NMDA receptor mobility: cultures versus acute brain slices or neonatal versus mature synapses?

Abstract

The determining factors for NMDAR functions in synapse formation, synaptic plasticity, learning and memory include receptor assembly and trafficking, subunit-specific associations with other proteins as well as subcellular localization at synaptic or extrasynaptic sites. NR2B-rich NMDARs occupy both locations in immature principal neurons. During postnatal maturation, NR2A expression increases, NR2A-containing NMDARs enrich in synapses and can be activated by single synaptic stimulation. In contrast, NR2B-rich NMDARs are activated by glutamate spillover or glutamate released from astrocytes (Kohr, 2006). For a long time, the anchoring of NMDARs opposite to presynaptic glutamate release sites has been taken as evidence for a relatively stable organization of the postsy-naptic membrane. Within the postsynaptic density (PSD), signalling molecules, scaffolding and adaptor proteins have been identified as associating with NMDARs and forming large macromolecular signalling complexes. Nevertheless, synaptic NMDARs can be replaced by extrasynaptic ones in 6- to 8-day-old (DIV6–8) autapses or organotypic cultures (Barria & Malinow, 2002; Tovar & Westbrook, 2002). This mobility could involve lateral diffusion and hence be distinct from exocytic–endocytic vesicular NMDAR trafficking (Lau & Zukin, 2007). Lateral mobility of NMDARs between synaptic and extrasynaptic pools was demonstrated in hippocampal autapses by use of the NMDAR open channel blocker MK-801. Repeated synaptic stimulation in the presence of MK-801 progressively and irreversibly blocked NMDAR-mediated EPSCs (NMDA EPSCs). As a subsequent substantial recovery of NMDA EPSCs occurred within minutes, Tovar & Westbrook (2002) suggested that at least 65% of the synaptic NMDARs are mobile. More recently, surface mobility of NMDARs was visualized in hippocampal dissociated cultures (Groc et al. 2006). NR2B-containing NMDARs switched between extrasynaptic and synaptic localizations about 36 times per minute at DIV8 and DIV15. At DIV15, more NR2A-containing NMDARs were present in the synapses, showing a slower diffusion rate than NR2B-containing NMDARs. Together, these studies in culture generated the view that NMDARs switch as rapidly as AMPARs and challenged trafficking experiments in intact brain tissue. In this issue of The Journal of Physiology Harris and Petit investigated the mobility of NMDARs at CA3-to-CA1 synapses in acute hippocampal slices of 2- to 3-week-old rats (P14–22). Initially, Harris & Pettit determined the spatial distribution of NMDARs within CA1 neurons using glutamate uncaging techniques to activate synaptic and extrasynaptic NMDARs within a circumscribed dendritic region before applying synaptic stimulation in the presence of MK-801 to block the synaptic NMDARs. Subsequently, glutamate uncaging activated the unblocked extrasynaptic NMDAR pool, which was found to be about one third of the total NMDAR pool at both proximal and distal dendritic regions, comparable to results from cultured neurons after DIV7. In acute slices, the subunit composition of the synaptic and extrasynaptic NMDARs was uniform (unlike DIV5–7 cultures), as judged by the sensitivity of NMDA currents to ifenprodil (3 μm), which is > 100-fold more selective for NR1–NR2B than for NR1–NR2A. Somewhat surprising, NMDA currents evoked by uncaging glutamate over the soma did not show higher ifenprodil sensitivity than currents evoked in dendrites, although NMDA field EPSPs showed the expected developmental decrease in ifenprodil sensitivity between P5 and P39–92. Finally and remarkably, extrasynaptic NMDARs in acute slices appeared not to exchange with synaptic NMDARs, as no recovery of NMDA EPSCs was observed after MK-801 blockade and washout. This important issue was examined after partial or complete MK-801 blockade by short or prolonged MK-801 incubations (2 versus 14 min). Recovery from MK-801 was tested at 46 versus 23 min, respectively, following a 20 min stimulation-free period. Considering the slice preparation, the MK-801 washout following the complete blockade of NMDA EPSCs was rather short, although prolonged washout could have caused some MK-801 unblock, thus feigning NMDAR mobility. Also, the number of mobility experiments was low (n= 4 at each incubation time), calling for more experiments at hippocampal and other synapses. In spite of this caveat, the mobility of NMDARs in acute slices appears to be less than in cultured autaptic neurons. Tovar & Westbrook (2002) used young autapses (starting at DIV6), which are more apt for NMDAR mobility, given that (i) extrasynaptic NMDARs outnumber synaptic NMDARs by 3 : 1 at DIV5–7 and (ii) molecules involved in synaptogenesis and synaptic maturation can be assumed to be incompletely expressed at that age. This immature synaptic architecture also occurs in acute slices of young rats, allowing activity-dependent, rapid NMDAR switching at P2–9 but not at P16–21 (Bellone & Nicoll, 2007), consistent with stable NMDAR pools at P14–22 (Harris & Pettit, 2007). Thus, rapid switching of synaptic NMDARs could be restricted to neonatal synapses, which are suitable to study the still unresolved mechanism for NMDAR mobility. In addition, it will be important to elucidate whether lateral mobility of NMDARs occurs in mature synapses and which role it plays during plasticity and remodeling.