Current and calcium responses to local activation of axonal NMDA receptors in developing cerebellar molecular layer interneurons

In developing cerebellar molecular layer interneurons (MLIs), NMDA increases spontaneous GABA release. This effect had been attributed to either direct activation of presynaptic NMDA receptors (preNMDARs) or an indirect pathway involving activation of somato-dendritic NMDARs followed by passive spread of somatic depolarization along the axon and activation of axonal voltage dependent Ca(2+) channels (VDCCs). Using Ca(2+) imaging and electrophysiology, we searched for preNMDARs by uncaging NMDAR agonists either broadly throughout the whole field or locally at specific axonal locations. Releasing either NMDA or glutamate in the presence of NBQX using short laser pulses elicited current transients that were highly sensitive to the location of the spot and restricted to a small number of varicosities. The signal was abolished in the presence of high Mg(2+) or by the addition of APV. Similar paradigms yielded restricted Ca(2+) transients in interneurons loaded with a Ca(2+) indicator. We found that the synaptic effects of NMDA were not inhibited by blocking VDCCs but were impaired in the presence of the ryanodine receptor antagonist dantrolene. Furthermore, in voltage clamped cells, bath applied NMDA triggers Ca(2+) elevations and induces neurotransmitter release in the axonal compartment. Our results suggest the existence of preNMDARs in developing MLIs and propose their involvement in the NMDA-evoked increase in GABA release by triggering a Ca(2+)-induced Ca(2+) release process mediated by presynaptic Ca(2+) stores. Such a mechanism is likely to exert a crucial role in various forms of Ca(2+)-mediated synaptic plasticity.     

Presynaptic NMDA receptors act as local high‐gain glutamate detector in developing cerebellar molecular layer interneurons

In the classical view, NMDA receptors (NMDARs) are located postsynaptically and play a pivotal role in excitatory transmission and synaptic plasticity. In developing cerebellar molecular layer interneurons (MLIs) however, NMDARs are known to be solely extra‐ or presynaptic and somewhat poorly expressed. Somatodendritic NMDARs are exclusively activated by glutamate spillover from adjacent synapses, but the mode of activation of axonal NMDARs remains unclear. Our data suggest that a volume transmission is likely to stimulate presynaptic NMDARs (preNMDARs) since NMDA puffs directed to the axon led to inward currents and Ca²⁺ transients restricted to axonal varicosities. Using local glutamate photoliberation, we show that pre‐ and post‐synaptic NMDARs share the same voltage dependence indicating their containing NR2A/B subunits. Ca²⁺ transients elicited by NMDA puffs are eventually followed by delayed events reminding of the spontaneous Ca²⁺ transients (ScaTs) described at the basket cell/Purkinje cell terminals. Moreover, the presence of Ca²⁺ transients at varicosities located more than 5 μm away from the uncaging site indicates that the activation of preNMDARs sensitizes the Ca²⁺ stores in adjacent varicosities, a process that is abolished in the presence of a high concentration of ryanodine. Altogether, the data demonstrate that preNMDARs act as high‐gain glutamate detectors.     

A cortical attractor network with Martinotti cells driven by facilitating synapses

The population of pyramidal cells significantly outnumbers the inhibitory interneurons in the neocortex, while at the same time the diversity of interneuron types is much more pronounced. One acknowledged key role of inhibition is to control the rate and patterning of pyramidal cell firing via negative feedback, but most likely the diversity of inhibitory pathways is matched by a corresponding diversity of functional roles. An important distinguishing feature of cortical interneurons is the variability of the short-term plasticity properties of synapses received from pyramidal cells. The Martinotti cell type has recently come under scrutiny due to the distinctly facilitating nature of the synapses they receive from pyramidal cells. This distinguishes these neurons from basket cells and other inhibitory interneurons typically targeted by depressing synapses. A key aspect of the work reported here has been to pinpoint the role of this variability. We first set out to reproduce quantitatively based on in vitro data the di-synaptic inhibitory microcircuit connecting two pyramidal cells via one or a few Martinotti cells. In a second step, we embedded this microcircuit in a previously developed attractor memory network model of neocortical layers 2/3. This model network demonstrated that basket cells with their characteristic depressing synapses are the first to discharge when the network enters an attractor state and that Martinotti cells respond with a delay, thereby shifting the excitation-inhibition balance and acting to terminate the attractor state. A parameter sensitivity analysis suggested that Martinotti cells might, in fact, play a dominant role in setting the attractor dwell time and thus cortical speed of processing, with cellular adaptation and synaptic depression having a less prominent role than previously thought.     

Analysis of relations between NMDA receptors and GABA release at olfactory bulb reciprocal synapses

In the mammalian olfactory bulb, signal processing is mediated by synaptic interactions between dendrites. Glutamate released from mitral cell dendrites excites dendritic spines of granule cells, which in turn release GABA back onto the mitral cell dendrites, forming a reciprocal synaptic pair. This feedback synaptic circuit was shown to be mediated predominantly by NMDA receptors. We further utilized caged Ca2+ compounds to obtain insight into the mechanism that couples NMDA receptor activation to GABA release. Feedback inhibition elicited by photo-release of caged Ca2+ in mitral cell secondary dendrites persisted when voltage-gated Ca2+ channels were blocked by cadmium (Cd2+) and nickel (Ni2+). These results indicate that Ca2+ influx through NMDA receptors can directly trigger presynaptic GABA release for local dendrodendritic feedback inhibition.     

Presynaptic NMDA receptors mediate IPSC potentiation at GABAergic synapses in developing rat neocortex

Background

NMDA receptors are traditionally viewed as being located postsynaptically, at both synaptic and extrasynaptic locations. However, both anatomical and physiological studies have indicated the presence of NMDA receptors located presynaptically. Physiological studies of presynaptic NMDA receptors on neocortical GABAergic terminals and their possible role in synaptic plasticity are lacking.

Methodology/Principal Findings

We report here that presynaptic NMDA receptors are present on GABAergic terminals in developing (postnatal day (PND) 12-15) but not older (PND21-25) rat frontal cortex. Using MK-801 in the recording pipette to block postsynaptic NMDA receptors, evoked and miniature IPSCs were recorded in layer II/III pyramidal cells in the presence of AMPA/KA receptor antagonists. Bath application of NMDA or NMDA receptor antagonists produced increases and decreases in mIPSC frequency, respectively. Physiologically patterned stimulation (10 bursts of 10 stimuli at 25 Hz delivered at 1.25 Hz) induced potentiation at inhibitory synapses in PND12-15 animals. This consisted of an initial rapid, large increase in IPSC amplitude followed by a significant but smaller persistent increase. Similar changes were not observed in PND21-25 animals. When 20 mM BAPTA was included in the recording pipette, potentiation was still observed in the PND12-15 group indicating that postsynaptic increases in calcium were not required. Potentiation was not observed when patterned stimulation was given in the presence of D-APV or the NR2B subunit antagonist Ro25-6981.

Conclusions/Significance

The present results indicate that presynaptic NMDA receptors modulate GABA release onto neocortical pyramidal cells. Presynaptic NR2B subunit containing NMDA receptors are also involved in potentiation at developing GABAergic synapses in rat frontal cortex. Modulation of inhibitory GABAergic synapses by presynaptic NMDA receptors may be important for proper functioning of local cortical networks during development.     

Pre and post synaptic NMDA effects targeting Purkinje cells in the mouse cerebellar cortex

N-methyl-D-aspartate (NMDA) receptors are associated with many forms of synaptic plasticity. Their expression level and subunit composition undergo developmental changes in several brain regions. In the mouse cerebellum, beside a developmental switch between NR2B and NR2A/C subunits in granule cells, functional postsynaptic NMDA receptors are seen in Purkinje cells of neonate and adult but not juvenile rat and mice. A presynaptic effect of NMDA on GABA release by cerebellar interneurons was identified recently. Nevertheless whereas NMDA receptor subunits are detected on parallel fiber terminals, a presynaptic effect of NMDA on spontaneous release of glutamate has not been demonstrated. Using mouse cerebellar cultures and patch-clamp recordings we show that NMDA facilitates glutamate release onto Purkinje cells in young cultures via a presynaptic mechanism, whereas NMDA activates extrasynaptic receptors in Purkinje cells recorded in old cultures. The presynaptic effect of NMDA on glutamate release is also observed in Purkinje cells recorded in acute slices prepared from juvenile but not from adult mice and requires a specific protocol of NMDA application.     

Long-term potentiation selectively expressed by NMDA receptors at hippocampal mossy fiber synapses

The mossy fiber to CA3 pyramidal cell synapse (mf-CA3) provides a major source of excitation to the hippocampus. Thus far, these glutamatergic synapses are well recognized for showing a presynaptic, NMDA receptor-independent form of LTP that is expressed as a long-lasting increase of transmitter release. Here, we show that in addition to this "classical" LTP, mf-CA3 synapses can undergo a form of LTP characterized by a selective enhancement of NMDA receptor-mediated transmission. This potentiation requires coactivation of NMDA and mGlu5 receptors and a postsynaptic calcium rise. Unlike classical LTP, expression of this mossy fiber LTP is due to a PKC-dependent recruitment of NMDA receptors specifically to the mf-CA3 synapse via a SNARE-dependent process. Having two mechanistically different forms of LTP may allow mf-CA3 synapses to respond with more flexibility to the changing demands of the hippocampal network.     

AMPA receptor-dependent clustering of synaptic NMDA receptors is mediated by Stargazin and NR2A/B in spinal neurons and hippocampal interneurons

Under standard conditions, cultured ventral spinal neurons cluster AMPA- but not NMDA-type glutamate receptors at excitatory synapses on their dendritic shafts in spite of abundant expression of the ubiquitous NMDA receptor subunit NR1. We demonstrate here that the NMDA receptor subunits NR2A and NR2B are not routinely expressed in cultured spinal neurons and that transfection with NR2A or NR2B reconstitutes the synaptic targeting of NMDA receptors and confers on exogenous application of the immediate early gene product Narp the ability to cluster both AMPA and NMDA receptors. The use of dominant-negative mutants of GluR2 further showed that the synaptic targeting of NMDA receptors is dependent on the presence of synaptic AMPA receptors and that synaptic AMPA and NMDA receptors are linked by Stargazin and a MAGUK protein. This system of AMPA receptor-dependent synaptic NMDA receptor localization was preserved in hippocampal interneurons but reversed in hippocampal pyramidal neurons.     

Glutamate-Bound NMDARs Arising from In Vivo-like Network Activity Extend Spatio-temporal Integration in a L5 Cortical Pyramidal Cell Model

In vivo, cortical pyramidal cells are bombarded by asynchronous synaptic input arising from ongoing network activity. However, little is known about how such ‘background’ synaptic input interacts with nonlinear dendritic mechanisms. We have modified an existing model of a layer 5 (L5) pyramidal cell to explore how dendritic integration in the apical dendritic tuft could be altered by the levels of network activity observed in vivo. Here we show that asynchronous background excitatory input increases neuronal gain and extends both temporal and spatial integration of stimulus-evoked synaptic input onto the dendritic tuft. Addition of fast and slow inhibitory synaptic conductances, with properties similar to those from dendritic targeting interneurons, that provided a ‘balanced’ background configuration, partially counteracted these effects, suggesting that inhibition can tune spatio-temporal integration in the tuft. Excitatory background input lowered the threshold for NMDA receptor-mediated dendritic spikes, extended their duration and increased the probability of additional regenerative events occurring in neighbouring branches. These effects were also observed in a passive model where all the non-synaptic voltage-gated conductances were removed. Our results show that glutamate-bound NMDA receptors arising from ongoing network activity can provide a powerful spatially distributed nonlinear dendritic conductance. This may enable L5 pyramidal cells to change their integrative properties as a function of local network activity, potentially allowing both clustered and spatially distributed synaptic inputs to be integrated over extended timescales.     

Disynaptic inhibition between neocortical pyramidal cells mediated by Martinotti cells

Reliable activation of inhibitory pathways is essential for maintaining the balance between excitation and inhibition during cortical activity. Little is known, however, about the activation of these pathways at the level of the local neocortical microcircuit. We report a disynaptic inhibitory pathway among neocortical pyramidal cells (PCs). Inhibitory responses were evoked in layer 5 PCs following stimulation of individual neighboring PCs with trains of action potentials. The probability for inhibition between PCs was more than twice that of direct excitation, and inhibitory responses increased as a function of rate and duration of presynaptic discharge. Simultaneous somatic and dendritic recordings indicated that inhibition originated from PC apical and tuft dendrites. Multineuron whole-cell recordings from PCs and interneurons combined with morphological reconstructions revealed the mediating interneurons as Martinotti cells. Martinotti cells received facilitating synapses from PCs and formed reliable inhibitory synapses onto dendrites of neighboring PCs. We describe this feedback pathway and propose it as a central mechanism for regulation of cortical activity.     

The action of some analogues of the excitatory amino acids in the dentate gyrus of the rat

We have confirmed that gamma-D-glutamylglycine and the L-isomer of 2-amino-4-phosphonobutyric acid, and have shown also that L-2-amino-5-phosphonovaleric (L-APV) acid, are antagonists of synaptic excitations of dentate granule cells induced from both lateral and medial perforant paths. The N-methyl-D-aspartic acid (NMDA) antagonist D-APV is without effect. The synaptic antagonists reduce the presynaptic fibre volley particularly in the lateral path, suggesting that a reduced transmitter output contributes to their action. NMDA receptors exist upon the granule cells, but they are not involved with these synaptic process.     

Adenosine A2A receptors are essential for long-term potentiation of NMDA-EPSCs at hippocampal mossy fiber synapses

The physiological conditions under which adenosine A2A receptors modulate synaptic transmission are presently unclear. We show that A2A receptors are localized postsynaptically at synapses between mossy fibers and CA3 pyramidal cells and are essential for a form of long-term potentiation (LTP) of NMDA-EPSCs induced by short bursts of mossy fiber stimulation. This LTP spares AMPA-EPSCs and is likely induced and expressed postsynaptically. It depends on a postsynaptic Ca2+ rise, on G protein activation, and on Src kinase. In addition to A2A receptors, LTP of NMDA-EPSCs requires the activation of NMDA and mGluR5 receptors as potential sources of Ca2+ increase. LTP of NMDA-EPSCs displays a lower threshold for induction as compared with the conventional presynaptic mossy fiber LTP; however, the two forms of LTP can combine with stronger induction protocols. Thus, postsynaptic A2A receptors may potentially affect information processing in CA3 neuronal networks and memory performance.     

Cellular and subcellular localization of the inhibitory glycine receptor in hippocampal neurons

Inhibitory glycine receptors are most abundant in spinal cord and brainstem, and glycinergic synapses have a well-established role in the regulation of locomotor behavior. Little is known about the function of glycine receptors in cortex and hippocampus, where GABA plays a dominant role in synaptic inhibition. Therefore, we have investigated tissue and cellular expression of glycine receptor alpha-subunits. Western blot and immunohistochemical analyses reveal the presence of glycine receptors in hippocampal tissue. Immunocytochemical experiments in hippocampal cultures show prominent cellular expression of glycine receptors in pyramidal neurons and GAD-positive interneurons similar to the calcium-binding protein VILIP-1 with widespread hippocampal distribution. On the subcellular level we found co-staining of GlyR and the presynaptic marker synapsin I. Furthermore, co-staining with GAD at synaptic terminals indicated partial co-localization of GABA- and glycine receptors.     

The Arg473Cys-neuroligin-1 mutation modulates NMDA mediated synaptic transmission and receptor distribution in hippocampal neurons

Synapses mediate communication between neurons, thus playing a fundamental role in information processing in the CNS. Neuroligins form a family of heterophilic synaptic cell adhesion molecules, and neuroligin 1 (NL1) has been shown to be involved in the formation of excitatory synapses and have been suggested to associate indirectly with NMDA receptors by common binding to PSD95. A mutation in neuroligin 3 (Arg451Cys-NL3, human sequence numbering) identified in autistic patients is associated with altered spine density and has reduced binding capacity for its presynaptic partner beta-neurexin. Here, we investigated the role of NL1 and the homologous NL1 mutation Arg473Cys-NL1 (R473C-NL1) in excitatory synaptic transmission and NMDA receptor distribution. We demonstrate that R473C-NL1, when expressed in cultured hippocampal neurons, can induce a dramatic increase in NMDA current amplitude and that this change is accompanied by NMDA receptor clustering in the postsynaptic cell.     

Glutamate Binding to the GluN2B Subunit Controls Surface Trafficking of N-Methyl-D-aspartate (NMDA) Receptors

Trafficking of NMDA receptors to the surface of neurons and to synapses is critical for proper brain function and activity-dependent plasticity. Recent evidence suggests that surface trafficking of other ionotropic glutamate receptors requires ligand binding for exit from the endoplasmic reticulum. Here, we show that glutamate binding to GluN2 is required for trafficking of NMDA receptors to the cell surface. We expressed a panel of GluN2B ligand binding mutants in heterologous cells with GluN1 or in rat cultured neurons and found that surface expression correlates with glutamate efficacy. Such a correlation was found even in the presence of dominant negative dynamin to inhibit endocytosis and surface expression correlated with Golgi localization, indicating differences in forward trafficking. Co-expression of wild type GluN2B did not enhance surface expression of the mutants, suggesting that glutamate must bind to both GluN2 subunits in a tetramer and that surface expression is limited by the least avid of the two glutamate binding sites. Surface trafficking of a constitutively closed cleft GluN2B was indistinguishable from that of wild type, suggesting that glutamate concentrations are typically not limiting for forward trafficking. YFP-GluN2B expressed in hippocampal neurons from GluN2B(-/-) mice rescued synaptic accumulation at similar levels to wild type. Under these conditions, surface synaptic accumulation of YFP-GluN2B mutants also correlated with apparent glutamate affinity. Altogether, these results indicate that glutamate controls forward trafficking of NMDA receptors to the cell surface and to synapses and raise the intriguing idea that NMDA receptors may be functional at intracellular sites.     

A computer model of unitary responses from associational/commissural and perforant path synapses in hippocampal CA3 pyramidal cells

Despite the central position of CA3 pyramidal cells in the hippocampal circuit, the experimental investigation of their synaptic properties has been limited. Recent slice experiments from adult rats characterized AMPA and NMDA receptor unitary synaptic responses in CA3b pyramidal cells. Here, excitatory synaptic activation is modeled to infer biophysical parameters, aid analysis interpretation, explore mechanisms, and formulate predictions by contrasting simulated somatic recordings with experimental data. Reconstructed CA3b pyramidal cells from the public repository NeuroMorpho.Org were used to allow for cell-specific morphological variation. For each cell, synaptic responses were simulated for perforant pathway and associational/commissural synapses. Means and variability for peak amplitude, time-to-peak, and half-height width in these responses were compared with equivalent statistics from experimental recordings. Synaptic responses mediated by AMPA receptors are best fit with properties typical of previously characterized glutamatergic receptors where perforant path synapses have conductances twice that of associational/commissural synapses (0.9 vs. 0.5 nS) and more rapid peak times (1.0 vs. 3.3 ms). Reanalysis of passive-cell experimental traces using the model shows no evidence of a CA1-like increase of associational/commissural AMPA receptor conductance with increasing distance from the soma. Synaptic responses mediated by NMDA receptors are best fit with rapid kinetics, suggestive of NR2A subunits as expected in mature animals. Predictions were made for passive-cell current clamp recordings, combined AMPA and NMDA receptor responses, and local dendritic depolarization in response to unitary stimulations. Models of synaptic responses in active cells suggest altered axial resistivity and the presence of synaptically activated potassium channels in spines.     

Selective cholinergic depletion in medial septum leads to impaired long term potentiation and glutamatergic synaptic currents in the hippocampus

Cholinergic depletion in the medial septum (MS) is associated with impaired hippocampal-dependent learning and memory. Here we investigated whether long term potentiation (LTP) and synaptic currents, mediated by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors in the CA1 hippocampal region, are affected following cholinergic lesions of the MS. Stereotaxic intra-medioseptal infusions of a selective immunotoxin, 192-saporin, against cholinergic neurons or sterile saline were made in adult rats. Four days after infusions, hippocampal slices were made and LTP, whole cell, and single channel (AMPA or NMDA receptor) currents were recorded. Results demonstrated impairment in the induction and expression of LTP in lesioned rats. Lesioned rats also showed decreases in synaptic currents from CA1 pyramidal cells and synaptosomal single channels of AMPA and NMDA receptors. Our results suggest that MS cholinergic afferents modulate LTP and glutamatergic currents in the CA1 region of the hippocampus, providing a potential synaptic mechanism for the learning and memory deficits observed in the rodent model of selective MS cholinergic lesioning.     

Fusion pore modulation as a presynaptic mechanism contributing to expression of long-term potentiation

Working on the idea that postsynaptic and presynaptic mechanisms of long-term potentiation (LTP) expression are not inherently mutually exclusive, we have looked for the existence and functionality of presynaptic mechanisms for augmenting transmitter release in hippocampal slices. Specifically, we asked if changes in glutamate release might contribute to the conversion of 'silent synapses' that show N-methyl-D-aspartate (NMDA) responses but no detectable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) responses, to ones that exhibit both. Here, we review experiments where NMDA receptor responses provided a bioassay of cleft glutamate concentration, using opposition between peak [glu](cleft )and a rapidly reversible antagonist, L-AP5. We discuss findings of a dramatic increase in peak [glu](cleft) upon expression of pairing-induced LTP (Choi). We present simulations with a quantitative model of glutamatergic synaptic transmission that includes modulation of the presynaptic fusion pore, realistic cleft geometry and a distributed array of postsynaptic receptors and glutamate transporters. The modelling supports the idea that changes in the dynamics of glutamate release can contribute to synaptic unsilencing. We review direct evidence from Renger et al., in accord with the modelling, that trading off the strength and duration of the glutamate transient can markedly alter AMPA receptor responses with little effect on NMDA receptor responses. An array of additional findings relevant to fusion pore modulation and its proposed contribution to LTP expression are considered.     

What mechanisms underlie involvement of different NMDA receptor subunits in the induction of hippocampal long-term potentiation and long-term depression?

There is a point of view that N-methyl-D-aspartate (NMDA) receptor subunit-specific signaling outcomes determine the direction of modifications of efficacy of synaptic transmission. Activation of NMDA receptors that contain the 2A subunit promotes LTP, while LTD requires activation of NMDA receptors containing 2B subunit. However, this hypothesis is inconsistent with some experimental data. For explanation of these data, we put forward an alternative hypothesis. According to this hypothesis, the activation of diverse subtypes of NMDA receptors can lead to ether LTP or LTD depending on the relation between posttetanic Ca2+ rise and increase in postsynaptic Ca2+ concentration produced by previous stimulation. Activation of NMDA receptors with 2B subunit can promote LTD of excitatory input to the pyramidal cell due to presence of these receptors on inhibitory interneurons, induction of the LTP in interneuron, and potentiation of inhibitory transmission between the interneuron and the target pyramidal cell.     

Distinct modes of AMPA receptor suppression at developing synapses by GluN2A and GluN2B: single-cell NMDA receptor subunit deletion in vivo

During development there is an activity-dependent switch in synaptic N-Methyl-D-aspartate (NMDA) receptor subunit composition from predominantly GluN2B to GluN2A, though the precise role of this?switch remains unknown. By deleting GluN2 subunits in single neurons during synaptogenesis, we find that both GluN2B and GluN2A suppress AMPA receptor expression, albeit by distinct means. Similar to GluN1, GluN2B deletion increases the number of functional synapses, while GluN2A deletion increases the strength of unitary connections without affecting the number of functional synapses. We propose a model of excitatory synapse maturation in which baseline activation of GluN2B-containing receptors prevents premature synapse maturation until correlated activity allows induction of functional synapses. This activity also triggers the switch to GluN2A, which dampens further potentiation. Furthermore, we analyze the subunit composition of synaptic NMDA receptors in CA1 pyramidal cells, provide electrophysiological evidence for?a large population of synaptic triheteromeric receptors, and estimate the subunit-dependent open probability.