Most excitatory synapses in the brain use the neurotransmitter glutamate to carry impulses between neurons. During fast transmission, glutamate usually activates a mixture of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the postsynaptic cell. Experimental scrutiny of NMDARs provides insight into their involvement in excitatory synaptic transmission and related processes such as as synaptic plasticity, neural development, and pain perception. There is increasing awareness that subtle variation in NMDAR properties is imparted by specific receptor subunits, and recent studies have started to provide perspective into some of the discrete tasks carried out by individual receptor subtypes.
N‐Methyl‐D‐aspartate (NMDA) receptor function can be modified by the action of several endogenous and exogenous modulatory processes. In the present study, we report that brief pulses of light potentiate NMDA, but not non‐NMDA glutamatergic receptor‐mediated whole‐cell and single channel currents in rat cortical neurones in vitro. In addition, light also potentiated NMDA receptor‐mediated whole‐cell responses in isolated rat retinal neurones. Potentiation of NMDA whole‐cell currents in cortical neurones was readily observed during and following a brief (< 2 s) exposure of neurones to wavelengths of less than 324 nm of relatively bright light (0·09 μW μm−2). In addition, prolonged exposures (> 30 s) to visible wavelengths (> 380 nm) or to attenuated light (1‐3 % transmittance of non‐attenuated light) were also sufficient to enhance NMDA receptor‐mediated responses. The light‐induced potentiation of NMDA receptor‐mediated currents persisted for several minutes, slowly reversing to control levels with a time constant of approximately 5 min. A subsequent exposure to light could potentiate NMDA receptor‐mediated currents for a second time. Light did not alter the apparent affinity of the NMDA receptor for the co‐agonists NMDA and glycine. Additionally, potentiation of the NMDA‐induced currents was not mediated by a change in the pH sensitivity of the receptor. In excised outside‐out membrane patches, the effects of light on NMDA‐activated unitary currents were manifested as a twofold increase in channel open frequency without alterations in single channel amplitude or open time. Our results suggest the presence of a light‐sensitive moiety within the NMDA receptor, or in a closely associated structure, which affects channel properties. This previously unrecognized form of NMDA receptor modulation may provide a tool for understanding the conformational changes associated with its gating. In addition, it is possible that light may affect NMDA receptor‐mediated function or dysfunction in the retina.
The development of cortical neurons in vivo and in vitro is accompanied by alterations in NMDA receptor subunit expression and concomitant modifications in the pharmacological profile of NMDA-activated ionic currents. For example, we observed that with decreasing NR2B/NR2A subunit expression ratio, the block of NMDA receptor-mediated whole-cell responses by the NR2B-selective antagonist haloperidol was also decreased. In mature cultures (Ͼ22 d in vitro), however, NMDA responses obtained from excised nucleated macropatches, which comprised a large portion of the soma, remained strongly antagonized by haloperidol. These results suggest that in more mature neurons NR1/NR2B receptors appear to be preferentially expressed in the cell body. As predicted from the whole-cell recording pharmacological profile, NMDA-induced toxicity was largely unaffected by haloperidol in mature cultures. However, haloperidol effectively blocked glutamate toxicity in the same cultures, suggesting that the neurotoxic actions of this amino acid were mostly due to the activation of somatic NMDA receptors. In experiments in which the potency of glutamate toxicity was increased by the transport inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid, the neuroprotective effects of haloperidol were significantly diminished. This was likely because of the fact that glutamate, now toxic at much lower concentrations, was able to reach and activate dendritic receptors under these conditions. These results strongly argue that exogenous glutamate and NMDA normally induce excitotoxicity at distinct cellular locations in mature mixed neuronal cultures and that NR1/NR2B receptors remain an important component in the expression of glutamate, but not NMDA-induced excitotoxicity.
Light has recently been shown to be a physical modulator of GABAA receptor activity. Here, we further characterize the effects of light on a native cortical and retinal population of GABAA receptors, and identify a possible mechanism for light induced potentiation using recombinant receptors. GABA-induced currents in cortical neurons were observed to be rapidly and reversibly potentiated following exposure to a brief flash of light (0.5-2 s; > 280 nm) directed via an optical fibre (50 micro m i.d.). GABAA receptor-mediated responses in retinal ganglion cells were also enhanced by light, while glycine-induced currents in these cells were unaffected by the same stimulus. We also determined that physiological levels of light, that is, those that would normally reach the retina, also enhanced GABA-induced currents. Finally, we observed that chemical reduction of recombinant alpha1beta2 and alpha1beta2gamma2S GABAA receptors by dithiothreitol substantially attenuated the effects of light. These results suggest that GABAA receptors can be reversibly modified by a brief pulse of light via an allosteric mechanism that is intimately linked to redox modulation.
Light has been shown to modulate NMDA receptor function. In this study, we have performed experiments aimed at elucidating the putative site of action of light within the receptor structure. Whole-cell recordings were performed in Chinese hamster ovary cells expressing various combinations of NMDA receptor subunits. Although there was no apparent difference in the actions of light between wild-type NR1-NR2A and NR1-NR2B subunit configurations, the light enhancement of NMDA-induced currents was either completely abolished or substantially diminished in the redox site mutants NR1a (C744A, C798A)-NR2B and NR1a (C744A, C798A)-NR2A. Further studies demonstrated that chemical reduction of NR1a-NR2B NMDA receptors decreased its sensitivity to light. In addition, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES), used to irreversibly bind free sulfhydryl groups and inactivate the redox site, abolished the effects of light on wild-type receptors. In contrast, no free sulfhydryls were available for MTSES following light stimulation, suggesting that light itself could not reduce the redox modulatory site. Our results suggest that a functionally intact, oxidized redox site is necessary for light-induced potentiation. Hence, light and redox modulation of the NMDA receptor may share a common intramolecular pathway for altering the function of this ion channel.
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