Neurochemical changes and neurotoxic effects of an acute treatment with sydnocarb, a novel psychostimulant: comparison with D-amphetamine.

Sydnocarb [(phenylisopropyl)N-phenylcarbamoylsydnonimine; SYD] was introduced to clinical practice in Russia as a psychostimulant drug used for the treatment of asthenia and apathy, which accompany schizophrenia and manic depression. It has been described as a psychostimulant with addiction liability and toxicity less than amphetamine (AMPH). The precise cellular mechanisms by which sydnocarb elicits its psychostimulant effect are still unclear. At present its neurochemical and neurotoxic effects are compared to those of AMPH in the striatum, the main input structure of the basal ganglia. The expression of c-fos protein in striatal neurons was much more increased after a single injection of D-AMPH (5 mg/kg) than after an equimolar concentration of SYD (23.8 mg/kg) in both the anterior and the posterior part of the striatum. Using in situ hybridization on striatal slices, we observed that AMPH increased the striatal levels of preprodynorphin (PPDYN) mRNAs in both parts of the striatum, while SYD did not affect basal levels of PPDYN mRNAs. Furthermore, AMPH and SYD increased striatal preprotachykinin (PPT-A) and preproenkephalin (PPE) mRNA levels. The effects of AMPH and SYD on PPT-A-mRNA levels were similar. A differential effect of AMPH and SYD was observed only on the PPE-mRNA levels measured in the anterior striatum where SYD increased these levels more than AMPH. The acute neurotoxicity of these two psychostimulants was analyzed by measuring their effects on the parameters of oxidative stress, such as nitric oxide (NO) generation, as well as specific indices of lipid peroxidation (i.e., thiobarbituric acid reactive substances; TBARS), while, on the other hand, the alpha-tocopherol level was taken as an index of antioxidant defense processes. Measuring generation of NO directly by electron paramagnetic resonance, it was observed that AMPH shows a more pronounced increase in comparison to SYD, in the striatum and in cortex. TBARS levels in the striatum and cortex were significantly less enhanced than AMPH after a single injection of SYD. Similarly, the alpha-tocopherol level was decreased only by AMPH in the striatum, and neither AMPH nor SYD had any effect in the cortex. Results show that a single injection of a high dose of AMPH is able to induce several neurotoxic effects. The study also demonstrates that SYD has mild neurochemical effects as well as fewer neurotoxic properties than AMPH.

Regulation of striatal neuropeptide mRNAs: effects of the 5-HT(2) antagonist SR46349B in adult rats with a neonatal 6-hydroxydopamine lesion.

The intrastriatal injection of 6-hydroxydopamine (6-OHDA) in newborn rats produces a marked striatal dopamine (DA) depletion, accompanied by a serotonin (5-HT) hyperinnervation and an up-regulation of 5-HT receptors. The aim of the present study was to investigate whether the increase in 5-HT(2) receptors could compensate for some of the DA lesion-induced effects, such as the increase in striatal preproenkephalin (PPE) and the decrease in preprotachykinin A (PPT-A) mRNA levels. Three months after the DA lesion, the effect of the selective 5-HT(2) antagonist SR46349B was investigated by a subacute treatment (10 mg/kg, IP, twice per day for 3.5 days). In sham-operated rats, the blockade of 5-HT(2) receptors decreased PPE mRNA levels in the striatum and, by contrast, had no effect on PPT-A mRNA levels. In rats with a unilateral neonatal DA lesion, SR46349B had no more effect on PPE mRNA levels in the intact striatum and was unable to modify the lesion induced-increase in PPE mRNA. The decrease in PPT-A mRNA levels induced by the neonatal DA lesion was not changed after SR46349B treatment in the posterior part of the lesioned striatum. Our results suggest that SR46349B indirectly decreases PPE mRNA levels in striatopallidal neurons in intact animals through a desinhibition of DA neuron activity. This is further evidenced by the lack of PPE mRNA changes in the DA lesioned striatum despite the up-regulation of 5-HT(2) receptor transmission induced in this model. Finally, the absence of any effect of 5-HT(2) antagonist on the expression of PPT-A mRNA in intact animals is discussed. The precise role of 5-HT(2) receptor on PPT-A mRNA biosynthesis after a neonatal lesion should be clarified by further experiments using 5-HT(2) agonists.

Morphological and biochemical adaptations to unilateral dopamine denervation of the neostriatum in newborn rats.

Basal ganglia of adult rats were examined for morphological and biochemical changes resulting from neonatal unilateral dopamine denervation of the striatum with increasing doses of 6-hydroxydopamine (4, 12 and 20 microg). Rotational behaviour induced by apomorphine (0.1 mg/kg) was observed in all rats injected with the high dose (20 microg) and totally absent in those injected with the low dose (4 microg). As assessed with tyrosine hydroxylase immunocytochemistry, the extent of dopamine denervation within the injected striatum was clearly related to the dose injected. In the mesencephalon, losses of tyrosine hydroxylase-immunoreactive cell bodies were proportional to the dose injected and the extent of neostriatal dopamine denervation. This retrograde cell loss predominated in the ventromedial and lateral parts of the substantia nigra pars compacta, with relative sparing of the ventral tegmental area. After the injection of the intermediate (12 microg) and the high (20 microg) doses, a network of thin tyrosine hydroxylase-immunoreactive fibres was visualized in the ventral part of the pars reticulata ipsilateral to the injected striatum, suggesting a neoinnervation of this structure by dopamine axons. After the high dose, the density of serotonin-immunoreactive fibres was enhanced in the anterior half of the lesioned striatum. Associated changes in dopamine and serotonin content and turnover were also documented on both sides, in the striatum and in two output structures of the basal ganglia, the globus pallidus and the substantia nigra. Dopamine content was decreased only on the injected side. After the low dose, equal reductions (-60%) were observed in the anterior striatum and the substantia nigra, whereas a more marked decrease was measured in the anterior striatum (-93%) than in the substantia nigra (-60% to -74%) after the intermediate and high doses. In the globus pallidus, dopamine tissue content was decreased (-51%) only after the high dose. Dopamine turnover was unchanged after the low dose in all structures examined and was increased in the striatum, on the lesioned side only, after the intermediate and high doses. Serotonin content was increased only on the injected side in the anterior striatum (+50% after the low and +92% after the high dose). Serotonin turnover was unchanged on the injected side but increased by +118% and by +81% in the contralateral anterior striatum after the low and high doses, respectively. It was also increased in both substantia nigra after the high dose. In conclusion, morphological changes similar to those described after a bilateral neonatal lesion were observed on the injected side in the model of the unilateral neonatal nigrostriatal dopamine denervation. Biochemical changes were, however, not restricted to the lesioned side. Notably, changes in serotonin turnover developed on the contralateral side. These morphological and biochemical adaptative changes need to be taken into account in considering the mechanisms implicated in the rotional behaviour measured in these animals.

Levels of stimulatory G protein are increased in the rat striatum after neonatal lesion of dopamine neurons.

After neonatal lesions of dopamine neurones, an enhanced behavioural responsiveness towards D1 agonists has been described, suggesting a D1 receptor hypersensitivity. In the present study, unilateral striatal dopamine denervation in newborn rats induced a pronounced rotational behaviour following apomorphine injection at the adult age, without any change in the density of D1 binding sites in the denervated striatum. The amount of stimulatory G(olf) alpha subunit was increased by 35% in the lesioned striatum. The large form and the short forms of Gs alpha were also increased by 26% and 9%, respectively. Since in striatal neurones, the coupling of D1 receptor to adenylate cyclase is mostly provided by G(olf) alpha, our results strongly suggest that D1 hypersensitivity described after neonatal dopamine lesions results from an increase in the levels of G(olf) alpha protein.

GABA interneurons in the rat medial frontal cortex: characterization by quantitative in situ hybridization of the glutamic acid decarboxylase (GAD67) mRNA.

In situ hybridization of mRNA encoding one isoform of glutamic acid decarboxylase (GAD67) was performed in the rat medial frontal cortex (MFC) to characterize GABA interneurons. Qualitatively, the labelling obtained with a [35S]cDNA probe was in register with neurons and was never associated with glial cells. No obvious differences in the density of labelled cells were observed between the different areas of the MFC examined (infralimbic, prelimbic, anterior cingulate and precentral medial) and between the various cortical layers. Grain counting was performed on single cells in the various layers of the prelimbic and the anterior cingulate area, two main areas of the MFC. According to their grain density, neurons were arbitrarily classified as low, high and very high GAD67 mRNA content. The neurons with the high GAD67 mRNA content corresponded to around 50% of the labelled cells in all the layers and in both areas. In the prelimbic area, the neuronal population with a low GAD67 mRNA content varied from 50% in layers I and II-III to 40% in layers V-VI whereas the very high GAD67 mRNA content neurons corresponded to around 5% of the labelled neurons in all layers. In the anterior cingulate area the neuronal population showing low GAD67 mRNA content varied from 35% in layers I and II-III to 20% in layers V-VI. In this area, neurons with a very high GAD67 mRNA content were more numerous than in the prelimbic area: they varied from 15% in layers I and II-III to 30% in layers V-VI. Parallel to the presence of very highly labelled cells, GAD enzymatic activity measured both in the presence and in the absence of pyridoxal 5'-phosphate was higher in the anterior cingulate area than in the prelimbic area. The heterogeneity of GAD67 mRNA content at the cellular level might underlie the existence of subpopulations of GABA interneurons in the MFC and suggests a higher GABAergic inhibitory control in the anterior cingulate area than in the prelimbic area.

Expression of GAD mRNA in GABA interneurons of the rat medial frontal cortex.

The distribution of glutamic acid decarboxylase (GAD) mRNA containing cells was studied in the rat medial frontal cortex (MFC). The neurons labelled by the 35S-labelled cDNA probe were distributed uniformly throughout all the layers and represented 16% of the total neuronal population. It was possible to distinguish two cell populations expressing high and low levels of GAD mRNA corresponding to 63% and 27% of labelled cells, respectively. Concerning the laminar distribution of these two populations of GAD mRNA containing neurons, no marked difference was observed between the various areas of the MFC.

Synergism between D1 and D2 dopamine receptors in the inhibition of the evoked release of [3H]GABA in the rat prefrontal cortex.

In order to examine a possible interaction between D1 and D2 receptors in the dopaminergic control of the electrically-evoked release of [3H]GABA in the rat prefrontal cortex, the effects of D1 and D2 dopamine agonists were studied in vitro on cortical slices. The D1 agonist SKF38393 (10(-5) M) inhibited the electrically-evoked release of [3H]GABA. This effect was totally reversed by both the D1 antagonist SCH23390 (10(-7) M) and the D2 antagonist sulpiride (10(-5) M). We previously observed that maximal D2-mediated inhibition of the electrically-evoked release of [3H]GABA was obtained with 10(-7) M RU24926 and 10(-8) M LY171555. Here we showed that the inhibition produced by these two D2 agonists is also abolished by 10(-7) M SCH23390. In dopamine-depleted slices from reserpine-treated animals, it was not possible to detect an effect of either RU24926 (10(-7) M) or SKF38393 (10(-5) M), suggesting a permissive role of endogenous dopamine in the effect of either D2 or D1 agonist. Finally, SKF38393 used at a subliminar concentration (10(-6) M) was able to potentiate the effect of a liminar concentration of RU24926 (1.5 x 10(-8) M). Taken together these results strongly suggest that in the rat prefrontal cortex a D1-D2 receptor synergism is involved in the dopaminergic control of the electrically-evoked release of [3H]GABA.

Opposing effects of dopamine D2 receptor stimulation on the spontaneous and the electrically evoked release of [3H]GABA on rat prefrontal cortex slices.

The spontaneous and the electrically evoked release of [3H]GABA were studied in vitro on slices of rat medial prefrontal cortex. The slices were preincubated with [3H]GABA and then superfused with a Krebs' solution. The superfusion with a Ca(2+)-free medium progressively increased the spontaneous [3H]GABA release and strongly decreased the electrically evoked release of [3H]GABA (-65%). The effects of three dopaminergic D2 receptor agonists (RU24926, lisuride and LY171555) were studied on both the spontaneous and the electrically evoked [3H]GABA release. The spontaneous release of [3H]GABA was increased by exposure to each of these three D2 agonists. RU24926 produced a dose-dependent increase from 10(-9) to 3 x 10(-8) M and the maximal effect was totally abolished by the dopaminergic D2 receptor antagonist sulpiride (10(-5) M). With lisuride a progressive increase of [3H]GABA release was observed and a plateau value was reached with concentrations between 10(-7) and 10(-6) M. These effects were totally reversed by 10(-5) M sulpiride. The dose-response relation for LY171555 was bell-shaped, with a maximal effect being obtained with 10(-9) M) LY171555. This effect decreased with a higher concentration (10(-8) M) and finally was no longer observed for 10(-7) M LY171555. The maximal increase induced by LY171555 was totally abolished by 10(-5) M sulpiride. In contrast, the electrically evoked release of [3H]GABA was inhibited by these three D2 agonists. The IC50 value of the inhibition was 4.1 x 10(-8) M for RU24926 and 2 x 10(-7) M for lisuride. Sulpiride (10(-5) M) totally abolished the effect of 10(-7) M RU24926. In the concentration range of lisuride examined, a 50% reduction of the lisuride inhibition was obtained in the presence of sulpiride (10(-5) M). The dose-response curve obtained with LY171555 had a U-shape, with a maximal inhibition reached with 10(-8) M, whereas no effect was observed with 10(-6) M. The inhibition induced by 10(-8) M LY171555 was completely antagonized by 10(-5) M sulpiride. The D2 agonist-induced inhibition of the electrically evoked release of [3H]GABA was mimicked by dopamine endogenously released by 10(-5) M amphetamine. This effect was reversed by 10(-5) M sulpiride. Our data provide further evidence for a dopaminergic control of GABA interneurons in the prefrontal cortex. This regulation implies the activation of D2 dopaminergic receptors. The possible mechanisms underlying the opposite effects of D2 agonists on the spontaneous and the electrically evoked release of [3H]GABA are discussed.

[D2 dopaminergic receptor activation enhances the spontaneous release of 3H-GABA in the prefrontal cortex of rats, in vitro. The facilitating role of D1 dopaminergic receptors]. / L'activation des récepteurs D2 dopaminergiques stimule la libération spontanée de 3H-GABA dans le cortex préfrontal du rat, in vitro. Rôle permissif des récepteurs D1 dopaminergiques.

The effects of three D2 dopaminergic agonists on the spontaneous release of 3H-GABA have been studied on rat prefrontal cortical slices. LY171555 (10(-9) M), RU24926 (3 x 10(-8) M) and lisuride (10(-7) M) respectively enhanced the spontaneous release by 25, 20.5 and 23%. These effects were totally reversed by the D2 antagonist sulpiride (10(-5) M). Furthermore, subliminar concentration of RU24926 (10(-9) M) and of the D1 agonist SKF38393 (10(-6) M) induced a clear enhancement of the spontaneous release of 3H-GABA when they were superfused simultaneously. Our results suggest that in the prefrontal cortex, the spontaneous release of 3H-GABA is under an activatory D2 dopaminergic control. The activation of D1 receptors seems to have an enabling effect on this regulation.

[Effects of the stimulation of D1 and D2 dopaminergic receptors on the electrically induced release of gamma-(3H)-aminobutyric acid in the prefrontal cortex of the rat]. / Effets de la stimulation des récepteurs D1 et D2 dopaminergiques sur la libération d'acide gamma-[3H] aminobutyrique induite électriquement dans le cortex préfrontal du rat.

The effects of D1 and D2 dopaminergic agonists and antagonists on the electrically-evoked release of gamma-[3H] aminobutyric acid (3H-GABA) have been studied on rat prefrontal cortex slices. The major part of the electrically-evoked release of 3H-GABA appeared to be Ca++ dependent since a 62% decrease was observed when calcium was removed from the superfusion medium. Two specific D2 dopaminergic agonists, RU 24926 (10(-7) M) and lisuride (10(-6) M), respectively induced a 32% and a 50% inhibition of the electrically-evoked release of 3H-GABA. The selective D2 dopaminergic antagonists sulpiride (10(-5) M) totally abolished the effect of RU 24926 and partially abolished the effect of lisuride. The selective D1 agonist SKF 38393 (10(-5) M) did not affect 3H-GABA release. These results suggest that in the rat prefrontal cortex in vitro, the dopaminergic modulation of 3H-GABA release is mediated through D2 but not D1 receptors. The activation of D2 dopaminergic receptors induces an inhibition of the electrically-evoked release of 3H-GABA.

Excitation of rat prefrontal cortical neurons by dopamine: an in vitro electrophysiological study.

The effects of dopamine (DA) on prefrontal pyramidal neurons were studied in vitro on rat cerebral cortex slices using intracellular recordings. Pyramidal neurons were first identified by Lucifer yellow and some of their basic bioelectrical properties were analysed. At resting potential, white matter stimulation mainly evoked depolarizing inhibitory postsynaptic potentials (IPSPs) which reversed between -60 and -50 mV and were almost totally abolished by bicuculline. Furthermore, pyramidal cells often exhibited spontaneous depolarizing IPSPs abolished by bicuculline. Under tetrodotoxin (TTX) this synaptic noise was partly blocked suggesting that it was due both to the spontaneous firing of presynaptic gamma-aminobutyric acid (GABA)ergic neurons and to a spontaneous quantal release from these afferent fibers. In pyramidal cells, DA enhanced the number of spikes evoked by depolarizing current pulses, and the input resistance was increased by 10-20%. DA also clearly increased the inhibitory synaptic noise. This effect was blocked by fluphenazine. In contrast, evoked IPSPs were not consistently affected by DA. Taken altogether, these results suggest, that in the prefrontal cortex, dopamine has a mild excitatory effect on both pyramidal cells and GABAergic interneurons impinging on them.

Inward rectification and low threshold calcium conductance in rat cerebellar Purkinje cells. An in vitro study.

The bioelectrical properties of Purkinje cells were analysed in sagittal slices of adult rat cerebellum by the use of intracellular recordings performed at a somatic level in current or in voltage clamp. The passive electrical constants of Purkinje cells were determined by measuring the time course and the amplitude of the voltage responses induced by hyperpolarizing current pulses. The mean value of input resistance was 21 +/- 1 M omega. Mean values of the membrane time constant and of the total electrotonic length of Purkinje cells were 19.5 +/- 1.7 ms and 0.59 +/- 0.01 ms respectively. A time dependent inward rectification was present in all cells. In current-clamp experiments it appeared as a sag in hyperpolarizing voltage responses which were followed by well developed anodal breaks. In voltage-clamped cells, the inward relaxation induced by hyperpolarizing commands fitted to a single exponential. It was already present near resting potential and could reach an amplitude of up to 4 nA for jumps near to -120 mV. This relaxation was provisionally termed Ih. Tail current relaxations also fitted to a single exponential when they were recorded in the presence of tetrodotoxin (TTX) and of Co. The inward relaxation induced by hyperpolarizing commands was readily blocked by Cs, whereas it was unaffected when Ba replaced Ca in the bath, except near rest where it was strongly reduced. The Ca channel blockers Cd, Co and D600 also markedly depressed or even suppressed the inward rectification near resting potential, and up to about -85 mV, whereas this blocking effect was much less apparent or even absent at more negative potentials. Ih was clearly enhanced when the external K concentration was raised up to 20 mM. In the presence of TTX and Co in the bath, inward relaxations induced by hyperpolarizing jumps were unaffected in Na-free solution, whereas the amplitude of tail currents was reduced. Furthermore, the reversal potential of Ih which ranged between -45 and -56 mV in the Co plus TTX containing solution, shifted toward more negative values in the Na-free medium. In contrast, Ih remained unchanged in low Cl solution. From these experiments, it is likely that K and Na are the main charge carriers of Ih. Furthermore, this current seems to be contaminated near resting potential by a Ca-dependent K current. Anodal breaks following hyperpolarizing commands were slightly attenuated when Cd or TTX were added to the bath.(ABSTRACT TRUNCATED AT 400 WORDS)

Beta-adrenergic induced potassium current in Xenopus oocyte: involvement of cyclic AMP.

The effect of a beta-adrenergic agonist, on full-grown Xenopus oocytes, still surrounded by their ovarian envelopes, has been studied by electrophysiological methods. The oocytes were hyperpolarized by isoproterenol. Under voltage clamp, the elicited outward current reversed at a membrane potential of - 95 mV, a value close to the K+ equilibrium potential. The isoproterenol induced current varied linearly with the membrane potential in the range studied (- 120 mV, - 30 mV). Half-maximum current was obtained at 3.10(-8) M isoproterenol. Propranolol (10(-7) M) completely suppressed the response to isoproterenol (10(-9) to 10(-5) M). 8-Br-cAMP induced a current which also reversed at - 95 mV. Methyl-isobutyl-xanthine (MIX), a potent inhibitor of phosphodiesterases, potentiated the current induced by isoproterenol. These experiments strongly suggest that the increase in K+ permeability due to catecholamines is mediated by cAMP.