BK potassium channels control transmitter release at CA3–CA3 synapses in the rat hippocampus

Augmentation is a component of short-term synaptic plasticity with a gradual onset and duration in seconds. To investigate this component at the corticogeniculate synapse, whole cell patch-clamp recordings were obtained from principal cells in a slice preparation of the rat dorsal lateral geniculate nucleus. Trains with 10 stimuli at 25 Hz evoked excitatory postsynaptic currents (EPSCs) that grew in amplitude, primarily from facilitation. Such trains also induced augmentation that decayed exponentially with a time constant τ= 4.6 ± 2.6 s (mean ± standard deviation). When the trains were repeated at 1–10 s intervals, augmentation markedly increased the size of the first EPSCs, leaving late EPSCs unaffected. The magnitude of augmentation was dependent on the number of pulses, pulse rate and intervals between trains. Augmented EPSCs changed proportionally to basal EPSC amplitudes following alterations in extracellular calcium ion concentration. The results indicate that augmentation is determined by residual calcium remaining in the presynaptic terminal after repetitive spikes, competing with fast facilitation. We propose that augmentation serves to maintain a high synaptic strength in the corticogeniculate positive feedback system during attentive visual exploration.     

Post-synaptic N-methyl-d-aspartate signalling in hippocampal neurons of rat: spillover increases the impact of each spike in a short burst discharge

High-frequency burst discharges in hippocampus typically consist of less than ten spikes fired at frequencies too high to be followed by a post-synaptic neuron. How significant are these numbers for synaptic signalling? We have measured the N-methyl-d-aspartate (NMDA) component of the excitatory post-synaptic current (EPSC(NMDA)) in hippocampal CA1 neurons of rat after burst discharge of variable duration. The synaptic facilitation is accompanied by a slow-down of the EPSC(NMDA) which develops on a spike-to-spike basis. Consequently the charge transferred by the after-burst EPSC(NMDA) is increased with each spike. The phenomenon is most probably due to the spillover-mediated recruitment of extrasynaptic NMDA receptors. In terms of post-synaptic signalling it dramatically increases the impact of each spike in a short burst discharge.     

The pool of fast releasing vesicles is augmented by myosin light chain kinase inhibition at the calyx of Held synapse

Synaptic strength is determined by release probability and the size of the readily releasable pool of docked vesicles. Here we describe the effects of blocking myosin light chain kinase (MLCK), a cytoskeletal regulatory protein thought to be involved in myosin-mediated vesicle transport, on synaptic transmission at the mouse calyx of Held synapse. Application of three different MLCK inhibitors increased the amplitude of the early excitatory postsynaptic currents (EPSCs) in a stimulus train, without affecting the late steady-state EPSCs. A presynaptic locus of action for MLCK inhibitors was confirmed by an increase in the frequency of miniature EPSCs that left their average amplitude unchanged. MLCK inhibition did not affect presynaptic Ca²⁺ currents or action potential waveform. Moreover, Ca²⁺ imaging experiments showed that [Ca²⁺]_i transients elicited by 100-Hz stimulus trains were not altered by MLCK inhibition. Studies using high-frequency stimulus trains indicated that MLCK inhibitors increase vesicle pool size, but do not significantly alter release probability. Accordingly, when AMPA-receptor desensitization was minimized, EPSC paired-pulse ratios were unaltered by MLCK inhibition, suggesting that release probability remains unaltered. MLCK inhibition potentiated EPSCs even when presynaptic Ca²⁺ buffering was greatly enhanced by treating slices with EGTA-AM. In addition, MLCK inhibition did not affect the rate of recovery from short-term depression. Finally, developmental studies revealed that EPSC potentiation by MLCK inhibition starts at postnatal day 5 (P5) and remains strong during synaptic maturation up to P18. Overall, our data suggest that MLCK plays a crucial role in determining the size of the pool of synaptic vesicles that undergo fast release at a CNS synapse.     

Contribution of individual spikes in burst-induced long-term synaptic modification

Long-term synaptic modification depends on the relative timing of individual pre- and postsynaptic spikes, but the rules governing the effects of multispike bursts remain to be fully understood. In particular, some studies suggest that the spike timing dependence of synaptic modification breaks down with high-frequency bursts. In this study, we characterized the effects of pre- and postsynaptic bursts on long-term modification of layer 2/3 synapses in visual cortical slices from young rats. We found that, while pairing-induced synaptic modification depends on the burst frequency, this dependence can be explained in terms of the timing of individual pre- and postsynaptic spikes. Later spikes in each burst are less effective in synaptic modification, but spike efficacy is regulated differently in pre- and postsynaptic bursts. Presynaptically, spike efficacy is progressively weakened, in parallel with short-term synaptic depression. Postsynaptically, spike efficacy is suppressed to a lesser extent, and it depends on postsynaptic potassium channel activation. Such timing-dependent interaction among multiple spikes can account for synaptic modifications induced by a variety of spike trains, including the frequency-dependent transition from depression to potentiation induced by a postsynaptic burst preceding a presynaptic burst.     

Presynaptic inhibition of corticothalamic feedback by metabotropic glutamate receptors

The thalamus relays sensory information to cortex, but this information may be influenced by excitatory feedback from cortical layer VI. The full importance of this feedback has only recently been explored, but among its possible functions are influences on the processing of sensory features, synchronization of thalamic firing, and transitions in response mode of thalamic relay cells. Uncontrolled, corticothalamic feedback has also been implicated in pathological thalamic rhythms associated with certain neurological disorders. We have found a form of presynaptic inhibition of corticothalamic synaptic transmission that is mediated by a Group II metabotropic glutamate receptor (mGluR) and activated by high-frequency corticothalamic activity. We tested putative retinogeniculate and corticogeniculate synapses for Group II mGluR modulation within the dorsal lateral geniculate nucleus of the ferret thalamus. Stimulation of optic-tract fibers elicited paired-pulse depression of excitatory postsynaptic currents (EPSCs), whereas stimulation of the optic radiations elicited paired-pulse facilitation. Paired-pulse responses were subsequently used to characterize the pathway of origin of stimulated synapses. Group II mGluR agonists (LY379268 and DCG-IV) applied to thalamic neurons under voltage-clamp conditions reduced the amplitude of corticogeniculate EPSCs. Stimulation with high-frequency trains produced a facilitating response that was reduced by Group II mGluR agonists, but was enhanced by the selective antagonist LY341495, revealing a presynaptic, mGluR-mediated reduction of high-frequency corticogeniculate feedback. Agonist treatment did not affect EPSCs from stimulation of the optic tract. NAAG (reported to be selective for mGluR3) was ineffective at the corticogeniculate synapse, implicating mGluR2 in the observed effects. Our data are the first to show a synaptically elicited form of presynaptic inhibition of corticothalamic synaptic transmission that is mediated by presynaptic action of mGluR2. This presynaptic inhibition may partially mute sensory feedback and prevent reentrant excitation from initiating abnormal thalamic rhythms.     

5-HT inhibits synaptic transmission in rat subthalamic nucleus neurons in vitro

The subthalamic nucleus (STN) plays a pivotal role in normal and abnormal motor function. We used patch pipettes to study effects of 5-HT on synaptic currents evoked in STN neurons by focal electrical stimulation of rat brain slices. 5-HT (10 microM) reduced glutamate-mediated excitatory postsynaptic currents (EPSCs) by 35+/-4%. However, a much higher concentration of 5-HT (100 microM) was required to inhibit GABA-mediated inhibitory postsynaptic currents (IPSCs) to a comparable extent. Concentration-response curves showed that the 5-HT inhibitory concentration 50% (IC50) for inhibition of IPSCs (20.2 microM) was more than fivefold greater than the IC50 for inhibition of EPSCs (3.4 microM). The 5-HT-induced reductions in EPSCs and IPSCs were accompanied by increases in paired-pulse ratios, indicating that 5-HT acts presynaptically to inhibit synaptic transmission. The 5-HT1B receptor antagonist NAS-181 significantly antagonized 5-HT-induced inhibitions of EPSCs and IPSCs. These studies show that 5-HT inhibits synaptic transmission in the STN by activating presynaptic 5-HT1B receptors.     

Subthalamic stimulation evokes complex EPSCs in the rat substantia nigra pars reticulata in vitro

The subthalamic nucleus (STN) plays an important role in movement control by exerting its excitatory influence on the substantia nigra pars reticulata (SNR), a major output structure of the basal ganglia. Moreover, excessive burst firing of SNR neurons seen in Parkinson's disease has been attributed to excessive transmission in the subthalamonigral pathway. Using the 'blind' whole-cell patch clamp recording technique in rat brain slices, we found that focal electrical stimulation of the STN evoked complex, long-duration excitatory postsynaptic currents (EPSCs) in SNR neurons. Complex EPSCs lasted 200–500 ms and consisted of an initial monosynaptic EPSC followed by a series of late EPSCs superimposed on a slow inward shift in holding current. Focal stimulation of regions outside the STN failed to evoke complex EPSCs. The late component of complex EPSCs was markedly reduced by ionotropic glutamate receptor antagonists (2-amino-5-phosphonopentanoic acid and 6-cyano-7-nitro-quinoxalone) and by a GABA_A receptor agonist (isoguvacine) when these agents were applied directly to the STN using a fast-flow microapplicator. Moreover, the complex EPSC was greatly enhanced by bath application of the GABA_A receptor antagonists picrotoxin or bicuculline. These data suggest that recurrent glutamate synapses in the STN generate polysynaptic, complex EPSCs that are under tonic inhibition by GABA. Because complex EPSCs are expected to generate bursts of action potentials in SNR neurons, we suggest that complex EPSCs may contribute to the pathological burst firing that is associated with the symptoms of Parkinson's disease.     

Cellular mechanisms of burst firing-mediated long-term depression in rat neocortical pyramidal cells

During wakefulness and sleep, neurons in the neocortex emit action potentials tonically or in rhythmic bursts, respectively. However, the role of synchronized discharge patterns is largely unknown. We have recently shown that pairings of excitatory postsynaptic potentials (EPSPs) and action potential bursts or single spikes lead to long-term depression (burst-LTD) or long-term potentiation, respectively. In this study, we elucidate the cellular mechanisms of burst-LTD and characterize its functional properties. Whole-cell patch-clamp recordings were obtained from layer V pyramidal cells in somatosensory cortex of juvenile rats in vitro and composite EPSPs and EPSCs were evoked extracellularly in layers II/III. Repetitive burst-pairings led to a long-lasting depression of EPSPs and EPSCs that was blocked by inhibitors of metabotropic glutamate group 1 receptors, phospholipase C, protein kinase C (PKC) and calcium release from the endoplasmic reticulum, and that required an intact machinery for endocytosis. Thus, burst-LTD is induced via a Ca²⁺- and phosphatidylinositol-dependent activation of PKC and expressed through phosphorylation-triggered endocytosis of AMPA receptors. Functionally, burst-LTD is inversely related to EPSP size and bursts dominate single spikes in determining the sign of synaptic plasticity. Thus burst-firing constitutes a signal by which coincident synaptic inputs are proportionally downsized. Overall, our data thus suggest a mechanism by which synaptic weights can be reconfigured during non-rapid eye movement sleep.     

Glial glutamate transporter 1 regulates the spatial and temporal coding of glutamatergic synaptic transmission in spinal lamina II neurons

Glutamatergic synaptic transmission is a dynamic process determined by the amount of glutamate released by presynaptic sites, the clearance of glutamate in the synaptic cleft, and the properties of postsynaptic glutamate receptors. Clearance of glutamate in the synaptic cleft depends on passive diffusion and active uptake by glutamate transporters. In this study, we examined the role of glial glutamate transporter 1 (GLT-1) in spinal sensory processing. Excitatory postsynaptic currents (EPSCs) of substantia gelatinosa neurons recorded from spinal slices of young adult rats were analyzed before and after GLT-1 was pharmacologically blocked by dihydrokainic acid. Inhibition of GLT-1 prolonged the EPSC duration and the EPSC decay phase. The EPSC amplitudes were increased in neurons with weak synaptic input but decreased in neurons with strong synaptic input upon inhibition of GLT-1. We suggest that presynaptic inhibition, desensitization of postsynaptic AMPA receptors, and glutamate "spillover" contributed to the kinetic change of EPSCs induced by the blockade of GLT-1. Thus, GLT-1 is a key component in maintaining the spatial and temporal coding in signal transmission at the glutamatergic synapse in substantia gelatinosa neurons.     

Transmitter release triggered by a local depolarization in motor nerve terminals of the frog: role of calcium entry and of depolarization

Quantal synaptic currents (EPSCs) were elicited at neuromuscular junctions of frogs, applying current pulses through the recording current-clamp electrode. Facilitation of a test-EPSC by a preceding EPSC of variable amplitude was determined. This facilitation after a prepulse and release during the prepulse were found to have different dependences on depolarizing current amplitude. In addition, if intraterminal calcium [( Ca]i) was raised by a train of EPSCs, small depolarizations with little additional Ca entry elicited release which depended strongly on pulse amplitude. It is concluded that, in addition to [Ca]i, depolarization of the terminal directly controls quantal release. This depolarization dependence of release is shown to be the probable cause for termination of release after a large Ca entry.     

Subthreshold contribution of N-methyl-d-aspartate receptors to long-term potentiation induced by low-frequency pairing in rat hippocampal CA1 pyramidal cells

Long-term potentiation (LTP) is a use-dependent and persistent enhancement of synaptic strength. In the CA1 region of the hippocampus, LTP has Hebbian characteristics and requires precisely timed interaction between presynaptic firing and postsynaptic depolarization. Although depolarization is an absolute requirement for plasticity, it is still not clear whether the postsynaptic response during LTP induction should be subthreshold or suprathreshold for the generation of somatic action potential. Here, we use the whole-cell patch-clamp technique and different pairing protocols to examine systematically the postsynaptic induction requirements for LTP. We induce LTP by changes only in membrane potential while keeping the afferent stimulation constant and at minimal levels. This approach permits differentiation of two types of LTP: LTP induced with suprathreshold synaptic responses (LTP(AP)) and LTP induced with subthreshold excitatory postsynaptic current (EPSCs; LTP(EPSC)). We found that LTP(AP) (>40%) required pairing of depolarization (V(m)>or=-40 mV, for 40-60 s) with four to six (0.1 Hz) single synaptically initiated action potentials. LTP(EPSC) was of smaller magnitude (<30%) and required pairing of depolarization to -50 mV (60 s) with six subthreshold EPSCs. The N-methyl-d-aspartate receptor (NMDAR) antagonists aminophosphonovaleric acid and 7-chlorokynurenic acid consistently blocked LTP(EPSC) but were ineffective in preventing LTP(AP). Robust, NMDAR-independent LTP is obtained by stronger postsynaptic depolarization that converts the EPSCs to suprathreshold somatic action potentials. Purely NMDAR-dependent LTP is obtained by pairing mild somatic depolarization with subthreshold afferent pulses to the postsynaptic cell. Our results indicate that the degree of postsynaptic depolarization in the presence of single afferent pulses determines the type and magnitude of LTP.     

Two distinct glutamatergic synaptic inputs to striatal medium spiny neurones of neonatal rats and paired-pulse depression.

Excitatory postsynaptic currents (EPSCs) were recorded from the medium spiny neurones of neonatal rat striatal slices using the whole-cell patch clamp method. EPSCs were selectively elicited in the presence of picrotoxin with a glass stimulating pipette placed in the striatum. We found two distinct unitary EPSCs that were evoked by stimulation of single presynaptic fibres. The major type of EPSC, termed 'S-type', failed frequently and had a small mean amplitude (2.05 pA). They probably represented cortical afferents. The other type of unitary EPSC, the 'H-type', seldom failed and was 13 times larger than the S-type. Spontaneous EPSCs with amplitudes similar to those of H-type EPSCs could be induced. H-type EPSCs were mediated by both non-NMDA and NMDA receptors. The two types of EPSCs could be evoked in the same neurons. The intensity of stimulation for H-type EPSCs was higher than that for S-type EPSCs. H-type EPSCs could be polysynaptically activated, suggesting the presence of glutamatergic interneurones in the striatum that generated H-type EPSCs. H-type EPSCs displayed particularly long-lasting paired-pulse depression, while that displayed by the S-type EPSCs was short. The paired-pulse depression of both EPSCs was Ca2+ dependent and involved presynaptic mechanisms. We have demonstrated that the medium spiny neurones of neonatal rats receive two different glutamatergic input systems having different amplitudes, origins and paired-pulse depression, reminiscent of cerebellar Purkinje cells. This suggests that the two types of EPSCs also play distinctive roles in striatal neuronal circuitry.     

Quantal encoding of information in a retinal ganglion cell

A retinal ganglion cell receives information about a white-noise stimulus as a flickering pattern of glutamate quanta. The ganglion cell reencodes this information as brief bursts of one to six spikes separated by quiescent periods. When the stimulus is repeated, the number of spikes in a burst is highly reproducible (variance < mean) and spike timing is precise to within 10 ms, leading to an estimate that each spike encodes about 2 bits. To understand how the ganglion cell reencodes information, we studied the quantal patterns by repeating a white-noise stimulus and recording excitatory currents from a voltage-clamped, brisk-sustained ganglion cell. Quanta occurred in synchronous bursts of 3 to 65; the resulting postsynaptic currents summed to form excitatory postsynaptic currents (EPSCs). The number of quanta in an EPSC was only moderately reproducible (variance = mean), quantal timing was precise to within 14 ms, and each quantum encoded 0.1-0.4 bit. In conclusion, compared to a spike, a quantum has similar temporal precision, but is less reproducible and encodes less information. Summing multiple quanta into discrete EPSCs improves the reproducibility of the overall quantal pattern and contributes to the reproducibility of the spike train.     

Synchronized afterdischarges in the hippocampus: Simulation studies of the cellular mechanism

Synchronized multiple bursts represent an epileptic neuronal behavior transitional between synchronized single bursts (interictal spikes) and self-sustained seizures. As described in the previous paper, synchronized multiple bursts occur in hippocampal slices treated with picrotoxin. Multiple bursts consist of an initial prolonged depolarizing burst followed by a rhythmical series of afterdischarges. Both the initial burst and the afterdischarges are synaptically elicited.Our previously described model of the interictal spike illustrates that the generation of a single synchronized burst requires a neuronal network possessing the following properties: intrinsic bursting capability of individual neurons, the presence of recurrent excitatory connections between principal neurons and the blockade of synaptic inhibition. The model demonstrates that the generation of single synchronized bursts involves the initial excitation of one or more neurons, and the subsequent sequential spread of excitation through a population of neurons via recurrent excitatory synapses.In the present study, we examined whether this same mechanism assumed in the previous model could also allow for the generation of synchronized afterdischarges in a population of neurons. We tested the effects of manipulating three network factors: synaptic strength, synaptic density and the refractoriness in the population members following a period of excitation. We discovered that the refractory period following prolonged excitation assumed in our previous model was insufficient to allow for afterdischarge generation. Once sufficient refractoriness was introduced, afterdischarges appeared in our network of neurons. In the present study, the required refractoriness was attributed to the properties of pyramidal cell axons. In principle, such refractoriness might be located elsewhere in the network. The possible contribution of axonal properties is emphasized because of the known intermittent conduction in other axons. Our present model also reproduced other experimental data. Thus, if the network was too small or if synaptic strength was too small, then only a single synchronized burst occurred. The basic assumptions of this model are both biologically plausible and experimentally testable.     

Dopamine modulates synaptic transmission in the nucleus of the solitary tract

10.1152/jn.00224.2002. Dopamine (DA) modulates the cardiorespiratory reflex by peripheral and central mechanisms. The aim of this study was to examine the role of DA in synaptic transmission of the nucleus tractus solitarius (NTS), the major integration site for cardiopulmonary reflexes. To examine DA's role, we used whole cell, voltage-clamp recordings in a rat horizontal brain stem slice. Solitary tract stimulation evoked excitatory postsynaptic currents (EPSCs) that were reduced to 70 +/- 5% of control by DA (100 microM). The reduction in EPSCs by DA was accompanied by a decrease in the paired pulse depression ratio with little or no change in input resistance or EPSC decay, suggesting a presynaptic mechanism. The D1-like agonist SKF 38393 Br (30 microM) did not alter EPSC amplitude, whereas the D2-like agonist, quinpirole HCl (30 microM), depressed EPSCs to 73 +/- 4% of control. The D2-like receptor antagonist, sulpiride (20 microM), abolished DA modulation of EPSCs. Most importantly, sulpiride alone increased EPSCs to 131 +/- 10% of control, suggesting a tonic D2-like modulation of synaptic transmission in the NTS. Examination of spontaneous EPSCs revealed DA reversibly decreased the frequency of events from 9.4 +/- 2.2 to 6.2 +/- 1.4 Hz. Sulpiride, however, did not alter spontaneous events. Immunohistochemistry of NTS slices demonstrated that D2 receptors colocalized with synaptophysin and substance P, confirming a presynaptic distribution. D2 receptors also localized to cultured petrosal neurons, the soma of presynaptic afferent fibers. In the petrosal neurons, D2 was found in cells that were TH-immunopositive, suggesting they were chemoreceptor afferent fibers. These results demonstrate that DA tonically modulates synaptic activity between afferent sensory fibers and secondary relay neurons in the NTS via a presynaptic D2-like mechanism.     

Requirement of dendritic calcium spikes for induction of spike-timing-dependent synaptic plasticity

Spike-timing-dependent synaptic plasticity (STDP) by definition requires the temporal association of pre- and postsynaptic action potentials (APs). Yet, in cortical pyramidal neurons pairing unitary EPSPs with single APs at low frequencies is ineffective at generating plasticity. Using recordings from synaptically coupled layer 5 pyramidal neurons, we show here that high-frequency (200 Hz) postsynaptic AP bursts, rather than single APs, are required for both long-term potentiation (LTP) induction and NMDA channel activation during EPSP–AP pairing at low frequencies. Furthermore, we find that AP bursts can lead to LTP induction and NMDA channel activation during EPSP–AP pairing at both positive and negative times. High-frequency AP bursts generated supralinear calcium signals in basal dendrites suggesting the generation of dendritic calcium spikes, as has been observed previously in apical dendrites during AP burst firing at frequencies greater than 100 Hz. Consistent with a role of these dendritic calcium spikes in LTP induction, pairing EPSPs with low frequency (50 Hz) AP bursts was ineffective in generating LTP. Furthermore, supralinear calcium signals in basal dendrites during AP bursts were blocked by low concentrations of the T- and R-type calcium channel antagonist nickel, which also blocked LTP and NMDA channel activation. These data suggest an important role of dendritic calcium spikes during AP bursts in determining both the efficacy and time window for STDP induction.     

Kindling induces transient NMDA receptor-mediated facilitation of high-frequency input in the rat dentate gyrus

To elucidate the gating mechanism of the epileptic dentate gyrus on seizure-like input, we investigated dentate gyrus field potentials and granule cell excitatory postsynaptic potentials (EPSPs) following high-frequency stimulation (10-100 Hz) of the lateral perforant path in an experimental model of temporal lobe epilepsy (i.e., kindled rats). Although control slices showed steady EPSP depression at frequencies greater than 20 Hz, slices taken from animals 48 h after the last seizure presented pronounced EPSP facilitation at 50 and 100 Hz, followed by steady depression. However, 28 days after kindling, the EPSP facilitation was no longer detectable. Using the specific N-methyl-D-aspartate (NMDA) and RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproponic acid (AMPA) receptor antagonists 2-amino-5-phosphonovaleric acid and SYM 2206, we examined the time course of alterations in glutamate receptor-dependent synaptic currents that parallel transient EPSP facilitation. Forty-eight hours after kindling, the fractional AMPA and NMDA receptor-mediated excitatory postsynaptic current (EPSC) components shifted dramatically in favor of the NMDA receptor-mediated response. Four weeks after kindling, however, AMPA and NMDA receptor-mediated EPSCs reverted to control-like values. Although the granule cells of the dentate gyrus contain mRNA-encoding kainate receptors, neither single nor repetitive perforant path stimuli evoked kainate receptor-mediated EPSCs in control or in kindled rats. The enhanced excitability of the kindled dentate gyrus 48 h after the last seizure, as well as the breakdown of its gating function, appear to result from transiently enhanced NMDA receptor activation that provides significantly slower EPSC kinetics than those observed in control slices and in slices from kindled animals with a 28-day seizure-free interval. Therefore, NMDA receptors seem to play a critical role in the acute throughput of seizure activity and in the induction of the kindled state but not in the persistence of enhanced seizure susceptibility.     

Synaptic transmission in pair recordings from CA3 pyramidal cells in organotypic culture.

We performed simultaneous whole cell recordings from pairs of monosynaptically coupled hippocampal CA3 pyramidal neurons in organotypic slices. Stimulation of an action potential in a presynaptic cell resulted in an AMPA-receptor-mediated excitatory postsynaptic current (EPSC) in the postsynaptic cell that averaged approximately 34 pA. The average size of EPSCs varied in amplitude over a 20-fold range across different pairs. Both paired-pulse facilitation and depression were observed in the synaptic current in response to two presynaptic action potentials delivered 50 ms apart, but the average usually was dominated by depression. In addition, the amplitude of the second EPSC depended on the amplitude of the first EPSC, indicating competition between successive events for a common resource that is not restored within the 50-ms interpulse interval. Variation in the synaptic strength among pairs could arise from a variety of sources. Our data from anatomic reconstruction, 1/CV2 analysis, paired-pulse analysis, and manipulations of calcium/magnesium ratio suggest that differences in quantal size and release probability do not appear to vary sufficiently to fully account for the observed differences in amplitude. Thus it seems most likely that the variability in EPSC amplitude between pairs arises primarily from differences in the number of functional synapses. Injections of the calcium chelator bis-(o-aminophenoxy)-N, N,N',N'-tetraacetic acid into the presynaptic neuron resulted in a rapid and nearly complete block of transmission, whereas injection of the slower-acting chelator EGTA resulted in a variable and partial block. In addition to demonstrating the feasibility of manipulating the intracellular presynaptic environment by injection into the presynaptic soma, these data, and the EGTA results in particular may suggest variability in the linkage between calcium entry sites an release sites in these synapses.     

Silent NMDA receptor-mediated synapses are developmentally regulated in the dorsal horn of the rat spinal cord

In vitro whole cell patch-clamp recording techniques were utilized to study silent pure-N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses in lamina II (substantia gelatinosa, SG) and lamina III of the spinal dorsal horn. To clarify whether these synapses are present in the adult and contribute to neuropathic pain, transverse lumbar spinal cord slices were prepared from neonatal, naive adult and adult sciatic nerve transected rats. In neonatal rats, pure-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) were elicited in SG neurons either by focal intraspinal stimulation (n = 15 of 20 neurons) or focal stimulation of the dorsal root (n = 2 of 7 neurons). In contrast, in slices from naive adult rats, no silent pure-NMDA EPSCs were recorded in SG neurons following focal intraspinal stimulation (n = 27), and only one pure-NMDA EPSC was observed in lamina III (n = 23). Furthermore, in rats with chronic sciatic nerve transection, pure-NMDA EPSCs were elicited by focal intraspinal stimulation in only 2 of 45 SG neurons. Although a large increase in Abeta fiber evoked mixed alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA receptor-mediated synapses was detected after sciatic nerve injury, Abeta fiber-mediated pure-NMDA EPSCs were not evoked in SG neurons by dorsal root stimulation. Pure-NMDA receptor-mediated EPSCs are therefore a transient, developmentally regulated phenomenon, and, although they may have a role in synaptic refinement in the immature dorsal horn, they are unlikely to be involved in receptive field plasticity in the adult.     

Frequency-dependent shift from paired-pulse facilitation to paired-pulse depression at unitary CA3-CA3 synapses in the rat hippocampus

Paired recordings between CA3 interconnected pyramidal neurons were used to study the properties of short-term depression occurring in these synapses under different frequencies of presynaptic firing (   n = 22  ). In stationary conditions (0.05-0.067 Hz) pairs of presynaptic action potentials (50 ms apart) evoked EPSCs whose amplitude fluctuated from trial to trial with occasional response failures. In 15/20 cells, paired-pulse ratio (PPR) was characterized by facilitation (PPF) while in the remaining five by depression (PPD). Increasing stimulation frequency from 0.05-0.067 Hz to 0.1-1 Hz induced low frequency depression (LFD) of EPSC amplitude with a gradual increase in the failure rate. Overall, 9/12 cells at 1 Hz became almost 'silent'. In six cells in which the firing rate was sequentially shifted from 0.05 to 0.1 and 1 Hz, changes in synaptic efficacy were so strong that PPR shifted from PPF to PPD. The time course of depression of EPSC1 could be fitted with single exponentials with time constants of 98 and 36 s at 0.1 and 1 Hz, respectively. In line with the inversion of PPR at 1 Hz, the time course of depression of EPSC2 was faster than EPSC1 (7 s). Recovery from depression could be obtained by lowering the frequency of stimulation to 0.025 Hz. These results could be explained by a model that takes into account two distinct release processes, one dependent on the residual calcium and the other on the size of the readily releasable pool of vesicles.