The Armc10/SVH gene: genome context, regulation of mitochondrial dynamics and protection against Aß-induced mitochondrial fragmentation.

Mitochondrial function and dynamics are essential for neurotransmission, neural function and neuronal viability. Recently, we showed that the eutherian-specific Armcx gene cluster (Armcx1-6 genes), located in the X chromosome, encodes for a new family of proteins that localise to mitochondria, regulating mitochondrial trafficking. The Armcx gene cluster evolved by retrotransposition of the Armc10 gene mRNA, which is present in all vertebrates and is considered to be the ancestor gene. Here we investigate the genomic organisation, mitochondrial functions and putative neuroprotective role of the Armc10 ancestor gene. The genomic context of the Armc10 locus shows considerable syntenic conservation among vertebrates, and sequence comparisons and CHIP-data suggest the presence of at least three conserved enhancers. We also show that the Armc10 protein localises to mitochondria and that it is highly expressed in the brain. Furthermore, we show that Armc10 levels regulate mitochondrial trafficking in neurons, but not mitochondrial aggregation, by controlling the number of moving mitochondria. We further demonstrate that the Armc10 protein interacts with the KIF5/Miro1-2/Trak2 trafficking complex. Finally, we show that overexpression of Armc10 in neurons prevents Aß-induced mitochondrial fission and neuronal death. Our data suggest both conserved and differential roles of the Armc10/Armcx gene family in regulating mitochondrial dynamics in neurons, and underscore a protective effect of the Armc10 gene against Aß-induced toxicity. Overall, our findings support a further degree of regulation of mitochondrial dynamics in the brain of more evolved mammals.

Glycogen synthase kinase-3 is involved in the regulation of the cell cycle in cerebellar granule cells.

Recent studies have demonstrated that neuronal reentry in the cell cycle and specifically the expression of the transcription factor E2F-1, constitutes a pathway that may be involved in neuronal apoptosis after serum and potassium withdrawal. Other enzymes such as glycogen synthase kinase-3beta (GSK-3beta) are also involved in this apoptotic stimulus, and thus in the process of neuronal cell death. Primary cerebellar granule cells (CGNs) were used in this study to determine whether pharmacological inhibition of GSK-3beta is involved in neuronal modulation of the cell cycle, and specifically in the regulation of E2F-1 and retinoblastoma protein (Rb). CGNs showed a dramatic increase in GSK-3beta activity after 2h of serum and potassium deprivation. Immunoblot and activity assays revealed that lithium and SB415286 inhibit fully the activation of GSK-3beta and attenuate the expression of cyclin D, cyclin E, pRb phosphorylation and the transcription factor E2F-1. These data were confirmed using AR-014418, a selective GSK-3beta inhibitor that prevents the expression of cell-cycle proteins. Our data indicate that GSK-3beta inhibition regulates, in part, the cell cycle in CGNs by inhibiting Rb phosphorylation and thus inhibiting E2F-1 activity. However, the selective inhibition of GSK-3beta with AR-A014418 had not effect on cell viability or apoptosis mediated by S/K withdrawal. Furthermore, our results suggest that selective GSK-3beta inhibition is not sufficient to protect against apoptosis in this S/K withdrawal model, indicating that Li(+) and SB415286 neuroprotective effects are mediated by the inhibition of additional targets to GSK3beta. Therefore, there is a connection between cell cycle and GSK-3beta activation and that these, along with other mechanisms, are involved in the molecular paths leading to the apoptotic process of rat CGNs triggered by S/K withdrawal.

Inhibition of cyclin-dependent kinases is neuroprotective in 1-methyl-4-phenylpyridinium-induced apoptosis in neurons.

The biochemical pathways involved in neuronal cell death in Parkinson's disease are not completely characterized. Mitochondrial dysfunction, specifically alteration of the mitochondrial complex I, is the primary target of the parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP+) induced apoptosis in neurons. In the present study, we examine the role of caspase-dependent and -independent routes in MPP+-induced apoptosis in rat cerebellar granule neurons (CGNs). We show a distinct increase in the expression of the cell cycle proteins cyclin D, cyclin E, cdk2, cdk4 and the transcription factor E2F-1 following a MPP+ treatment of CGNs. Flavopiridol (FLAV), a broad inhibitor of cyclin-dependent kinases (CDKs), attenuated the neurotoxic effects of MPP+ and significantly attenuates apoptosis mediated by MPP+ 200 microM. Likewise, the antioxidant vitamin E (vit E) increases neuronal cell viability and attenuates apoptosis induced by MPP+. Moreover, the expression levels of cyclin D and E2F-1 induced by this parkinsonian neurotoxin were also attenuated by vit E. Since, the broad-spectrum caspase inhibitor zVAD-fmk did not attenuate MPP+-induced apoptosis in CGNs, our data provide a caspase-independent mechanism mediated by neuronal reentry in the cell cycle and increased expression of the pro-apoptotic transcription factor E2F-1. Our results also suggest a potential role of oxidative stress in neuronal reentry in the cell cycle mediated by MPP+. Finally, our data further support the therapeutic potential of flavopiridol, for the treatment of Parkinson's disease.

Differential responses to anxiogenic drugs in a mouse model of panic disorder as revealed by Fos immunocytochemistry in specific areas of the fear circuitry.

Sensitivity to pharmacological challenges has been reported in patients with panic disorder. We have previously validated transgenic mice overexpressing the neurotrophin-3 (NT-3) receptor, TrkC (TgNTRK3), as an engineered murine model of panic disorder. We could determine that TgNTRK3 mice presented increased cellularity in brain regions, such as the locus ceruleus, that are important neural substrates for the expression of anxiety in severe anxiety states. Here, we investigated the sensitivity to induce anxiety and panic-related symptoms by sodium lactate and the effects of various drugs (the alpha2-adrenoceptor antagonist, yohimbine and the adenosine antagonist, caffeine), in TgNTRK3 mice. We found enhanced panicogenic sensitivity to sodium lactate and an increased intensity and a differential pattern of Fos expression after the administration of yohimbine or caffeine in TgNTRK3. Our findings validate the relevance of the NT-3/TrkC system to pathological anxiety and raise the possibility that a specific set of fear-related pathways involved in the processing of anxiety-related information may be differentially activated in panic disorder.

Inhibition of phosphatidylcholine synthesis is associated with excitotoxic cell death in cerebellar granule cell cultures.

Glucose deprivation (GD) enhances the sensitivity of cerebellar granule cells to die by excitotoxicity. Neither 70 min of GD, a treatment that depletes cell energy resources, nor exposure to 20 microM glutamate (GLU) for 30 min, induce significant cell death in cultures of cerebellar granule cells. However, the combined treatment with GLU and GD induces choline (Cho) release before excitotoxic cell death. We investigated whether the neurotoxic effect of this treatment is related with inhibition of phosphatidylcholine (PC) synthesis. We found that exposure to GLU for 30 min, to GD for 70 min, and to the combination of both, inhibited PC synthesis at the end of treatment by 71%, 92% and 91%, respectively. The inhibition of PC synthesis was accompanied by a decrease in the incorporation of [(3)H]Cho into phosphocholine and by an increase of the intracellular content of free [(3)H]Cho, indicating that these treatments inhibit the synthesis of PC by inhibiting choline kinase activity. However, only the combined treatment with GLU and GD induced a prolonged inhibition of PC synthesis that extended after the end of treatment. These results show that excitotoxic death is associated with sustained inhibition of PC synthesis and suggest that this effect of the combined treatment with GLU and GD on PC synthesis is produced by an action on an enzymatic step downstream of choline kinase activity.

Overactivation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate but not kainate receptors inhibits phosphatidylcholine synthesis before excitotoxic neuronal death.

Glutamate receptor overactivation induces excitotoxic neuronal death, but the contribution of glutamate receptor subtypes to this excitotoxicity is unclear. We have previously shown that excitotoxicity by NMDA receptor overactivation is associated with choline release and inhibition of phosphatidylcholine synthesis. We have now investigated whether the ability of non-NMDA ionotropic glutamate receptor subtypes to induce excitotoxicity is related to the ability to inhibit phosphatidylcholine synthesis. alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-induced a concentration-dependent increase in extracellular choline and inhibited phosphatidylcholine synthesis when receptor desensitization was prevented. Kainate released choline and inhibited phosphatidylcholine synthesis by an action at AMPA receptors, because these effects of kainate were blocked by the AMPA receptor antagonist LY300164. Selective activation of kainate receptors failed to release choline, even when kainate receptor desensitization was prevented. The inhibition of phosphatidylcholine synthesis evoked by activation of non-desensitizing AMPA receptors was followed by neuronal death. In contrast, specific kainate receptor activation, which did not inhibit phosphatidylcholine synthesis, did not produce neuronal death. Choline release and inhibition of phosphatidylcholine synthesis were induced by AMPA at non-desensitizing AMPA receptors well before excitotoxicity. Furthermore, choline release by AMPA required the entry of Ca(2+) through the receptor channel. Our results show that AMPA, but not kainate, receptor overactivation induces excitotoxic cell death, and that this effect is directly related to the ability to inhibit phosphatidylcholine synthesis. Moreover, these results indicate that inhibition of phosphatidylcholine synthesis is an early event of the excitotoxic process, downstream of glutamate receptor-mediated Ca(2+) overload.

Overexpression of neuronal pentraxin 1 is involved in neuronal death evoked by low K(+) in cerebellar granule cells.

Mature cerebellar granule cells in culture die by a process that requires new RNA and protein synthesis when deprived of depolarizing concentrations of potassium. We investigated gene expression during the early phase of the cell death program evoked by potassium deprivation. Using a differential gene display technique, we isolated a cDNA that was increased by potassium deprivation. This cDNA was homologous to the 3' mRNA end of neuronal pentraxin 1 (NP1), a gene encoding a secreted glycoprotein whose expression is restricted to the nervous system. Reverse-Northern and Northern blot analyses confirmed that treatment with low potassium induces overexpression of NP1 mRNA, with a subsequent increase in NP1 protein levels. Time-course studies indicated that overexpression of NP1 protein reaches a maximum after 4 h of exposure to potassium deprivation and 4 h before significant cell death. Incubation of cerebellar granule cells with an antisense oligodeoxyribonucleotide directed against NP1 mRNA reduced low potassium-evoked NP1 protein levels by 60% and attenuated neuronal death by 50%, whereas incubation with the corresponding sense oligodeoxyribonucleotide was ineffective. Furthermore, acute treatment with lithium significantly inhibited both overexpression of NP1 and cell death evoked by low potassium. These results indicate that NP1 is part of the gene expression program of apoptotic cell death activated by nondepolarizing culture conditions in cerebellar granule cells.

Glycine(B) receptor antagonists and partial agonists prevent memory deficits in inhibitory avoidance learning.

Activation of N-methyl-d-aspartate (NMDA) receptors has been hypothesized to mediate certain forms of learning and memory. This hypothesis is based on the ability of competitive and uncompetitive NMDA receptor antagonists to disrupt learning. We investigated the effects of glycine site antagonists and partial agonists on deficits of acquisition (learning) and consolidation (memory) in a single trial inhibitory avoidance learning paradigm. Posttraining administration of either hypoxia (exposure to 7% oxygen) or the convulsant drug pentylenetetrazole (PTZ) (45 mg/kg) to mice impaired consolidation without producing neuronal cell death. Pretreatment with the competitive glycine antagonist 7-chlorokynurenic acid (7KYN) and the glycine partial agonists 1-aminocyclopropanecarboxylic acid (ACPC) and (+)HA-966 prevented memory deficits induced by hypoxia and PTZ, but did not affect scopolamine-induced learning impairment. In addition, ACPC prevented consolidation deficits evoked by a nonexcitotoxic concentration of l-trans-pyrrolidine-2, 4-dicarboxylate, a competitive inhibitor of glutamate transport that increases extracellular levels of glutamate. Moreover, (+)HA-966, 7KYN, and ACPC facilitated both acquisition and consolidation of inhibitory avoidance training, an effect that was dose-dependent and reversed by glycine. These results indicate that memory deficits induced by both hypoxia and PTZ involve NMDA receptor activation. Furthermore, the present findings demonstrate that glycine site antagonists and partial agonists prevent memory deficits of inhibitory avoidance learning by affecting consolidation, but not acquisition processes.

Choline release and inhibition of phosphatidylcholine synthesis precede excitotoxic neuronal death but not neurotoxicity induced by serum deprivation.

N-Methyl-d-aspartate (NMDA) receptor overactivation has been proposed to induce excitotoxic neuronal death by enhancing membrane phospholipid degradation. In previous studies, we have shown that NMDA releases choline and reduces membrane phosphatidylcholine in vivo. We now observed that glutamate and NMDA induce choline release in primary neuronal cortical cell cultures. This effect is Ca(2+)-dependent and is blocked by MK-801 ((+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate). In cortical neurons, the NMDA receptor-mediated choline release precedes excitotoxic cell death but not neuronal death induced by either osmotic lysis or serum deprivation. Glutamate, at concentrations that release arachidonic acid, does not release choline in cerebellar granule cells, unless these cells are rendered susceptible to excitotoxic death by energy deprivation. The NMDA-evoked release of choline is not mediated by phospholipases A(2) or C. Moreover, NMDA does not activate phospholipase D in cortical cells. However, NMDA inhibits incorporation of [methyl-(3)H]choline into both membrane phosphatidylcholine and sphingomyelin. These results show that the increase in extracellular choline induced by NMDA receptor activation is directly related with excitotoxic cell death and indicate that choline release is an early event of the excitotoxic process produced by inhibition of phosphatidylcholine synthesis and not by activation of membrane phospholipid degradation.

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.

Protection by imidazol(ine) drugs and agmatine of glutamate-induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor.

This study was designed to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate-induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells. Exposure (30 min) of energy deprived cells to L-glutamate (1-100 microM) caused a concentration-dependent neurotoxicity, as determined 24 h later by a decrease in the ability of the cells to metabolize 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) into a reduced formazan product. L-glutamate-induced neurotoxicity (EC50=5 microM) was blocked by the specific NMDA receptor antagonist MK-801 (dizocilpine). Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20 microM (EC100) L-glutamate with the rank order (EC50 in microM): antazoline (13)>cirazoline (44)>LSL 61122 [2-styryl-2-imidazoline] (54)>LSL 60101 [2-(2-benzofuranyl) imidazole] (75)>idazoxan (90)>LSL 60129 [2-(1,4-benzodioxan-6-yl)-4,5-dihydroimidazole](101)>RX82 1002 (2-methoxy idazoxan) (106)>agmatine (196). No neuroprotective effect of these drugs was observed in a model of apoptotic neuronal cell death (reduction of extracellular K+) which does not involve stimulation of NMDA receptors. Imidazol(ine) drugs and agmatine fully inhibited [3H]-(+)-MK-801 binding to the phencyclidine site of NMDA receptors in rat brain. The profile of drug potency protecting against L-glutamate neurotoxicity correlated well (r=0.90) with the potency of the same compounds competing against [3H]-(+)-MK-801 binding. In HEK-293 cells transfected to express the NR1-1a and NR2C subunits of the NMDA receptor, antazoline and agmatine produced a voltage- and concentration-dependent block of glutamate-induced currents. Analysis of the voltage dependence of the block was consistent with the presence of a binding site for antazoline located within the NMDA channel pore with an IC50 of 10-12 microM at 0 mV. It is concluded that imidazol(ine) drugs and agmatine are neuroprotective against glutamate-induced necrotic neuronal cell death in vitro and that this effect is mediated through NMDA receptor blockade by interacting with a site located within the NMDA channel pore.

Influence of aging on thromboxane A2 and prostacyclin levels in rat hippocampal brain slices.

We have investigated the influence of age (3, 18, 24 months) on Thromboxane A2 (TXA2) and Prostacyclin (PGI2) levels in hippocampal slices from F344/NHSD rats. A significant increase in TXA2 and PGI2 levels was observed in 18 and 24 months old compared to 3 months old animals. A significant reduction in the ratio TXA2/PGI2 produced by a higher increase in PGI2 was observed in 24 month old animals. The reduction in the TXA2/PGI2 ratio has been related to vasodilatory and antiaggregating effects that may contribute to protect the brain against neuronal damage.

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.

Reactive oxygen production by glutamate agonists in dissociated cerebellar cells: a flow cytometric study.

1. The effect of glutamate, N-methyl-D-aspartate (NMDA) and kainate on radical oxygen species (ROS) production and calcium influx was studied in dissociated cerebellar granule cells with the use of flow cytometry. 2. Glutamate and NMDA induced an intracellular ROS increase by an activation of NMDA receptors. 3. (+)MK-801 inhibited the effect on ROS production of both agonists (IC50 values of 1.52 +/- 0.05 and 0.71 +/- 0.02 microM, respectively). 4. (+)MK-801 inhibited the intracellular calcium increase induced by glutamate and NMDA, whereas 6-cyano-7-nitroquinoxaline-2,3-dione inhibited that induced by kainate. 5. NG-Nitro-L-arginine, but not nitrendipine, inhibited the ROS production induced by glutamate agonists. Consequently, nitric oxide synthase might play an important role in the neurotoxic process induced by excitatory amino acids.

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.

Effect of 1-aminocyclopropanecarboxylic acid on N-methyl-D-aspartate-stimulated [3H]-noradrenaline release in rat hippocampal synaptosomes.

1. The effect of 1-aminocyclopropanecarboxylic acid (ACPC), a partial agonist at the glycine site of the N-methyl-D-aspartate (NMDA) receptor complex that exhibits neuroprotective, anxiolytic and antidepressant-like actions, was investigated in a functional assay for presynaptic NMDA receptors. 2. NMDA (100 microM) produced a 36% increase of tritium efflux above basal efflux in rat hippocampal synaptosomes preincubated with [3H]-noradrenaline ([3H]-NA), reflecting a release of tritiated noradrenaline. This effect was prevented by 10 microM 7-chlorokynurenic acid, an antagonist of the glycine site of the NMDA receptor. 3. Glycine enhanced the effect of NMDA with Emax and EC50 values of 84 +/- 11% and 1.82 +/- 0.04 microM, respectively. ACPC potentiated the effect of NMDA on tritium overflow with a lower EC50 (43 +/- 6 nM) and a lower maximal effect (Emax = 40 +/- 9%) than glycine. Furthermore, ACPC (0.1 microM) shifted the EC50 of glycine from 1.82 microM to > or = 3 mM. 4. These results show that ACPC can reduce the potentiation by glycine of NMDA-evoked [3H]-NA release and hence, may act as an antagonist at the glycine site of presynaptic hippocampal NMDA receptors when the concentration of glycine is high.