phenobarbital IgG on Cyclobarbital-Induced Sleeping Time and Hepatic Enzyme Activities of C3H Mice

Effects of normal rabbit. IgG (N-IgG) and phenobarbital specific IgG (Ab-IgG) on cyclobarbital-induced sleeping time were studied. Ab-IgG was specifically purified by Immunoadsorbent from rabbit antisera obtained by immunization with p-azophenobarbital bovine gamma globulin. N-IgG was purified from normal rabbit sera by DEAE-cellulose chromatography. The aggregate-free IgG were passively immunized to C3H mice 1 hr before intraperitoneal injection of cyclobarbital which has a high binding affinity to the antibodies. The pretreatment with N-IgG prolonged significantly cyclobarbital-induced sleeping time as compared with that of saline treated group, however, in the Ab-IgG treated group the duration of the sleeping was much the same as that seen in the saline group. To determine whether Ab-IgG has any influence on barbiturate tolerance, effects of N-IgG and Ab-IgG on the activities of hepatic enzymes in 9, 000 g supernatant (alkaline RNase and aminopyrine demethylase) of the mice were examined at 40 hours after cyclobarbital administration. The activities of both enzymes which could he induced by cyclobarbital were decreased significantly in Ab-IgG treated group as compared with those of N-IgG group. These results are discussed with relation to barbiturate tolerance.