The sensitivity of the brain to barbiturate during chronic administration and withdrawal of barbitone sodium in the rat

1. The sensitivity of the central nervous system to barbiturate was determined in rats during the chronic administration of barbitone sodium and after its withdrawal.

2. The brain barbiturate concentration determined on awakening from a hypnotic dose administered intraperitoneally was found to increase throughout the period of barbitone administration.

3. A similar gradual development of central nervous system tolerance was indicated by measuring the duration of anaesthesia following an intraventricular injection of pentobarbitone.

4. The change in sensitivity of the brain which occurred during the period of barbitone administration was not demonstrable from the measurement of sleeping time following intraperitoneal injection of barbitone or pentobarbitone.

5. After withdrawal, the sensitivity of the brain to barbiturate gradually returned to normal.

6. It was concluded that the hypersensitivity to pentobarbitone, but not to barbitone, which develops after withdrawal of barbitone sodium is due to a decreased drug-metabolizing capacity.

     

Development of tolerance to the prolongation of Hexobarbitone sleeping time caused by cannabidiol

1 The effects of acute and subacute cannabidiol (CBD) administration on hexobarbitone sleeping time and on some constituents of the hepatic microsomal drug-metabolizing system were assessed in the mouse.

2 Acutely administered CBD prolonged sleeping time; but with subacute treatment, tolerance to the effect rapidly developed.

3 Brain hexobarbitone concentration upon awakening was unchanged by either acute or subacute CBD treatment, which suggests that neither the prolongation of sleeping time nor the tolerance is the result of a change in sensitivity of the central nervous system to the barbiturate.

4 Acute CBD treatment increased the half-time of hexobarbitone in the brain, which returned toward normal with the development of tolerance.

5 Acutely, CBD caused a 30% decrease in hepatic cytochrome P-450 level; with tolerance, the cytochrome concentration returned to normal.

6 The evidence suggests that the CBD-induced prolongation of barbiturate sleeping time and the tolerance to this effect are the result of changes in the rate of drug metabolism, which are related to changes in the amount of cytochrome P-450.

7 The effects of CBD on the hepatic microsomal drug-metabolizing enzyme system are different from those attributed to SKF 525-A and piperonyl butoxide because the cannabinoid does not decrease cytochrome P-450 quantitatively by complex formation, it does not produce a recovery overshoot in the cytochrome concentration and, finally, it does not cause an induction of the hexobarbitone-metabolizing enzymes.

     

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.     

Barbitone-induced tolerance to the effects of sedative-hypnotics and related compounds on operant behaviour in the rat

1 Pretreatment doses of barbitone, pentobarbitone, ethanol, and phenytoin (diphenylhydantoin) in non-tolerant rats produced increases in operant responding at low doses and at higher doses resulted in decreases in responding.

2 Daily barbitone injections (100 mg/kg, i.p.) resulted in the development of functional tolerance to both the stimulant and depressant effects of barbitone on responding.

3 Barbitone tolerance development did not result in any change in the brain or plasma pharmacokinetics of barbitone.

4 Barbitone-tolerant rats were cross-tolerant to the behavioural effects of pentobarbitone, ethanol, and phenytoin. The dose-effect curves for all of these drugs were shifted to the right in tolerant rats, compared to non-tolerant rats.

5 Comparison of the brain and plasma levels of these drugs in non-tolerant and tolerant rats provided a means of separating functional cross-tolerance from dispositional cross-tolerance. Barbitone-tolerant rats appeared to be functionally cross-tolerant to ethanol in that there was no change in the brain and blood ethanol levels at times when the degree of behavioural impairment was substantially reduced. In contrast to ethanol, cross-tolerance to phenytoin appeared to be due to a decrease in the brain and plasma levels (dispositional tolerance). Cross-tolerance to pentobarbitone appeared to be comprised of both functional and dispositional cross-tolerance.

6 The usefulness of a multidisciplinary approach in the analysis of sedative hypnotic tolerance and cross-tolerance is discussed. It is concluded that without the concurrent determination of both brain and plasma drug levels it would not be possible to distinguish between functional and dispositional tolerance.

     

Mechanisms contributing to barbiturate intolerance in rats

1. Female rats, 3 weeks after pretreatment with 200 or 400 (mg/kg)/day barbitone for 2 or 30 days, exhibited a prolonged sleeping time and a reduced awakening barbiturate brain level when challenged with either barbitone or pentobarbitone. After 3 additional weeks, the latter responses had returned, or were returning to, control values.2. Barbitone pretreatment schedules had no residual effect upon in vitro hepatic pentobarbitone-metabolizing activity measured 3 or 6 weeks later, except in one instance, when hepatic enzyme activity was significantly enhanced 3 weeks after 30 daily doses of 200 mg/kg barbitone. In this case, however, an enhanced barbiturate sleeping time, together with a reduced awakening barbiturate brain level, were observed.3. It is concluded that barbitone administered intraperitoneally in doses of 200 to 400 (mg/kg)/day for 2 or 30 days induces a non-hepatogenic intolerance to barbiturates, related to an increased sensitivity of the central nervous system to these drugs. This central intolerance is seen 3 weeks, but not 6 weeks, after pretreatment. Furthermore, this central intolerance has been observed to co-exist with an hepatic tolerance, a situation which could result in a reduced LD(50) coupled with an increase in ED(50).     

Determination of halothane-induced sleeping time in the rat: effect of prior administration of centrally active drugs.

A method is described for the determination of halothane-induced sleeping time in the rat. 2 The sleeping time exhibited a diurnal variation which was due, at least in part, to a change in the sensitivity of the central nervous system (CNS) to the anaesthetic. 3 Tolerance to halothane did not develop in rats repeatedly exposed to the anaesthetic over a period of over 48 hours. 4 Repeated sleeping time determinations have been used to follow changes in the sensitivity of the CNS to the anaesthetic occurring with time. 5 A tolerance to halothane was induced by pretreatment of rats with doses of amylobarbitone, pentobarbitone or meprobamate sufficient to keep animals anaesthetized for approximately 12 hours. This tolerance was followed by a period of halothane-hypersensitivity. 6 Halothane-tolerant animals awakened with higher brain halothane concentrations and were also tolerant to intracerebroventricularly administered pentobarbitone. 7 Halothane-hypertensive rats awakened with lower brain halothane concentrations and were also hypersensitivity to intracerebroventricularly administered pentobarbitone. 8 The possibility that the induction of cross-tolerance to halothane may be indicative of a drug's potential to produce dependence is discussed.     

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 barbiturate 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.     

The effects of oestrogens and progestins on the response of mice to barbiturates

1. Mestranol (oestrogen) prolonged, whilst lynestrenol (progestin) reduced, the duration of pentobarbitone and hexobarbitone sleep in mice, whilst the effects of barbitone were not altered.

2. The effects of these steroids on pentobarbitone sleep were dose-related, did not show tachyphylaxis, and produced optimal effects after only 4 days pretreatment.

3. The effects of lynestrenol were abolished by SKF 525A, whilst those of mestranol were markedly potentiated, suggesting a different mechanism and/or locus of action for mestranol and SKF 525A.

4. Examination of plasma levels of pentobarbitone in mice pretreated with mestranol, lynestrenol or SKF 525A showed that lynestrenol increased whilst mestranol and SKF 525A reduced the rate of clearance of barbiturate from the plasma.

5. The effects of lynestrenol disappeared when pentobarbitone was prevented from inducing hypothermia, whilst some significant prolongation of pentobarbitone sleep persisted in mestranol treated mice. This suggested that the ability to potentiate hypothermia was not the sole mechanism by which the effects of pentobarbitone were enhanced by mestranol.

6. It is concluded these steroids alter the duration of action of pentobarbitone (and hexobarbitone) by changing the rate of barbiturate metabolism. In the case of mestranol, this might be a combination of an effect upon basal metabolic rate (enhancing hypothermia) and a direct effect on the liver. An effect upon renal clearance cannot be excluded by these results.

     

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.     

Effects of 4-aminopyridine on acetylcholine output from the cerebral cortex of the rat in vivo

1 The effects of 4-aminopyridine (4AP) on the output of acetylcholine (ACh) from the cerebral cortex were investigated in unanaesthetized freely moving rats and in anaesthetized rats by means of the `cup technique'. ACh was determined by bioassay on the dorsal muscle of the leech.

2 In unanaesthetized rats intraperitoneal injection of 4AP (3 mg/kg) had no effect on the cortical output of ACh.

3 After injection of morphine (10 mg/kg s.c.), which depressed the spontaneous output of ACh, 4AP increased the cortical output to a level significantly higher than that determined before morphine injection.

4 In rats anaesthetized with either urethane or pentobarbitone, drugs known to decrease cortical output of ACh, 4AP (i.v. or i.p.) elicited a significant increase in the output of ACh. The time-courses of the 4AP-induced effects were different depending on the anaesthetic drug used: an immediate increase slowly fading in urethane anaesthesia and a gradual increase after delayed onset in pentobarbitone-anaesthetized rats.

5 In some urethane-anaesthetized rats, respiratory frequency was kept constant (tracheotomy, connection to respirator, bilateral vagotomy) and prazosin (1 mg/kg i.v.) was administered to reduce the 4AP-induced increase of blood pressure. Cortical output of ACh was not related to changes in blood pressure. Moreover, the 4AP-induced increase in cortical ACh output was not related to changes in respiratory frequency.

6 In summary systemic administration of 4AP in subconvulsive doses (1 and 3 mg/kg) increased cortical output of ACh in rats anaesthetized with urethane or pentobarbitone or after injection of morphine, but not in untreated freely moving rats. It is suggested that the anaesthetic agents and morphine may cause an imbalance between excitatory and inhibitory central pathways, and that this imbalance may play a role in their depressant effect on cortical output of ACh and/or in the 4AP-induced facilitation described in this paper.

     

Naloxone interaction with some CNS depressants

Mice were either pretreated with naloxone (10 mg/kg i.p.) and then injected with a hypnotic, or given a similar dose of naloxone simultaneously with a hypnotic (combination treatment). Neither the naloxone pretreatment nor the combination treatment changed the time of on-set or the sleeping duration of urethane, chloral hydrate or ketamine. However, the naloxone pretreatment markedly decreased the on-set time and prolonged the sleeping duration of phenobarbitone and barbitone; naloxone in combination prolonged the sleeping duration of pentobarbitone, phenobarbitone and barbitone.     

Central respiratory and circulatory effects of Gymnodinium breve toxin in anaesthetized cats

1 In cats anaesthetized with pentobarbitone, observations were made on respiration, spontaneous and evoked diaphragmatic electromyograms, blood pressure, heart rate, indirectly-induced contractions of the anterior tibialis muscle and nictitating membrane, and electrical excitability of the inspiratory centre in the medulla oblongata.

2 Gymnodinium breve toxin (GBTX) was administered intravenously, intra-arterially to the brain, and intracerebroventricularly. Physiological effects were recorded while alveolar P_CO2 was controlled at a constant level except when changes in gas tension were made in order to measure CO₂-ventilatory responsiveness.

3 Adequate doses of GBTX given intravenously by bolus injection elicited a non-tachyphylactic reflex response triad of apnoea, hypotension and bradycardia mediated by the vagus nerves independently of arterial baroreceptor and chemoreceptor innervation.

4 After vagotomy, additional amounts of GBTX (i.v.) resulted in apneustic breathing, hypertension and tachycardia. The cardiovascular effects were abolished by ganglionic blockade with hexamethonium.

5 Smaller doses of GBTX were required intra-arterially and intracerebroventricularly than by the intravenous route of injection to produce respiratory irregularity and cardiovascular hyperactivity.

6 Evoked motor responses, electrical excitability of the medulla oblongata and CO₂-ventilatory responsiveness were largely spared even though GBTX caused marked disturbances in respiratory rhythmicity and cardiovascular functions.

7 It is concluded that GBTX acts reflexly on vagally innervated receptors to evoke a Bezold-Jarisch effect but that the toxin further acts centrally to cause irregular breathholding and hypertension with tachycardia, leading ultimately to respiratory and circulatory failure.

     

Pharmacological and electrophysiological studies of morphine and enkephalin on rat supraspinal neurones and cat spinal neurones

1 The actions of morphine, methionine and leucine enkephalin, administered electrophoretically, were studied on supraspinal neurones in the cortex and brainstem of the rat anaesthetized with urethane and on spinal Renshaw cells and dorsal horn interneurones in the cat anaesthetized with pentobarbitone.

2 The majority of Renshaw cells and cortical and brainstem neurones were excited by all three compounds although some supraspinal neurones were depressed.

3 Naloxone reversibly antagonized both excitatory and depressant actions of morphine and enkephalin. Acetylcholine-induced excitation but not amino acid-induced excitation was also antagonized by naloxone.

4 Neither morphine nor the enkephalins had any naloxone-reversible action on dorsal horn neurones when ejected from conventional multibarrelled electrodes. However, morphine but not enkephalin, administered into the substantia gelatinosa region of the spinal cord selectively reduced responses to noxious stimuli of neurones in deeper laminae. Naloxone administered into the same region antagonized this action of morphine.

5 Intravenous morphine also antagonized responses of dorsal horn neurones to noxious stimuli and subsequent intravenous naloxone reversed this effect.

6 It was concluded that the excitatory and inhibitory effects of morphine and enkephalin on central neurones may be mediated by actions on different opiate receptors and that depression of noxious responses of dorsal horn neurones may be relevant to the analgesic action of morphine.

     

The effects of drugs on barbiturate withdrawal convulsions in the rat

The effects of drugs on barbitone withdrawal convulsions in rats have been examined. Morphine and mebanazine had no effect on audiogenically induced barbiturate withdrawal seizures. Alcohol, although suppressing the seizures, did not maintain drug dependence. Chlorpromazine prolonged the recovery period after the induction of the convulsions. Meprobamate, chlordiazepoxide and primidone substituted for the barbiturate and maintained drug dependence. 5-Hydroxytryptamine, when administered intraventricularly, tryptophan, α-methyl-p-tyrosine and ethosuximide reduced the severity of the withdrawal seizures. Reserpine and p-chlorophenylalanine greatly increased the severity of the seizures. Anxiolytic sedatives substituted for barbitone in dependent animals, other drugs studied affected barbiturate withdrawal convulsions in a way similar to other convulsive processes.     

The effects of certain anaesthetic and anti-convulsant drugs on the CSF potassium fluxes of the dog

1 A technique has been developed for open-ended perfusion of the cerebroventricular system of the unanaesthetized dog.

2 Perfusion with an artificial CSF solution containing inulin and ⁴²K allowed the potassium fluxes out of and into the CSF to be monitored over a period of 2 to 3 hours.

3 Sodium thiopentone and sodium pentobarbitone, in doses producing light anaesthesia, caused varying degrees of depression (up to 50%) in the CSF potassium fluxes, influx being consistently more affected than efflux. These effects are attributed to a decrease in the potassium exchange between extracellular and intracellular compartments in the brain.

4 Diazepam depressed both potassium fluxes by up to 10% while there was some evidence that diphenylhydantoin depressed only potassium influx.

5 Paraldehyde, in contrast to the other drugs, when given at a dose level sufficient to produce light anaesthesia, stimulated CSF potassium fluxes, particularly efflux.

     

Action of intracerebroventricular dibutyryl cyclic AMP on amobarbital anaesthesia in rats

The ability of intracerebroventricular exogenous administration of N⁶,O²'-dibutyryl analog of adenosine 3':5'-cyclic monophosphate (db cyclic AMP) to shorten the duration of amobarbital anaesthesia in a dose-related manner has been shown in the rat. Alteration of the duration of sleeping time (ST) appeared to be specific to db cyclic AMP since neither adenosine nor guanosine nucleotides administered intracerebroventricularly produced any appreciable effects. Similarly, intracerebroventricular injected succinate or pyruvate did not alter amobarbital-induced ST. None of the behavioral symptoms observed following the intracerebroventricular injection of db cyclic AMP in the unanaesthetized conscious rat were evident in the amobarbital-anaesthetized rat.     

Mechanism of induction of hepatic microsomal drug metabolizing enzymes by a series of barbiturates.

The inducing effect of certain barbiturates (secobarbitone, thiopentone, pentobarbitone, allobarbitone, phenobarbitone and barbitone) on the levels of the hepatic microsomal drug-metabolizing enzymes has been studied in the rat both in vivo and in vitro. The extent of induction was related to the plasma half-lives of the barbiturates; compounds with low rates of metabolism and long half-lives were the most potent inducing agents. The latter (phenobarbitone, pentobarbitone and allobarbitone) were shown by spectral technique to interact with cytochrome P-450 suggesting that their mechanism of enzyme induction was 'substrate induction' in type. Barbiturates containing an allyl group (secobarbitone and allobarbitone) had a weaker inducing effect than expected, possibly due to their destruction of cytochrome P-450. Despite its short plasma half-life of 0-5 h thiopentone was a relatively potent inducer probably due to its metabolism to pentobarbitone, which has a much longer plasma half-life (1-3 h). Barbitone is an effective inducer of the drug-metabolizing enzymes, yet does not interact spectrally with cytochrome P-450; this is in accord with the observations that although there are increases in NADPH-cytochrome c reductase and cytochrome b5, following administration of barbitone there is no increase in cytochrome P-450. Barbiturate pretreatment does not affect the activities of glucose-6-phosphatase, glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase.     

Methyl xanthines, Adenosine 3′,5′-cyclic monophosphate and the spinal transmission of nociceptive information

1 In spinal cats anaesthetized with either α-chloralose or sodium pentobarbitone, a study was made of the effects of adenosine 3′,5′-cyclic monophosphate (cyclic AMP), mono- and di-butyryl cyclic AMP and the methyl xanthines, theophylline and isobutyl methyl xanthine (IBMX), on the responses of neurones of laminae I, IV and V to noxious and innocuous skin stimuli. The compounds were administered from micropipettes positioned in the substantia gelatinosa. IBMX was also given intravenously.

2 When administered in the substantia gelatinosa, neither cyclic AMP, its butyryl derivatives, nor the methyl xanthines had any effect on the excitation of neurones of spinal laminae IV and V by noxious heating of the skin or deflection of hairs. When the nociceptive responses of cells had been reduced by electrophoretic morphine, methyl xanthines and cyclic AMP failed to modify the effects of morphine on these deeper neurones. Electrophoretically administered naloxone reversed the effect of morphine.

3 Intravenously administered IBMX (1 to 2 mg/kg) produced large transient increases in the firing rate of both C fibres and the excitation of dorsal horn neurones by noxious heating of the skin. These increases coincided with decreases in the mean systemic blood pressure, and probably resulted from increased temperatures being attained in the dermis by each noxious stimulus. When dorsal horn neurones were activated by electrical stimulation of the tibial nerve by a stimulus adequate to excite C fibres, intravenous IBMX produced a small or no increase in the number of spikes per stimulus.

4 These results in the spinal cord do not support the hypothesis that the inhibition of synthesis of cyclic AMP is relevant to the analgesic action of morphine in mammals.

     

Effect of chronic administration and withdrawal of sodium barbitone on protein synthesis of rat brain

After chronic administration of sodium barbitone to rats, a marked increase incorporation of [14C]-Leucine into isolated nerve endings was observed. Withdrawal of the drug resulted in a decreased incorporation with respect to values obtained with chronic ingesting animals. On the other hand chronic administration of the barbiturate produced a decreased incorporation in mitochondrial and microsomal fractions. These results are discussed in relation to the development of tolerance and abstinence syndrome to this drug.     

Toxicity of ethanol-barbiturate mixtures

The simultaneous administration of ethanol at doses of either 2, 3, or 4 g/kg intraperitoneally produced a dose-related decrease in the intraperitoneal LD50 for thiopentone, pentobarbitone, amylobarbitone, phenobarbitone and barbitone in rats. The most marked ethanol-barbiturate interaction was with the long-acting, poorly metabolized, less potent barbiturates phenobarbitone and barbitone. Similarly, a non-hypnotic dose of ethanol (3 g/kg, i.p.) produced a much greater prolongation of the sleeping time with non-hypnotic doses of phenobarbitone and barbitone, than with threshold doses of the shorter acting barbiturates. Various postulates are advanced to explain the underlying mechanism of the barbiturate-ethanol interaction.