The present study investigated the effects of anaesthesia on protein levels of all three NMDA receptor subunits in different subcellular compartments of mouse cortical neurones in vivo. We found that anaesthesia led to a significant reduction of NR1 protein abundance in the surface pool. Anaesthesia also increased NR1 protein levels in the intracellular pool. In parallel with NR1, NR2B proteins were reduced in the surface pool and enhanced in the intracellular pool. In contrast to NR1 and NR2B, NR2A was stable in both compartments in response to anaesthesia. In addition, anaesthesia did not alter total protein levels of the three subunits in cortical neurones. These results for the first time provide evidence that the subcellular location of NMDA receptors, specifically NR2B-containing NMDA receptors, in cortical neurones is subject to modulation by anaesthesia, and general anaesthesia causes a significant redistribution of the receptor from the surface pool to the intracellular pool. Of note, this study was conducted on cerebral cortical neurones. It is currently unclear if the redistribution of NMDA receptors observed in cortical neurones also occurs to other brain regions during anaesthesia. In addition, we have determined that the redistribution of NR1/NR2B was independent of the effect of hypothermia. However, the potentially confounding effects of hypotension, hypoxia, hypercarbia, and acidosis have not been investigated in the current study, and need to be evaluated in the future.
The important finding in the present work is the redistribution of NR2B-containing NMDA receptors between the surface and the intracellular pools of cortical neurones after anaesthesia. This redistribution is characterized by the loss of the receptor in the surface pool and the accumulation of the receptor in the intracellular pool. The mechanism(s) responsible for this redistribution are unclear. Presumably, the delivery process of the receptor from the intracellular organelles to the surface membrane, that is, an externalization trafficking process, is preferentially suppressed. Alternatively, an internalization trafficking process removing surface receptors to intracellular sites is selectively accelerated. Either suppressed externalization or accelerated internalization or both could cause a redistribution phenomenon characterized by the subtraction of the receptor in the surface pool in combination with the proportional addition of the receptor in the intracellular pool, whereas total receptor proteins remain unchanged.7,16
Future studies will be required to elucidate possible molecular mechanisms underlying the anaesthesia-induced redistribution of NMDA receptors.
This study did not explore the functional consequences of the reduction of surface NMDA receptors during anaesthesia. It is also unclear if the loss of NMDA receptors occurred at synaptic or extrasynaptic sites. The affected receptors could be extrasynaptic with little immediate impact on synaptic transmission. If it occurred at synaptic sites, the loss of surface NMDA receptors is believed to weaken NMDA receptor function and reduce the efficacy and strength of excitatory synapses containing NMDA receptors.9,11
With regard to cellular mechanisms underlying general anaesthesia, potentiation of inhibitory synaptic transmission seems to be a common pathway mediating many forms of general anaesthesia induced by a variety of general anaesthetics.21,22
Specifically, general anaesthetics, including chloral hydrate, augment GABAA
-mediated inhibitory transmission to produce anaesthesia. A large number of reports have documented that chloral hydrate or trichloroethanol, an active metabolite of chloral hydrate within minutes of injection, potentiates the function of GABAA
receptors in a manner similar to barbiturate or steroid anaesthetics in heterologous cells expressing GABAA
receptors or in hippocampal neurones.23–26
In contrast to the well-defined role of GABAA
receptors, little is known about a direct role of NMDA receptors in producing anaesthesia.27,28
Chloral hydrate reduced basal levels of glutamate by 70% in the striatum.29
Trichloroethanol also inhibited glutamatergic transmission in hippocampal slices.23
However, the potency of this inhibition appears to be lower than its potency for enhancing GABAA
The concentrations (1–10 mM) at which trichloroethanol inhibited glutamate receptor-mediated currents are higher than those (0.2–0.5 mM) required for producing threshold effect on GABAA
Thus, the depression of NMDA receptor-mediated glutamatergic transmission may not significantly contribute to anaesthesia specifically induced by chloral hydrate, even though it contributes to anaesthesia induced by the dissociative anaesthetics such as ketamine and phencyclidine.30–32
Recently, we found that the general anaesthetic propofol inhibited phosphorylation of NR1 subunits at two specific serine sites (serine 896 and serine 897) and impaired NMDA receptor-mediated Ca2+
influx in cultured rat cortical neurones.33
We also found that propofol affected AMPA receptor phosphorylation34
and inhibited activation of extracellular signal-regulated protein kinases in hippocampal neurones.35
In this study, we revealed that general anaesthesia caused the loss of surface-expressed NMDA receptors. These new biochemical data join a large volume of early electrophysiological results to support the profound effect of various general anaesthetics on NMDA receptors.21
Although the role of depressed NMDA receptors in inducing anaesthesia remains to be defined, NMDA receptors might well contribute to some specific biological actions of anaesthetics. Future studies need to elucidate the importance of NMDA receptors in processing a specific cellular response to a given anaesthetic agent.