Using ephaptic coupling to estimate the synaptic cleft resistivity of the calyx of Held synapse

At synapses, the pre- and postsynaptic cells get so close that currents entering the cleft do not flow exclusively along its conductance, gcl. A prominent example is found in the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB), where the presynaptic action potential can be recorded in the postsynaptic cell in the form of a prespike. Here, we developed a theoretical framework for ephaptic coupling via the synaptic cleft, and we tested its predictions using the MNTB prespike recorded in voltage-clamp. The shape of the prespike is predicted to resemble either the first or the second derivative of the inverted presynaptic action potential if cleft currents dissipate either mostly capacitively or resistively, respectively. We found that the resistive dissipation scenario provided a better description of the prespike shape. Its size is predicted to scale with the fourth power of the radius of the synapse, explaining why intracellularly recorded prespikes are uncommon in the central nervous system. We show that presynaptic calcium currents also contribute to the prespike shape. This calcium prespike resembled the first derivative of the inverted calcium current, again as predicted by the resistive dissipation scenario. Using this calcium prespike, we obtained an estimate for gcl of ~1 μS. We demonstrate that, for a circular synapse geometry, such as in conventional boutons or the immature calyx of Held, gcl is scale-invariant and only defined by extracellular resistivity, which was ~75 Ωcm, and by cleft height. During development the calyx of Held develops fenestrations. We show that these fenestrations effectively minimize the cleft potentials generated by the adult action potential, which might otherwise interfere with calcium channel opening. We thus provide a quantitative account of the dissipation of currents by the synaptic cleft, which can be readily extrapolated to conventional, bouton-like synapses.

Using ephaptic coupling to estimate the synaptic cleft resistivity of the calyx of Held synapse.

At synapses, the pre- and postsynaptic cell get so close that currents entering the cleft do not flow exclusively along its conductance, gcl. A prominent example is found in the calyx of Held synapse in the medial nucleus of the trapezoid body, where the presynaptic action potential can be recorded in the postsynaptic cell in the form of a prespike. Here, we developed a theoretical framework for ephaptic coupling via the synaptic cleft. We found that the capacitive component of the prespike recorded in voltage clamp is closely approximated by the second time derivative of the presynaptic action potential. Its size scales with the fourth power of the radius of the synapse, explaining why intracellularly recorded prespikes are uncommon in the CNS. We show that presynaptic calcium currents can contribute to the prespike and that their contribution is closely approximated by the scaled first derivative of these currents. We confirmed these predictions in juvenile rat brainstem slices, and used the presynaptic calcium currents to obtain an estimate for gcl of ~1 μS. We demonstrate that for a typical synapse geometry, gcl is scale-invariant and only defined by extracellular resistivity, which was ~75 Ωcm, and by cleft height. During development the calyx of Held develops fenestrations. These fenestrations effectively minimize the cleft potentials generated by the adult action potential, which would otherwise interfere with calcium channel opening. We thus provide a quantitative account of the dissipation of currents by the synaptic cleft, which can be readily extrapolated to conventional, bouton-like synapses.

Large, stable spikes exhibit differential broadening in excitatory and inhibitory neocortical boutons

SUMMARYPresynaptic action potential spikes control neurotransmitter release and thus interneuronal communication. However, the properties and the dynamics of presynaptic spikes in the neocortex remain enigmatic because boutons in the neocortex are small and direct patch-clamp recordings have not been performed. Here we report direct recordings from boutons of neocortical pyramidal neurons and interneurons. Our data reveal rapid and large presynaptic action potentials in layer 5 neurons and fast-spiking interneurons reliably propagating into axon collaterals. For in-depth analyses we validate boutons of mature cultured neurons as models for excitatory neocortical boutons, demonstrating that the presynaptic spike amplitude was unaffected by potassium channels, homeostatic long-term plasticity, and high-frequency firing. In contrast to the stable amplitude, presynaptic spikes profoundly broadened for example during high-frequency firing in layer 5 pyramidal neurons but not in fast-spiking interneurons. Thus, our data demonstrate large presynaptic spikes and fundamental differences between excitatory and inhibitory boutons in the neocortex.

The Potassium Channel Subunit Kvβ1 is Required for Synaptic Facilitation

Analysis of the presynaptic action potential’s (APsyn) role in synaptic facilitation in hippocampal pyramidal neurons has been difficult due to size limitations of axons. We overcame these size barriers by combining high resolution optical recordings of membrane potential, exocytosis and Ca2+ in cultured hippocampal neurons. These recordings revealed a critical and selective role for Kv1 channel inactivation in synaptic facilitation of excitatory hippocampal neurons. Presynaptic Kv1 channel inactivation was mediated by the Kvβ1 subunit, and had a surprisingly rapid onset that was readily apparent even in brief physiological stimulation paradigms including paired-pulse stimulation. Genetic depletion of Kvβ1 blocked all broadening of the APsyn during high frequency stimulation and eliminated synaptic facilitation without altering the initial probability of vesicle release. Thus using all quantitative optical measurements of presynaptic physiology, we reveal a critical role for presynaptic Kv channels in synaptic facilitation at small presynaptic terminals of the hippocampal neurons upstream of exocytic machinery.SignificanceNerve terminals generally engage in two opposite and essential forms of synaptic plasticity (facilitation or depression) during high frequency stimulation that play critical roles in learning and memory. Measurements of the electrical impulses (action potentials) underlying these two forms of plasticity has been difficult in small nerve terminals due to their size. In this study we deployed a combination of optical measurements of vesicle fusion and membrane voltage to overcome this previous size barrier. Here, we found a unique molecular composition of Kv1 channel β-subunits that causes broadening of the presynaptic action essential to synaptic facilitation. Disruption of the Kvβ1 inactivation mechanism switches excitatory nerve terminals into a depressive state, without any disruption to initial probability of vesicle fusion.

KCNQ channels enable reliable presynaptic spiking and synaptic transmission at high-frequency

SUMMARYThe presynaptic action potential (AP) results in calcium influx which triggers neurotransmitter release. For this reason, the AP waveform is crucial in determining the timing and strength of synaptic transmission. The calyx of Held nerve terminals of rat show minimum changes in AP waveform during high-frequency AP firing. We found that the stability of the calyceal AP waveform requires KCNQ K+ channel activated during high-frequency spiking activity. High-frequency presynaptic spikes gradually led to accumulation of KCNQ channels in open states which kept interspike membrane potential sufficiently negative to maintain Na+ channel availability. Accordingly, blocking KCNQ channels during stimulus trains led to inactivation of presynaptic Na+, and to a lesser extent KV1 channels, thereby reducing the AP height and broadening AP duration. Thus, while KCNQ channels are generally thought to prevent hyperactivity of neurons, we find that in axon terminals these channels function to facilitate high-frequency firing needed for sensory coding.HIGHLIGHTSKCNQ channels are activated during high-frequency firingThe activity of KCNQ channels helps the recovery of Na+ and KV1 channels from inactivation and maintains action potential waveformReliable presynaptic action potential waveform preserves stable Ca2+ influx and reliable synaptic signaling

Glycolysis selectively shapes the presynaptic action potential waveform

Mitochondria are major suppliers of cellular energy in neurons; however, utilization of energy from glycolysis vs. mitochondrial oxidative phosphorylation (OxPhos) in the presynaptic compartment during neurotransmission is largely unknown. Using presynaptic and postsynaptic recordings from the mouse calyx of Held, we examined the effect of acute selective pharmacological inhibition of glycolysis or mitochondrial OxPhos on multiple mechanisms regulating presynaptic function. Inhibition of glycolysis via glucose depletion and iodoacetic acid (1 mM) treatment, but not mitochondrial OxPhos, rapidly altered transmission, resulting in highly variable, oscillating responses. At reduced temperature, this same treatment attenuated synaptic transmission because of a smaller and broader presynaptic action potential (AP) waveform. We show via experimental manipulation and ion channel modeling that the altered AP waveform results in smaller Ca2+ influx, resulting in attenuated excitatory postsynaptic currents (EPSCs). In contrast, inhibition of mitochondria-derived ATP production via extracellular pyruvate depletion and bath-applied oligomycin (1 μM) had no significant effect on Ca2+ influx and did not alter the AP waveform within the same time frame (up to 30 min), and the resultant EPSC remained unaffected. Glycolysis, but not mitochondrial OxPhos, is thus required to maintain basal synaptic transmission at the presynaptic terminal. We propose that glycolytic enzymes are closely apposed to ATP-dependent ion pumps on the presynaptic membrane. Our results indicate a novel mechanism for the effect of hypoglycemia on neurotransmission. Attenuated transmission likely results from a single presynaptic mechanism at reduced temperature: a slower, smaller AP, before and independent of any effect on synaptic vesicle release or receptor activity.