A. Herbet

Specificity of dopaminergic neuronal degeneration induced by intracerebral injection of 6-hydroxydopamine in the nigrostriatal dopamine system

The neurotoxic specificity of injections of 6-hydroxydopamine (6-OHDA) into areas containing either dopamine (DA) cell bodies (substantia nigra) or DA axon terminals (striatum) was studied. This selective effect was compared to the unspecific effects of copper sulfate (CuSO4) injection and electrocoagulation. One to two days after unilateral nigral injection of 2 mug of either 6-OHDA or CuSO4 into the nigra the volume of the unspecific lesions around the tip of the cannula was very similar. Only the 6-OHDA-induced lesions were associated with elective degeneration of the nigral DA neurons. Ten days after the administration of the same compounds the gliosis in the substantia nigra was much more extensive in CuSO4-than in 6-OHDA-treated rats; however, the reduction of DA concentrations in the ipsilateral striatum was only noticeable after 6-OHDA (-62%). A somewhat similar decrease of striatal DA levels (-52%) was observed after large electrocoagulation of the substantia nigra. Ten days after 6-OHDA (8mug) or electrolytic lesion of the striatum the Km for DA, serotonin and choline uptakes were similar in the striata of both sides, suggesting that the uptake process in the non-damaged neurons of the lesioned side was functionally normal. Following electrolytic lesion of the striatum, serotonin and choline Vmax values were decreased to about the same extent as the striatal reduction in weight and DA levels. When directly administered into the striatum 6-OHDA also produced a decline in DA concentration and Vmax but in contrast did not affect serotonin and choline uptake (Vmax), suggesting that the drug specifically destroyed dopaminergic neurons. The present data confirm that selective DA denervation can be achieved when appropriate amounts of the drug are injected into brain tissue in order to limit the unspecific lesion.

[3H]HARMALINE AS A SPECIFIC LIGAND OF MAO A—I. PROPERTIES OF THE ACTIVE SITE OF MAO A FROM RAT AND BOVINE BRAINS

A high‐affinity (Kd= 5.9 nM) specific binding site for [3H]harmaline was detected in membranes from rat and bovine brains. Studies of the regional and subcellular distributions of this binding indicated its close association with monoamine oxidase type A activity (MAO A) measured with [3H]serotonin ([3H]5‐HT) as the substrate. Maximal binding capacity and MAO A activity were found in mitochondrial enriched fractions. Mitochondria of synaptosomal or extra‐synaptosomal origin exhibited very similar properties with respect to [3H]harmaline binding characteristics and MAO A activity.

Serotonin-sensitive adenylate cyclase and [3H]serotonin binding sites in the CNS of the rat—II: Respective regional and subcellular distributions and ontogenetic developments

The 5-HT receptor linked to adenylate cyclase and the high affinity binding site of [3H]5-HT were compared on the basis of their localization and ontogenetic changes in the CNS of the rat. Subcellular fractionation of cerebral tissues from newborn rats showed a good correlation between the distributions of 5-HT-sensitive cyclase and [3H]5-HT binding sites, with the mitochondrial fraction exhibiting the highest specific adenylate cyclase activity and density of binding sites. There was also a good correlation between the regional distributions of the cyclase and the binding in the CNS of newborn rats. However, when the regional distribution of [3H]5-HT binding in newborns was compared to that of adults, no correlation was found, showing that large changes were occurring during ontogenesis. In the cortex and hippocampus, there was little change in the amount of 5-HT-sensitive adenylate cyclase during development whereas [3H]5-HT binding increased approximately 7-fold from birth to adulthood. Only in the striatum was there a positive correlation between the changes in the cyclase and the binding. The injection of kainic acid into the striatum of 10-day-old rats produced large decreases in both DA-and 5-HT-sensitive adenylate cyclase activities. The specific binding of [3H]5-HT was also reduced in the injected striatum but to a smaller extent. Therefore, the 5-HT-sensitive adenylate cyclase but not the [3H]5-HT-high affinity binding site appeared to be preferentially associated with neurons destroyed by kainic acid, i.e. neurons intrinsic to the striatum. The findings that the 5-HT-sensitive adenylate cyclase and the [3H]5-HT binding sites can develop independently and are localized, at least partly, on different types of cells provide additional evidence for the existence of multiple types of 5-HT receptors in the CNS of the rat.

[3H]Metergoline: A New Ligand of Serotonin Receptors in the Rat Brain

Abstract: A specific binding site for [3H]metergoline characterized by a KD of 0.5–1.0 nM was detected in microsomal and synaptic plasma membranes from various areas of the adult rat brain. Experiments with 5,7‐dihydroxy‐tryptamine‐ and kainic acid‐induced lesions indicated that this specific binding site was localized post‐synaptically with respect to serotoninergic neurons. The pharmacological characteristics of [3H]metergoline binding to microsomal membranes from the whole forebrain strongly suggest that this ligand labels a class of serotonin receptors. This was particularly obvious in the hippocampus in which serotonin was about 400 times more potent than dopamine and norad‐renaline for displacing bound [3H]metergoline. In the striatum, serotonin was only 10 times as potent as dopamine in inhibiting [3H]metergoline binding, suggesting that this ligand may also bind to dopamine receptors. Striking similarities between the binding sites for [3H]metergoline and [3H]serotonin were observed in the hippocampus. Thus, not only the total numbers of binding sites for these two ligands in control rats but also their respective increases following intracerebral 5,7‐dihydroxytryptamine treatment were very similar. Therefore, at least in the hippocampus, [3H]metergoline might well be the appropriate ligand for studying the characteristics of the ‘antagonist form’ of serotonin receptors postulated by Bennett and Snyder.

Regional differences in the sensitivity of cholinergic neurons to dopaminergic drugs and quipazine in the rat striatum.

Marked differences were found in the activity of choline acetylase (ChAc) in various discrete areas of the rat striatum. The richest cholinergic innervation was observed in the centrolateral part of the structure. A similar distribution was obtained by measuring acetycholine (ACh) levels in punches taken from frozen frontal serial slices. As revealed by the analysis of the topographical distributions of ChAc activity, ACh, 5-HT and DA, the regional cholinergic innervation differed markedly from that of aminergic terminals. Changes in ACh levels induced by drugs could be estimated in microdiscs of tissues punched from frozen slices. Apomorphine and haloperidol, which increased and decreased ACh levels respectively, induced similar effects in the various striatal areas examined. By contrast quipazine, a drug acting on 5-HT uptake and release and on serotoninergic receptors, selectively increased ACh levels in some areas of the striatum but not in others. The regional changes in ACh levels induced by quipazine were satisfactorily correlated with the regional distribution of 5-HT but not with that of DA. These results suggest that a limited population of striatal cholinergic neurons is under the inhibitory control of serotoninergic neurons. They also indicate that some striatal cholinergic neurons influenced by dopaminergic neurons are not controlled by serotoninergic neurons.

[3H]Harmaline as a specific ligand of MAO A--II. Measurement of the turnover rates of MAO A during ontogenesis in the rat brain.

The combined measurement of MAO A activity (using [3H]5‐HT as a specific substrate) and [3H]harmaline binding capacity indicated that the concentration of MAO A in brain was higher in 14‐28 day old rats than in adult animals. The turnover rates of this enzyme in the forebrain and the brain stem of young (14‐28 day old) and adult rats were calculated by following the recovery of MAO A activity and of [3H]harmaline binding capacity after an acute treatment with pargyline (75mg/kg i.p.). Both the fractional rate constant for MAO A degradation and its synthesis rate per g of fresh tissue were significantly higher in young animals. However, the calculation of the absolute synthesis rates of MAO A per brain area gave very similar values in young and adult animals: 1.3‐1.5 × 1013 molecules of MAO A synthesized per day in the forebrain and 2.3‐2.9 × 1012 molecules per day in the brain stern.

In vitro and in vivo disposition of 3H-methiothepin in brain tissues

SummaryA single treatment with a large dose of methiothepin (20 mg/kg, i.p.) induced, as early as the 2nd day after injection, a significant increase (+20–35%) in the number of specific binding sites for 3H-5-HT in forebrain areas, particularly the hippocampus. Experiments with 3H-methiothepin indicated that the drug remained firmly bound to brain membranes thus maintaining a local concentration high enough to effectively block 5-HT receptors for 10–12 h after its peripheral administration. Accordingly, it can be concluded that the occupancy of central 5-HT receptor sites by methiothepin for several hours was sufficient to induce a supersensitivity phenomenon within the two following days.Although 3H-methiothepin was a useful marker for analyzing the disposition and the kinetics of the 5-HT antagonist in brain tissues, it could not be used as a specific ligand of 5-HT receptors in brain since under in vitro as well as in vivo conditions most of 3H-methiothepin bound to non-specific sites, especially to the lipid component of the membranes.

Serotonin-Receptors Coupled with an Adenylate Cyclase in the Rat Brain: Non-Identity with 3H-5-HT Binding Sites

Recently, two biochemical approaches have been used for studying serotonin (5-HT) receptors in the central nervous system (CNS). One consists of measuring the stimulation of a specific adenylate cyclase by 5-HT (Ahn and Makman,1978; Daszuta et al., 1979; Enjalbert et al., 1978 a,b; Fillion et al.,1979; Nelson et al.,1979 a,b; Pagel et al.,1976; Von Hungen et ai.,1975) while the other involves the labelling of specific receptors with 3H-5-HT (Bennett and Snyder,1976; Fillion et al.,1976; 1978; Nelson et al.1978; 1979 a,b; Schwarcz et ai.,1977), 3H-LSD (Bennett and Snyder,1976; Fillion et al.1978) or 3H-spiroperidol (Creese and Snyder,1978; Hamon et al.,1979 b; Leysen et al., (1978). An important question is whether the specific 5-HT receptors detected with these different biochemical tools are identical or not. So far, this question has not been solved. In this report, the kinetic characteristics, ontogenetic development, topographical and subcellular distributions and the pharmacological properties of the 5-HT-sensitive adenylate cyclase and 3H-5-HT binding sites have been compared.

Lack of involvement of dopaminergic and GABA neurones in the inhibitory effect of harmaline on the activity of striatal cholinergic neurones in the rat

SummaryHarmaline (10 mg/kg i.p. 45 min) increased acetylcholine (ACh) levels in the rat striatum but did not affect the ACh content of the parietal cortex, the hippocampus or certain limbic nuclei. This change in striatal ACh levels may reflect a decreased utilization of the transmitter. The selective effect of harmaline on striatal cholinergic neurones is probably mediated by interneuronal processes. Various hypotheses were examined:1.The harmaline-induced increase in striatal ACh levels was not abolished after destruction of the nigrostriatal dopaminergic neurones. Harmaline (10−5 M or 10−4 M) and its metabolites (harmalol, harmine) also failed to modify the activity of the striatal dopamine-sensitive adenylate cyclase. This excludes any involvement of dopaminergic mechanisms in the action of harmaline on striatal cholinergic neurones.2.The harmaline-induced increase in striatal ACh levels could not be prevented by diazepam (5 mg/kg i.p. 65 min). In contrast the increase in ACh content elicited by picrotoxin (25 mg/kg i.p. 60 min) was abolished by diazepam. Furthermore, the harmaline-and picrotoxin-induced rises in striatal ACh levels were additive. These results suggest that GABA mechanisms are not involved in the effect of harmaline on striatal cholinergic neurones.3.Parachlorophenylalanine pretreatment (48 and 24 h, 300 mg/kg i.p.) which reduced striatal serotonin (5-HT) levels by 95%, failed to affect ACh levels in the striatum. The inhibitor of 5-HT synthesis did not prevent the harmaline-induced rise in ACh levels. Harmaline (10−5 M or 10−4 M), harmine and harmalol, did not stimulate the 5-HT sensitive adenylate cyclase in the colliculi of newborn rats. It is thus unlikely that harmaline reduces the activity of striatal cholinergic neurones by its effects on 5-HT transmission. Moreover, other monoamine oxidase inhibitors (pargyline and tranylcypromine) were also ineffective on cholinergic neurones.