Synaptotagmin 7 Sculpts Short-Term Plasticity at a High Probability Synapse.

Synapses with high release probability (Pr ) tend to exhibit short-term synaptic depression. According to the prevailing model, this reflects the temporary depletion of release-ready vesicles after an initial action potential (AP). At the high-Pr layer 4 to layer 2/3 (L4-L2/3) synapse in rodent somatosensory cortex, short-term plasticity appears to contradict the depletion model: depression is absent at interstimulus intervals (ISIs) <50 ms and develops to a maximum at ∼200 ms. To understand the mechanism(s) underlying the biphasic time course of short-term plasticity at this synapse, we used whole-cell electrophysiology and two-photon calcium imaging in acute slices from male and female juvenile mice. We tested several candidate mechanisms including neuromodulation, postsynaptic receptor desensitization, and use-dependent changes in presynaptic AP-evoked calcium. We found that, at single L4-L2/3 synapses, Pr varies as a function of ISI, giving rise to the distinctive short-term plasticity time course. Furthermore, the higher-than-expected Pr at short ISIs depends on expression of synaptotagmin 7 (Syt7). Our results show that two distinct vesicle release processes summate to give rise to short-term plasticity at this synapse: (1) a basal, high-Pr release mechanism that undergoes rapid depression and recovers slowly (τ = ∼3 s) and (2) a Syt7-dependent mechanism that leads to a transient increase in Pr (τ = ∼100 ms) after the initial AP. We thus reveal how these synapses can maintain a very high probability of neurotransmission for multiple APs within a short time frame. Key words : depression; facilitation; short-term plasticity; synaptotagmin 7.

Synaptic NMDA receptor activity at resting membrane potentials.

NMDA receptors (NMDARs) are crucial for glutamatergic synaptic signaling in the mammalian central nervous system. When activated by glutamate and glycine/D-serine, the NMDAR ion channel can open, but current flux is further regulated by voltage-dependent block conferred by extracellular Mg2+ ions. The unique biophysical property of ligand- and voltage-dependence positions NMDARs as synaptic coincidence detectors, controlling a major source of synaptic Ca2+ influx. We measured synaptic currents in layer 2/3 neurons after stimulation in layer 4 of somatosensory cortex and found measurable NMDAR currents at all voltages tested. This NMDAR current did not require concurrent AMPAR depolarization. In physiological ionic conditions, the NMDAR current response at negative potentials was enhanced relative to ionic conditions typically used in slice experiments. NMDAR activity was also seen in synaptic recordings from hippocampal CA1 neurons, indicating a general property of NMDAR signaling. Using a fluorescent Ca2+ indicator, we measured responses to stimulation in layer 4 at individual synaptic sites, and Ca2+ influx could be detected even with AMPARs blocked. In current clamp recordings, we found that resting membrane potential was hyperpolarized by ∼7 mV and AP firing threshold depolarized by ∼4 mV in traditional compared to physiological ionic concentrations, and that NMDARs contribute to EPSPs at resting membrane potentials. These measurements demonstrate that, even in the presence of extracellular Mg2+ and absence of postsynaptic depolarization, NMDARs contribute to synaptic currents and Ca2+ influx.

Incomplete inactivation and rapid recovery of voltage-dependent sodium channels during high-frequency firing in cerebellar Purkinje neurons.

Purkinje neurons can spike very rapidly for sustained periods. We examined the cycle of sodium channel gating during high-frequency firing of Purkinje neurons, focusing on the kinetics of sodium channel inactivation and recovery during and after spikes. To analyze sodium channel availability during spiking, we recorded the firing patterns of acutely dissociated Purkinje neurons in current clamp and used these records as command voltages in voltage-clamp experiments in the same cell, adding step depolarizations at various points to assay availability. Sodium channel availability decreased abruptly during the spike, as expected, but never reached zero. During spontaneous firing (∼ 40 Hz at 37°C), availability decreased from nearly 90% before the spike to about 10-20% after the spike. With fast steady firing stimulated by current injection (∼ 300 Hz at 37°C), the availability decreased from about 60% between spikes to roughly 15-20% after the spike. Thus even at the fastest firing rates, sodium channel inactivation is incomplete after a spike, leaving a substantial fraction of sodium channels immediately available for activation. Also, inactivation recovered quickly during the early interspike interval (time constant ∼ 1 ms at 37°C), but developed slowly during the depolarization of the late interspike interval, ensuring high availability until spike threshold. These features of sodium channel gating, especially the availability remaining after the spike, reduce the refractory period and facilitate rapid repetitive firing.

Incomplete block of NMDA receptors by intracellular MK-801.

NMDA receptors (NMDARs) are essential components in glutamatergic synaptic signaling. The NMDAR antagonist MK-801 has been a valuable pharmacological tool in evaluating NMDAR function because it binds with high affinity to the NMDAR ion channel pore and is non-competitive with ligand binding. MK-801 has also been used to selectively inhibit NMDAR current in only the cell being recorded by including the drug in the intracellular recording solution. Here, we report that intracellular MK-801 (iMK-801) only partially inhibits synaptic NMDAR currents at +40 mV at both cortical layer 4 to layer 2/3 and hippocampal Schaffer collateral to CA1 synapses. Furthermore, iMK-801 incompletely inhibits heterologously expressed NMDAR currents at -60 mV, consistent with a model of iMK-801 having a very slow binding rate and consequently ∼30,000 times lower affinity than MK-801 applied to the extracellular side of the receptor. While iMK-801 can be used as a qualitative tool to study reduced postsynaptic NMDAR function, it cannot be assumed to completely block NMDARs at concentrations typically used in experiments.

Postsynaptic, not presynaptic NMDA receptors are required for spike-timing-dependent LTD induction.

Long-term depression (LTD) between cortical layer 4 spiny stellate cells and layer 2/3 pyramidal cells requires the activation of NMDA receptors (NMDARs). In young rodents, this form of LTD has been repeatedly reported to require presynaptic NMDARs for its induction. Here we show that at this synapse in the somatosensory cortex of 2- to 3-week-old rats and mice, postsynaptic, not presynaptic NMDARs are required for LTD induction. First, we find no evidence for functional NMDARs in L4 neuron axons using two-photon laser scanning microscopy and two-photon glutamate uncaging. Second, we find that genetic deletion of postsynaptic, but not presynaptic NMDARs prevents LTD induction. Finally, the pharmacology of the NMDAR requirement is consistent with a nonionic signaling mechanism.

Transient sodium current at subthreshold voltages: activation by EPSP waveforms.

Tetrodotoxin (TTX)-sensitive sodium channels carry large transient currents during action potentials and also "persistent" sodium current, a noninactivating TTX-sensitive current present at subthreshold voltages. We examined gating of subthreshold sodium current in dissociated cerebellar Purkinje neurons and hippocampal CA1 neurons, studied at 37°C with near-physiological ionic conditions. Unexpectedly, in both cell types small voltage steps at subthreshold voltages activated a substantial component of transient sodium current as well as persistent current. Subthreshold EPSP-like waveforms also activated a large component of transient sodium current, but IPSP-like waveforms engaged primarily persistent sodium current with only a small additional transient component. Activation of transient as well as persistent sodium current at subthreshold voltages produces amplification of EPSPs that is sensitive to the rate of depolarization and can help account for the dependence of spike threshold on depolarization rate, as previously observed in vivo.

Cleavage of TRPM7 releases the kinase domain from the ion channel and regulates its participation in Fas-induced apoptosis.

Transient receptor potential melastatin-like 7 (TRPM7) is a channel protein that also contains a regulatory serine-threonine kinase domain. Here, we find that Trpm7-/- T cells are deficient in Fas-receptor-induced apoptosis and that TRPM7 channel activity participates in the apoptotic process and is regulated by caspase-dependent cleavage. This function of TRPM7 is dependent on its function as a channel, but not as a kinase. TRPM7 is cleaved by caspases at D1510, disassociating the carboxy-terminal kinase domain from the pore without disrupting the phosphotransferase activity of the released kinase but substantially increasing TRPM7 ion channel activity. Furthermore, we show that TRPM7 regulates endocytic compartmentalization of the Fas receptor after receptor stimulation, an important process for apoptotic signaling through Fas receptors. These findings raise the possibility that other members of the TRP channel superfamily are also regulated by caspase-mediated cleavage, with wide-ranging implications for cell death and differentiation.

Sodium entry during action potentials of mammalian neurons: incomplete inactivation and reduced metabolic efficiency in fast-spiking neurons.

We measured the time course of sodium entry during action potentials of mouse central neurons at 37 degrees C to examine how efficiently sodium entry is coupled to depolarization. In cortical pyramidal neurons, sodium entry was nearly completely confined to the rising phase of the spike: only approximately 25% more sodium enters than the theoretical minimum necessary for spike depolarization. However, in fast-spiking GABAergic neurons (cerebellar Purkinje cells and cortical interneurons), twice as much sodium enters as the theoretical minimum. The extra entry occurs because sodium channel inactivation is incomplete during the falling phase of the spike. The efficiency of sodium entry in different cell types is primarily a function of action potential shape and not cell-type-specific differences in sodium channel kinetics. The narrow spikes of fast-spiking GABAergic neurons result in incomplete inactivation of sodium channels; this reduces metabolic efficiency but likely enhances the ability to fire spikes at high frequency.