J.W. van der Laan

DT‐n‐PROPYLACETATE AND GABA DEGRADATION. PREFERENTIAL INHIBITION OF SUCCINIC SEMIALDEHYDE DEHYDROGENASE AND INDIRECT INHIBITION OF GABA‐TRANSAMINASE

The kinetic constants for 4‐aminobutyrate: 2‐oxoglutarate aminotransferase (GABA‐trans‐aminase) and succinate‐semialdehyde: NAD+ oxidoreductase (SSA‐DH) have been determined using rat brain homogenate.

Changes in locomotor-activity patterns as a measure of spontaneous morphine withdrawal: no effect of clonidine

Abstract The anti-withdrawal action of clonidine was studied in a model of spontaneous morphine withdrawal in rats. After withdrawal the behaviour of the animals was registered continuously for several days. In the initial phase of induction of dependence the locomotor activity was enhanced during daytime. Partial tolerance to this increase developed in the course of 3 weeks. In morphine-withdrawn animals the activity decreased strongly at night, and this effect was maximal on the second night after removal of morphine. After four nights the nightly activity was restored. Treatment with clonidine (100 μg/kg s.c. twice daily) changed neither the observed decrease in nightly locomotor activity nor other withdrawal symptoms such as a decrease in food intake and loss of body weight. In non-dependent animals clonidine induced a biphasic effect in locomotor activity, i.e. a decrease in the first few hours of the night and an increase in the second part of the night. The latter can be interpreted as a rebound phenomenon occuring after only three injections. It was concluded that clonidine was not effective as an anti-withdrawal compound in a model for spontaneous morphine abstinence. The low incidence of symptoms relating to a hyperactive sympathetic system during spontaneous withdrawal may be an explanation for the absence of an effect of clonidine.

A dual role for GABA in quasi-morphine abstinence behavior induced by di-n-propylacetate involving both initiation and termination.

Di-n-propylacetate (DPA) induces a behavioral syndrome in rats resembling morphine abstinence behavior. The inhibitory action of DPA on GABA degradation, resulting in an enhanced release of GABA, is probably responsible for this behavioral effect, since GABA antagonists, like bicuculline and picrotoxin, have been shown to suppress this behavior. However, the time-course of the DPA-induced behavior is much shorter than that of the DPA-induced increase of GABA concentrations. Therefore, we have studied the influence of enhanced GABA levels caused by a first injection of DPA and the behavior evoked by a second injection of DPA at different time intervals after the first injection. The results indicate that GABA fulfills a role in both the initiation and termination of DPA-induced behavior. The mechanism responsible for this dual action of GABA is ascribed to a differential sensitivity to DPA of the nerve terminal and glial metabolic compartments of GABA in the brain. The increase of GABA in the nerve terminal caused by DPA is probably responsible for the initiation of the quasiabstinence behavior, whereas the overflow of GABA into the synaptic cleft may be responsible for the suppression of this behavior via stimulation of presynaptic autoreceptors. Another mechanism responsible for the rapid termination of the DPA-evoked behavior could be the formation of DPA metabolites which antagonize this behavior. From the results of experiments using some primary metabolites of DPA, a role for these metabolites in the termination of the DPA-induced behavior seems unlikely.

Chronic infusion of clonidine does not alleviate spontaneous morphine withdrawal symptoms in rats

Opiate withdrawal is associated with behavioural symptoms and a sympathetic hyperactivity, the latter being sensitive to clonidine. The central question is whether behavioural symptoms would be also sensitive to clonidine. A rat model was used in which the locomotor activity was measured 24 h a day during the morphine withdrawal phase. Spontaneous withdrawal of morphine reduced strongly the high nocturnal locomotor activity, concomitently decreasing food intake and body weight. Chronic infusion of clonidine (30–120 µg/kg/day) using osmotic minipumps had no effect on the withdrawal symptoms. Higher dosages (250–1000 µg/kg/day) potentiated rather than alleviated the withdrawal symptoms, suggesting anα1-adrenergic effect of clonidine rather than anα2-action. Therefore, we studied the action of a more specificα2-agonist UK-14.304. UK-14.304 was less potent than clonidine in naive animals. It slightly alleviates the decrease of nocturnal activity during spontaneous morphine withdrawal. Furthermore, we have tested whether the effects of high dosages of clonidine could be altered by a specificα1-antagonist doxazosine. Doxazosine reduced only slightly the potentiation in the decrease in food intake by clonidine during morphine withdrawal. For the other symptoms no interaction between doxazosine and clonidine was found. The data suggest that the use of clonidine in the detoxification of opiate dependent people is based on the suppression of the sympathetic hyperactivity rather than on symptoms with a more behavioural character.

Tolerance and withdrawal after chronic lorazepam treatment in rats

Abstract The purpose of the study was to develop an animal model for benzodiazepine tolerance and dependence on the basis of oral administration, using lorazepam as the test drug. We have used the continuous measurement of locomotor activity in home cages to obtain a narrow estimation of the time course of withdrawal related hyperactivity as an observer-independent symptom. Acute administration of lorazepam (9.5–37.5 mg/kg body weight/day) resulted in the first week in a dose-dependent muscle relaxation on the accelerod and sedation in the open field. The most striking manifestation of sedation, however, was the decrease of nocturnal locomotor activity in home cages. After 5 weeks of administration tolerance to the sedative effect had developed. In a second study , using a lower dose range (2.5–9.5 mg/kg body weight/day), a decrease of nocturnal locomotor activity was also observed as was the development of tolerance. The latter can be partly explained by dispositional tolerance, i.e., decreased serum concentrations after administration of lorazepam for more than 2- weeks. Withdrawal of lorazepam in the experiment using high doses led to three symptoms, i.e., a decrease in food intake, loss of body weight and an increase in daytime locomotor activity. The decrease in food intake and the loss of body weight were maximal on the first day of withdrawal. The increase in the daytime locomotor activity was present in the high dose experiment only, with a maximum on days 2–3 and a duration of at least 1 week. The increase however, was not dependent on the dose previously administered. The symptoms ‘loss of body weight’ and ‘decrease in food intake’ appeared to be more sensitive in benzodiazepine withdrawal: they were dose dependently present over the whole dosage range (2.5–37.5 mg/kg body weight/day). It is concluded that the model represents a sensitive model to measure lorazepam tolerance and dependence in animals. Comparative studies with other benzodiazepines are in progress.

Dipropylacetate-induced quasi-morphine abstinence behaviour in the rat: Participation of the locus coeruleus system

Abstract The role of the noradrenergic system in quasi-morphine abstinence behaviour induced by di- n -propylacetate (DPA) in rats has been studied. Depletion of noradrenaline (NA), by treatment with FLA-63, decreased the number of body shakes and the extent of horizontal activity evoked by DPA. Almost total suppression of these symptoms was obtained by injection of 20 ng morphine bilaterally into the locus coeruleus (LC). Destruction of the LC system by electrolytic lesion of the LC or by injection of 6-hyrodroxy-dopamine into the dorsal bundle revealed that degeneration of the NA system by at least 80% appears to be necessary to decrease the number of body shakes. It is concluded that the noradrenergic LC system fulfills a modulatory role in quasi-morphine abstinence behaviour induced by DPA.

Effects of branched-chain fatty acids on GABA-degradation and behavior: Further evidence for a role of GABA in quasi-morphine abstinence behavior

An increase in GABA-ergic activity has been implicated in the initiation of quasi-morphine abstinence behavior by di-n-propylacetate (DPA). Two structural analogues of DPA, namely, the branched-chain-fatty acid 2-methyl, 2-ethylcaproic acid and 2,2-dimethylvaleric acid have now been used to study this relationship between behavioral and biochemical effects. A correlation appeared to exist between the K1 of these compounds for succinic semi-aldehyde dehydrogenase, the second enzyme in the degradation of GABA, and the doses exerting a maximum effect on behavior. On the other hand concurrent inhibition of GABA-transaminase seemed to suppress the behavioral effects of the fatty acids. This apparent paradox can possibly be explained by supposing a different action of the fatty acids in distinct compartments of the brain, suggesting an important role for increased GABA-ergic activity in the neuronal compartment in the initiation of the quasi-morphine abstinence behavior.

The concentration of morphine in serum of rats made dependent using a drug-admixed food method

The present study reports on the induction of physical dependence in rats using morphine-admixed food and addresses the question of the resulting concentration of morphine in serum. The stability of morphine in food is good, since no decrease in concentration could be observed. The concentration of morphine in serum during the experiment was measured using a radioimmunoassay technique. A correlation was found between the food intake during a 7-hour period and the concentration of morphine in the serum at the end of that period, both for a 1 g/kg and a 2 g/kg batch of morphine-admixed food. The concentration of morphine in serum was also found to be dose-related during a period of 6-23 days when the rats were fed for a prolonged period. After long-term administration of 1 g/kg morphine in food a steady-state level of about 0.5 mg/l serum was obtained. Similarly with 2 g/kg morphine in food a steady-state level of 0.8-1.1 mg/l serum was reached. After withdrawal of morphine the serum concentration of morphine dropped to 0.1 mg/l within 24 hours and to below the detection limit within 48 hours. During the withdrawal period sharp drops were noted in body weight (20%) and food intake (50%) after one day.

Differences in the duration of sedative and anxiolytic effects of desmethyldiazepam in two outbred Wistar strains

Different sensitivities to benzodiazepines have been described for various strains of both rats and mice suggesting that variations in biological features of the animals are responsible for these differences. Since all reports concern inbred strains, we studied two outbred Wistar strains which are used routinely in several research disciplines. The pharmacodynamics of desmethyldiazepam (DMD), the main active metabolite of diazepam in man, were compared for male rats of the Riv:TOX strain (from the National Institute of Public Health and Environmental Protection) and the Crl:(WI)BR strain. The duration of sedative action of DMD after oral administration, as derived from suppression of the nocturnal locomotor activity, was longer in the Riv:TOX strain than in the Crl:(WI)BR strain. Accordingly, suppression of novelty-induced corticosterone release as an index of anxiolytic action was observed 11 hours after DMD administration in Riv:TOX rats but not in Crl:(WI)BR rats. At that time, serum DMD concentration was shown to be higher in the Riv:TOX strain than in the Crl:(WI)BR strain. The data are discussed in relation with possible metabolic differences between the two strains.

GABA degradation and its possible relationship to the “morphine abstinence” syndrome

Abstract Dipropylacetate (Valproate) is used as a therapeutic agent in the treatment of petit mal epilepsia. Its mechanism of action has been ascribed to an inhibition of GABA-T. However, inhibition of SSA-DH, an enzyme involved in the second step of the degradation of GABA, by DPA has also been reported. The kinetics of GABA-T are consistent with Ping Pong Bi Bi mechanism. A K eq of 0.04 for GABA-T was found, indicating that the reaction was strongly biased towards GABA. DPA inhibited SSA-DH much strongly biased towards GABA. Acute IP administration of DPA to rats evoked a behavioural syndrome resembling the morphine abstinence syndrome including “wet dog shakes”. This effect of DPA was prevented by the administration of low doses of morphine (ED 50 =0.5 mg/kg IP). The inhibitory effect of morphine could be antagonized by naloxone. Since the principal action of DPA is an inhibition of SSA-DH, structural analogues of DPA were tested for inhibition of SSA-DH and for induction of the behavioural syndrome. A qualitative relationship between inhibition of SSA-DH and induction of abstinence behaviour was present.