PCR and patch-clamp analysis of single neurons.

The combination of patch-clamp and molecular biology techniques has made it possible to characterize the pharmacological and biophysical properties of ion channels in single neurons and to screen for expression of specific mRNAs in the same cell. Following whole-cell recording, the cytoplasm of the cell is harvested, and RNA is reverse transcribed into cDNA and amplified in PCR with primers specific for individual ion channel subunits. Additional experiments can then be designed to relate structure and function at the protein level more directly, since cells appear to regulate the composition of ion channels at least partly at the posttranscriptional stage.

Synergistic effects of HIV coat protein and NMDA receptor-mediated neurotoxicity.

Exposure of rat retinal cultures to HIV-1 coat protein gp120 for several minutes increases [Ca2+]i in approximately half of the ganglion cells; this effect is associated with delayed-onset neuronal injury, similar to that previously reported in NMDA receptor-mediated neurotoxicity. Here we show that NMDA antagonists can prevent both the rise in [Ca2+]i and subsequent neuronal damage engendered by 20 pM gp120. However, whole-cell patch-clamp recordings demonstrate that gp120 does not directly evoke an NMDA-like response or enhance glutamate/NMDA-activated currents. Moreover, complete protection from gp120-induced [Ca2+]i increases and neurotoxicity is afforded by incubation with glutamate-pyruvate transaminase, which breaks down endogenous glutamate as verified by HPLC. Since, under standard conditions in these cultures, neither glutamate nor a low picomolar concentration of gp120 is deleterious on its own, our results suggest that their neurotoxicity is synergistic.

Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex.

Nitric oxide (NO) is an important messenger both systemically and in the CNS. In digital Ca2+ imaging and patch-clamp experiments, clinically available nitroso compounds that generate NO are shown to inhibit responses mediated by the NMDA subtype of the glutamate receptor on rat cortical neurons in vitro. A mechanism of action for this effect was investigated by using the specific NO-generating agent S-nitrosocysteine. We propose that free sulfhydryl groups on the NMDA receptor-channel complex react to form one or more S-nitrosothiols in the presence of NO. If vicinal thiol groups react in this manner, they can form a disulfide bond(s), which is thought to constitute the redox modulatory site of the receptor, resulting in a relatively persistent blockade of NMDA responses. These reactions with NO can afford protection from NMDA receptor-mediated neurotoxicity. Our results demonstrate a new pathway for NO regulation of physiological function that is not via cGMP, but instead involves reactions with membrane-bound thiol groups on the NMDA receptor-channel complex.

An NMDA receptor signaling complex with protein phosphatase 2A.

Regulation of protein phosphatase 2A (PP2A) activity and NMDA receptor (NMDAR) phosphorylation state contribute to the modulation of synaptic plasticity, yet these two mechanisms have not been functionally linked. The NMDAR subunit NR3A is equipped with a unique carboxyl domain that is different from other NMDAR subunits. We hypothesized that the NR3A C-terminal intracellular domain might serve as synaptic anchor for the phosphatase in the developing CNS. A cDNA library was screened by the yeast two-hybrid method using the NR3A carboxyl domain as the bait. The catalytic subunit of the serine-threonine PP2A was found to be associated with the NR3A carboxyl domain. Immunoprecipitation studies indicated that the NR3A subunit formed a stable complex with PP2A in the rat brain in vivo. Association of PP2A with NMDARs led to an increase in the phosphatase activity of PP2A and the dephosphorylation of serine 897 of the NMDAR subunit NR1. Stimulation of NMDARs led to the dissociation of PP2A from the complex and the reduction of PP2A activity. A peptide corresponding to the PP2A-NR3A binding domain functioned as a negative regulator of PP2A activity. These data suggest that NMDARs are allosteric modulators of PP2A, which in turn controls their phosphorylation state. The data delineate a mechanistic model of the dynamic regulation of a PP2A-NMDAR signaling complex, mediated by the interaction of NR3A and PP2A, and suggest a novel NMDAR-mediated signaling mechanism in addition to the traditional ionotropic functions of NMDARs.

Assembly with the NR1 subunit is required for surface expression of NR3A-containing NMDA receptors.

Functional NMDA receptors are heteromultimeric complexes of the NR1 subunit in combination with at least one of the four NR2 subunits (A-D). Coexpression of NR3A, an additional subunit of the NMDA receptor family, modifies NMDA-mediated responses. It is unclear whether NR3A interacts directly with NR1 and/or NR2 subunits and how such association might regulate the intracellular trafficking and membrane expression of NR3A. Here we show that NR3A coassembles with NR1-1a and NR2A to form a receptor complex with distinct single-channel properties and a reduced relative calcium permeability. NR3A associates independently with both NR1-1a and NR2A in the endoplasmic reticulum, but only heteromeric complexes containing the NR1-1a NMDA receptor subunit are targeted to the plasma membrane. Homomeric NR3A complexes or complexes composed of NR2A and NR3A were not detected on the cell surface and are retained in the endoplasmic reticulum. Coexpression of NR1-1a facilitates the surface expression of NR3A-containing receptors, reduces the accumulation of NR3A subunits in the endoplasmic reticulum, and induces the appearance of intracellular clusters where both subunits are colocalized. Our data demonstrate a role for subunit oligomerization and specifically assembly with the NR1 subunit in the trafficking and plasma membrane targeting of the receptor complex.

Stroke therapy in traditional Chinese medicine (TCM): prospects for drug discovery and development.

Brain injuries resulting from stroke are a major and increasing public health problem in both developed and developing countries worldwide. China's extensive experience in the use of traditional Chinese medicines (TCMs) in stroke therapy indicates that TCM preparations are effective, with few or no side-effects. There are more than 100 traditional medicines in use for stroke therapy in China. Some of their therapeutic effects in stroke have been confirmed by recent clinical studies. A large number of compounds have been isolated from TCMs and most of these resources have not yet been characterized for pharmacological purposes. Here, this article explains how TCM provides an extensive and knowledge-rich foundation for implementing a strategically focused pharmacological research programme aimed at the development of new drugs.

NMDA receptors: from genes to channels.

N-methyl-D-aspartate receptors belong to the family of ionotropic glutamate receptors. NMDA receptors were named after the specific glutamate-like synthetic agonist N-methyl-D-aspartate. In the past decade, an increasing number of functional sites have been discovered and used to refine the operational definition of NMDA receptors. The goal to characterize the molecular substrate underlying the heretofore strictly operationally defined NMDA receptors has come into reach following the cloning of a number of cDNAs coding for NMDA receptor subunits. However, in their review, Nikolaus Sucher and colleagues show that caution should be exercised in comparing the pharmacological properties of recombinant NMDA receptors to those of native neurones. Future work on NMDA receptors will be challenged to reconcile disparate effects obtained with recombinant versus native receptors.

Co-expression of AMPA/kainate receptor-operated channels with high and low Ca2+ permeability in single rat retinal ganglion cells.

The patch-clamp technique was used to record whole-cell currents induced by alpha-amino-3-hydroxyl-5-methyl-isoxazol-4-propionic acid (AMPA) or kainate in solitary rat retinal ganglion cells (n = 125) in vitro. Two groups of retinal ganglion cells could be distinguished according to their responses to kainate or AMPA in extracellular solutions with Ca2+ as the only permeant cation. The ratio of the steady-state currents evoked by a given concentration of AMPA compared to kainate was low (0.08) in the first group and high (0.61) in the second group of retinal ganglion cells. The Ca2+ permeability through AMPA/kainate receptor-operated channels was low (PCa2+/PCs+ < 0.1) in the first group (n = 74, 59%) and moderate (PCa2+/PCs+ = 0.53) in the second group (n = 51, 41%) of retinal ganglion cells. The fraction of the total current induced by stimulation of non-N-methyl-D-aspartate receptors that is flowing through Ca2+ permeable AMPA/kainate channels in single cells with high Ca2+ permeability was estimated by comparing the current-voltage relationship in extracellular solutions with either Ca2+ or Na+ as the sole charge carrier. The contribution of Ca(2+)-permeable channels to the non-N-methyl-D-aspartate receptor induced whole-cell current in single Ca2+ permeable cells (n = 12) ranged from 40 to 70%, correlating with the intermediate level of Ca2+ permeability (PCa2+/PCs+ = 0.22-0.80) measured by an independent method in these cells. Thus, single Ca(2+)-permeable cells appear to express at least two types of AMPA/kainate receptor-operated channels with high or low Ca2+ permeability. Using the polymerase chain reaction, transcripts for the glutamate receptor subunits 1-4, including their "flip" and "flop" versions, were identified in retinal ganglion cells. Together, these findings suggest that among rat retinal ganglion cells there are differences in the pattern of expression of AMPA/kainate receptor-operated channels. Moreover, individual cells co-express multiple heterologous non-N-methyl-D-aspartate receptors with distinct functional properties. The functional diversity of these receptors may play an important role in controlling Ca2+ entry into neurons. We speculate that the low Ca2+ permeability and the preference for kainate in one group of retinal ganglion cells may be due to the predominant expression of non-N-methyl-D-aspartate receptors containing the edited form of the glutamate receptor subunit 2 flop splice variant.

Cryopreservation of postnatal rat retinal ganglion cells: persistence of voltage- and ligand-gated ionic currents.

Established methods for cryopreservation of living cells were modified for freeze-storage of postnatal retinal ganglion cells from rat. Retinal cell suspensions containing fluorescently labeled ganglion cells were frozen after addition of 8% dimethyl sulfoxide and stored at -80 degrees C for up to 66 days. Viability of identified retinal ganglion cells was assessed by their ability to take up and cleave fluorescein diacetate to fluorescein. No significant difference was found in the number of living retinal ganglion cells when cells obtained from the same dissociation were counted before and after freezing (6.65 +/- 2.37 x 10(4) vs 7.05 +/- 3.67 x 10(4) retinal ganglion cells per ml, respectively; mean +/- S.D., n = 4). In culture following cryopreservation, the cells appeared morphologically normal, and developed neurites and growth cones similar to their freshly dissociated counterparts. Since very little is known about the electrophysiology and membrane properties of neurons after cryopreservation, we used the whole-cell configuration of the patch-clamp technique to study voltage- and ligand-gated conductances in cryopreserved retinal ganglion cells. The cryopreserved retinal ganglion cells studied under current-clamp maintained resting potentials of -60.9 +/- 6.6 mV (n = 10) and upon depolarization fired action potentials. During voltage-clamp in the whole-cell mode, depolarizing voltage steps activated Na(+)-(INa), Ca(2+)-(ICa), and K(+)-currents in all cells tested (n = 122). INa could be reversibly blocked by 1 microM tetrodotoxin added to the external solution. ICa was blocked by external 250 microM Cd2+ or 3 mM Co2+. In some cells, ICa consisted of both a transient and prolonged component. The outward K(+)-current consisted of Ca(2+)-dependent and -independent components. The Ca(2+)-insensitive portion of the K+ outward current was separated into four distinct components based upon pharmacological sensitivity and biophysical properties. In many cells, a rapidly inactivating current similar to the A-type K(+)-current (IA) observed in freshly cultured retinal ganglion cells was isolated by its greater sensitivity to 4-aminopyridine (5 mM) than to tetraethylammonium (20 mM). A tetraethylammonium-sensitive current with a more prolonged time course reminiscent of IK, the delayed rectifier, was also found. When the 4-aminopyridine- and tetraethylammonium-insensitive portions of the outward current were further analysed with voltage protocols, an additional slowly decaying potassium current became apparent. The inhibitory amino acids, GABA (20 microM) and glycine (100 microM), activated chloride-selective currents that were selectively blocked by bicuculline methiodide (10 microM) and strychnine (5 microM), respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

N-methyl-D-aspartate receptors are critical for mediating the effects of glutamate on intracellular calcium concentration and immediate early gene expression in cultured hippocampal neurons.

The mechanisms by which activation of excitatory amino acid receptors is coupled to the regulation of gene transcription were studied using cultured hippocampal neurons from neonatal rats. Voltage recording, calcium imaging, specific RNA analysis and immunocytochemistry were carried out on sister cultures. This allowed analysis of the expression of functional glutamate receptor subtypes, examination of their role in controlling intracellular free calcium ([Ca2+]i), and determination of their relative contributions to the transcriptional regulation of six immediate early genes c-fos, fosB, c-jun, junB, zif/268 (also termed Egr-1; NGFI-A; Krox-24) and nur/77 (also termed NGFI-B). Expression of all six immediate early genes was induced in hippocampal neurons by glutamate treatment. Nuclear run-on assays demonstrated that this induction occurred at the transcriptional level. Activation of the N-methyl-D-aspartate subtype of glutamate receptor was necessary and sufficient for the transcriptional response. Non-N-methyl-D-aspartate receptors, while present in cultured hippocampal neurons, contributed relatively little to the regulation of transcription. Calcium imaging showed that glutamate-induced changes in [Ca2+]i were almost entirely mediated by N-methyl-D-aspartate receptors, rather than by L-type voltage-sensitive calcium channels. Previous studies have shown that stimulation with selective agonists of either N-methyl-D-aspartate receptors, non-N-methyl-D-aspartate receptors, or L-type calcium channels can lead to an increase in [Ca2+]i and c-fos expression. Here we demonstrate that in our hippocampal culture system glutamate controls [Ca2+]i and induces immediate early gene transcription primarily by activating N-methyl-D-aspartate receptors.

Redox modulation of the NMDA receptor by NO-related species.

The chemical reactions of NO are largely dictated by its redox state. Increasing evidence suggests that the various redox states of the NO group exist endogenously in biological tissues. In the case of NO+ equivalents, the mechanism of reaction often involves S-nitrosylation (transfer of the NO group to a cysteine sulfhydryl to form an RS-NO); further oxidation of critical thiols can possibly form disulfide bonds. We have physiological and chemical evidence that NMDA receptor activity can be modulated by S-nitrosylation, resulting in a decrease in channel opening. Recent data suggest that NO-, probably in the singlet (or high-energy) state, can also react with critical sulfhydryl group(s) of the NMDA receptor to down-regulate its activity; in the triplet (lower-energy) state NO- may oxidize these NMDA receptor sulfhydryl groups by formation of an intermediate such as peroxynitrite. It has also been reported that NO can react with thiol but only under specific circumstances, e.g., if an electron acceptor such as O2 is present, as well at catalytic amounts of metals like copper, and if the conditions do not favor the kinetically preferred reaction with O2.- to yield peroxynitrite. Mounting evidence in many fields suggests that S-nitrosylation can regulate the biological activity of a great variety of proteins, perhaps analogous to phosphorylation. Thus, this chemical reaction is gaining acceptance as a newly-recognized molecular switch to control protein function via reactive thiol groups such as those encountered on the NMDA receptor.

Glutathione prevents N-methyl-D-aspartate receptor-mediated neurotoxicity.

N-methyl-D-aspartate(NMDA) antagonists afford possible therapeutic modalities for stroke, trauma, and various neurodegenerative conditions thought to be mediated by excessive stimulation of NMDA receptors in the brain. To date, however, no drug has been demonstrated to be a safe NMDA antagonist at a dosage necessary for neuroprotection. In the present study, we use an in-vitro preparation to show that glutathione is capable of attenuating neuronal damage mediated by NMDA receptor activation. Both oxidized and reduced glutathione are protective, and an extracellular mechanism of action on the NMDA receptor-channel complex is suggested.

Redox modulation of NMDA receptor-mediated Ca2+ flux in mammalian central neurons.

NMDA receptor-mediated Ca2+ flux was studied in cultured rat retinal ganglion cells and neocortical neurons. Intracellular free calcium ([Ca2+]i was measured with fura-2 fluorescence imaging. Baseline [Ca2+]i was 59 +/- 5 nM. In low [Mg2+]o, 200 microM NMDA reversibly increased [Ca2+]i to 421 +/- 70 nM. This rise in [Ca2+]i was blocked by the NMDA antagonists APV (200 microM) or [Mg2+]o (1 mM), but only slightly inhibited by the non-NMDA antagonist CNQX (10 microM). Chemical reduction with dithiothreitol (DTT) had no effect on resting [Ca2+]i. However, DTT increased the NMDA-induced rise in [Ca2+]i approximately 1.6-fold; the oxidizing agent dithiobisnitrobenzoic acid (DTNB) reversed this effect. In patch-clamp experiments, DTT increased NMDA-activated whole-cell conductance approximately 1.7-fold in low and high [Ca2+]o. The Ca2+/Na+ permeability ratio of approximately 7 for NMDA channels remained unaltered by chemical reduction. Thus, redox modulation of the NMDA receptor/channel complex results in a dramatic alteration in current magnitude but no change in ionic permeabilities.

Calcium channel antagonists attenuate NMDA receptor-mediated neurotoxicity of retinal ganglion cells in culture.

Dihydropyridine calcium channel antagonists block a prolonged or 'L-type' component of voltage-dependent Ca2+ current in patch-clamp recordings of postnatal rat retinal ganglion cells. In the present study on these neurons, calcium channel antagonists were found at 500-1000 nM concentrations to attenuate the early rise in [Ca2+]i and the subsequent toxic effects of exogenous glutamate, N-methyl-D-aspartate (NMDA), or an endogenous glutamate-related compound present in the retinal cultures. Previous data have shown that the neurotoxicity engendered by these agents can also be prevented by selective NMDA antagonists. The present observations raise the possibility, at least in this preparation, that activation of both voltage-dependent calcium channels and NMDA receptor-operated channels contribute to the injurious effects triggered by molecules binding to the NMDA receptor.

Neural nicotinic acetylcholine responses in sensory neurons from postnatal rat.

The whole-cell configuration of the patch-clamp technique was used to study nicotinic acetylcholine (ACh) responses in freshly dissociated dorsal root ganglion (DRG) cells from postnatal rat. At negative holding potentials with physiological solutions in the bath and the pipette, ACh (20 microM), nicotine (5 microM) or DMPP (20 microM) activated inward currents in 51% of the cells. Average current density was higher in 1-month-old compared to newborn animals. Nicotinic agonist-induced currents were unaffected by atropine (10 microM) but reversibly blocked by hexamethonium (20 microM). Although labeling with fluorescent alpha-bungarotoxin (BGT) demonstrated the presence of toxin binding sites on DRG cells, DMPP-induced inward currents were unaffected by micromolar BGT. Neuronal bungarotoxin (100 nM), in contrast, led to a largely irreversible block of the nicotinic responses. These results show that postnatal DRG cells express functional nicotinic acetylcholine receptors (nAChR) of a neuronal type.

Redox modulation of NMDA receptor-mediated toxicity in mammalian central neurons.

Acute neurological injury from hypoxia-ischemia, hypoglycemia, and trauma is thought to be predominantly mediated by activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor in the brain and the subsequent influx of calcium ions through receptor-operated channels. Several chronic degenerative diseases, such as Huntington's disease and the amyotrophic lateral sclerosis-Parkinsonism-dementia complex found on Guam, may share a similar pathogenesis due to a glutamate-like toxin. This laboratory recently reported that exposure to a reducing agent, such as dithiothreitol (DTT), selectively increases ionic current flow through NMDA-activated channels in several types of central neurons; conversely, oxidizing agents reverse this effect. To investigate the novel influence of redox modulation on NMDA neurotoxicity, in the present in vitro study we monitored survival of an identified central neuron, the retinal ganglion cell, approximately 24 h after a brief exposure to DTT. To determine the degree of killing specifically related to activation of the NMDA receptor, 2-amino-5-phosphonovalerate (APV, a selective NMDA antagonist) was added to sibling cultures. APV-preventable, glutamate-induced death was increased 70 +/- 9% with DTT treatment. This effect was totally blocked by the concomitant addition of an oxidizing agent, 5,5-dithiobis-2-nitrobenzoic acid (DTNB). These findings suggest that the enhanced killing following chemical reduction with DTT is mediated at the NMDA receptor site, and that the redox state of the NMDA receptor is crucial for the survival of neurons facing glutamate-related injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of N-methyl-D-aspartate receptor subunit mRNAs in the rat pheochromocytoma cell line PC12.

The expression of the N-methyl-D-aspartate (NMDA) receptor subunit mRNAs NMDAR1 and NMDAR2A-D was characterized in undifferentiated and nerve growth factor (NGF)-differentiated PC12 cells using Northern blotting, RNase protection assays (RPA) and polymerase chain reaction (PCR). PC12 cells expressed predominately the splice variant NMDAR1-4a and smaller amounts of NMDAR1-1a, NMDAR1-2a and NMDAR1-3a. No splice isoforms containing exon 5 were detected. The NMDAR2C subunit was detected in PC12 cells by Northern blotting and trace amounts of NMDAR2A, B and D were detected by PCR. PC12 cells may be a useful model system for the study of the transcriptional and post-transcriptional regulation of expression of the NMDA receptor subunit genes, including the alternative splicing of NMDAR1 pre-mRNAs.

Molecular basis of glutamate toxicity in retinal ganglion cells.

Loss of retinal ganglion cells (RGCs) is a hallmark of many ophthalmic diseases including glaucoma, retinal ischemia due to central artery occlusion, anterior ischemic optic neuropathy and may be significant in optic neuritis, optic nerve trauma, and AIDS. Recent research indicates that neurotoxicity is caused by excessive stimulation of receptors for excitatory amino acids (EAAs). In particular, the amino acid glutamate has been shown to act as a neurotoxin which exerts its toxic effect on RGCs predominantly through the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. NMDA-receptor-mediated toxicity in RGCs is dependent on the influx of extracellular Ca2+. The increase in [Ca2+]i acts as a second messenger that sets in motion the cascade leading to eventual cell death. Glutamate stimulates its own release in a positive feedback loop by its interaction with the non-NMDA receptor subtypes. Ca(2+)-induced Ca2+ release and further influx of Ca2+ through voltage-gated Ca2+ channels after glutamate-induced depolarization contribute to glutamate toxicity. In vitro and in vivo studies suggest that the use of selective NMDA receptor antagonists or Ca2+ channel blockers should be useful in preventing or at least abating neuronal loss in the retina. Of particular importance for future clinical use of NMDA receptor antagonists in the treatment of acute vascular insults is the finding that some drugs can prevent glutamate-induced neurotoxicity, even when administered a few hours after the onset of retinal ischemia.

Neuroprotective versus neurodestructive effects of NO-related species.

Nitric oxide (NO.) can lead to damaging or protective actions in the central nervous system. Here we consider the chemistry of the NO group and its redox-related species that can lead to these exactly opposite ends. In the neurodestructive mode, NO. reacts with superoxide anion (02.-) to form peroxynitrite (ONOO-), which leads to neuronal injury. In contrast, the reaction of the NO group with cysteine sulfhydryls on the NMDA receptor leads to a decrease in receptor/channel activity. avoidance of excessive Ca2+ entry, and thus neuroprotection. Site-directed mutagenesis of recombinant NMDA receptor subunits has recently increased our knowledge of such redox modulation by NO. Transfer of the NO group to cysteine sulfhydryls on the NMDA receptor or other proteins, known as S-nitrosylation, is becoming recognized as a ubiquitous regulatory reaction, skin to phosphorylation, and represents a form of redox modulation in diverse tissues including the brain.