Stuart G. Cull-Candy

NMDA receptor subunits: diversity, development and disease

N-methyl-D-aspartate receptors (NMDARs) are present at many excitatory glutamate synapses in the central nervous system and display unique properties that depend on their subunit composition. Biophysical, pharmacological and molecular methods have been used to determine the key features conferred by the various NMDAR subunits, and have helped to establish which NMDAR subtypes are present at particular synapses. Recent studies are beginning to address the functional significance of NMDAR diversity under normal and pathological conditions.

Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors.

1. To investigate the origin and functional significance of a recently described tonic GABAA receptor‐mediated conductance in cerebellar granule cells we have made recordings from cells in cerebellar slices from rats of different ages (postnatal days P4 to P28). 2. During development there was a dramatic change in the properties of GABA‐mediated synaptic transmission. The contribution to GABAA receptor‐mediated charge transfer from the tonic conductance (GGABA), relative to that resulting from discrete spontaneous postsynaptic currents (sPSCs), was increased from 5% at P7 to 99% at P21. GGABA was reduced by bicuculline, tetrodotoxin and by lowering extracellular Ca2+, and was initially present only in those cells which exhibited sPSCs. 3. At P7 sPSCs were depolarizing, occasionally triggering a single action potential. By P18 the GABA reversal potential was shifted close to the resting potential and GGABA produced a shunting inhibition. Removal of GGABA by bicuculline increased granule cell excitability in response to current injection. 4. This novel tonic inhibition is present despite the low number of Golgi cell synapses on individual granule cells and appears to result from ‘overspill’ of synaptically released GABA leading to activation of synaptic and extrasynaptic GABAA receptors.

Role of Distinct NMDA Receptor Subtypes at Central Synapses

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 α-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. 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 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the postsynaptic cell. NMDA receptors (NMDARs), in particular, have generated a great deal of interest, having been implicated in such processes as synaptic plasticity, neural development, and pain perception. Members of this class of receptors have unique molecular properties that allow them to serve as molecular coincidence detectors and to promote long-term changes in neuronal function. There is increasing awareness that subtle variation in NMDAR properties is imparted by specific receptor subunits. This Review covers recent research that has started to provide perspective into some of the discrete tasks carried out by individual receptor subtypes.

Adaptive regulation of neuronal excitability by a voltage-independent potassium conductance

Many neurons receive a continuous, or ‘tonic’, synaptic input, which increases their membrane conductance, and so modifies the spatial and temporal integration of excitatory signals. In cerebellar granule cells, although the frequency of inhibitory synaptic currents is relatively low, the spillover of synaptically released GABA (γ-aminobutyric acid) gives rise to a persistent conductance mediated by the GABA A receptor that also modifies the excitability of granule cells. Here we show that this tonic conductance is absent in granule cells that lack the α6 and δ-subunits of the GABAA receptor. The response of these granule cells to excitatory synaptic input remains unaltered, owing to an increase in a ‘leak’ conductance, which is present at rest, with properties characteristic of the two-pore-domain K+ channel TASK-1 (refs 9,10,11,12). Our results highlight the importance of tonic inhibition mediated by GABAA receptors, loss of which triggers a form of homeostatic plasticity leading to a change in the magnitude of a voltage-independent K + conductance that maintains normal neuronal behaviour.

Differences in Synaptic GABAA Receptor Number Underlie Variation in GABA Mini Amplitude

In many neurons, responses to individual quanta of transmitter exhibit large variations in amplitude. The origin of this variability, although central to our understanding of synaptic transmission and plasticity, remains controversial. To examine the relationship between quantal amplitude and postsynaptic receptor number, we adopted a novel approach, combining patch-clamp recording of synaptic currents with quantitative immunogold localization of synaptic receptors. Here, we report that in cerebellar stellate cells, where variability in GABA miniature synaptic currents is particularly marked, the distribution of quantal amplitudes parallels that of synaptic GABA(A) receptor number. We also show that postsynaptic GABA(A) receptor density is uniform, allowing synaptic area to be used as a measure of relative receptor content. Flurazepam, which increases GABA(A) receptor affinity, prolongs the decay of all miniature currents but selectively increases the amplitude of large events. From this differential effect, we show that a quantum of GABA saturates postsynaptic receptors when <80 receptors are present but results in incomplete occupancy at larger synapses.

Synaptic activity at calcium-permeable AMPA receptors induces a switch in receptor subtype.

Activity-dependent change in the efficacy of transmission is a basic feature of many excitatory synapses in the central nervous system. The best understood postsynaptic modification involves a change in responsiveness of AMPAR (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor)-mediated currents following activation of NMDA ( N-methyl-D-aspartate) receptors or Ca 2+-permeable AMPARs. This process is thought to involve alteration in the number and phosphorylation state of postsynaptic AMPARs. Here we describe a new form of synaptic plasticity—a rapid and lasting change in the subunit composition and Ca2+ permeability of AMPARs at cerebellar stellate cell synapses following synaptic activity. AMPARs lacking the edited GluR2 subunit not only exhibit high Ca 2+ permeability but also are blocked by intracellular polyamines. These properties have allowed us to follow directly the involvement of GluR2 subunits in synaptic transmission. Repetitive synaptic activation of Ca2+-permeable AMPARs causes a rapid reduction in Ca2+ permeability and a change in the amplitude of excitatory postsynaptic currents, owing to the incorporation of GluR2-containing AMPARs. Our experiments show that activity-induced Ca 2+ influx through GluR2-lacking AMPARs controls the targeting of GluR2-containing AMPARs, implying the presence of a self-regulating mechanism.

Intracellular spermine confers rectification on rat calcium‐permeable AMPA and kainate receptors.

1. Whole‐cell recordings were made from cerebellar granule cells cultured in high‐K+ medium to induce expression of Ca(2+)‐permeable AMPA receptors. Current‐voltage (I‐V) plots of agonist‐evoked responses showed varying degrees of inward rectification, but became linear within 5‐10 min. 2. Recombinant Ca(2+)‐permeable kainate receptors, composed of GluR6(Q)/KA‐2 subunits, exhibited rectifying whole‐cell I‐V plots that became linear in outside‐out patches. 3. Loss of rectification in granule cells was prevented by including 100 microM spermine in the pipette; the degree of rectification was then correlated with Ca2+ permeability. 4. Spermine also prevented loss of rectification in patches containing GluR6(Q)/KA‐2 receptors (IC50, 1.7 microM). 5. We suggest that spermine, or a similar cellular constituent, may act as a cytoplasmic factor conferring inward rectification on Ca(2+)‐permeable non‐NMDA receptors, and that ‘washout’ of this factor underlies the observed loss of rectification.

Regulation of Ca2+-permeable AMPA receptors: synaptic plasticity and beyond.

AMPA-type glutamate receptors (AMPARs) mediate most fast excitatory synaptic transmission in the brain. Diversity in excitatory signalling arises, in part, from functional differences among AMPAR subtypes. Although the rapid insertion or deletion of AMPARs is recognised as important for the expression of conventional forms of long-term synaptic plasticity--triggered, for example, by Ca2+ entry through NMDA-type glutamate receptors--only recently has attention focused on novel forms of plasticity that are regulated by, or alter the expression of, Ca2+-permeable AMPARs. The dynamic regulation of these receptors is important for normal synaptic function and in disease states.

Non-NMDA glutamate receptor occupancy and open probability at a rat cerebellar synapse with single and multiple release sites.

1. Excitatory postsynaptic currents (EPSCs) were recorded under whole‐cell voltage clamp from granule cells in slices of rat cerebellum. EPSCs from individual mossy fibre inputs were identified by their all‐or‐none appearance in response to a graded stimulus. Excitatory synaptic transmission was investigated at room temperature (approximately 24 degrees C) and at near‐physiological temperature (approximately 34 degrees C) by analysing current fluctuations in the peak and decay of the non‐N‐methyl‐D‐aspartate (non‐NMDA) component of EPSCs. 2. In a subset of synapses the mean EPSC amplitude remained unchanged as the probability of transmitter release was substantially lowered by raising the extracellular [Mg2+] and lowering [Ca2+]. These synapses were considered to have only one functional release site. Single‐site synapses had small EPSCs (139 +/‐ 16 pS, n = 5, at 24 degrees C) with a large coefficient of variation (c.v. = 0.23 +/‐ 0.02, n = 5) and an amplitude distribution that was well fitted by a Gaussian distribution in four out of five cases. The EPSC latency had a unimodal distribution and its standard deviation had a temperature dependence with a temperature coefficient (Q10; range, 24‐35 degrees C) of 2.4 +/‐ 0.4 (n = 4). 3. Peak‐scaled non‐stationary fluctuation analysis of single‐site EPSCs indicated that the mean conductance of the underlying non‐NMDA channels was 12 +/‐ 2 pS (n = 4) at 35 degrees C. Upper and lower limits for mean channel open probability (Po), calculated from fluctuations in the EPSC peak amplitude, were 0.51 and 0.38, respectively. These estimates, together with the open probability of the channel when bound by transmitter, suggest that only about 50% of the non‐NMDA channels were occupied following the release of a quantum of transmitter. 4. At some multi‐site synapses EPSCs had a low c.v. (0.4 +/‐ 0.01, n = 5) at 34 degrees C and non‐stationary fluctuation analysis gave a parabolic variance‐mean current relationship. This suggests that practically all of the non‐NMDA receptors were occupied by glutamate at the peak of EPSC. The channel open probability (Po = 0.84 +/‐ 0.03, n = 5) at these ‘saturated’ multi‐site synapses will therefore equal the open probability of the channel when bound by transmitter (Po,max). 5. Non‐stationary fluctuation analysis of EPSCs from ‘saturating’ multi‐site synapses indicated that 170 +/‐ 40 postsynaptic non‐NMDA channels were exposed to transmitter at the peak of the EPSC. The mean conductance of the synaptic channels was 10 +/‐ 2 pS (n = 5) at 34 degrees C. 6. At synapses with multiple release sites the EPSC decay time became faster when release probability was lowered (by reducing the external [Ca2+]/[Mg2+] ratio), indicating that the transmitter concentration profile depended on release probability. No such speeding of the EPSC decay was observed at single‐site synapses. 7. Our results suggest that release of a packet of transmitter from a single release site does not saturate postsynaptic non‐NMDA receptors at cerebellar mossy fibre‐granule cell synapses. However, at multi‐site synapses transmitter released from neighbouring sites can overlap, changing the transmitter concentration profile in the synaptic cleft. We conclude that the level of postsynaptic receptor occupancy can depend on the probability of transmitter release at individual multi‐site synapses.

Locus of frequency-dependent depression identified with multiple-probability fluctuation analysis at rat climbing fibre-Purkinje cell synapses

1 EPSCs were recorded under whole‐cell voltage clamp at room temperature from Purkinje cells in slices of cerebellum from 12‐ to 14‐day‐old rats. EPSCs from individual climbing fibre (CF) inputs were identified on the basis of their large size, paired‐pulse depression and all‐or‐none appearance in response to a graded stimulus. 2 Synaptic transmission was investigated over a wide range of experimentally imposed release probabilities by analysing fluctuations in the peak of the EPSC. Release probability was manipulated by altering the extracellular [Ca2+] and [Mg2+]. Quantal parameters were estimated from plots of coefficient of variation (CV) or variance against mean conductance by fitting a multinomial model that incorporated both spatial variation in quantal size and non‐uniform release probability. This ‘multiple‐probability fluctuation’ (MPF) analysis gave an estimate of 510 ± 50 for the number of functional release sites (N) and a quantal size (q) of 0.5 ± 0.03 nS (n= 6). 3 Control experiments, and simulations examining the effects of non‐uniform release probability, indicate that MPF analysis provides a reliable estimate of quantal parameters. Direct measurement of quantal amplitudes in the presence of 5 mM Sr2+, which gave asynchronous release, yielded distributions with a mean quantal size of 0.55 ± 0.01 nS and a CV of 0.37 ± 0.01 (n= 4). Similar estimates of q were obtained in 2 mM Ca2+ when release probability was lowered with the calcium channel blocker Cd2+. The non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 1 μM) reduced both the evoked current and the quantal size (estimated with MPF analysis) to a similar degree, but did not affect the estimate of N. 4 We used MPF analysis to identify those quantal parameters that change during frequency‐dependent depression at climbing fibre‐Purkinje cell synaptic connections. At low stimulation frequencies, the mean release probability (P̄r) was unusually high (0.90 ± 0.03 at 0.033 Hz, n= 5), but as the frequency of stimulation was increased, pr fell dramatically (0.02 ± 0.01 at 10 Hz, n= 4) with no apparent change in either q or N. This indicates that the observed 50‐fold depression in EPSC amplitude is presynaptic in origin. 5 Presynaptic frequency‐dependent depression was investigated with double‐pulse and multiple‐pulse protocols. EPSC recovery, following simultaneous release at practically all sites, was slow, being well fitted by the sum of two exponential functions (time constants of 0.35 ± 0.09 and 3.2 ± 0.4 s, n= 5). EPSC recovery following sustained stimulation was even slower. We propose that presynaptic depression at CF synapses reflects a slow recovery of release probability following release of each quantum of transmitter. 6 The large number of functional release sites, relatively large quantal size, and unusual dynamics of transmitter release at the CF synapse appear specialized to ensure highly reliable olivocerebellar transmission at low frequencies but to limit transmission at higher frequencies.