Trafficking of NMDA Receptors During Status Epilepticus: Therapeutic Implications

Summary

We used two models of status epilepticus (SE) to study trafficking of NMDA receptors (NMDAR). SE is associated with increased surface expression of NR1 subunits of NMDAR, and with an increase of NMDA synaptic and extrasynaptic currents suggesting an increase in number of functional NMDAR on dentate granule cells. The therapeutic implications of these results are discussed.

Despite the emergence of several new anticonvulsants, status epilepticus (SE) is still associated with significant morbidity and mortality. Pharmacoresistance to benzodiazepines and other drugs develops quickly (Mazarati et al., 1998). Trafficking and inactivation explains benzodiazepine pharmacoresistance (Naylor et al 2005) but not the enhanced glutamatergic excitation or the effectiveness of NMDA antagonists in late stages of SE (Mazarati and Wasterlain, 1999)

Here we found during SE a relocation of NMDA receptor subunits to the cell surface with an increase in postsynaptic response in dentate granule cells, suggesting a potential mechanism for seizure maintenance during SE and for SE-related excitotoxicity

We used a standard lithium/pilocarpine model of SE and a model of SE after intrahippocampal injection of neurokinin B (Naylor et al 2013) to study NMDAR trafficking and its therapeutic implications. After I hour of SE, rats were perfused/fixed for immunocytochemistry or hippocampal slices were prepared for whole-cell patch clamp of dentate granule cells.(Naylor et al 2013).

NR1, the essential subunit of NMDA receptors combines primarily with NR2A and/or NR2B subunits in the hippocampus. In granule and pyramidal cells from control hippocampi, NR1 subunit-like immunoreactivity (LI) localizes to the cell interior. After 1 hour of SE, much of this NR1 subunit-LI has relocated to discrete puncta which outline the cell membrane, and frequently colocalize with the synaptic marker synaptophysin-LI. In the soma of hippocampal granule cells (Fig. 1A), the number of overlaps between NR1 subunit-LI and synaptophysin-LI increases from 1.85 ± 0.18 per soma in controls to 7.15 ± 0.28 in SE (p < .001). Proximal dendrites, CA3 and CA1 pyramids show similar increases.

Figure 1.

A-D: Distribution of NR1-LI in asymmetric synapses of the granule cell layer of the dentate gyrus from control (A, B) and SE animals (C, D). Arrows indicate the postsynaptic density (bars=150 nm). Asymmetric synapses show central/synaptic immunogold labeling (A, C) or a mix of central/synaptic and lateral/perisynaptic immunogold labeling of postsynaptic densities (B, D). SE increases the percentage of immunogold-labeled asymmetric synapses compared to controls (E). The lateral distribution of NR1 on the surface of synapses also increases with SE (F). (Error bars are ±SEM). G: Miniature excitatory post-synaptic NMDA currents (NMDA-mEPSCs) from representative dentate gyrus granule cells of SE (gray) and control (black). demonstrating larger amplitude and area-under-the curve (AUC) after SE. H: Ifenpodil(3 μM)-sensitive component of NMDA tonic currents of granule cells increases after SE. Both control and SE cells have significant baseline shifts with the subsequent addition of the non-selective NMDA receptor antagonist, APV.

By electron microscopy, the number of asymmetric synapses in the dentate granule cell layer which were immunoreactive for NR1 increased from 8.4 ± 2.8 % in controls to 26.9 ± 4.7 % in SE (Fig. 1 A-D), confirming the light microscopy results. The percentage of asymmetric synapses with more than 1 gold particle increased from 0.5 ± 0.3% in controls to 4.7 ± 1.2% after SE (p < 0.05) (Fig. 1E). A greater proportion of NR1 particles were located in the periphery of asymmetric synapses after SE (Fig. 1F), suggesting the possibility of a perisynaptic location and of lateral entry of subunits into synapses (45.3 ± 3.2% for controls versus 58.5 ± 2.7% for SE; p < .05).

In SE induced by intrahippocampal neurokinin B, similar increases were observed by immunocytochemistry. This increase in synaptic and/or perisynaptic NR1 subunits is likely to reflect movement of whole NMDAR to the membranes, since isolated subunits remain in the Golgi apparatus and lack the machinery for trafficking to the membrane. To examine whether this immunocytochemical change did indeed reflect an increase in functional NMDAR on the cell surface, we recorded miniature excitatory NMDA currents (mEPSCs) from dentate granule cells in hippocampal slices obtained from animals in lithium-pilocarpine SE for 1 hour (Fig. 1G). Mean traces of NMDA-mEPSCs recorded from dentate gyrus granule cells in slices from SE animals (Fig. 2) displayed increase peak amplitude (-20.2 ± 2.7 pA for SE vs –16.4 ± 0.64 pA for controls; p < .001).

To interpret the basis for the increase in amplitude and AUC with SE, a model using mean-variance analysis of NMDA receptors suggested that the number of NMDA receptors increased during SE from 8 ± 1 per synapse in controls to 11 ± 2 per synapse with SE (p < .001).

In addition to synaptic relocation of NMDA receptors, we found relatively minor changes affecting NMDA-mEPSC kinetics, which predict that no change occurred in channel conductance with SE.

With ifenprodil in the perfusate, a positive tonic current baseline shift from of -76.0 ± 41.1 pA to -66.8 ± 34.5 pA (p<.05; n = 17) for SE granule cells held at -60 mv revealed a depolarizing tonic current of +9.2 pA, presumably mediated by NMDA receptors containing NR2B subunits (Fig. 1H). No tonic current shift was observed in controls (-2.6 ± 12.9 pA; n.s.; n = 5).

After one hour of SE, dentate granule cells display a 38% increase in the number of physiologically active NMDA receptors per somatic synapse, a two-fold increase in the number of NR1 subunit-LI in the vicinity of the presynaptic marker synaptophysin, and a three-fold increase in NR1-like immunoreactivity associated with postsynaptic densities. The NR1 immunoreactivity is likely a marker for functional heteromeric NMDA receptor complexes because all hippocampal NMDA receptors contain NR1 subunits, and solitary NR1 subunits are retained in the ER (Prybylowski et al., 2002). These changes could in part account for the enhanced glutamatergic excitation during SE (Wasterlain et al., 2000), and for the therapeutic efficacy of NMDA antagonists in some models of SE (Bertram and Lothman, 1990; Mazarati and Wasterlain, 1999). This movement of NMDA receptor subunits may reflect trafficking of NMDAR towards perisynaptic areas followed by lateral movement into synapses, and coincides with an internalization of synaptic GABA_A receptor. Both changes appear maladaptive in a seizing brain, and further exacerbate an imbalance between synaptic excitation and inhibitionn. We do not know whether physiological recordings underestimate those changes because the slice rests for 90-120 minutes (without seizures) before recordings, or whether, immunocytochemisty may detect extrasynaptic surface receptors that do not contribute to NMDA-mEPSC responses.

The increase of synaptic NMDA receptors may characterize an important transition when augmented synaptic excitation, coupled with a major loss of synaptic inhibition, provides the framework for progression from single seizures to self-sustaining SE. While loss of inhibition may be important with initiation of SE, augmented glutamatergic excitation is important for the maintenance of SE (Mazarati et al 1999). These findings have therapeutic implications: the standard initial treatment of SE (benzodiazepines) is insufficient because increased glutamatergic excitation is untouched. Furthermore, benzodiazepines are unlikely to fully restore GABAergic inhibition given the dramatic reduction in number of postsynaptic receptors available for allosteric activation. Combinations of a benzodiazepine with an antagonist at NMDA receptors and another drug which increases inhibition at a non-benzodiazepine site would more fully address the effects of SE-induced receptor trafficking. Indeed, such combinations seem to be more effective than monotherapy at stopping SE (Wasterlain et al., 2011).

Over-activity of NMDA receptors can increase excitotoxic neuronal death, and NMDA receptor blockade may also prove neuroprotective during SE, (Fujikawa, 1995; Mazarati et al., 2000, Frasca et al 2011). and modify SE-associated epileptogenesis (Mazarati et al., 2002b) Since early treatment seems very effective in SE (Silbergleit et al 2012), polytherapinitial treatment of SE with drug combinations including an NMDA antagonists deserves further study.