Developmental regulation of NMDA receptor-mediated synaptic currents at a central synapse.

The central nervous system has extraordinary plasticity in early life. This is thought to involve N-methyl-D-aspartate (NMDA) receptors which, along with the non-NMDA receptors, mediate fast excitatory synaptic transmission. Although NMDA receptors may be transiently enhanced early in life, it has not been possible to demonstrate directly a functional change in the NMDA receptor-mediated synaptic response because of the voltage-dependence of the NMDA conductance and the overlapping inhibitory synaptic conductances. Here I report that the duration of evoked NMDA-receptor-mediated excitatory postsynaptic currents (e.p.s.cs) in the superior colliculus is several times longer at early developmental stages compared to that measured in older animals. In contrast, the amplitude of NMDA-receptor-mediated miniature e.p.s.cs does not change during development. The kinetic response of excised membrane patches to a brief activation of NMDA receptors is similar to that of the NMDA e.p.s.c, which suggests that the time course of the NMDA e.p.s.c. in the superior colliculus reflects slow NMDA channel properties as in the hippocampus. Therefore, these data indicate that the molecular properties of NMDA receptors are developmentally regulated and thus may be controlling the ability of synapses to change in early life.     

Channel kinetics determine the time course of NMDA receptor-mediated synaptic currents

Synaptic release of glutamate results in a two component excitatory postsynaptic current (e.p.s.c.) at many vertebrate central synapses. Non-N-methyl-D-aspartate receptors mediate a component that has a rapid onset and decay while the component mediated by N-methyl-D-aspartate (NMDA) receptors has a slow rise-time and a decay of several hundred milliseconds, 100 times longer than the mean open time of NMDA channels. The slow decay of the NMDA-mediated e.p.s.c. could be due to residual glutamate in the synaptic cleft resulting in repeated binding and activation of NMDA receptors. However, in cultured hippocampal neurons, we find that the NMDA receptor antagonist D-2-amino-5-phosphonopentanoate has no effect on the slow e.p.s.c. when rapidly applied after activation of the synapse, suggesting that rebinding of glutamate does not occur. In addition, a brief pulse of glutamate to an outside-out membrane patch results in openings of NMDA channels that persist for hundreds of milliseconds, indicating that glutamate can remain bound for this period. These results imply that a brief pulse of glutamate in the synaptic cleft is sufficient to account for the slow e.p.s.c.     

Synaptic excitation produces a long-lasting rebound potentiation of inhibitory synaptic signals in cerebellar Purkinje cells.

Persistent changes in synaptic efficacy are thought to underlie the formation of learning and memory in the brain. High-frequency activation of an afferent excitatory fibre system can induce long-term potentiation, and conjunctive activation of two distinct excitatory synaptic inputs to the cerebellar Purkinje cells can lead to long-term depression of the synaptic activity of one of the inputs. Here we report a new form of neural plasticity in which activation of an excitatory synaptic input can induce a potentiation of inhibitory synaptic signals to the same cell. In cerebellar Purkinje cells stimulation of the excitatory climbing fibre synapses is followed by a long-lasting (up to 75 min) potentiation of gamma-aminobutyric acid A (GABAA) receptor-mediated inhibitory postsynaptic currents (i.p.s.cs), a phenomenon that we term rebound potentiation. Using whole-cell patch-clamp recordings in combination with fluorometric video imaging of intracellular calcium ion concentration, we find that a climbing fibre-induced transient increase in postsynaptic calcium concentration triggers the induction of rebound potentiation. Because the response of Purkinje cells to bath-applied exogenous GABA is also potentiated after climbing fibre-stimulation with a time course similar to that of the rebound potentiation of i.p.s.cs, we conclude that the potentiation is caused by a calcium-dependent upregulation of postsynaptic GABAA receptor function. We propose that rebound potentiation is a mechanism by which in vivo block of climbing fibre activity induces an increase in excitability in Purkinje cells. Moreover, rebound potentiation of i.p.s.cs is a cellular mechanism which, in addition to the long-term depression of parallel fibre synaptic activity, may have an important role for motor learning in the cerebellum.     

NMDA and non-NMDA receptors are co-localized at individual excitatory synapses in cultured rat hippocampus

A CENTRAL assumption about long-term potentiation in the hippocampus is that the two classes of glutamate-receptor ion channel, the N-methyl-D-aspartate (NMDA) and the kainate/quisqualate (non-NMDA) subtypes, are co-localized at individual excitatory synapses. This assumption is important because of the perceived interplay between NMDA and non-NMDA receptors in the induction and expression of long-term potentiation: the NMDA class, by virtue of its voltage-dependent channel block by magnesium and calcium permeability, provides the trigger for the induction of long-term potentiation, whereas the actual enhancement of synaptic efficacy is thought to be provided by the non-NMDA class. If both receptor subtypes are present at the one synapse, such cross-modulation could occur rapidly and locally through diffusible factors. By measuring miniature synaptic currents in cultured hippocampal neurons we show that the majority (approximately 70%) of the excitatory synapses on a postsynaptic cell possess both kinds of receptor, although to different extents. Of the remaining excitatory synapses, approximately 20% contain only the non-NMDA subtype and the rest possess only NMDA receptors. This finding provides direct evidence for co-localization of glutamate-receptor subtypes at individual synapses, and also points to the possibility that long-term potentiation might be differentially expressed at each synapse according to the mix of receptor subtypes at that synapse.     

Embryonic acetylcholine receptors guarantee spontaneous contractions in rat developing muscle

Many proteins are expressed in distinct embryonic and adult forms. However, in most cases we do not know why the embryonic form of proteins is required. This question can be readily addressed for the acetylcholine receptor (AChR) because developmentally specified modifications of this ligand-gated ion channel can be directly related to changes in membrane currents. In developing rat soleus muscle, spontaneous transmitter release causes miniature end-plate currents (m.e.p.cs) to flow into the muscle cell. We show here that these m.e.p.cs in neonatal soleus trigger spontaneous contractions. By injecting m.e.p.cs into young fibres, we showed that only embryonic m.e.p.cs can trigger such contractions; adult m.e.p.cs do not last long enough. Developing muscle fibres must be active for synapse and muscle differentiation. Our experiments indicate that the embryonic form of the AChR is essential for spontaneous contractile activity and may therefore be required for normal neuromuscular development.     

ATP receptor-mediated synaptic currents in the central nervous system.

Until now, the only well documented, fast excitatory neurotransmitter in the brain has been glutamate. Although there is evidence for adenosine 5'-triphosphate (ATP) acting as a transmitter in the peripheral nervous system, suggestions for such a role in the central nervous system have so far not been supported by any direct evidence. Here we report the recording of evoked and miniature synaptic currents in the rat medial habenula. The fast rise time of the currents showed that they were mediated by a ligand-activated ion channel rather than a second messenger system, thus limiting the known transmitter candidates. Evidence was found for the presence on the cells of glutamate, gamma-aminobutyric acid, acetylcholine and ATP receptors, but not for 5-hydroxytryptamine (5HT3) or glycine receptors. The evoked currents were unaffected by blockers of glutamate, gamma-aminobutyric acid or acetylcholine receptors but were blocked by the ATP receptor-blocker, suramin and the desensitizing ATP receptor-agonist alpha,beta-methylene-ATP. Our evidence identifies for the first time synaptic currents in the brain, mediated directly by ATP receptors.     

Potentiation of NMDA receptor currents by arachidonic acid.

Arachidonic acid is released by phospholipase A2 when activation of N-methyl-D-aspartate (NMDA) receptors by neurotransmitter glutamate raises the calcium concentration in neurons, for example during the initiation of long-term potentiation and during brain anoxia. Here we investigate the effect of arachidonic acid on glutamate-gated ion channels by whole-cell clamping isolated cerebellar granule cells. Arachidonic acid potentiates, and makes more transient, the current through NMDA receptor channels, and slightly reduces the current through non-NMDA receptor channels. Potentiation of the NMDA receptor current results from an increase in channel open probability, with no change in open channel current. We observe potentiation even with saturating levels of agonist at the glutamate- and glycine-binding sites on these channels; it does not result from conversion of arachidonic acid to lipoxygenase or cyclooxygenase derivatives, or from activation of protein kinase C. Arachidonic acid may act by binding to a site on the NMDA receptor, or by modifying the receptor's lipid environment. Our results suggest that arachidonic acid released by activation of NMDA (or other) receptors will potentiate NMDA receptor currents, and thus amplify increases in intracellular calcium concentration caused by glutamate. This may explain why inhibition of phospholipase A2 blocks the induction of long-term potentiation.     

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 (gamma-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 alpha6 and delta-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.     

NMDA receptors of dentate gyrus granule cells participate in synaptic transmission following kindling

In the mammalian central nervous system, receptors for the excitatory amino-acid neurotransmitters are divided into three subtypes depending on their sensitivity to three specific agonists: kainate, quisqualate and N-methyl-D-aspartate (NMDA). The ionophores operated by NMDA are gated by Mg2+ in a voltage-dependent manner and allow passage of several cations, including Ca2+ which may be important in plastic alterations of neuronal excitability. Indeed, specific antagonists of NMDA receptors effectively block spatial learning, long-term potentiation and some animal models of chronic epilepsy. Despite their abundance on central neurons, NMDA receptors, with a few noteworthy exceptions, do not generally seem to be involved in low-frequency synaptic transmission. Here we report for the first time that NMDA receptors of the dentate gyrus, where they do not normally contribute to the generation of synaptic potentials, become actively involved in synaptic transmission following long-lasting neuronal changes induced by daily electrical stimulation (kindling) of the amygdala or hippocampal commissures. In contrast to controls, the excitatory postsynaptic potentials (e.p.s.ps) of granule cells in hippocampal slices obtained from kindled animals displayed characteristics typical of an NMDA-receptor-mediated component. The involvement of NMDA receptors in synaptic transmission may underlie the long-lasting changes in neuronal function induced by kindling.     

Glutamate-induced long-term potentiation of the frequency of miniature synaptic currents in cultured hippocampal neurons.

Glutamate application at synapses between hippocampal neurons in culture produces long-term potentiation of the frequency of spontaneous miniature synaptic currents, together with long-term potentiation of evoked synaptic currents. The mini frequency potentiation is initiated postsynaptically and requires activity of NMDA receptors. Although the frequency of unitary quantal responses increases strongly, their amplitude remains little changed with potentiation. Tests of postsynaptic responsiveness rule out recruitment of latent glutamate receptor clusters. Thus, postsynaptic induction can lead to enhancement of presynaptic transmitter release. The sustained potentiation of mini frequency is expressed even in the absence of Ca2+ entry into presynaptic terminals.     

Integration of quanta in cerebellar granule cells during sensory processing

To understand the computations performed by the input layers of cortical structures, it is essential to determine the relationship between sensory-evoked synaptic input and the resulting pattern of output spikes. In the cerebellum, granule cells constitute the input layer, translating mossy fibre signals into parallel fibre input to Purkinje cells. Until now, their small size and dense packing have precluded recordings from individual granule cells in vivo. Here we use whole-cell patch-clamp recordings to show the relationship between mossy fibre synaptic currents evoked by somatosensory stimulation and the resulting granule cell output patterns. Granule cells exhibited a low ongoing firing rate, due in part to dampening of excitability by a tonic inhibitory conductance mediated by GABA(A) (gamma-aminobutyric acid type A) receptors. Sensory stimulation produced bursts of mossy fibre excitatory postsynaptic currents (EPSCs) that summate to trigger bursts of spikes. Notably, these spike bursts were evoked by only a few quantal EPSCs, and yet spontaneous mossy fibre inputs triggered spikes only when inhibition was reduced. Our results reveal that the input layer of the cerebellum balances exquisite sensitivity with a high signal-to-noise ratio. Granule cell bursts are optimally suited to trigger glutamate receptor activation and plasticity at parallel fibre synapses, providing a link between input representation and memory storage in the cerebellum.     

Dual-component NMDA receptor currents at a single central synapse

Present thinking about the way that the NMDA (N-methyl-D-aspartate) class of glutamate receptor operates at central synapses relies mainly on information obtained from single-channel and whole-cell recordings from cultured neurons stimulated by exogenous NMDA receptor agonists. The mechanisms that operate in the postsynaptic membrane of a normal neuron following release of the natural transmitter are far less clear. An important problem is that most normal neurons receive many excitatory synapses (10(3)-10(5) per cell) and these synapses are located on slender dendritic elements far away from the somatic recording site, making the study of discrete synaptic events difficult. Typically, when populations of synapses are activated, NMDA receptor-mediated synaptic potentials appear as slowly rising, long-lasting waves superimposed on faster, non-NMDA-receptor potentials. Although believed to be critical for NMDA receptor function, this slow time-course would not be predicted from single-channel kinetics and its origin remains puzzling. We have now analysed the events occurring at the level of a single excitatory synapse using a simple, small, neuron--the cerebellar granule cell--which has an unusually simple glutamatergic input. By applying high-resolution whole-cell recording techniques to these cells in situ, we were able to study the nature of elementary NMDA receptor-mediated synaptic currents. Contrary to expectations, the prominent currents are fast but are followed by slow ones. Both types of current are strongly voltage-dependent but differ subtly in this respect. Furthermore, the currents are absent unless glycine is provided.     

High-fidelity transmission of sensory information by single cerebellar mossy fibre boutons

Understanding the transmission of sensory information at individual synaptic connections requires knowledge of the properties of presynaptic terminals and their patterns of firing evoked by sensory stimuli. Such information has been difficult to obtain because of the small size and inaccessibility of nerve terminals in the central nervous system. Here we show, by making direct patch-clamp recordings in vivo from cerebellar mossy fibre boutons-the primary source of synaptic input to the cerebellar cortex-that sensory stimulation can produce bursts of spikes in single boutons at very high instantaneous firing frequencies (more than 700 Hz). We show that the mossy fibre-granule cell synapse exhibits high-fidelity transmission at these frequencies, indicating that the rapid burst of excitatory postsynaptic currents underlying the sensory-evoked response of granule cells can be driven by such a presynaptic spike burst. We also demonstrate that a single mossy fibre can trigger action potential bursts in granule cells in vitro when driven with in vivo firing patterns. These findings suggest that the relay from mossy fibre to granule cell can act in a 'detonator' fashion, such that a single presynaptic afferent may be sufficient to transmit the sensory message. This endows the cerebellar mossy fibre system with remarkable sensitivity and high fidelity in the transmission of sensory information.     

Glutamate activates multiple single channel conductances in hippocampal neurons

There is considerable evidence that glutamate is the principal neurotransmitter that mediates fast excitatory synaptic transmission in the vertebrate central nervous system. This single transmitter seems to activate two or three distinct types of receptors, defined by their affinities for three selective structural analogues of glutamate, NMDA (N-methyl-D-aspartate), quisqualate and kainate. All these agonists increase membrane permeability to monovalent cations, but NMDA also activates a conductance that permits significant calcium influx and is blocked in a voltage-dependent manner by extracellular magnesium. Fast synaptic excitation seems to be mediated mainly by kainate/quisqualate receptors, although NMDA receptors are sometimes activated. We have investigated the properties of these conductances using single-channel recording in primary cultures of hippocampal neurons, because the hippocampus contains all subtypes of glutamate receptors and because long-term potentiation of synaptic transmission occurs in this structure. We find that four or more distinct single-channel currents are evoked by applying glutamate to each outside-out membrane patch. These conductances vary in their ionic permeability and in the agonist most effective in causing them to open. Clear transitions between all the conductance levels are observed. Our observations are compatible with the model that all the single channel conductances activated by glutamate reflect the operation of one or two complex molecular entities.     

Mediation of thalamic sensory input by both NMDA receptors and non-NMDA receptors

Excitatory amino acids such as L-glutamate and L-aspartate are well established as neurotransmitter candidates in the mammalian central nervous system, and three types of receptor for these substances have been proposed, characterized by the agonists N-methyl-D-aspartate (NMDA), kainate and quisqualate. All these receptors have been suggested to have synaptic roles in excitatory transmission in the brain. Here I demonstrate that NMDA receptors play a crucial role in the observed response of ventrobasal thalamus (VB) neurones to natural stimulation of somatosensory afferents, but do not appear to be responsible for the short-latency excitation seen on electrical stimulation of the afferents which is apparently mediated by excitatory amino-acid receptors of the non-NMDA type. This result indicates an involvement of NMDA and non-NMDA receptors in the responses of VB neurones to stimulation of somatosensory somatosensory afferents, depending on the mode of stimulation of the pathway.     

ATP mediates fast synaptic transmission in mammalian neurons.

In addition to its diverse functions inside cells, ATP can act at several types of cell-surface receptor. One of these (P2X-purinoceptor) is believed to be a ligand-gated cation channel. The presence of P2X receptors on autonomic, sensory and central neurons suggests that ATP might be released to act as a fast excitatory synaptic transmitter. Here we record excitatory synaptic potentials and currents from cultured coeliac ganglion neurons which are mimicked by ATP, blocked by the P2-purinoceptor antagonist suramin, desensitized by alpha,beta-methylene-ATP and unaffected by antagonists acting at nicotine, 5-hydroxytryptamine, N-methyl-D-aspartate (NMDA), non-NMDA glutamate, gamma-aminobutyric acid (GABA), noradrenaline or adenosine receptors. We conclude that ATP is the neurotransmitter at this neuroneuronal synapse.     

GABA autoreceptors regulate the induction of LTP.

Understanding the mechanisms involved in long-term potentiation (LTP) should provide insights into the cellular and molecular basis of learning and memory in vertebrates. It has been established that in the CA1 region of the hippocampus the induction of LTP requires the transient activation of the N-methyl-D-aspartate (NMDA) receptor system. During low-frequency transmission, significant activation of this system is prevented by gamma-aminobutyric acid (GABA) mediated synaptic inhibition which hyperpolarizes neurons into a region where NMDA receptor-operated channels are substantially blocked by Mg2+ (refs. 5, 6). But during high-frequency transmission, mechanisms are evoked that provide sufficient depolarization of the postsynaptic membrane to reduce this block and thereby permit the induction of LTP. We now report that this critical depolarization is enabled because during high-frequency transmission GABA depresses its own release by an action on GABAB autoreceptors, which permits sufficient NMDA receptor activation for the induction of LTP. These findings demonstrate a role for GABAB receptors in synaptic plasticity.     

Frequency-dependent involvement of NMDA receptors in the hippocampus: a novel synaptic mechanism

Acidic amino acids, such as l-glutamate, are believed to be excitatory neurotransmitters in the mammalian brain and exert effects on several different receptors named after the selective agonists kainate, quisqualate and N-methyl-D-aspartate (NMDA). The first two receptors collectively termed non-NMDA receptors, have been implicated in the mediation of synaptic transmission in many excitatory pathways in the central nervous system (CNS), whereas NMDA receptors, with few exceptions do not appear to be involved; this is typified in the hippocampus where there is a high density of NMDA receptors yet selective NMDA receptor antagonists, such as D-2-amino-5-phosphonovalerate (APV), do not affect synaptic potentials. NMDA receptors have, however, been shown to be involved in long-term potentiation (LTP) in the hippocampus, a form of synaptic plasticity which may be involved in learning and memory. NMDA receptors have also been found to contribute to epileptiform activity in this region. We now describe how NMDA receptors can participate during high-frequency synaptic transmission in the hippocampus, their involvement during low-frequency transmission being greatly suppressed by Mg2+. A frequency dependent alleviation of this blockade provides a novel synaptic mechanism whereby a single neurotransmitter can transmit very different information depending on the temporal nature of the input. This mechanism could account for the involvement of NMDA receptors in the initiation of LPT and their contribution, in part, to epileptic activity.     

NMDA application potentiates synaptic transmission in the hippocampus

The NMDA (N-methyl-D-aspartate) class of glutamate receptor plays a critical role in a variety of forms of synaptic plasticity in the vertebrate central nervous system. One extensively studied example of plasticity is long-term potentiation (LTP), a remarkably long-lasting enhancement of synaptic efficiency induced in the hippocampus by brief, high-frequency stimulation of excitatory synapses. LTP is a strong candidate for a cellular mechanism of learning and memory. The site of LTP induction appears to be the postsynaptic cell and induction requires both activation of NMDA receptors by synaptically released glutamate and depolarization of the postsynaptic membrane. It is proposed that this depolarization relieves a voltage-dependent Mg2+ block of the NMDA receptor channel, resulting in increased calcium influx which is the trigger for the induction of LTP. This model predicts that application of a large depolarizing dose of NMDA should be sufficient to evoke LTP. In agreement with a previous study, we have found that NMDA or glutamate application does potentiate synaptic transmission in the hippocampus. This agonist-induced potentiation is, however, decremental and short-lived, unlike LTP. It is occluded shortly after the induction of LTP and a similar short-term potentiation can be evoked by synaptically released glutamate. We thus propose that LTP has two components, a short-term, decremental component which can be mimicked by NMDA receptor activation, and a long-lasting, non-decremental component which, in addition to requiring activation of NMDA receptors, requires stimulation of presynaptic afferents.     

Mechanisms of slow postsynaptic potentials

Classical views of synaptic transmission have to be modified to take account of slow postsynaptic potentials (p.s.ps), which are often mediated by novel ionic and molecular mechanisms and which are sometimes evoked by neuropeptides. Understanding slow p.s.ps will provide insights into the mechanisms of biological signal transduction and long-term signalling in the nervous system.