The sinoatrial node (SAN), the primary cardiac pacemaker, consists of a head domain and a junction/tail domain that exhibit different functional properties. However, the underlying molecular mechanism defining these two pacemaker domains remains elusive. Nkx2-5 is a key transcription factor essential for the formation of the working myocardium, but it was generally thought to be detrimental to SAN development. However, Nkx2-5 is expressed in the developing SAN junction, suggesting a role for Nkx2-5 in SAN junction development and function. In this study, we present unambiguous evidence that SAN junction cells exhibit unique action potential configurations intermediate to those manifested by the SAN head and the surrounding atrial cells, suggesting a specific role for the junction cells in impulse generation and in SAN-atrial exit conduction. Singlecell RNA-seq analyses support this concept. Although Nkx2-5 inactivation in the SAN junction did not cause a malformed SAN at birth, the mutant mice manifested sinus node dysfunction. Thus, Nkx2-5 defines a population of pacemaker cells in the transitional zone. Despite Nkx2-5 being dispensable for SAN morphogenesis during embryogenesis, its deletion hampers atrial activation by the pacemaker.

Synaptic vesicles need to be recycled and refilled rapidly to maintain high-frequency synaptic transmission. However, little is known about the control of neurotransmitter transport into synaptic vesicles, which determines the contents of synaptic vesicles and the strength of synaptic transmission. Here, we report that Na 1 substantially accumulated in the calyx of Held terminals of juvenile mice of either sex during high-frequency spiking. The activity-induced elevation of cytosolic Na 1 activated vesicular Na 1 /H 1 exchanger, facilitated glutamate loading into synaptic vesicles, and increased quantal size of asynchronous released vesicles but did not affect the vesicle pool size or release probability. Consequently, presynaptic Na 1 increased the EPSCs and was required to maintain the reliable high-frequency signal transmission from the presynaptic calyces to the postsynaptic medial nucleus of the trapezoid body (MNTB) neurons. Blocking Na 1 /H 1 exchange activity decreased the postsynaptic current and caused failures in postsynaptic firing. Therefore, during high-frequency synaptic transmission, when large amounts of glutamate are released, Na 1 accumulated in the terminals, activated vesicular Na 1 /H 1 exchanger, and regulated glutamate loading as a function of the level of vesicle release.

One emerging concept in neuroscience states that synaptic vesicles and the molecular machinery underlying spontaneous transmitter release are different from those underlying action potential-driven synchronized transmitter release. Differential neuromodulation of these two distinct release modes by metabotropic glutamate receptors (mGluRs) constitutes critical supporting evidence. However, the mechanisms underlying such a differential modulation are not understood. Here, we investigated the mechanisms of the modulation by group I mGluRs (mGluR Is) on spontaneous glutamate release in the medial nucleus of the trapezoid body (MNTB), an auditory brainstem nucleus critically involved in sound localization. Whole-cell patch recordings from brainstem slices of mice of both sexes were performed. Activation of mGluR I by 3,5-dihydroxyphenylglycine (3,5-DHPG; 200 lM) produced an inward current at 260 mV and increased spontaneous glutamate release in MNTB neurons. Pharmacological evidence indicated involvement of both mGluR1 and mGluR5, which was further supported for mGluR5 by immunolabeling results. The modulation was eliminated by blocking Na V channels (tetrodotoxin, 1 lM), persistent Na 1 current (I NaP ; riluzole, 10 lM), or Ca V channels (CdCl 2 , 100 lM). Presynaptic calyx recordings revealed that 3,5-DHPG shifted the activation of I NaP to more hyperpolarized voltages and increased I NaP at resting membrane potential. Our data indicate that mGluR I enhances spontaneous glutamate release via regulation of I NaP and subsequent Ca 21 -dependent processes under resting condition.

The presynaptic action potential (AP) is required to drive calcium influx into nerve terminals, resulting in neurotransmitter release. Accordingly, the AP waveform is crucial in determining the timing and strength of synaptic transmission. The calyx of Held nerve terminals of rats of either sex showed minimum changes in AP waveform during high-frequency AP firing. We found that the stability of the calyceal AP waveform requires KCNQ (KV7) K+channel activation 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. Blocking KCNQ channels during stimulus trains led to inactivation of presynaptic Na+, and to a lesser extent KV1 channels, thereby reducing the AP amplitude and broadening AP duration. Moreover, blocking KCNQ channels disrupted the stable calcium influx and glutamate release required for reliable synaptic transmission at high frequency. Thus, while KCNQ channels are generally thought to prevent hyperactivity of neurons, we find that in axon terminals these channels function to facilitate reliable high-frequency synaptic signaling needed for sensory information processing.SIGNIFICANCE STATEMENTThe presynaptic spike results in calcium influx required for neurotransmitter release. For this reason, the spike waveform is crucial in determining the timing and strength of synaptic transmission. Auditory information is encoded by spikes phase locked to sound frequency at high rates. The calyx of Held nerve terminals in the auditory brainstem show minimum changes in spike waveform during high-frequency spike firing. We found that activation of KCNQ K+channel builds up during high-frequency firing and its activation helps to maintain a stable spike waveform and reliable synaptic transmission. While KCNQ channels are generally thought to prevent hyperexcitability of neurons, we find that in axon terminals these channels function to facilitate high-frequency synaptic signaling during auditory information processing.

Retrieval of synaptic vesicles via endocytosis is essential for maintaining sustained synaptic transmission, especially for neurons that fire action potentials at high frequencies. However, how neuronal activity regulates synaptic vesicle recycling is largely unknown. Here we report that Na 1 substantially accumulated in the mouse calyx of Held terminals of either sex during repetitive high-frequency spiking. Elevated presynaptic Na 1 accelerated both slow and rapid forms of endocytosis and facilitated endocytosis overshoot, but did not affect the readily releasable pool size, Ca 21 influx, or exocytosis. To examine whether this facilitation of endocytosis is related to the Na 1 -dependent vesicular content change, we dialyzed glutamate into the presynaptic cytosol or blocked the vesicular glutamate uptake with bafilomycin and found that the rate of endocytosis was not affected by regulating the vesicular glutamate content. Endocytosis is critically dependent on intracellular Ca 21 , and the activity of Na 1 /Ca 21 exchanger (NCX) may be altered when the Na 1 gradient is changed. However, neither NCX inhibitor nor change of extracellular Na 1 concentration affected the endocytosis rate. Moreover, two-photon Ca 21 imaging showed that presynaptic Na 1 did not affect the action potential-evoked intracellular Ca 21 transient and decay. Therefore, we revealed a novel mechanism of cytosolic Na 1 in accelerating vesicle endocytosis. During high-frequency synaptic transmission, when large numbers of synaptic vesicles were fused, the rapid buildup of presynaptic cytosolic Na 1 promoted vesicle recycling and sustained synaptic transmission.