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Key points Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Survivors may develop a severe myasthenic syndrome or paralysis, associated with increased plasma levels of cyclohexanol, an insecticide solvent metabolite. Analysis of synaptic transmission at neuromuscular junctions in isolated mouse skeletal muscle, using isometric tension recording and microelectrode recording of endplate voltages and currents, showed that cyclohexanol reduced postsynaptic sensitivity to acetylcholine neurotransmitter (reduced quantal size) while simultaneously enhancing evoked transmitter release (increased quantal content). Patch recording from transfected cell lines, together with molecular modelling, indicated that cyclohexanol causes selective, allosteric antagonism of postsynaptic nicotinic acetylcholine receptor and block of presynaptic K+ -channel function. The data provide insight into the cellular and molecular mechanisms of neuromuscular weakness following intentional ingestion of agricultural organophosphorus insecticides. Our findings also extend understanding of the effects of alcohols on synaptic transmission and homeostatic synaptic function. Abstract Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Some survivors develop a severe, acute or delayed myasthenic syndrome. In animal models, similar myasthenia has been associated with increasing plasma concentration of one insecticide solvent metabolite, cyclohexanol. We investigated possible mechanisms using voltage and current recordings from mouse neuromuscular junctions (NMJs) and transfected human cell lines. Cyclohexanol (10-25 mM) reduced EPP amplitudes by 10-40% and enhanced depression during repetitive (2-20 Hz) stimulation by up to 60%. EPP decay was prolonged more than two fold. MEPPs were attenuated by more than 50%. Cyclohexanol inhibited whole-cell currents recorded from CN21 cells expressing human postjunctional acetylcholine receptors (hnAChR) with an IC50 of 3.74 mM. Cyclohexanol (10-20 mM) also caused prolonged episodes of reduced-current, multi-channel bursting in outside-out patch recordings from hnAChR expressed in transfected HEK293T cells, reducing charge transfer by more than 50%. Molecular modelling indicated cyclohexanol binding (-6kcal.mol-1 ) to a previously-identified alcohol binding site on nicotinic AChR α-subunits. Cyclohexanol also increased quantal content of evoked transmitter release by ∼50%. In perineurial recordings, cyclohexanol selectively inhibited pre-synaptic K+ -currents. Modelling indicated cyclohexanol binding (-3.8 kcal.mole-1 ) to voltage-sensitive K+ -channels at the same site as tetraethylammonium (TEA). TEA (10 mM) blocked K+ -channels more effectively than cyclohexanol but EPPs were more prolonged in 20 mM cyclohexanol. The results explain the pattern of neuromuscular dysfunction following ingestion of organophosphorus insecticides containing cyclohexanol precursors and suggest that cyclohexanol may facilitate investigation of mechanisms regulating synaptic strength at NMJs. Graphical Abstract: Cyclohexanol, the principal metabolite of a common agricultural insecticide solvent cyclohexanone, acts both presynaptically and postsynaptically to impair neuromuscular transmission. [1] Cyclohexanol binds to sites in the pore of voltage-sensitive K-channels that also bind the K+ -channel blocker tetraethylammonium (TEA), with the effect of reducing presynaptic motor nerve terminal K+ -currents and increasing quantal content of evoked transmitter release. [2] Simultaneously, cyclohexanol binds allosterically to an alcohol binding site on postsynaptic nicotinic acetylcholine receptors, altering ligand-gating characteristics and reducing charge transfer. The combined, overall effects of [1] and [2] result in reduced amplitude, prolonged depolarization and enhanced synaptic depression of endplate potentials (EPPs), weakening tetanic tension responses and causing fade in twitch responses to low frequency nerve stimulation. These effects mimic myasthenia or paralysis that occur in self-harming individuals who have swallowed significant quantities of organophosphorus insecticide, a significant public health issue in rural Asia. The present findings therefore provide an explanation for these clinical signs; and indicate potential utility of cyclohexanol in further study of mechanisms of neuromuscular synaptic homeostasis. This article is protected by copyright. All rights reserved.

The in vitro derivation of regionally defined human neuron types from patient-derived stem cells is now established as a resource to investigate human development and disease. Characterisation of such neurons initially focused on the expression of developmentally regulated transcription factors and neural markers, in conjunction with the development of protocols to direct and chart the fate of differentiated neurons. However, crucial to the understanding and exploitation of this technology is to determine the degree to which neurons recapitulate the key functional features exhibited by their native counterparts, essential for determining their usefulness in modelling human physiology and disease in vitro. Here, we review the emerging data concerning functional properties of human pluripotent stem cell-derived excitatory cortical neurons, both in the context of maturation and regional specificity. This article is protected by copyright. All rights reserved.

Key points Neurodegenerative disorders can exhibit dysfunctional mitochondrial respiratory chain complex IV activity.Conditional deletion of cytochrome c oxidase, the terminal enzyme in the respiratory electron transport chain of mitochondria, from hippocampal dentate granule cells in mice does not affect low‐frequency dentate to CA3 glutamatergic synaptic transmission.High‐frequency dentate to CA3 glutamatergic synaptic transmission and feedforward inhibition are significantly attenuated in cytochrome c oxidase‐deficient mice.Intact presynaptic mitochondrial function is critical for the short‐term dynamics of mossy fibre to CA3 synaptic function. Abstract Neurodegenerative disorders are characterized by peripheral and central symptoms including cognitive impairments which have been associated with reduced mitochondrial function, in particular mitochondrial respiratory chain complex IV or cytochrome c oxidase activity. In the present study we conditionally removed a key component of complex IV, protohaem IX farnesyltransferase encoded by the COX10 gene, in granule cells of the adult dentate gyrus. Utilizing whole‐cell patch‐clamp recordings from morphologically identified CA3 pyramidal cells from control and complex IV‐deficient mice, we found that reduced mitochondrial function did not result in overt deficits in basal glutamatergic synaptic transmission at the mossy‐fibre synapse because the amplitude, input–output relationship and 50 ms paired‐pulse facilitation were unchanged following COX10 removal from dentate granule cells. However, trains of stimuli given at high frequency (> 20 Hz) resulted in dramatic reductions in short‐term facilitation and, at the highest frequencies (> 50 Hz), also reduced paired‐pulse facilitation, suggesting a requirement for adequate mitochondrial function to maintain glutamate release during physiologically relevant activity patterns. Interestingly, local inhibition was reduced, suggesting the effect observed was not restricted to synapses with CA3 pyramidal cells via large mossy‐fibre boutons, but rather to all synapses formed by dentate granule cells. Therefore, presynaptic mitochondrial function is critical for the short‐term dynamics of synapse function, which may contribute to the cognitive deficits observed in pathological mitochondrial dysfunction., Key points Neurodegenerative disorders can exhibit dysfunctional mitochondrial respiratory chain complex IV activity.Conditional deletion of cytochrome c oxidase, the terminal enzyme in the respiratory electron transport chain of mitochondria, from hippocampal dentate granule cells in mice does not affect low‐frequency dentate to CA3 glutamatergic synaptic transmission.High‐frequency dentate to CA3 glutamatergic synaptic transmission and feedforward inhibition are significantly attenuated in cytochrome c oxidase‐deficient mice.Intact presynaptic mitochondrial function is critical for the short‐term dynamics of mossy fibre to CA3 synaptic function.

Parvalbumin containing inhibitory interneurons (PV + INs) are a key element controlling co-ordinated activity of neuronal ensembles in the mammalian central nervous system (CNS). This article is protected by copyright. All rights reserved

Homeostatic processes that regulate electrical activity in neurones are now an established aspect of physiology and rest on a large body of experimental evidence that points to roles in development, learning and memory, and disease. However, the concepts underlying homeostasis are too often summarized in ways that restrict their explanatory power and obviate important subtleties. Here, we present a review of the underlying theory of homeostasis--control theory--in an attempt to reconcile some existing conceptual problems in the context of neuronal physiology. In addition to clarifying the underlying theory, this review highlights the remaining challenges posed when analysing homeostatic phenomena that underlie the regulation of neuronal excitability. Moreover, we suggest approaches for future experimental and computational work that will further our understanding of neuronal homeostasis and the fundamental neurophysiological functions it serves.

In order to maintain stable functionality in the face of continually changing input, neurones in the CNS must dynamically modulate their electrical characteristics. It has been hypothesized that in order to retain stable network function, neurones possess homeostatic mechanisms which integrate activity levels and alter network and cellular properties in such a way as to counter long-term perturbations. Here we describe a simple model system where we investigate the effects of sustained neuronal depolarization, lasting up to several days, by exposing cultures of primary hippocampal pyramidal neurones to elevated concentrations (10–30 mm) of KCl. Following exposure to KCl, neurones exhibit lower input resistances and resting potentials, and require more current to be injected to evoke action potentials. This results in a rightward shift in the frequency-input current (FI) curve which is explained by a simple linear model of the subthreshold I–V relationship. No changes are observed in action potential profiles, nor in the membrane potential at which action potentials are evoked. Furthermore, following depolarization, an increase in subthreshold potassium conductance is observed which is accounted for within a biophysical model of the subthreshold I–V characteristics of neuronal membranes. The FI curve shift was blocked by the presence of the L-type Ca2+ channel blocker nifedipine, whilst antagonism of NMDA receptors did not interfere with the effect. Finally, changes in the intrinsic properties of neurones are reversible following removal of the depolarizing stimulus. We suggest that this experimental system provides a convenient model of homeostatic regulation of intrinsic excitability, and permits the study of temporal characteristics of homeostasis and its dependence on stimulus magnitude.

Heteromeric NMDARs are composed of coagonist glycine-binding NR1 subunits and glutamate-binding NR2 subunits. The majority of functional NMDARs in the mammalian central nervous system (CNS) contain two NR1 subunits and two NR2 subunits of which there are four types (A–D). We show that the potency of a variety of endogenous and synthetic glycine-site coagonists varies between recombinant NMDARs such that the highest potency is seen at NR2D-containing and the lowest at NR2A-containing NMDARs. This heterogeneity is specified by the particular NR2 subunit within the NMDAR complex since the glycine-binding NR1 subunit is common to all NMDARs investigated. To identify the molecular determinants responsible for this heterogeneity, we generated chimeric NR2A/2D subunits where we exchanged the S1 and S2 regions that form the ligand-binding domains and coexpressed these with NR1 subunits in Xenopus laevis oocytes. Glycine concentration–response curves for NMDARs containing NR2A subunits including the NR2D S1 region gave mean glycine EC50 values similar to NR2A(WT)-containing NMDARs. However, receptors containing NR2A subunits including the NR2D S2 region or both NR2D S1 and S2 regions gave glycine potencies similar to those seen in NR2D(WT)-containing NMDARs. In particular, two residues in the S2 region of the NR2A subunit (Lys719 and Tyr735) when mutated to the corresponding residues found in the NR2D subunit influence glycine potency. We conclude that the variation in glycine potency is caused by interactions between the NR1 and NR2 ligand-binding domains that occur following agonist binding and which may be involved in the initial conformation changes that determine channel gating.

N-methyl-d-aspartate receptors (NMDARs) display differences in their sensitivity to the channel blockers Mg2+ and memantine that are dependent on the identity of the NR2 subunit present in the receptor–channel complex. This study used two-electrode voltage-clamp recordings from Xenopus laevis oocytes expressing recombinant NMDARs to investigate the actions of Mg2+ and memantine at the two NMDARs displaying the largest differences in sensitivity to these blockers, namely NR1/NR2A and NR1/NR2D NMDARs. In addition, NR2A/2D chimeric subunits have been employed to examine the effects of pore-forming elements and ligand-binding domains (LBD) on the potency of the block produced by each of these inhibitors. Our results show that, as previously documented, NR2D-containing NMDARs are less sensitive to voltage-dependent Mg2+ block than their NR2A-containing counterparts. The reduced sensitivity is determined by the M1M2M3 membrane-associated regions, as replacing these regions in NR2A subunits with those found in NR2D subunits results in a ∼10-fold reduction in Mg2+ potency. Intriguingly, replacing the NR2A LBD with that from NR2D subunits results in a ∼2-fold increase in Mg2+ potency. Moreover, when responses mediated by NR1/NR2A NMDARs are evoked by the partial agonist homoquinolinate, rather than glutamate, Mg2+ also displays an increased potency. Memantine block of glutamate-evoked currents is most potent at NR1/NR2D NMDARs, but no differences are observed in its ability to inhibit NR2A-containing or NR2A/2D chimeric NMDARs. We suggest that the potency of block of NMDARs by Mg2+ is influenced not only by pore-forming regions but also the LBD and the resulting conformational changes that occur following agonist binding.

We have examined the function of a conserved serine residue (Ser670) in the S2 ligand-binding region of the NR2A N-methyl-d-aspartate (NMDA) receptor subunit, using recombinant NR1/NR2A receptors expressed in Xenopus laevis oocytes. Mutation of Ser670 to glycine (S670G) in NR2A reduced the potency of glutamate by 124-fold. Single-channel conductance and the duration of apparent open periods of NR2A(S670G) receptor mutants were, however, indistinguishable from wild-type NMDA receptors. NR1/NR2A(S670G) shut-time distributions were best described by a mixture of six exponential components, and the four shortest shut intervals of each distribution were considered to occur within a channel activation (burst). Bursts of single-channel openings were fitted with a mixture of four exponential components. The longest two components carried the majority of the charge transfer and had mean durations of 9.6 ± 0.5 and 29.6 ± 1.5 ms. The overall channel open probability during a burst was high (mean, 0.83 ± 0.06). Consistent with a shortening of NMDA receptor-channel burst lengths was the observation of an increased deactivation rate of macroscopic currents evoked by brief applications of glutamate to outside-out membrane patches. Correlations between shut times and adjacent open times were observed in all data records. Noticeably, shorter than average openings tended to occur next to long closed periods, whereas longer than average openings tended to occur next to short closings. Our single-channel data, together with modelling using a kinetic scheme to describe channel activations, support our hypothesis that the S670G point mutation reduces the dwell time of glutamate in its binding site.

Expression of N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) in the CA1 region of the hippocampus can be divided into an early (1–2 h), protein synthesis-independent phase and a late (>4 h), protein synthesis-dependent phase. In this study we have addressed whether the de novo protein synthesis required for the expression of late-LTP can be sustained solely from the translation of mRNAs located in the dendrites of CA1 pyramidal neurones. Our results show that late-LTP, lasting at least 5 h, can be maintained in hippocampal slices where the dendrites located in stratum radiatum have been isolated from their cell bodies by a microsurgical cut. The magnitude of the potentiation of the slope of field EPSPs in these ‘isolated’ slices was similar to that recorded in ‘intact’ slices. Incubation of the slices with the mRNA translation inhibitor cycloheximide or the mammalian target of rapamycin (mTOR) inhibitor rapamycin blocked late-LTP in both ‘intact’ and ‘isolated’ slice preparations. In contrast, incubation of slices with the transcription inhibitor, actinomycin D, resulted in a reduction of sustained potentiation, at 4 h, in ‘intact’ slices while in ‘isolated’ slices the magnitude of potentiation was similar to that seen in untreated slices. These results indicate that late-LTP can be induced and maintained in ‘isolated’ dendritic preparations via translation of pre-existing mRNAs.

NR2A and NR2B are the predominant NR2 NMDA receptor subunits expressed in cortex and hippocampus. The relative expression level of NR2A and NR2B is regulated developmentally and these two subunits have been suggested to play distinct roles in long-term synaptic plasticity. We have used patch-clamp recording of recombinant NMDA receptors expressed in HEK293 cells to characterize the activation properties of both NR1/NR2A and NR1/NR2B receptors. Recordings from outside-out patches that contain a single active channel show that NR2A-containing receptors have a higher probability of opening at least once in response to a brief synaptic-like pulse of glutamate than NR2B-containing receptors (NR2A, 0.80; NR2B, 0.56), a higher peak open probability (NR2A, 0.50; NR2B, 0.12), and a higher open probability within an activation (NR2A, 0.67; NR2B, 0.37). Analysis of the sequence of single-channel open and closed intervals shows that both NR2A- and NR2B-containing receptors undergo multiple conformational changes prior to opening of the channel, with at least one of these steps being faster for NR2A than NR2B. These distinct properties produce profoundly different temporal signalling profiles for NR2A- and NR2B-containing receptors. Simulations of synaptic responses demonstrate that at low frequencies typically used to induce long-term depression (LTD; 1 Hz), NR1/NR2B makes a larger contribution to total charge transfer and therefore calcium influx than NR1/NR2A. However, under high-frequency tetanic stimulation (100 Hz; > 100 ms) typically used to induce long-term potentiation (LTP), the charge transfer mediated by NR1/NR2A considerably exceeds that of NR1/NR2B.

NR1/NR2D NMDA receptors display unusually slow deactivation kinetics which may be critical for their role as extrasynaptic receptors. A threonine to alanine point mutation has been inserted at amino acid position 692 of the NR2D subunit (T692A). Recombinant NR1a/NR2D(T692A) NMDA receptors have been expressed in Xenopus laevis oocytes and their pharmacological and single-channel properties examined using two-electrode voltage-clamp and patch-clamp recording techniques. Glutamate dose–response curves from NR1a/NR2D(T692A) receptor channels produced an approximately 1600-fold reduction in glutamate potency compared to wild-type NR1a/NR2D receptors. There was no change in Hill slopes or gross reduction in mean maximal currents recorded in oocytes expressing either wild-type or mutant receptors. The mutation did not affect the potency of the co-agonist glycine. The shifts in potency produced by NR2D(T692A) containing receptors when activated by other glutamate-site agonists such as aspartate or NMDA were 30- to 60-fold compared to wild-type. Single-channel conductance levels of NR1a/NR2D(T692A) mutant receptors were indistinguishable from wild-type NR2D-containing channels. Additionally NR1a/NR2D(T692A) receptors showed the transitional asymmetry that is characteristic of NR2D-containing NMDA receptors. Rapid applications of glutamate on outside-out patches containing NR1a/NR2D(T692A) receptors produced macroscopic current deactivations that were about 60-fold faster than wild-type NR1a/NR2D receptors. Our results suggest that this conserved threonine residue plays a crucial role in ligand binding to NMDA NR2 receptor subunits and supports the idea that the slow decay kinetics associated with NR1a/NR2D NMDA receptors can be explained by the slow dissociation of glutamate from this NMDA receptor subtype.

We have assessed, using whole-cell patch-clamp recording and RNA-sequencing (RNA-seq), the properties and composition of GABAA receptors (GABAARs) and strychnine-sensitive glycine receptors (GlyRs) expressed by excitatory cortical neurons derived from human embryonic stem cells (hECNs). The agonists GABA and muscimol gave EC50 values of 278 μm and 182 μm, respectively, and the presence of a GABAAR population displaying low agonist potencies is supported by strong RNA-seq signals for α2 and α3 subunits. GABAAR-mediated currents, evoked by EC50 concentrations of GABA, were blocked by bicuculline and picrotoxin with IC50 values of 2.7 and 5.1 μm, respectively. hECN GABAARs are predominantly γ subunit-containing as assessed by the sensitivity of GABA-evoked currents to diazepam and insensitivity to Zn(2+), together with the weak direct agonist action of gaboxadol; RNA-seq indicated a predominant expression of the γ2 subunit. Potentiation of GABA-evoked currents by propofol and etomidate and the lack of inhibition of currents by salicylidine salycylhydrazide (SCS) indicate expression of the β2 or β3 subunit, with RNA-seq analysis indicating strong expression of β3 in hECN GABAARs. Taken together our data support the notion that hECN GABAARs have an α2/3β3γ2 subunit composition - a composition that also predominates in immature rodent cortex. GlyRs expressed by hECNs were activated by glycine with an EC50 of 167 μm. Glycine-evoked (500 μm) currents were blocked by strychnine (IC50 = 630 nm) and picrotoxin (IC50 = 197 μm), where the latter is suggestive of a population of heteromeric receptors. RNA-seq indicates GlyRs are likely to be composed of α2 and β subunits.

We have examined the deactivation kinetics of native N-methyl-D-aspartate receptors (NMDARs) containing NR1 and NR2D subunits by patch-clamp recording from Purkinje cells in cerebellar slices from young rats. NMDAR-mediated whole-cell currents were elicited in response to bath application of 20 microM NMDA and 50 microM glycine. The NMDAR-mediated currents were small, with an average whole-cell conductance of approximately 750 pS. Following the rapid application of brief pulses (1-10 ms) of 1 mM glutamate to outside-out membrane patches, we observed a low-conductance type of single-channel activity which lasted up to 30 s after the removal of agonist. Analysis of individual channel openings revealed asymmetry of transitions between the main- and subconductance states - a characteristic of NR1/NR2D-containing NMDARs. The averaged macroscopic current exhibited a decay time course which was well described by a single exponential function with a time constant of approximately 3 s. We conclude that native NR1/NR2D-containing NMDARs, like their recombinant counterparts, display very slow deactivation kinetics. This feature should provide a means for identification of these receptors at synapses, and indicates that they do not contribute to the synaptic NMDAR currents so far described.

It has been estimated that following the synaptic release of glutamate, the concentration in the synaptic cleft rises rapidly to around 1 mM and then decays with a time constant of about 1 ms (Clements, Lester, Tong, Jahr & Westbrook, 1992). Brief (1-5 ms) pulses of 1 mM glutamate to outside-out membrane patches containing N-methyl-D-aspartate (NMDA) channels yield currents that decay over a few hundred milliseconds, which resemble the NMDA component of an excitatory postsynaptic current (EPSC) (Lester, Clements, Westbrook & Jahr, 1990; Edmonds & Colquhoun, 1992). Given the short duration of time that glutamate is available for binding to postsynaptic receptors following release, and the slow rise and fall of currents through NMDA receptor channels, it is unlikely that an individual NMDA receptor would be activated more than once during a single synaptic event. The nature of these individual channel ‘activations’ is therefore considered to underlie the shape of the synaptic current (Lester et al. 1990; Gibb & Colquhoun, 1992; Lester & Jahr, 1992). The term ‘activation’ defines the sequence of events that takes place between the first opening that follows binding of agonist and the last opening before complete dissociation of agonist; the experimental estimate of this event is a ‘super-cluster’ (see Discussion for more rigorous definitions). The fact that two different molecules, a co-agonist and an agonist, are necessary to open the NMDA channel suggests that glycine as well as glutamate may determine the duration of NMDA single-channel activations. In all the experiments described here, however, glycine was used at a saturating concentration. Thus we expect the binding and unbinding of glutamate to the NMDA receptor complex (together with subsequent conformation changes) to determine the duration of receptor activations, as is presumably the case during synaptic transmission. Even under these conditions, however, the nature of such activations is complex, as illustrated by the equilibrium recordings of Gibb & Colquhoun (1991, 1992). They are far less well understood than, for instance, those of the nicotinic receptor at the neuromuscular junction (Colquhoun & Sakmann, 1985; Edmonds, Gibb & Colquhoun, 1995a,b), and no kinetic scheme is available that describes them adequately. In this paper, we investigate the macroscopic response of NMDA receptors elicited by short (1 ms) pulses of glutamate and the structure of the super-clusters of channel activity elicited by very low glutamate concentrations, and explore the relationship between these two sorts of measurement. We have examined these issues for two recombinant NMDA receptors, namely NR1a/NR2A channels and NR1a/NR2D channels. In recordings from transfected human embryonic kidney (HEK) 293 cells, these two subunit combinations display the fastest and slowest offset rates, respectively, following the termination of a 300 ms application of 100 μM glutamate (Monyer, Burnashev, Laurie, Sakmann & Seeburg, 1994), or following brief pulses of glutamate (Vicini et al. 1998). We find that the macroscopic current decay following a 1 ms concentration jump on NR1a/NR2D channels decays much more slowly than that mediated by NR1a/NR2A receptors. We reach the same conclusion by comparing NR1a/NR2A and NR1a/NR2D super-cluster durations recorded in the steady-state experiments employing very low agonist concentrations. A preliminary report of some of our findings has appeared previously (Wyllie, Behe, Edmonds & Colquhoun, 1997).

1. Patch-clamp recording methods have been used to compare the pharmacological properties and single-channel characteristics of non-NMDA receptor channels in cerebellar type-2 astrocytes and granule cells. 2. In type-2 astrocytes whole-cell concentration-response curves for glutamate, quisqualate, AMPA and kainate gave EC50 values of 5.8, 3.8, 7.6 and 160 microM and Hill slopes of 1.65, 1.18, 1.64 and 1.65, respectively, resembling estimates for granule cell receptors. 3. The non-NMDA receptor antagonists CNQX and diCl-HQC (see Methods) inhibited whole-cell kainate currents in both cell types. The IC50 for CNQX antagonism of the kainate response was 536 nM in type-2 astrocytes, and 500 nM in granule cells. The IC50 for diCl-HQC was 3.5 microM in astrocytes and 3.7 microM in granule cells. 4. CNQX acted as a competitive antagonist of whole-cell kainate responses in type-2 astrocytes and granule cells giving Schild plots with a slope near 1. The equilibrium constant, K, for CNQX binding was 524 nM in astrocytes and 489 nM in granule cells. 5. Quisqualate and AMPA responses showed rapid desensitization in type-2 astrocytes with a ratio of steady-state to peak response of 0.09. Concanavalin A reduced this desensitization. 6. Non-NMDA channels in type-2 astrocytes and granule cells showed a low permeability to Ca2+ ions with a reversal potential, for kainate-activated whole-cell currents in isotonic Ca2+, of approximately -25 mV for astrocytes and -45 mV for granule cells. 7. Outside-out patches from type-2 astrocytes exhibited a range of single-channel conductances that were superficially similar to the glutamate-activated conductances in granule cells. However, the type-2 astrocytes were devoid of NMDA receptors, hence all of these conductances originated from non-NMDA channels. Their slope conductances were approximately 11, 21, 32, 42 and 52 pS. Amplitudes were verified with mean low-variance plots and single-channel current-voltage curves, which were linear. 8. There was also evidence of lower conductance kainate-activated channels in astrocyte patches. From noise analysis their estimated mean conductance was 1.9 pS, as described for the 'low-conductance' type kainate responses in cerebellar neurones. 9. Apparent open times, shut times and burst lengths of AMPA-activated (3-10 microM) channels were examined in patches from type-2 astrocytes, and kinetic properties of the 40 and 50 pS levels were compared with the lower levels. 10. Our results indicate some marked pharmacological similarities between non-NMDA receptor channels in type-2 astrocytes and granule cells.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Application of non-NMDA (non-N-methyl-D-aspartate) receptor agonists onto outside-out patches of cerebellar granule cells gave two characteristic types of response (in different patches) which we have referred to as 'high conductance' and 'low conductance' responses. At a qualitative level these patches could be readily distinguished by the size of the noise increase accompanying their membrane currents. 2. In high conductance patches both AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate gave discrete single-channel conductances (10-30 pS), while in low conductance patches, AMPA produced small discrete events (6-10 pS), and kainate opened channels with conductances too small to be directly resolved. All patches examined contained NMDA receptor channels with characteristic 50 and 40 pS conductance levels. 3. Despite the marked differences in single-channel conductances, kainate dose-response curves constructed for high and low conductance patches had similar EC50 values of approximately 150 microM. 4. Spectral analysis of low conductance kainate responses gave an estimated channel conductance of approximately 1.5 pS. In these same low conductance patches AMPA produced discrete openings with two conductance levels; their mean conductances (and relative proportions) were 6 (87%) and 10 pS (13%). 5. In high conductance patches, glutamate (10-30 microM), AMPA (3-10 microM), and kainate (10-30 microM), each activated non-NMDA channels with three multiple conductance levels. The amplitudes of these conductance levels (approximately 10, 20 and 30 pS) were similar for each of the agonists, and their relative proportions (i.e. areas in the amplitude histograms) were constant for all three agonists. In addition, the relative proportion of levels was constant between patches, and all three levels were invariably present. These observations are all consistent with the idea that the three multiple conductances originate from a single receptor channel, activated by AMPA, kainate and glutamate. 6. Non-NMDA single-channel current-voltage (I-V) plots showed outward rectification in high conductance patches. For all three multiple conductance levels the ratio of outward to inward single-channel slope conductance was 1.8 +/- 0.1 and this rectification remained present in symmetrical Na+ solutions. 7. In high conductance patches, the events produced by a rapid application of 20-50 microM glutamate were compared with those activated during steady-state application.(ABSTRACT TRUNCATED AT 400 WORDS)

N-methyl-d-aspartate receptors (NMDARs) display differences in their sensitivity to the channel blockers Mg(2+) and memantine that are dependent on the identity of the NR2 subunit present in the receptor-channel complex. This study used two-electrode voltage-clamp recordings from Xenopus laevis oocytes expressing recombinant NMDARs to investigate the actions of Mg(2+) and memantine at the two NMDARs displaying the largest differences in sensitivity to these blockers, namely NR1/NR2A and NR1/NR2D NMDARs. In addition, NR2A/2D chimeric subunits have been employed to examine the effects of pore-forming elements and ligand-binding domains (LBD) on the potency of the block produced by each of these inhibitors. Our results show that, as previously documented, NR2D-containing NMDARs are less sensitive to voltage-dependent Mg(2+) block than their NR2A-containing counterparts. The reduced sensitivity is determined by the M1M2M3 membrane-associated regions, as replacing these regions in NR2A subunits with those found in NR2D subunits results in a approximately 10-fold reduction in Mg(2+) potency. Intriguingly, replacing the NR2A LBD with that from NR2D subunits results in a approximately 2-fold increase in Mg(2+) potency. Moreover, when responses mediated by NR1/NR2A NMDARs are evoked by the partial agonist homoquinolinate, rather than glutamate, Mg(2+) also displays an increased potency. Memantine block of glutamate-evoked currents is most potent at NR1/NR2D NMDARs, but no differences are observed in its ability to inhibit NR2A-containing or NR2A/2D chimeric NMDARs. We suggest that the potency of block of NMDARs by Mg(2+) is influenced not only by pore-forming regions but also the LBD and the resulting conformational changes that occur following agonist binding.

We have examined the function of a conserved serine residue (Ser670) in the S2 ligand-binding region of the NR2A N-methyl-d-aspartate (NMDA) receptor subunit, using recombinant NR1/NR2A receptors expressed in Xenopus laevis oocytes. Mutation of Ser670 to glycine (S670G) in NR2A reduced the potency of glutamate by 124-fold. Single-channel conductance and the duration of apparent open periods of NR2A(S670G) receptor mutants were, however, indistinguishable from wild-type NMDA receptors. NR1/NR2A(S670G) shut-time distributions were best described by a mixture of six exponential components, and the four shortest shut intervals of each distribution were considered to occur within a channel activation (burst). Bursts of single-channel openings were fitted with a mixture of four exponential components. The longest two components carried the majority of the charge transfer and had mean durations of 9.6 +/- 0.5 and 29.6 +/- 1.5 ms. The overall channel open probability during a burst was high (mean, 0.83 +/- 0.06). Consistent with a shortening of NMDA receptor-channel burst lengths was the observation of an increased deactivation rate of macroscopic currents evoked by brief applications of glutamate to outside-out membrane patches. Correlations between shut times and adjacent open times were observed in all data records. Noticeably, shorter than average openings tended to occur next to long closed periods, whereas longer than average openings tended to occur next to short closings. Our single-channel data, together with modelling using a kinetic scheme to describe channel activations, support our hypothesis that the S670G point mutation reduces the dwell time of glutamate in its binding site.

NR1/NR2D NMDA receptors display unusually slow deactivation kinetics which may be critical for their role as extrasynaptic receptors. A threonine to alanine point mutation has been inserted at amino acid position 692 of the NR2D subunit (T692A). Recombinant NR1a/NR2D(T692A) NMDA receptors have been expressed in Xenopus laevis oocytes and their pharmacological and single-channel properties examined using two-electrode voltage-clamp and patch-clamp recording techniques. Glutamate dose–response curves from NR1a/NR2D(T692A) receptor channels produced an approximately 1600-fold reduction in glutamate potency compared to wild-type NR1a/NR2D receptors. There was no change in Hill slopes or gross reduction in mean maximal currents recorded in oocytes expressing either wild-type or mutant receptors. The mutation did not affect the potency of the co-agonist glycine. The shifts in potency produced by NR2D(T692A) containing receptors when activated by other glutamate-site agonists such as aspartate or NMDA were 30- to 60-fold compared to wild-type. Single-channel conductance levels of NR1a/NR2D(T692A) mutant receptors were indistinguishable from wild-type NR2D-containing channels. Additionally NR1a/NR2D(T692A) receptors showed the transitional asymmetry that is characteristic of NR2D-containing NMDA receptors. Rapid applications of glutamate on outside-out patches containing NR1a/NR2D(T692A) receptors produced macroscopic current deactivations that were about 60-fold faster than wild-type NR1a/NR2D receptors. Our results suggest that this conserved threonine residue plays a crucial role in ligand binding to NMDA NR2 receptor subunits and supports the idea that the slow decay kinetics associated with NR1a/NR2D NMDA receptors can be explained by the slow dissociation of glutamate from this NMDA receptor subtype.

In the current economic climate it is all too apparent that what goes up can also go down; the same is true for the strength of glutamatergic excitatory postsynaptic potentials/currents (EPSPs/EPSCs). What is less apparent is what underlies changes when they occur. There can be no doubt that the exquisite detail of synaptic connectivity in the mammalian brain is suggestive of its power to store and process information. Such a static image of the nervous system – complex though it may be in isolation – acquires a deeper significance when we witness its ability to change: synapses are plastic and synaptic activity controls plasticity. This dynamic re-wiring of synaptic connections is now the pre-eminent physiological model for learning and memory. Indeed, dissecting the rules that govern synaptic plasticity has kept investigators busy since the seminal paper by Tim Bliss and Terje Lomo published in The Journal of Physiology (Bliss & Lomo, 1973) in which they showed that bursts of high frequency, ‘tetanic’, stimulation of the perforant path of the hippocampus led to a persistent increase in the strength of synaptic connections, a phenomenon known as long-term potentiation (LTP). Since then, a host of other ‘learning rules’ for excitatory synapses has emerged. These include long-term depression (LTD), a weakening of synaptic strength which results from low frequency stimulation, and ‘theta-burst LTP’, arising from trains of stimulation that mimic the endogenous theta band activity in the hippocampus. More recently, evidence has emerged that the precise timing of repeated pre- and postsynaptic action potentials can strengthen or weaken EPSPs/EPSCs, so-called spike timing-dependent plasticity (STDP). What subcellular mechanisms underlie these learning rules? In the hippocampus, especially at CA1–Schaeffer collateral synapses, we already know that many forms of plasticity, including LTP, LTD and STDP, depend on the activation of N-methyl-d-aspartate receptors (NMDARs) and are expressed as changes in the amplitude of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated EPSPs/EPSCs. This cause-and-effect scenario requires signalling molecules resident at the synapse to couple NMDAR activation to mechanisms that regulate AMPAR expression. Systematic attempts to identify the proteins involved in this signalling complex have resulted in a catalogue of hundreds of molecules (Collins et al. 2006). Among these are several members of the membrane-associated guanylate kinase (MAGUK) family, including PSD-93 and PSD-95, two proteins that interact specifically with NMDARs and which are the focus of a paper in this issue of The Journal of Physiology by Carlisle et al. (2008). MAGUKs represent a family of signalling molecules that are evolutionarily conserved across species as diverse as drosophila, mouse and human (Funke et al. 2005). All multicellular organisms need such molecules to couple intracellular communication to differentiation and coordinated growth. Nowhere is such a precise interplay between ultrastructural changes and signalling more crucial than at synapses, where both PSD-93/95 are found. In this issue of The Journal of Physiology, Carlisle et al. (2008) have uncovered distinct roles for each of these proteins in LTP and LTD by examining the plasticity phenotype in PSD-93/95 mutant mice. The main ‘take home’ messages from this study can be summarized as follows. When compared to wild-type littermates, mice expressing a mutant form of PSD-95 (Migaud et al. 1998) display (1) impaired basal AMPAR-mediated synaptic transmission, (2) enhanced tetanus-, theta-burst-, and STDP-induced LTP, and (3) no LTD. In contrast mice lacking PSD-93 display (1) normal levels of basal AMPAR-mediated synaptic transmission, (2) normal tetanus-induced but impaired theta-burst- and STDP-induced LTP, and (3) normal levels NMDAR-dependent LTD. As with many studies of synaptic plasticity, details matter, and this summary needs expanding upon. First, two earlier studies using the same form of PSD-95 mutant mice concluded that basal AMPAR-mediated transmission is normal (Migaud et al. 1998; Elias et al. 2006). While the discrepancy between Elias et al. (2006) and the current study may be accounted for by the age of the mice used, a reassessment of the other study (Migaud et al. 1998) conducted by Carlisle et al. (2008) confirms reduced AMPAR-mediated basal synaptic transmission. Perhaps this reduced basal AMPAR expression in PSD-95 mutants engenders a greater capacity for increasing synaptic strength. Certainly it appears from this and earlier studies (Migaud et al. 1998) that potentiation is not only stronger, but can also result from stimulation protocols that do not normally give rise to changes in synaptic strength, such as single spike-pairing STDP. Does PSD-95 therefore participate in a signalling pathway that simultaneously limits the strength of potentiation and its sensitivity to the temporal order of pre- and postsynaptic events? If so, it is somewhat surprising that acute slices show impaired basal transmission: one might expect that ‘experiences’ of PSD-95 mutants (during their lives) may have triggered inappropriate synaptic strengthening and therefore an increase in basal synaptic transmission. Thus while LTP in vitro and mechanisms of experience-dependent plasticity in vivo share common features (Whitlock et al. 2006) it remains difficult to interpret results such as this in both contexts. In this respect it would be useful to know the temporal characteristics of late-phase (protein synthesis-dependent) LTP in hippocampal slices obtained from PSD-95 mutants. PSD-93 mutants show more subtle changes in synaptic transmission/plasticity compared to PSD-95 mutants. Basal synaptic transmission, tetanus-induced LTP and low frequency-induced LTD are indistinguishable from wild-type and it is only in theta burst-induced LTP and STDP that deficits are apparent. This might indicate that the levels of intracellular calcium triggered by the various protocols are crucial for understanding the role of PSD-93 in particular. In conclusion, the study by Carlisle et al. (2008) demonstrates that PSD-93 and PSD-95 have opposing effects on plasticity in the CA1 region of the hippocampus; however a challenge that emerges is to understand how expression levels of MAGUKs are regulated if proteins such as these are part of the currency used to determine the ‘synaptic economy’.