L. Kerkerian-Le Goff

Ultrastructural features of the choline acetyltransferase-containing neurons and relationships with nigral dopaminergic and cortical afferent pathways in the rat striatum

The aim of this study was first to specify the morphology and neuronal environment of the large cholinergic neurons, and second to determine the distribution and mode of termination of the corticostriatal and dopaminergic inputs on these neurons in the rat striatum. Immunocytochemical procedures with a monoclonal antibody against choline acetyltransferase, Golgi staining and standard electron microscopic techniques were used to specify the ultrastructural features of the putatively cholinergic classical large neurons. The large/choline acetyltransferase-positive neurons are characterized by a voluminous, eccentric, and deeply indented nucleus leaving a large cytoplasmic area, and by the presence of an abundant granular endoplasmic reticulum and of many polysomes and free ribosomes. Serial ultrathin sectioning further indicated the presence of nematosomes or nucleolus-like bodies within the nucleus and the cytoplasm of the large neurons. In addition, these neurons were found to be in direct apposition with up to four surrounding neurons showing features typical of medium-sized spiny neurons. These data support the view that the putatively cholinergic neurons may have an intense metabolic activity and may be involved in striatal clusters. When choline acetyltransferase immunostaining was coupled with the identification of degenerating corticostriatal afferents after lesion of the cerebral cortex, degenerating terminals were seen to form synapses of an asymmetrical type on distal labelled dendrites, but these contacts were very rare. On the other hand, nigrostriatal dopaminergic axons, identified by means of either the degeneration method or tyrosine hydroxylase immunostaining, were often found to run directly for long distances around the choline acetyltransferase-positive cell bodies. Occasionally, dopaminergic terminals formed possible symmetrical synapses on choline acetyltransferase-positive cell bodies or proximal dendrites. These data provide evidence that the putatively cholinergic neurons are directly contacted by corticostriatal and dopaminergic nigrostriatal afferents. The respective positions and nature of the two types of contacts further provide morphological support for the hypothesis that postsynaptic interactions may occur between the corticostriatal and dopaminergic nigrostriatal afferents at the level of the cholinergic neurons.

Characterization of striatal lesions produced by glutamate uptake alteration: Cell death, reactive gliosis, and changes in GLT1 and GADD45 mRNA expression

This study investigated the time course of the striatal lesions produced by continuous local injection of the glutamate uptake inhibitor, L‐trans‐pyrrolidine‐2,4‐dicarboxylate (PDC) at the rate of 25 nmol/h in rats. The extent of the neurodegeneration area (defined as the lesion area) did not significantly vary with the duration of the PDC treatment between 3 and 14 days, but was markedly reduced 3 months after cessation of the 14‐day treatment, probably reflecting striatal atrophy. After the 3‐day treatment, the lesion zone showed calcium precipitates and marked microglial reaction contrasting with the reduction of astroglial labeling and loss of the glutamate transporter GLT1 mRNA expression; however reactive astrocytes were observed around the lesion. After the 14‐day treatment, the lesion zone presented reactive astrocytes and microglia without calcification, and a partial recovery of GLT1 mRNA expression. Interestingly, the growth arrest DNA damage‐inducible GADD45 mRNA expression was induced around the lesion after 3 days but inside the lesion after 14 days of treatment. Three months after the 14‐day treatment, the astroglial reactivity persisted within the lesion whereas most of the other markers examined tended to normalize. These data suggest that defective glutamate transport induces primary death of neurons and dysfunction of astrocytes. They strongly implicate reactive astrocytes with GLT1 and GADD45 transcripts in preventing secondary neuronal death. GLIA 29:222–232, 2000.

Nigrostriatal denervation does not affect glutamate transporter mRNA expression but subsequent levodopa treatment selectively increases GLT1 mRNA and protein expression in the rat striatum.

There is growing evidence that the loss of the nigrostriatal dopaminergic neurones induces an overactivity of the corticostriatal glutamatergic pathway which seems to be central to the physiopathology of parkinsonism. Moreover, glutamatergic mechanisms involving NMDA receptors have been shown to interfere with the therapeutical action of levodopa. Given the key role played by uptake processes in glutamate neurotransmission, this study examined the effects of nigrostriatal deafferentation and of levodopa treatment on the striatal expression of the glutamate transporters GLT1, GLAST and EAAC1 in the rat. No significant changes in striatal mRNA levels of these transporters were detected after either levodopa treatment (100 mg/kg; i.p., twice a day for 21 days) or unilateral lesion of the nigrostriatal pathway by intranigral 6‐hydroxydopamine injection. In contrast, animals with the lesion subsequently treated with levodopa showed a selective increase (36%) in GLT1 mRNA levels in the denervated striatum versus controls. These animals also showed increased GLT1 protein expression, as assessed by immunostaining and western blotting. These data provide the first evidence that levodopa therapy may interfere with striatal glutamate transmission through change in expression of the primarily glial glutamate transporter GLT1. We further suggest that levodopa‐induced GLT1 overexpression may represent a compensatory mechanism preventing neurotoxic accumulation of endogenous glutamate.

Effects of PKA and PKC modulators on high affinity glutamate uptake in primary neuronal cell cultures from rat cerebral cortex

In this study, the effects of various agents known to alter protein phosphorylation, via protein kinase C or A, on high affinity glutamate uptake were investigated in primary neuronal cell cultures of rat cerebral cortex. Incubating the culture dishes with chelerythrine or H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide), which inhibit PKC and PKA, respectively, dramatically decreased the glutamate uptake in a dose-dependent manner. Saturation kinetic analysis showed that chelerythrine and H89 decreased the Vmax (chelerythrine: -61%, P < 0.06; -59%, P < 0.05) without affecting the Km of the transport process as compared to the control values. These inhibitory effects were counteracted by the corresponding protein kinase activators, i.e. PMA (phorbol-12-myristate 13-acetate) in the case of PKC and forskolin in the case of PKA, although these protein kinase activators alone did not significantly affect the glutamate uptake. These results provide evidence that, in primary cultures of neuronal cells, the high affinity glutamate uptake may be regulated by both PKA and PKC-mediated phosphorylation processes.

Activation of the Adenylate Cyclase-dependent Protein Kinase Pathway Increases High Affinity Glutamate Uptake Into Rat Striatal Synaptosomes

This study examined the effects of various agents known to alter protein phosphorylation through protein kinase A or C on high affinity glutamate uptake measured in vitro on rat striatal homogenates. Incubation of synaptosomes with the phosphatase inhibitor, okadaic acid, dramatically increased glutamate uptake indicating that underlying phosphorylation mechanisms may be involved in the regulation of this transport process. The protein kinase C activator, phorbol-12,13-dibutyrate, or inhibitor, staurosporine, did not significantly modify glutamate uptake. In contrast, forskolin, which activates adenylate cyclase, induced a dose-dependent increase in glutamate uptake. Saturation kinetic analysis indicated that forskolin increased the Vmax without modifying the Km of the transport process as compared to control values. The effect of forskolin was mimicked by dibutyryl adenosine monophosphate, an analog of cAMP which activates protein kinase A, and counteracted by high doses of N-[2-(methylamino) ethy1]-5-isoquinoline sulfonamide, a protein kinase A inhibitor. These results suggest that an adenylate cyclase-dependent protein kinase may be involved in the post-translational regulation of glutamate transporters.

Relationships between striatin-containing neurons and cortical or thalamic afferent fibres in the rat striatum. An ultrastructural study by dual labelling.

Striatin, a recently isolated rat brain calmodulin-binding protein belonging to the WD-repeat protein family, is thought to be part of a calcium signal transduction pathway presumably specific to excitatory synapses, at least in the striatum. This study was aimed to specify the cellular and subcellular localization of striatin, and to determine the possible synaptic relationships between the two main excitatory afferent pathways, arising from the cerebral cortex and the thalamus, and the striatin-containing elements, in the rat striatum. Anterograde tract-tracing by means of biotinylated dextran amine injection in the frontoparietal cerebral cortex or the parafascicular nucleus of the thalamus was combined with immunogold detection of striatin. Striatin-immunoreactivity was confined to the neuronal somatodendritic compartment, including spines. Whereas 90-95% of the striatal neurons were striatin-positive, only about 50% of the sections of dendritic spines engaged in asymmetrical synaptic contacts exhibited striatin labelling. Among the sections of striatin-immunopositive dendritic spines, the number of immunogold particles ranged from one to more than seven, indicating an heterogeneity of the spine labelling. Moreover, within each class of spines presenting at least two silver-gold particles, the distribution of the particles varied from a clear-cut alignment under the postsynaptic densities (24-33% of spines) to a location distant from the synaptic area. In the cell bodies and dendrites, striatin labelling was usually not associated with the cytoplasmic membrane nor with the postsynaptic densities. In the striatum ipsilateral to the tracer injections, only 34.8% of the synaptic contacts formed by corticostriatal afferents involved striatin-positive elements (slightly labelled dendritic spines), whereas 56.7% of the synaptic contacts formed by thalamostriatal boutons were made on striatin-positive targets (mostly dendrites). In both cases, striatin labelling was usually not associated with the postsynaptic density. Most of the immunoreactive dendritic spines were in contact with unidentified afferents. These data reveal that striatin is expressed in the vast majority of the cell bodies of striatal spiny neurons, but is heterogeneously distributed among the dendritic spines of those neurons. Data also indicate a preferential relationship between striatin-containing structures and afferents from the parafascicular thalamic nucleus with respect to the frontoparietal cerebral cortex. But, at the dendritic spine level, striatin may be involved in signal transduction mechanisms involving as yet unidentified excitatory afferents to striatal neurons.

Differential Effects of Corticostriatal and Thalamostriatal Deafferentation on Expression of the Glutamate Transporter GLT1 in the Rat Striatum

Abstract: This study compared the effects of the disruption of the two main presumably glutamatergic striatal inputs, the corticostriatal and thalamostriatal pathways, on GLT1 expression in the rat striatum, using in situ hybridization and immunohistochemistry. Unilateral ibotenate‐induced thalamic lesion produced no significant changes in striatal GLT1 mRNA labeling and immunostaining as assessed at 5 and 12 days postlesion. In contrast, significant increases in both parameters were measured after bilateral cortical lesion by superficial thermocoagulation. GLT1 mRNA levels increased predominantly in the dorsolateral part of the striatum; there, the increases were significant at 5 (+84%), 12 (+101%), and 21 (+45%) but not at 35 days postlesion. GLT1 immunostaining increased significantly and homogeneously by 17‐26% at 12 and 21 days postlesion. The increase in GLT1 expression at 12 days postlesion was further confirmed by western blot analysis; in contrast, a 36% decrease in glutamate uptake activity was measured at the same time point. These data indicate that striatal GLT1 expression depends on corticostriatal but not thalamostriatal innervation. Comparison of our results with previous data showing that cortical lesion by aspiration down‐regulates striatal GLT1 expression further suggests that differential changes in GLT1 expression, and thus presumably in glial cell function, may occur in the target striatum depending on the way the cortical neurons degenerate.

The “Orphan” Na+/Cl--Dependent Transporter, Rxt1, Is Primarily Localized Within Nerve Endings of Cortical Origin in the Rat Striatum

Abstract : Previous studies have shown that the striatum expresses very low levels of Na+/Cl‐‐dependent “orphan” transporter Rxt1 transcripts but contains high levels of protein. This study investigated the origin of Rxt1 expression in rat striatum. Striatal Rxt1 contents assessed by immunocytochemistry or western blotting were found to be significantly reduced after corticostriatal denervation but not after striatal or thalamic lesion with kainic acid or selective 6‐hydroxydopamine‐induced nigrostriatal deafferentation. Corticostriatal neurons retrogradely labeled by intrastriatal fluorogold injections were shown to express Rxt1 mRNA. Combination of anterograde biotin‐dextran amine labeling of the corticostriatal pathway with Rxt1 immunogold detection at the ultrastructural level demonstrated the presence of Rxt1 in about one‐third of the corticostriatal synaptic terminals and in numerous unidentified synaptic terminals. All the Rxt1‐positive terminals formed asymmetrical contacts on spines. These data provide evidence that striatal Rxt1 immunoreactivity is mainly of extrinsic origin and more specifically associated with the corticostriatal pathway. Rxt1 appears as a selective presynaptic marker of synapses formed by presumably excitatory amino acid afferents, but it segregates a subclass of these synapses, thereby revealing a functional heterogeneity among excitatory amino acid systems.

A Dual Immunoperoxidase Labelling Method to Study the Ultrastructural Relationships between Choline Acetyltransferase- and Neuropeptide Y-Containing Neurons in the Rat Striatum

In the striatum, neuropeptide Y (NPY) and choline acetyltransferase (ChAT) are to be found in populations of aspiny neurons with distinct morphological features. Indeed, immunocytochemical studies using selective antibodies have indicated that NPY-labelled neurons are medium-sized type IV aspiny neurons (Vuillet et al., 1989b), whereas ChAT-labelled neurons belong mainly to the category of large neurons in the striatum (Bolam et al., 1984). Neurochemical studies in combination with lesion experiments and morphological studies have provided evidence that both NPY- and ChAT- containing neuronal populations represent interneurons, receive afferents from extrinsic origin (Chang, 1988; Vuillet et al., 1989a; 1989b), and provide synaptic input to medium-sized spiny neurons that are predominantly striatal output neurons (Izzo and Bolam., 1988; Vuillet et al, 1989b). These data suggest that both cholinergic and NPY interneurons in the striatum may play an integrative role which may be of particular relevance in the control of sensorimotor function. The question of whether or not synaptic interactions exist between those NPY and cholinergic interneurons then appears of great interest for further functional resolution of the striatal network. Two major difficulties emerge to answer this question: both types of neurons represent less than one percent of the total striatal neuron population; in addition, besides the paucity of NPY terminals, both ChAT and NPY axonal boutons form predominantly symmetrical type synapses that are difficult to characterize.

Further Considerations on the Cellular Mechanisms of Neuronal Plasticity

Recent evidences indicate that functional recovery after brain injury in adults is dependent on extreme adaptive capacities of mature neuronal circuits. Neuroplasticity at the cellular level would involve three sets of mechanisms referring to structural, metabolic and genomic adaptive changes. Most of studies have examined the effects of elimination of afferent pathways to particular brain regions and more recently the effects of intracerebral transplantations. For a long time, neuroplasticity then appeared as a compensatory structural or functional reorganization of neuronal networks triggered by brain damage. However, the concept has recently emerged that the adaptive capacities of the adult brain at ceIlular level are also expressed in the absence of denervation, and would represent a basic mechanism for learning and memory processes as weIl as for behavioural adjustements to pennanent changes in the external context.