C. Gauchy

Glutamatergic Control of Dopamine Release in the Rat Striatum: Evidence for Presynaptic N-Methyl-D-Aspartate Receptors on Dopaminergic Nerve Terminals

Abstract: The N‐methyl‐D‐aspartate (NMDA) receptor‐mediated regulation of the release of newly synthesized [3H]dopamine ([3H]DA) was studied in vitro, both on rat striatal slices using a new microsuperfusion device and on rat striatal synaptosomes. Under Mg2+‐free medium conditions, the NMDA (5 × 10−5M)‐evoked release of [3H]DA from slices was found to be partly insensitive to tetrodotoxin (TTX). This TTX‐resistant stimulatory effect of NMDA was blocked by either Mg2+ (10−3M) or the noncompetitive antagonist MK‐801 (10−6M). In addition, the TTX‐resistant NMDA‐evoked response could be potentiated by glycine (10−6M) in the presence of strychnine (10−6M). The coapplication of NMDA (5 × 10−5M) and glycine (10−6M) stimulated the release of [3H]DA from striatal synaptosomes. This effect was blocked by Mg2+ (10−3M) or MK‐801 (10−5M). These results indicate that some of the NMDA receptors involved in the facilitation of DA release are located on DA nerve terminals. These presynaptic receptors exhibit pharmacological properties similar to those described in electro‐physiological studies for postsynaptic NMDA receptors.

Distinct presynaptic regulation of dopamine release through NMDA receptors in striosome- and matrix-enriched areas of the rat striatum

Striosome- and matrix-enriched striatal zones were defined in coronal and sagittal brain sections of the rat, on the basis of 3H-naloxone binding to mu-opiate receptors (a striosome-specific marker). Then, using a new in vitro microsuperfusion device, the NMDA (50 microM)- evoked release of newly synthesized 3H-dopamine (3H-DA) was examined in these four striatal areas under Mg(2+)-free conditions. The amplitudes of the responses were different in striosomal (171 +/- 6% and 161 +/- 5% of the spontaneous release) than in matrix areas (223 +/- 6% and 248 +/- 12%), even when glycine (1 or 100 microM) was coapplied (in the presence of 1 microM strychnine). In the four areas, the NMDA-evoked release of 3H-DA was blocked completely by Mg2+ (1 mM) or (+)-5-methyl- 10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine maleate (MK-801; 1 microM) and almost totally abolished by kynurenate (100 microM). Because the tetrodotoxin (TTX)-resistant NMDA-evoked release of 3H-DA was similar in striosome- (148 +/- 5% and 152 +/- 6%) or matrix- enriched (161 +/- 5% and 156 +/- 7%) areas, the indirect (TTX- sensitive) component of NMDA-evoked responses, which involves striatal neurons and/or afferent fibers, seems more important in the matrix- than in the striosome-enriched areas. The modulation of DA release by cortical glutamate and/or aspartate-containing inputs through NMDA receptors in the matrix appears thus to be partly distinct from that observed in the striosomes, providing some functional basis for the histochemical striatal heterogeneity.

Spatial organization of patch and matrix compartments in the rat striatum.

The visualization of mu opiate receptors by [3H]naloxone binding was used to determine precisely the spatial organization of the patch compartment in the rat striatum and its reproducibility in different animals. Three-dimensional reconstruction of the patch network was made using maps of autoradiographic data obtained from successive coronal, sagittal or horizontal sections. The extreme rostral pole of the striatum (A 11) was characterized by a large patch territory exhibiting complex and tortuous fields with several extensions. In the intermediate part of the structure (A 9.0-10.0), about 20 serial parallel continuous patch channels running in a mediolateral axis, obliquely oriented and displaying in some cases connecting branches, could be observed. However, no channels could be distinguished in the rostrocaudal direction. More caudally, patches were rare and of small size. In addition, the laterocaudal region of the striatum was almost exclusively represented by a large matrix field. Finally, a fine discontinuous band of [3H]naloxone binding was seen in all sections, bordering and limiting the dorsolateral part of the striatum. The topographical and spatial distribution of the patch compartment was similar in all animals investigated. However, due to the tortuous shape and the labyrinthine organization of the patches, the precise degree of reproducibility from one animal to another could not be established. Nevertheless, the prominent patch compartment observed in the rostral pole of the striatum, the patch channels, oriented in the mediolateral axis as well as the large laterocaudal matrix field were observed in all cases. These results were compared with previous data obtained in the cat in which patch (striosome) channels oriented along a rostrocaudal axis are also observed.

Acute effects of morphine on dopamine synthesis and release and tyrosine metabolism in the rat striatum

The effect of the acute administration of morphine (60 mg/kg) on the metabolism of dopamine (DA) and tyrosine in the rat striatum were investigated using in vivo and in vitro methods. 30 min after morphine injection, the initial accumulation of 3H-DA in tissues seen after the i.v. injection of L-3,5-H-tyrosine was enhanced as well as the accumulation of 3HH2O and 3H-DA in tissues and medium of striatal slices incubated with the 3H precursor. These results and the estimation of the conversion index of tyrosine into DA (3H-DA/tyrosine specific activity) indicated that morphine stimulated DA synthesis. This effect was not seen 2 hr later. The enhanced DA synthesis was associated with a parallel increase in the release of newly synthesized 3H-DA as indicated by the greater accumulation of 3H-DA in incubating medium of slices of morphine-pretreated rats. Therefore the increased endogenous levels of DA seen after morphine are related to the increased synthesis of the transmitter. Apart from its effect on dopaminergic neuron activity, morphine also induced marked changes in tyrosine metabolism, particularly at 2 or 2.5 hr after its injection. Tyrosine levels in plasma and in striatum were about 160% of the control levels at this time: these changes in the size of the precursor pool may explain the enhanced accumulation of 3H-tyrosine seen in plasma and striatum 10 min after the 3H-amino acid injection. Marked changes in endogenous levels of DA and tyrosine occurred in the striatum of control animals from 9.00 to 12.00 am. Acute treatment with morphine significantly affected these diurnal variations.

Isolation and radioenzymic estimation of picogram quantities of dopamine and norepinephrine in biological samples.

Abstract— A method was developed to measure small amounts of dopamine or norepincphrine in biological samples. It was based on the radioenzymic assay originally described by Engelmanet al. (1968) and improved by other workers: dopamine and norepinephrine are O‐methylated by catechol‐O‐ methyl‐transferase in the presence of [3H]S‐adenosylmethioniiie as la belled methyl donor. O‐Methylated derivatives are extracted and estimated. The optimal conditions for the O‐methylation and the extractions were determined. The most important improvement in the method consisted in the selective isolation of catecholamines on microcolumns of alumina under conditions that allowed their subsequent enzymic assay. The assay described can thus be used on any size of biological sample. The assay was not affected by the prescnce of various agents (tissue extracts, CSF salts and particularly calcium, drugs etc.) which were shown to interfere with the O‐methylation process, and it allowed the measurement of quantities of dopamine and norepinephrine as low as 0.15 and 0.1 pmol respectively, whatever the origin of the sample (CSF superfusates, cerebral tissue).

Three-dimensional organization of the striosomal compartment and patchy distribution of striatonigral projections in the matrix of the cat caudate nucleus

Acetylcholinesterase staining on successive frontal or sagittal sections was used to determine the three-dimensional organization of the striosomal and matrix compartments in the adult cat caudate nucleus. Reconstruction drawings of the acetylcholinesterase-poor zones (striosomes) indicated that the striosomal compartment is a labyrinthine network organized in the rostrocaudal and mediolateral axis which is reproducible from one animal to another. Four main anteroposterior channels converging in the mediorostral pole of the caudate nucleus were distinguished. Seven to eight diagonally oriented channels crossing the previous ones were seen also in the mediolateral axis on the central core of the caudate nucleus. The pattern of organization of the numerous and tortuous striosomal channels was more complicated medially, while the lateral part of the caudate nucleus was represented mainly by the matrix compartment. In addition, a sub-compartmentation of the matrix was demonstrated by retrograde tracing studies made by injecting either horseradish peroxidase-wheat germ agglutinin, [14C]amino acids or a mixture of horseradish peroxidase-wheat germ agglutinin and [14C]amino acids in several areas of the substantia nigra pars reticulata. Labelled patches were seen with both tracers, their topographical localization depended on the nigral injection site but reconstruction analysis indicated that the populations of cells which innervate the substantia nigra pars reticulata originate in the two third lateral parts of the caudate nucleus all along its rostrocaudal extension. Examination of horseradish peroxidase-wheat germ agglutinin labelled cells indicated that not all cells were labelled in patches suggesting a further sub-compartmentation of these patches. Finally, a comparison of the topographical distributions of labelled patches and of striosomes revealed that most patches were located in the extrastriosomal matrix.

Calpain product of WT‐CRMP2 reduces the amount of surface NR2B NMDA receptor subunit

The brain is particularly vulnerable to ischaemia; however, neurons can become tolerant to ischaemic insult. This tolerance has been shown to involve activation of NMDA receptors, but its mechanisms have not yet been fully elucidated. Using a preconditioning protocol, we show that neurons surviving to a transient NMDA exposure become resistant to the glutamatergic agonist. Using a proteomic approach, we found that alterations of the protein pattern of NMDA‐resistant neurons are restricted mainly to the five collapsin response mediator proteins (CRMPs). A sustained increase in calpain activity following NMDA treatment is responsible for the production of cleaved CRMPs. Finally, we provide evidence for the involvement of the cleaved form of WT‐CRMP2 in the down‐regulation of NR2B. Our data suggests that, beside their role in neuronal morphogenesis, CRMPs may contribute to neuronal plasticity.

Glycine potentiates the NMDA-induced release of dopamine through a strychnine-insensitive site in the rat striatum

A new procedure, involving a push-pull cannula, was used to estimate the release of [3H]dopamine ([3H]DA) synthesized from [3H]tyrosine in rat striatal slices. NMDA (5 x 10(-5) M) stimulated [3H]DA release in the absence of Mg2+, and this effect was abolished in the presence of Mg2+ (10(-3) M), MK-801 (10(-6) M) or kynurenate (10(-4) M). Glycine markedly potentiated the NMDA-evoked response and reversed the inhibitory effect of kynurenate in the absence of Mg2+ and in the presence of strychnine (10(-6) M).

Spontaneous and potassium-evoked release of 3H-GABA newly synthesized from 3H-glutamine in slices of the rat substantia nigra

Abstract A technique has been developed to measure 3 H-GABA not only in tissues but also in medium of slices of the rat substantia nigra (SN) incubated for 15 min with 3 H-glutamine. The quantity of 3 H-GABA in tissues was about 30 to 35 times that released in the medium. Nevertheless, the amount of the 3 H-transmitter spontaneously released was about 10 to 15 times the blank value. GAD activity in the SN was decreased by 40 and 80% respectively ten days after the kainic acid lesion of the ipsilateral striatum or hemitransection. These effects were associated with parallel reductions in the amounts of 3 H-GABA accumulated in tissues and released in medium. The spontaneous release of newly synthesized 3 H-GABA was increased in absence of calcium and reduced with an excess of calcium (10 −2 M). Tetrodotoxin (10 −5 , 5.10 −6 M) reduced by 40% the spontaneous release of 3 H-GABA. These various effects were not associated with significant change in the total accumulation of 3 H-GABA in tissues + medium. Finally depolarization of the slices with potassium (30 mM) increased the release of 3 H-GABA (300%). This effect was abolished in absence of calcium and was not associated with a significant change in the amount of 3 H-GABA accumulated in tissues.

In vivo continuous estimation of 3H-dopamine synthesis and release in the cat caudate nucleus

Synthesis and release of 3H-DA were examined during the continuous superfusion of a limited area of the ventricular surface of the cat caudate nucleus with l-3,5-3H-tyrosine, using a cup technique. 3H−H2O, an index of the conversion of l-3,5-3H-tyrosine into 3H-Dopa, and 2H-DA were estimated in serial superfusate fractions. Alpha-methyl-paratyrosine (α-MpT) (10−4 M) inhibited rapidly and simultaneously both 3H−H2O formation and 3H-DA release. Transection of the nigro-striatal dopaminergic pathway immediately blocked 3H-DA release but 3H−H2O levels were reduced by 30% one hour later.SummarySynthesis and release of 3H-DA were examined during the continuous superfusion of a limited area of the ventricular surface of the cat caudate nucleus with l-3,5-3H-tyrosine, using a cup technique. 3H−H2O, an index of the conversion of l-3,5-3H-tyrosine into 3H-Dopa, and 2H-DA were estimated in serial superfusate fractions. Alpha-methyl-paratyrosine (α-MpT) (10−4 M) inhibited rapidly and simultaneously both 3H−H2O formation and 3H-DA release. Transection of the nigro-striatal dopaminergic pathway immediately blocked 3H-DA release but 3H−H2O levels were reduced by 30% one hour later.