Role of high-affinity choline uptake on extracellular choline and acetylcholine evoked by NMDA.

Previous studies have shown that NMDA evokes a calcium-dependent and region-specific increase in extracellular choline that is associated with a reduction of membrane phosphatidylcholine and precedes neuronal cell death. We investigated, using in vivo microdialysis, the contribution of high-affinity choline uptake on the increase in extracellular choline evoked by NMDA. Dialysis was performed in the presence of Neostigmine (0.5 microM), an acetylcholinesterase inhibitor, in prefrontal cortex or hippocampus of freely moving rats. Drugs were administered through the dialysis probe. In cholinergic denervation experiments, rats were subjected to sham or AMPA-induced lesion of cholinergic nuclei at least 2 weeks before microdialysis. Excitotoxic lesion of the medial septum / ventral diagonal band nuclei reduced hippocampal choline acetyltransferase activity by 74%, [(3)H]hemicholinium-3 binding by 32%, and completely abolished potassium-evoked acetylcholine release. Despite this reduction of presynaptic cholinergic function, perfusion of NMDA (300 microM) by retrodialysis produced an increase in hippocampal extracellular choline (249 +/- 22% of basal levels) that was similar to that observed in sham controls (301 +/- 35%). Inhibition of choline uptake with hemicholinium-3 in nonlesioned rats produced a sustained increase in dialysate choline (163 +/- 8%) and reduced acetylcholine to 33 +/- 2% of basal levels, consistent with a depletion of the acetylcholine pool due to precursor deficit. Simultaneous perfusion of hemicholinium-3 and NMDA produced a synergistic increase in dialysate choline (664 +/- 95% of basal levels), indicating that part of the choline released by NMDA is taken up. In contrast, NMDA antagonized the decrease of acetylcholine produced by hemicholinium-3. These results show that NMDA-evoked choline release is not mediated by inhibition of high-affinity choline uptake and indicate that choline released by NMDA can be used to sustain acetylcholine synthesis when there is a precursor deficit secondary to uptake inhibition.

Region-specific and calcium-dependent increase in dialysate choline levels by NMDA.

NMDA receptor-induced excitotoxicity has been hypothesized to mediate abnormal choline (Cho) metabolism that is involved in alterations in membrane permeability and cell death in certain neurodegenerative disorders. To determine whether NMDA receptor overactivation modulates choline metabolism in vivo, we investigated the effects of NMDA on interstitial choline concentrations using microdialysis. Perfusion of NMDA by retrodialysis increased dialysate choline (approximately 400%) and reduced dialysate acetylcholine (Ach) (approximately 40%). Choline levels remained increased for at least 2.5 hr, but acetylcholine returned to pretreatment values 75 min after NMDA perfusion. The NMDA-evoked increase in dialysate choline was calcium and concentration dependent and was prevented with 1 mM AP-5, a competitive NMDA antagonist, but was not altered by mepacrine, a phospholipase A2 inhibitor. NMDA increased extracellular choline levels four- to fivefold in prefrontal cortex and hippocampus, produced a slight increase in neostriatum, and did not modify dialysate choline in cerebellum. Perfusion with NMDA for 2 hr produced a delayed, but not acute, reduction in choline acetyltransferase activity in the area surrounding the dialysis probe. Consistent with a lack of acute cholinergic neurotoxicity evoked by this treatment, basal acetylcholine levels were unaltered by 2 hr of continuous NMDA perfusion. Prolonged NMDA perfusion produced a 34% decrease in phosphatidylcholine content in the lipid fraction of the tissue surrounding the dialysis probe. These results show that NMDA modulates choline metabolism, eliciting a receptor-mediated, calcium-dependent, and region-specific increase in extracellular choline from membrane phospholipids that is not mediated by phospholipase A2 and precedes delayed excitotoxic neuronal cell death.

Glycine site antagonists and partial agonists inhibit N-methyl-D-aspartate receptor-mediated [3H]arachidonic acid release in cerebellar granule cells.

Activation of N-methyl-D-aspartate (NMDA) receptors is known to produce arachidonic acid release, which has been implicated in excitotoxicity. Antagonists and partial agonists at the glycine site of the NMDA receptor, despite exhibiting functional differences in electrophysiological studies, inhibit glutamate-induced neurotoxicity and ischemia-induced neurodegeneration. The objective of this study was to investigate the effects of both glycine site antagonists and partial agonists on NMDA receptor-mediated [3H]arachidonic acid (AA) release evoked by glutamate, NMDA or a competitive inhibitor of the glutamate/aspartate uptake carrier. The [3H]AA release evoked by a maximally effective concentration of glutamate (100 microM) was blocked by the glycine site antagonists 7-chlorokynurenic acid (7-CKYN) and 5,7-dichlorokynurenic acid (5,7-DCKYN) and by a low intrinsic efficacy glycine partial agonist (+)-1-hydroxy-3-aminopyrrolid-2-one [(+)-HA-966]. 1-Aminocyclopropanecarboxylic acid (ACPC), a high intrinsic efficacy glycine partial agonist, did not modify [3H]AA release evoked by 100 microM glutamate. However, ACPC blocked (in a glycine reversible manner) the [3H]AA release induced by NMDA (100 microM) with an IC50 of 131 +/- 2 microM. Furthermore, L-trans-pyrrolidine-2,4-dicarboxylate (PDC), a competitive inhibitor of the glutamate transporter, also released [3H]AA (Emax and EC50 of 127 +/- 4% and 30 +/- 1 microM, respectively). ACPC, 7-CKYN and (+/-)-2-amino-7-phosphonoheptanoic acid (AP-7), a competitive NMDA receptor antagonist, inhibited [3H]AA release evoked by PDC. These results demonstrate that both glycine site antagonists and partial agonists can inhibit NMDA receptor-mediated [3H]AA release in cerebellar granule cells, an action consistent with the neuroprotective effects of these compounds.

Effects of NMDA-R1 antisense oligodeoxynucleotide administration: behavioral and radioligand binding studies.

The effects of an antisense phosphodiester oligodeoxynucleotide (ODN) directed to the NR1 subunit of the NMDA receptor mRNA and of its corresponding sense ODN were investigated in mice. Treatment with the antisense ODN significantly increased the time mice spent in the open arms of an elevated maze while the total number of arm entries was unaltered. Furthermore, seizure latencies after the administration of an ED100 dose of NMDA (150 mg/kg) were significantly higher in antisense treated animals compared to vehicle controls. At the same time, treatment with NR1 antisense ODN significantly reduced the Bmax of [3H]CGS-19755 binding (2101 fmol/mg protein) compared to both vehicle (2787 fmol/mg protein) and sense (2832 +/- 39 fmol/mg protein) controls without any significant change in KD (33 nM). A corresponding reduction of [3H]CGP-39653 binding was also observed after treatment with NR1 antisense compared to both sense and vehicle controls. In contrast, neither antisense nor sense ODNs altered the proportion of high affinity glycine sites or the potency of glycine at either high or low affinity glycine binding sites to inhibit [3H]CGP-39653 binding. These results show that in vivo treatment with NR1 antisense ODNs to the NMDA receptor complex reduces antagonist binding at NMDA receptors and has pharmacological effects similar to those observed with some NMDA receptor antagonists. These results also suggest that treatment with antisense ODNs may provide another means to investigate allosteric modulation of receptor subtypes in vivo.

Modulation of NMDA-induced cytosolic calcium levels by ACPC in cultured cerebellar granule cells.

The effect of 1-aminocyclopropanecarboxylic acid (ACPC) on the potentiation by glycine of N-methyl-D-aspartate (NMDA)-evoked increases in intracellular free calcium concentration [Ca2+]i was examined in cultured rat cerebellar granule cells. NMDA (50 microM) produced a rapid and sustained increase in [Ca2+]i from 72 +/- 3 to 205 +/- 18 nM. Addition of exogenous glycine potentiated (EC50 -2 microM) the effects of NMDA, increasing [Ca2+]i to an Emax of 323 +/- 5 nM. ACPC increased the EC50 of glycine from 2 microM (no ACPC) to 17 microM (400 microM ACPC). Concomitant with reduced potency of glycine, ACPC also inhibited the Emax of glycine to enhance NMDA-evoked cytosolic free calcium to values (224 +/- 1 nM) approaching those observed in the nominal absence of glycine. These results show that ACPC, a compound previously reported to prevent excitotoxic cell death, inhibits the glycine-induced increase of Ca2+ entry through NMDA receptors in cerebellar granule cells.

1-Aminocyclopropane-carboxylic acid reduces NMDA-induced hippocampal neurodegeneration in vivo.

The effect of systemic treatment with 1-aminocyclopro-panecarboxylic acid (ACPC), a partial agonist at the glycine site of the NMDA receptor, on convulsions and neurodegeneration induced by intrahippocampal injection of NMDA was investigated in mice. Five days after intrahippocampal NMDA infusion, 80-100% pyramidal cell death was observed in the CA1 region of the hippocampus. Pretreatment with ACPC prevented the lethal effects of NMDA and significantly reduced seizure induction. ACPC reduced cell death to 40% of that induced by a dose of NMDA (6 nmol) that damaged 80% of hippocampal CA1 neurones in untreated animals. These findings provide further evidence that ACPC can reduce NMDA receptor function in vivo and suggest that partial agonists at the glycine site of the NMDA receptor complex may be useful anticonvulsant and neuroprotective agents.