Philippe Soubrié

Attenuation of induced-anxiety in rats by chlordiazepoxide: Role of raphe dorsalis benzodiazepine binding sites and serotoninergic neurons

In chronically implanted awake rats, microinjections of chlordiazepoxide (5 x 10(-7) M) into the dorsal raphé significantly attenuated the inhibition of lever-pressing for food elicited by a signal of punishment. This effect is abolished by prior application of 5,7-dihydroxytryptamine into the dorsal raphé (3 weeks after the infusion of the neurotoxin, dorsal raphé tryptophan hydroxylase activity was reduced to 25% of control values). Furthermore, the disinhibitory effect of intra raphé chlordiazepoxide can be mimicked or potentiated by intra raphé dorsalis application of serotonin (10(-7) or 10(-8) M, respectively). Further evidence for a crucial interaction between benzodiazepines and serotoninergic processes are provided by in vitro experiments showing that chlordiazepoxide or diazepam (10(-5) M) are able to facilitate the K+-evoked [3H]serotonin release from rat midbrain slices. Finally, a high density of [3H]flunitrazepam binding sites was found in the dorsal (and the median) raphé nucleus, the Kd and Bmax values being not altered by prior infusion of 5,7-dihydroxytryptamine. These in vitro data suggest possible means by which intra raphé (and perhaps peripherally administered) benzodiazepines may affect the activity of serotoninergic neurons and thereby produce their effects on experimental anxiety.

In vivo release of met-enkephalin in the cat brain

Abstract Push-pull cannulae were implanted into the globus pallidus (anterior part), the caudate nucleus (medio-dorsal part) and the substantia nigra of halothane-anaesthesized cats to study the characteristics of the in vivo release of met-enkephalin. Met-enkephalin was measured by radioimmunoassay (sensitivity: 0.1 pg) and identified by Bio-Gel P 2 chromatography and high-pressure liquid chromatography. Under resting conditions, met-enkephalin was detected in perfusates from these three regions; in the globus pallidus, the rate of spontaneous release of met-enkephalin was 5–6 times higher than that observed in the caudate nucleus and the substantia nigra. The local application of either an excess (60 mM) of K + or of glutamate (10 μM) produced a marked increase in the rate of met-enkephalin release both from the globus pallidus and the caudate nucleus. Further analyses in the globus pallidus indicated that the depolarizing agents, veratridine (50 μM) and batrachotoxin (0.1 μM), produced a large increment in the rate of met-enkephalin release; this effect was completely prevented by the local application of tetrodotoxin (2 μM). The chemical (with 60 mM K + or 10 μM glutamate), electrical or mechanical stimulation of the dorsomedial part of the caudate nucleus failed to significantly alter the rate of met-enkephalin release in the ipsilateral globus pallidus, in spite of the high sensitivity of enkephalinergic nerve terminals in the globus pallidus itself to local stimuli. This observation argues against the existence of a major caudatopallidal enkephalinergic pathway.

Evidence against the involvement of serotonergic neurons in the anti-punishment activity of diazepam in the rat

The effects of manipulating central serotonergic transmission were assessed on the anti-punishment effects of diazepam (2 mg/kg IP) in rats. In a paradigm involving the inhibition of pressing for food induced by the delivery of a signal previously associated with electric foot-shocks, lesioning serotonergic neurons of the dorsal raphé with the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT; 1 μg in 0.4 μl) neither affected behavioral inhibition in control rats nor modified the ability of diazepam to release responding. Furthermore, suppression of pressing for food induced by a fixed ratio 7 schedule of shock presentation was reduced by bilateral infusion of 5,7-DHT (2 μg in 0.5 μl) into the substantia nigra, but the ability of diazepam to increase punished responding was preserved. Finally, blockade of benzodiazepine-induced decrease in serotonin release by application of the benzodiazepine receptor antagonist Ro 15-1788 (10−5–10−4 M in 0.2 μl) into the dorsal raphé did not alter the releasing effect of diazepam on suppression of pressing for food caused by a signal of punishment. At these concentrations. Ro 15-1788 was devoid of any effect on behavioral inhibition in control rats. Taken together, these results indicate that the anti-punishment activity of benzodiazepines can be dissociated from the reduction in tryptaminergic transmission produced by these drugs.

The progressive ratio schedule as a model for studying the psychomotor stimulant activity of drugs in the rat

Male Wistar rats were trained to press a lever with food reinforcement according to a continuously reinforced schedule (CRF). Afterwards, rats were subjected to three experimental sessions (30 min each) during which responding was rewarded according to a progressive ratio schedule (following an initial 2-min CRF period, the number of presses necessary for the pellet delivery was doubled every second minute). Responding during the first half of each session, i.e., pressing for food, was maintained at a significant level, whereas it was almost suppressed during the second part of the session. As compared to controls (200±20 presses/30 min) animals given amfonelic acid (0.5, 1 mg/kg IP), methylphenidate (4, 8 mg/kg IP), caffeine (16 mg/kg IP), cocaine (4 mg/kg IP), oxolinic acid (32 mg/kg IP), nomifensine (4 mg/kg IP), DR 250 (2, 4 mg/kg IP) and d-amphetamine (0.25, 0.5, 1 mg/kg IP) showed an increased rate of responding ranging from 400 to 950 presses/30 min. In contrast, apomorphine, MK 486+l-dopa, trihexyphenidyl, imipramine, salbutamol and diazepam did not increase responding. These results suggested that this test is highly sensitive for psychomotor stimulants and perhaps for their ability to enhance the reinforcing value of the reward or stimuli associated with the reward. Such activity seemed related to a catecholaminergic substrate since the increase of responding induced by amphetamine was blocked by pimozide, d,l-propranolol and prazosin.

The involvement of nigral serotonin innervation in the control of punishment-induced behavioral inhibition in rats

In rats, serotonergic innervation of the substantia nigra plays a role in the control of experimentally-elicited anxiety: punishment-induced inhibition is lessened following bilateral intra-nigral infusion of 5,7-dihydroxytryptamine (2 micrograms; 0.5 microliter). A significant correlation (0.62) is found between the loss of nigral, but not hippocampal, tryptophan hydroxylase activity and the release of behavior in two situations of shock-induced suppression of responding. Likewise, infusion of this neurotoxin (1 microgram; 0.4 microliter) into the nucleus raphé dorsalis causes an attenuation of punishment-induced suppression. These findings suggest an involvement of serotonergic raphé-nigral neurons in experimentally-elicited anxiety.

Effects of neuroleptic drugs, clonidine and lithium on the expression of conditioned behavioral excitation in rats

Rats with a history of daily (21 days) amphetamine (2.5 mg/kg) treatment showed enhanced activity when under placebo in their amphetamine-associated environment. We found that this conditioned effect was reduced by haloperidol (0.06; 0.125; 0.25 mg/kg), pimozide (0.25; 0.5 mg/kg) and sulpiride (8; 16; 32 mg/kg) but only at doses similar to or, in the case of pimozide, higher than those required to antagonize the unconditioned stimulant effects of amphetamine (2.5 mg/kg). Conversely, we observed that clonidine (7; 15; 30; 60 μg/kg) or lithium regimen (between days 15 and 21) leading to lithium plasma levels of 1.3±0.1 mEq/1, abolished amphetamine-conditioned hyperactivity but did not affect the unconditioned stimulation of amphetamine or locomotor activity in control rats. Moreover, we found that hyperactivity induced by the daily anticipation of food delivery shared identical pharmacological sensitivity with the behavioural excitation produced by a conditioning history with amphetamine. In light of the antimanic properties of lithium and clonidine and the ability of this latter drug to reduce noradrenergic transmission, our findings raise the posibility that incentive activity may model noradrenergic-dependent aspects of mania.

Diazepam-induced release of behavior in an extinction procedure: Its reversal by Ro 15-1788

The effects of the benzodiazepine receptor antagonist Ro 15-1788, an imidazobenzodiazepine derivative, were studied with respect to three pharmacological activities exerted by diazepam in rats. Two of these, release of shock-induced suppression of drinking and attenuation of non-reward-induced cessation of responding for food, reflect the anxiolytic property of benzodiazepines. The amnesic-like effect of diazepam was also investigated. Ro 15-1788 (in doses ranging from 4 to 16 mg/kg p.o.) completely reversed diazepam (2 mg/kg)-induced release of behavior in both punishment and non-reward procedures. In contrast, Ro 15-1788 reduced but did not completely abolish diazepam-induced amnesia. These data suggest that the anticonflict and anti-frustration effects of benzodiazepines probably involve similar receptor types which nevertheless differ from those chiefly implicated in the amnesic-like activity of benzodiazepines.

Application ofl-glutamic acid and substance P to the substantia nigra modulates in vivo [3H]serotonin release in the basal ganglia of the cat

L-Glutamic acid or substance P were applied to the caudate nucleus (CN) or substantia nigra (SN) and their effects on local, spontaneous, in vivo [3H]serotonin ([3H]5-HT) release as well as [3H]5-HT release in the contralateral CN and SN were studied using cats implanted with push-pull cannulae. L-Glutamic acid (5 x 10(-5) M), when applied to the CN or SN inhibited the local release of [3H]5-HT but did not affect release in the contralateral CN and SN. In the SN, the L-glutamic acid effect was blocked by L-glutamic acid diethylester. Substance P (10(-7) M) applied to the SN induced an increase in [3H]5-HT release that was delayed in onset. Furthermore, [3H]5-HT release was elevated in the contralateral CN immediately upon the application of substance P to the SN. These results suggest that L-glutamic acid and substance P may control 5-HT transmission in the basal ganglia.

In vivo evidence for gabaergic control of serotonin release in the cat substantia nigra

Push-pull cannulae were used for estimating the release of endogenously synthesized [3H]serotonin in both substantia nigra and caudate nuclei of halothane-anaesthetized cats. The unilateral nigral application of GABA (10-5 M) reduced the local release of [3H]serotonin picrotoxin induced an opposite effect. Both treatments failed to modify [3H]serotonin release in the caudate nuclei or in the contralateral substantia nigra. These results suggest that GABAergic neurons innervating the substantia nigra may regulate nigral serotonin transmission. The possibility that such a regulation could be presynaptic (direct or through other nigral neurotransmitters) or related to a change in the activity of the nigro-raphe projection is discussed.

Distinct modifications by neurokinin1 (SR140333) and neurokinin2 (SR48968) tachykinin receptor antagonists of the N-methyl-d-aspartate-evoked release of acetylcholine in striosomes and matrix of the rat striatum

The effects of SR140333 and SR48968 (neurokinin1 and neurokinin2 tachykinin receptor antagonists, respectively) on the N-methyl-D-aspartate-evoked release of [3H]acetylcholine (previously formed from [3H]choline) were investigated in striosome-enriched areas and in the matrix of the rat striatum using an in vitro microsuperfusion method. In both striatal compartments, SR140333 and SR48968 did not modify the 50 microM N-methyl-D-aspartate-evoked release of [3H]acetylcholine. However, in low concentrations, both SR140333 (0.1 microM to 1 pM) and SR48968 (0.1 microM to 0.1 nM) markedly enhanced the 1 mM N-methyl-D-aspartate (+10 microM D-serine)-evoked release of [3H]acetylcholine in striosome-enriched areas. These responses were dopamine-dependent since they were not observed any more following the local blockade of D2 receptors by sulpiride or of dopamine synthesis by alpha-methyl-p-tyrosine. A dopamine-dependent disinhibitory effect (of lower amplitude) on the 1 mM N-methyl-D-aspartate (+10 microM D-serine)-evoked release of [3H]acetylcholine was also induced by SR48968 (0.1 microM to 0.1 nM) (but not by SR140333) in the matrix. In addition, in the matrix, as shown only in the presence of alpha-methyl-p-tyrosine, both SR140333 and SR48968 reduced the 1 mM N-methyl-D-aspartate (+10 microM D-serine)-evoked response and these non-dopamine-mediated inhibitory effects only occurred at the highest tested concentration (0.1 microM) of the antagonists. Indicating the specificity of these responses, the effects of SR140333 were reproduced by RP67580, another neurokinin1 receptor antagonist and, as expected from previous binding studies, corresponding SR140333 and SR48968 enantiomers were without effect. These results suggest that under potent stimulation of N-methyl-D-aspartate receptors, endogenously released substance P and neurokinin A (or related tachykinins) regulate differently the N-methyl-D-aspartate-evoked release of [3H]acetylcholine in striosomes and in the matrix. The inhibitory effects of these tachykinins on the evoked release of [3H]acetylcholine are mediated by dopamine. On the contrary, their facilitatory responses are only observed in the matrix under blockade of dopamine transmission.