Denise Samuel

Localization of N‐Methyl‐d‐Aspartate Receptors in the Rat Striatum: Effects of Specific Lesions on the [3H]3‐(2‐Carboxypiperazin‐4‐yl)propyl‐1‐Phosphonic Acid Binding

Abstract: The binding of [3H]3‐(2‐carboxypiperazin‐4‐yl)propyl‐1‐phosphonic acid ([3H]CPP), a rigid analogue of 2‐amino‐7‐phosphonoheptanoic acid (AP7) and reported to be a selective N‐methyld‐aspartate (NMDA) antagonist, was studied in rat striatal membranes using a centrifugation procedure to separate bound and free radioligand. [3H]CPP bound with high affinity (Kd= 272 nM) in a saturable, reversible, and protein concentration‐dependent manner, Specific binding was suggested to involve a single class of noninteracting binding sites. The most potent [[3H]CPP binding inhibitors tested were CPP, l‐glutamate, 2‐amino‐5‐phosphonovalerate, and AP7, NMDA, l‐aspartate, and α‐aminoadipate were also shown to be efficient in inhibiting the binding, whereas quisqualate, d,l‐2‐amino‐4‐phosphonobutyrate, kainate, l‐glutamate diethylester, and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid were found to be essentially inactive. These data are therefore consistent with the view that [3H]CPP selectively binds to NMDA receptors in the rat striatum. Lesions of intrastriatal neurons using local injections of kainic acid revealed a marked decrease in [3H]CPP binding, suggesting an almost exclusively postsynaptic location of binding sites in the striatum. Conversely, bilateral lesion of corticostriatal glutamatergic fibers resulted in an increased number of [3H]CPP striatal binding sites, providing evidence for a putative supersensitivity response to this striatal deafferentation. Interestingly, lesion of the nigrostriatal dopaminergic neurons using intranigral 6‐hydroxydopamine injections resulted, 2–3 weeks later, in a similar increase in the number of [3H]CPP striatal binding sites. These data suggest the occurrence of functional receptor‐receptor interregulations at the postsynaptic level between dopaminergic and NMDA receptors in complement with the interactions occurring at the presynaptic level between glutamatergic and dopaminergic nerve terminals.

3α,5α-tetrahydroprogesterone (allopregnanolone) and γ-aminobutyric acid: Autocrine/paracrine interactions in the control of neonatal PSA-NCAM+ progenitor proliferation

The earliest identified neonatal neural progenitors are cells that express the polysialylated form of the neural cell adhesion molecule (PSA‐NCAM). One of these progenitors is the early PSA‐NCAM+ progenitor (ePSA‐NCAM+ progenitor; Gago et al. [ 2003 ] Mol Cell Neurosci 22:162–178), which corresponds to a multipotential cell with a default differentiation through glial lineages. The ePSA‐NCAM+ progenitor can synthesize the neurosteroid progesterone (PROG) and its reduced metabolite 3α,5α‐tetrahydroprogesterone (3α,5α‐THP, or allopregnanolone; Gago et al. [ 2001 ] Glia 36:295–308). The latter is a potent positive allosteric modulator of γ‐aminobutyric acid type A (GABAA) receptors. In the present work, we demonstrate that PROG and 3α,5α‐THP both stimulate ePSA‐NCAM+ progenitor proliferation. PROG exerted its mitogenic effect indirectly, through its conversion to 3α,5α‐THP, since it could be abolished by an inhibitor of the 5α‐reductase (L685‐273) and mimicked by 3α,5α‐THP. A dose‐response curve revealed a bell‐shaped effect of 3α,5α‐THP on ePSA‐NCAM+ progenitor proliferation, with greatest stimulation at nanomolar concentrations. The mitogenic effect of 3α,5α‐THP was mediated by GABAA receptors, insofar as it could be blocked by the selective antagonist bicuculline. ePSA‐NCAM+ progenitors indeed expressed mRNAs for GABAA receptor subunits, and GABA enhanced cell proliferation, an effect that was also bicuculline sensitive. Moreover, these cells synthesized GABA, which was involved in a tonic stimulation of their proliferation. These results reveal complex autocrine/paracrine loops in the control of ePSA‐NCAM+ progenitor proliferation, involving both neurosteroid and GABA signaling, and suggest a novel key role for 3α,5α‐THP in the development of the nervous system.

Glutamate transport alteration triggers differentiation-state selective oxidative death of cultured astrocytes: a mechanism different from excitotoxicity depending on intracellular GSH contents

Recent evidence has been provided for astrocyte degeneration in experimental models of neurodegenerative insults associated with glutamate transport alteration. To determine whether astrocyte death can directly result from altered glutamate transport, we here investigated the effects of l‐trans‐pyrrolidine‐2,4‐dicarboxylate (PDC) on undifferentiated or differentiated cultured rat striatal astrocytes. PDC induced death of differentiated astrocytes without affecting undifferentiated astrocyte viability. Death of differentiated astrocytes was also triggered by another substrate inhibitor but not by blockers of glutamate transporters. The PDC‐induced death was delayed and apoptotic, and death rate was dose and treatment duration‐dependent. Although preceded by extracellular glutamate increase, this death was not mediated through glutamate receptor stimulation, as antagonists did not provide protection. It involves oxidative stress, as a decrease in glutathione contents and a dramatic raise in reactive oxygen species preceded cell loss, and as protection was provided by antioxidants. PDC induced a similar percentage of GSH depletion in the undifferentiated astrocytes, but only a slight increase in reactive oxygen species. Interestingly, undifferentiated astrocytes exhibited twofold higher basal GSH content compared with the differentiated ones, and depleting their GSH content was found to render them susceptible to PDC. Altogether, these data demonstrate that basal GSH content is a critical factor of astrocyte vulnerability to glutamate transport alteration with possible insights onto concurrent death of astrocytes and gliosis in neurodegenerative insults.

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.

γ-Aminobutyric Acid Function in the Rat Striatum Is Under the Double Influence of Nigrostriatal Dopaminergic and Thalamostriatal Inputs: Two Modes of Regulation?

Abstract: Glutamic acid decarboxylase (GAD), γ‐[3H]‐aminobutyric acid ([3H]GABA) high‐affinity uptake into synaptosomes, and endogenous GABA content were measured in the rat striatum 2–3 weeks following 6‐hydroxydopamine injection in the ipsilateral substantia nigra to destroy the nigrostriatal dopaminergic pathway and after kainic acid injection into the centromedial‐parafascicular complex of the ipsilateral thalamus to lesion the thalamostriatal input. Both lesions resulted in apparent GAD increase concomitant with a decreased [3H]GABA uptake into striatal synaptosomes. GABA content was increased selectively following the dopaminergic lesion. Kinetic analysis of the uptake process for [3H]GABA showed selectively a decreased Vmax following the dopaminergic lesion; in animals with thalamic lesion, however, the change only concerned the Km, which showed a decreased affinity of the transport sites for [3H]GABA. Determination of Km and Vmax for GAD action on its substrate glutamic acid showed an increased affinity of GAD for glutamic acid in the case of the dopaminergic lesion without any change in Vmax, whereas the thalamic lesion resulted in GAD increase concomitant with a selective increase in Vmax. These data suggest that striatal GABA neurons are under the influence of nigrostriatal dopaminergic neurons which may reduce the GABA turnover, whereas the exact nature of the powerful control also revealed on these neurons following thalamic lesion remains to be determined. Both lesions induced adaptive neurochemical responses of striatal GABA neurons, possibly reflecting in the case of the dopaminergic deprivation an increased GABA turnover.

Developmental expression and activity of high affinity glutamate transporters in rat cortical primary cultures

The expression and activity of glutamate transporters (EAAC1, GLAST and GLT1) were examined during the development of cortical neuron-enriched cultures. Protein content and mitochondrial respiration both increased during the first 7 days, later stabilized and decreased from DIV14. Glutamate transport and extracellular concentration were relatively constant from DIV3 to 18. The kinetic parameters of glutamate transport were at DIV7: K(m)=19+/-3 microM and V(max)=1068+/-83 pmol/mg protein/min and at DIV14: K(m)=40.8+/-9.3 microM and V(max)=1060+/-235 pmol/mg protein/min. The shift in K(m) towards higher values suggest a more important participation of GLAST after DIV14. At DIV7 and 14, glutamate transport was poorly sensitive to dihydrokaïnate (DHK) suggesting a weak participation of GLT1 in glutamate transport. Western blot experiments and immunocytochemistry showed that EAAC1 was expressed by neurons whatever the stage of the culture. GLAST was found in astrocytes as soon as DIV3 and labeling increased during the development of the culture. There was little neuronal GLT1 immunoreactivity at DIV7, only detected by immunocytochemistry. From DIV10 to 18, an increasing astrocytic expression of GLT1 was observed, also detected by Western blotting. These results show that: (1) glutamate uptake remains stable all along the development of the cultures although the pattern of expression of the different transporters is changing, suggesting that glutamate transport is highly regulated; (2) neuronal EAAC1 may play a critical role during the early stages of the culture when it is expressed alone; and (3) the developmental expression pattern of glutamate transporters in cortical neuron-enriched cultures is quite similar to that observed in vivo during early postnatal development.

Effects of Ionotropic Excitatory Amino Acid Receptor Antagonists on Glutamate Transport and Transport‐Mediated Changes in Extracellular Excitatory Amino Acids in the Rat Striatum

Abstract: This study examined the effects of intrastriatal administration of ionotropic excitatory amino acid receptor antagonists on biochemical markers of excitatory amino acid transmission in the rat striatum. High‐affinity glutamate uptake was measured ex vivo on striatal homogenates 15 min after the local administration of either 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX), a non‐NMDA receptor antagonist, or dl‐2‐amino‐5‐phosphonopentanoic acid (AP5), a competitive NMDA antagonist, at various doses (10–500 pmol injected). DNQX induced a dose‐dependent increase in glutamate uptake rate, related to an increase in the Vmax of the transport process, whereas no significant change in glutamate uptake was detected after AP5 administration. Similar results were obtained from animals subjected to excitotoxic lesion of striatal neurons by kainate administration 15 days before the injection of DNQX or AP5. In a parallel series of experiments using in vivo microdialysis we showed that DNQX (10−5M) in the dialysis probe diminished by ∼30–40% the increases in the concentrations of glutamate and aspartate elicited by l‐trans‐pyrrolidine‐2,4‐dicarboxylic acid (1 mM). These data suggest that presynaptic glutamate transmission in the rat striatum may undergo facilitatory autoregulatory processes involving ionotropic non‐NMDA receptors and highlight the view that transporters for glutamate may be potent regulatory sites for glutamatergic transmission.

Effects of riluzole (2-amino-6-trifluoromethoxy benzothiazole) on striatal neurochemical markers in the rat, with special reference to the dopamine, choline, GABA and glutamate synaptosomal high affinity uptake systems

Summary— Riluzole, a new compound with anticonvulsant properties, was found to induce a dose‐dependent decrease in the uptake of 3H‐dopamine, 3H‐GABA and 3H‐glutamate into striatal synaptosmes when added to the incubation medium or after in vivo administration, whereas an inlubition of 3H‐choline uptake was detected only in the in vitro experiments. Interestingly, riluzole affected 3H‐dopamine and 3H‐glutamate uptake differentially since 3H‐dopamine uptake was found to be more sensitive to the compound. Moreover, riluzole inhibited 3H‐dopamine uptake competitively and 3H‐glutamate uptake non‐competitively, which further suggests that the action of the compound is selective. After in vivo injection, riluzole did not affect the striatal dopamine, DOPAC, serotonin, 5HIAA, glutamate, aspartate or GABA contents. Since this compound was previously reported to induce a decrease in the spontaneous release of glutamate, serotonin, dopamine and possibly acetylcholine, the hypothesis is put forward that riluzole may, at least at high concentrations, have general effects on the striatal nerve terminals affecting both the uptake and release processes. This action may be correlated with the recently identified blocking properties of the compound on the sodium channels, as previously shown for local anaesthetics.

Involvement of the glutamatergic metabotropic receptors in the regulation of glutamate uptake and extracellular excitatory amino acid levels in the striatum of chloral hydrate-anesthetized rats

The microdialysis technique was used to assess in vivo the putative functional role of metabotropic excitatory amino acid receptors in regulating extracellular levels of the excitatory amino acids glutamate and aspartate in the striatum of chloral hydrate-anesthetized rats. Addition of the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) (10(-3) or 4 x 10(-3) M) in the dialysis probe did not modify the basal extracellular levels of glutamate and aspartate but induced a significant dose-dependent decrease in the KCl-elicited elevation of glutamate and aspartate extracellular levels. The effect of MCPG on glutamate extracellular concentration under K+ stimulation was reduced by the simultaneous superfusion of the metabotropic glutamate receptor agonist (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine) (L-CCG) (10(-3) M) which had no significant effect when tested alone. In contrast, L-CCG alone significantly potentiated the KCl-elicited elevation of aspartate extracellular concentrations but failed to modify the MCPG effect on this amino acid concentration. In a parallel series of experiments, high-affinity glutamate uptake was measured ex vivo 20 min after an in vivo injection of 10 pmol of MCPG in the striatum. MCPG was found unable to modify the glutamate uptake rate. In vitro, MCPG (1-1000 microM) again had no effect on glutamate transport rate. These data suggest that metabotropic excitatory amino acid receptors (1) may act to increase the extracellular levels of glutamate and aspartate under depolarizing conditions, and (2) may not have a major role in the regulation of high affinity glutamate uptake under basal conditions. In addition, it can be assumed that the control of glutamate and aspartate extracellular levels may involve different metabotropic receptors.