The Na+ leak channel NALCN controls spontaneous activity and mediates synaptic modulation by α2-adrenergic receptors in auditory neurons.

Cartwheel interneurons of the dorsal cochlear nucleus (DCN) potently suppress multisensory signals that converge with primary auditory afferent input, and thus regulate auditory processing. Noradrenergic fibers from locus coeruleus project to the DCN, and α2-adrenergic receptors inhibit spontaneous spike activity but simultaneously enhance synaptic strength in cartwheel cells, a dual effect leading to enhanced signal-to-noise for inhibition. However, the ionic mechanism of this striking modulation is unknown. We generated a glycinergic neuron-specific knockout of the Na+ leak channel NALCN in mice and found that its presence was required for spontaneous firing in cartwheel cells. Activation of α2-adrenergic receptors inhibited both NALCN and spike generation, and this modulation was absent in the NALCN knockout. Moreover, α2-dependent enhancement of synaptic strength was also absent in the knockout. GABAB receptors mediated inhibition through NALCN as well, acting on the same population of channels as α2 receptors, suggesting close apposition of both receptor subtypes with NALCN. Thus, multiple neuromodulatory systems determine the impact of synaptic inhibition by suppressing the excitatory leak channel, NALCN.

Calcium-Sensitive Subthreshold Oscillations and Electrical Coupling in Principal Cells of Mouse Dorsal Cochlear Nucleus.

In higher sensory brain regions, slow oscillations (0.5-5 Hz) associated with quiet wakefulness and attention modulate multisensory integration, predictive coding, and perception. Although often assumed to originate via thalamocortical mechanisms, the extent to which subcortical sensory pathways are independently capable of slow oscillatory activity is unclear. We find that in the first station for auditory processing, the cochlear nucleus, fusiform cells from juvenile mice (of either sex) generate robust 1-2 Hz oscillations in membrane potential and exhibit electrical resonance. Such oscillations were absent prior to the onset of hearing, intrinsically generated by hyperpolarization-activated cyclic nucleotide-gated (HCN) and persistent Na+ conductances (NaP) interacting with passive membrane properties, and reflected the intrinsic resonance properties of fusiform cells. Cx36-containing gap junctions facilitated oscillation strength and promoted pairwise synchrony of oscillations between neighboring neurons. The strength of oscillations were strikingly sensitive to external Ca2+, disappearing at concentrations >1.7 mM, due in part to the shunting effect of small-conductance calcium-activated potassium (SK) channels. This effect explains their apparent absence in previous in vitro studies of cochlear nucleus which routinely employed high-Ca2+ extracellular solution. In contrast, oscillations were amplified in reduced Ca2+ solutions, due to relief of suppression by Ca2+ of Na+ channel gating. Our results thus reveal mechanisms for synchronous oscillatory activity in auditory brainstem, suggesting that slow oscillations, and by extension their perceptual effects, may originate at the earliest stages of sensory processing.

The Na+ leak channel NALCN controls spontaneous activity and mediates synaptic modulation by α2-adrenergic receptors in auditory neurons.

Cartwheel interneurons of the dorsal cochlear nucleus (DCN) potently suppress multisensory signals that converge with primary auditory afferent input, and thus regulate auditory processing. Noradrenergic fibers from locus coeruleus project to the DCN, and α2-adrenergic receptors inhibit spontaneous spike activity but simultaneously enhance synaptic strength in cartwheel cells, a dual effect leading to enhanced signal-to-noise for inhibition. However, the ionic mechanism of this striking modulation is unknown. We generated a glycinergic neuron-specific knockout of the Na+ leak channel NALCN, and found that its presence was required for spontaneous firing in cartwheel cells. Activation of α2-adrenergic receptors inhibited both NALCN and spike generation, and this modulation was absent in the NALCN knockout. Moreover, α2-dependent enhancement of synaptic strength was also absent in the knockout. GABAB receptors mediated inhibition through NALCN as well, acting on the same population of channels as α2 receptors, suggesting close apposition of both receptor subtypes with NALCN. Thus, multiple neuromodulatory systems determine the impact of synaptic inhibition by suppressing the excitatory leak channel, NALCN.

Regulation of Neurotransmitter Release by K+ Channels.

K+ channels play potent roles in the process of neurotransmitter release by influencing the action potential waveform and modulating neuronal excitability and release probability. These diverse effects of K+ channel activation are ensured by the wide variety of K+ channel genes and their differential expression in different cell types. Accordingly, a variety of K+ channels have been implicated in regulating neurotransmitter release, including the Ca2+- and voltage-gated K+ channel Slo1 (also known as BK channel), voltage-gated K+ channels of the Kv3 (Shaw-type), Kv1 (Shaker-type), and Kv7 (KCNQ) families, G-protein-gated inwardly rectifying K+ (GIRK) channels, and SLO-2 (a Ca2+-. Cl-, and voltage-gated K+ channel in C. elegans). These channels vary in their expression patterns, subcellular localization, and biophysical properties. Their roles in neurotransmitter release may also vary depending on the synapse and physiological or experimental conditions. This chapter summarizes key findings about the roles of K+ channels in regulating neurotransmitter release.

Comprehensive analysis of cellular specializations that initiate parallel auditory processing pathways in mice.

The cochlear nuclear complex (CN) is the starting point for all central auditory processing and comprises a suite of neuronal cell types that are highly specialized for neural coding of acoustic signals. To examine how their striking functional specializations are determined at the molecular level, we performed single-nucleus RNA sequencing of the mouse CN to molecularly define all constituent cell types and related them to morphologically- and electrophysiologically-defined neurons using Patch-seq. We reveal an expanded set of molecular cell types encompassing all previously described major types and discover new subtypes both in terms of topographic and cell-physiologic properties. Our results define a complete cell-type taxonomy in CN that reconciles anatomical position, morphological, physiological, and molecular criteria. This high-resolution account of cellular heterogeneity and specializations from the molecular to the circuit level illustrates molecular underpinnings of functional specializations and enables genetic dissection of auditory processing and hearing disorders with unprecedented specificity.

Electrical signaling in cochlear efferents is driven by an intrinsic neuronal oscillator.

Efferent neurons are believed to play essential roles in maintaining auditory function. The lateral olivocochlear (LOC) neurons-which project from the brainstem to the inner ear, where they release multiple transmitters including peptides, catecholamines, and acetylcholine-are the most numerous yet least understood elements of efferent control of the cochlea. Using in vitro calcium imaging and patch-clamp recordings, we found that LOC neurons in juvenile and young adult mice exhibited extremely slow waves of activity (∼0.1 Hz). These seconds-long bursts of Na+ spikes were driven by an intrinsic oscillator dependent on L-type Ca2+ channels and were not observed in prehearing mice, suggesting an age-dependent mechanism underlying the intrinsic oscillator. Using optogenetic approaches, we identified both ascending (T-stellate cells of the cochlear nucleus) and descending (auditory cortex) sources of synaptic excitation, as well as the synaptic receptors used for such excitation. Additionally, we identified potent inhibition originating in the glycinergic medial nucleus of trapezoid body (MNTB). Conductance-clamp experiments revealed an unusual mechanism of electrical signaling in LOC neurons, in which synaptic excitation and inhibition served to switch on and off the intrinsically generated spike burst mechanism, allowing for prolonged periods of activity or silence controlled by brief synaptic events. Protracted bursts of action potentials may be essential for effective exocytosis of the diverse transmitters released by LOC fibers in the cochlea.

Vestibular Organ Dissection and Whole-Mount Immunolabeling in Mouse.

The vestibular sensory apparatus contained in the inner ear is a marvelous evolutionary adaptation for sensing movement in 3 dimensions and is essential for an animal's sense of orientation in space, head movement, and balance. Damage to these systems through injury or disease can lead to vertigo, Meniere's disease, and other disorders that are profoundly debilitating. One challenge in studying vestibular organs is their location within the boney inner ear and their small size, especially in mice, which have become an advantageous mammalian model. This protocol describes the dissection procedure of the five vestibular organs from the inner ear of adult mice, followed by immunohistochemical labeling of a whole mount preparation using antibodies to label endogenous proteins such as calretinin to label Type I hair cells or to amplify genetically expressed fluorescent proteins for confocal microscopic imaging. Using typical lab equipment and reagents, a patient technician, student, or postdoc can learn to dissect and immunolabel mouse vestibular organs to investigate their structure in health and disease.

Central circuitry and function of the cochlear efferent systems.

The cochlear efferent system comprises multiple populations of brainstem neurons whose axons project to the cochlea, and whose responses to acoustic stimuli lead to regulation of auditory sensitivity. The major groups of efferent neurons are found in the superior olivary complex and are likely activated by neurons of the cochlear nucleus, thus forming a simple reflex pathway back to the cochlea. The peripheral actions of only one of these efferent cell types has been well described. Moreover, the efferent neurons are not well understood at the cellular- and circuit-levels. For example, ample demonstration of descending projections to efferent neurons raises the question of whether these additional inputs constitute a mechanism for modulation of relay function or instead play a more prominent role in driving the efferent response. Related to this is the question of synaptic plasticity at these synapses, which has the potential to differentially scale the degree of efferent activation across time, depending on the input pathway. This review will explore central nervous system aspects of the efferent system, the physiological properties of the neurons, their synaptic inputs, their modulation, and the effects of efferent axon collaterals within the brainstem.

KCNQ Channels Enable Reliable Presynaptic Spiking and Synaptic Transmission at High Frequency.

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 STATEMENT The 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.

Descending Axonal Projections from the Inferior Colliculus Target Nearly All Excitatory and Inhibitory Cell Types of the Dorsal Cochlear Nucleus.

The dorsal cochlear nucleus (DCN) integrates auditory nerve input with nonauditory sensory signals and is proposed to function in sound source localization and suppression of self-generated sounds. The DCN also integrates activity from descending auditory pathways, including a particularly large feedback projection from the inferior colliculus (IC), the main ascending target of the DCN. Understanding how these descending feedback signals are integrated into the DCN circuit and what role they play in hearing requires knowing the targeted DCN cell types and their postsynaptic responses. In order to explore these questions, neurons in the DCN that received descending synaptic input from the IC were labeled with a trans-synaptic viral approach in male and female mice, which allowed them to be targeted for whole-cell recording in acute brain slices. We tested their synaptic responses to optogenetic activation of the descending IC projection. Every cell type in the granule cell domain received monosynaptic, glutamatergic input from the IC, indicating that this region, considered an integrator of nonauditory sensory inputs, processes auditory input as well and may have complex and underappreciated roles in hearing. Additionally, we found that DCN cell types outside the granule cell regions also receive descending IC signals, including the principal projection neurons, as well as the neurons that inhibit them, leading to a circuit that may sharpen tuning through feedback excitation and lateral inhibition.SIGNIFICANCE STATEMENT Auditory processing starts in the cochlea and ascends through the dorsal cochlear nucleus (DCN) to the inferior colliculus (IC) and beyond. Here, we investigated the feedback projection from IC to DCN, whose synaptic targets and roles in auditory processing are unclear. We found that all cell types in the granule cell regions, which process multisensory feedback, also process this descending auditory feedback. Surprisingly, all except one cell type in the entire DCN receive IC input. The IC-DCN projection may therefore modulate the multisensory pathway as well as sharpen tuning and gate auditory signals that are sent to downstream areas. This excitatory feedback loop from DCN to IC and back to DCN could underlie hyperexcitability in DCN, widely considered an etiology of tinnitus.

Trigeminal Contributions to the Dorsal Cochlear Nucleus in Mouse.

The dorsal cochlear nucleus (DCN) is the first site of multisensory integration in the auditory pathway of mammals. The DCN circuit integrates non-auditory information, such as head and ear position, with auditory signals, and this convergence may contribute to the ability to localize sound sources or to suppress perceptions of self-generated sounds. Several extrinsic sources of these non-auditory signals have been described in various species, and among these are first- and second-order trigeminal axonal projections. Trigeminal sensory signals from the face and ears could provide the non-auditory information that the DCN requires for its role in sound source localization and cancelation of self-generated sounds, for example, head and ear position or mouth movements that could predict the production of chewing or licking sounds. There is evidence for these axonal projections in guinea pigs and rats, although the size of the pathway is smaller than might be expected for a function essential for a prey animals' survival. However, evidence for these projections in mice, an increasingly important species in auditory neuroscience, is lacking, raising questions about the universality of such proposed functions. We therefore investigated the presence of trigeminal projections to the DCN in mice, using viral and transgenic approaches. We found that the spinal trigeminal nucleus indeed projects to DCN, targeting granule cells and unipolar brush cells. However, direct axonal projections from the trigeminal ganglion itself were undetectable. Thus, secondary brainstem sources carry non-auditory signals to the DCN in mice that could provide a processed trigeminal signal to the DCN, but primary trigeminal afferents are not integrated directly by DCN.

Distinct forms of synaptic plasticity during ascending vs descending control of medial olivocochlear efferent neurons.

Activity in each brain region is shaped by the convergence of ascending and descending axonal pathways, and the balance and characteristics of these determine the neural output. The medial olivocochlear (MOC) efferent system is part of a reflex arc that critically controls auditory sensitivity. Multiple central pathways contact MOC neurons, raising the question of how a reflex arc could be engaged by diverse inputs. We examined functional properties of synapses onto brainstem MOC neurons from ascending (ventral cochlear nucleus, VCN) and descending (inferior colliculus, IC) sources in mice using an optogenetic approach. We found that these pathways exhibited opposing forms of short-term plasticity, with the VCN input showing depression and the IC input showing marked facilitation. By using a conductance-clamp approach, we found that combinations of facilitating and depressing inputs enabled firing of MOC neurons over a surprisingly wide dynamic range, suggesting an essential role for descending signaling to a brainstem nucleus.

Incomplete removal of extracellular glutamate controls synaptic transmission and integration at a cerebellar synapse.

Synapses of glutamatergic mossy fibers (MFs) onto cerebellar unipolar brush cells (UBCs) generate slow excitatory (ON) or inhibitory (OFF) postsynaptic responses dependent on the complement of glutamate receptors expressed on the UBC's large dendritic brush. Using mouse brain slice recording and computational modeling of synaptic transmission, we found that substantial glutamate is maintained in the UBC synaptic cleft, sufficient to modify spontaneous firing in OFF UBCs and tonically desensitize AMPARs of ON UBCs. The source of this ambient glutamate was spontaneous, spike-independent exocytosis from the MF terminal, and its level was dependent on activity of glutamate transporters EAAT1-2. Increasing levels of ambient glutamate shifted the polarity of evoked synaptic responses in ON UBCs and altered the phase of responses to in vivo-like synaptic activity. Unlike classical fast synapses, receptors at the UBC synapse are virtually always exposed to a significant level of glutamate, which varies in a graded manner during transmission.

Identification of an inhibitory neuron subtype, the L-stellate cell of the cochlear nucleus.

Auditory processing depends upon inhibitory signaling by interneurons, even at its earliest stages in the ventral cochlear nucleus (VCN). Remarkably, to date only a single subtype of inhibitory neuron has been documented in the VCN, a projection neuron termed the D-stellate cell. With the use of a transgenic mouse line, optical clearing, and imaging techniques, combined with electrophysiological tools, we revealed a population of glycinergic cells in the VCN distinct from the D-stellate cell. These multipolar glycinergic cells were smaller in soma size and dendritic area, but over ten-fold more numerous than D-stellate cells. They were activated by auditory nerve and T-stellate cells, and made local inhibitory synaptic contacts on principal cells of the VCN. Given their abundance, combined with their narrow dendritic fields and axonal projections, it is likely that these neurons, here termed L-stellate cells, play a significant role in frequency-specific processing of acoustic signals.

Selective targeting of unipolar brush cell subtypes by cerebellar mossy fibers.

In vestibular cerebellum, primary afferents carry signals from single vestibular end organs, whereas secondary afferents from vestibular nucleus carry integrated signals. Selective targeting of distinct mossy fibers determines how the cerebellum processes vestibular signals. We focused on vestibular projections to ON and OFF classes of unipolar brush cells (UBCs), which transform single mossy fiber signals into long-lasting excitation or inhibition respectively, and impact the activity of ensembles of granule cells. To determine whether these contacts are indeed selective, connectivity was traced back from UBC to specific ganglion cell, hair cell and vestibular organ subtypes in mice. We show that a specialized subset of primary afferents contacts ON UBCs, but not OFF UBCs, while secondary afferents contact both subtypes. Striking anatomical differences were observed between primary and secondary afferents, their synapses, and the UBCs they contact. Thus, each class of UBC functions to transform specific signals through distinct anatomical pathways.

The Calyx of Held: A Hypothesis on the Need for Reliable Timing in an Intensity-Difference Encoder.

The calyx of Held is the preeminent model for the study of synaptic function in the mammalian CNS. Despite much work on the synapse and associated circuit, its role in hearing remains enigmatic. We propose that the calyx is one of the key adaptations that enables an animal to lateralize transient sounds. The calyx is part of a binaural circuit that is biased toward high sound frequencies and is sensitive to intensity differences between the ears. This circuit also shows marked sensitivity to interaural time differences, but only for brief sound transients ("clicks"). In a natural environment, such transients are rare except as adventitious sounds generated by other animals moving at close range. We argue that the calyx, and associated temporal specializations, evolved to enable spatial localization of sound transients, through a neural code congruent with the circuit's sensitivity to interaural intensity differences, thereby conferring a key benefit to survival.

Rapid Rebalancing of Excitation and Inhibition by Cortical Circuitry.

Excitation is balanced by inhibition to cortical neurons across a wide range of conditions. To understand how this relationship is maintained, we broadly suppressed the activity of parvalbumin-expressing (PV+) inhibitory neurons and asked how this affected the balance of excitation and inhibition throughout auditory cortex. Activating archaerhodopsin in PV+ neurons effectively suppressed them in layer 4. However, the resulting increase in excitation outweighed Arch suppression and produced a net increase in PV+ activity in downstream layers. Consequently, suppressing PV+ neurons did not reduce inhibition to principal neurons (PNs) but instead resulted in a tightly coordinated increase in both excitation and inhibition. The increase in inhibition constrained the magnitude of PN spiking responses to the increase in excitation and produced nonlinear changes in spike tuning. Excitatory-inhibitory rebalancing is mediated by strong PN-PV+ connectivity within and between layers and is likely engaged during normal cortical operation to ensure balance in downstream neurons.

Double-Nanodomain Coupling of Calcium Channels, Ryanodine Receptors, and BK Channels Controls the Generation of Burst Firing.

Action potentials clustered into high-frequency bursts play distinct roles in neural computations. However, little is known about ionic currents that control the duration and probability of these bursts. We found that, in cartwheel inhibitory interneurons of the dorsal cochlear nucleus, the likelihood of bursts and the interval between their spikelets were controlled by Ca2+ acting across two nanodomains, one between plasma membrane P/Q Ca2+ channels and endoplasmic reticulum (ER) ryanodine receptors and another between ryanodine receptors and large-conductance, voltage- and Ca2+-activated K+ (BK) channels. Each spike triggered Ca2+-induced Ca2+ release (CICR) from the ER immediately beneath somatic, but not axonal or dendritic, plasma membrane. Moreover, immunolabeling demonstrated close apposition of ryanodine receptors and BK channels. Double-nanodomain coupling between somatic plasma membrane and hypolemmal ER cisterns provides a unique mechanism for rapid control of action potentials on the millisecond timescale.

Slow AMPAR Synaptic Transmission Is Determined by Stargazin and Glutamate Transporters.

AMPARs mediate the briefest synaptic currents in the brain by virtue of their rapid gating kinetics. However, at the mossy fiber-to-unipolar brush cell synapse in the cerebellum, AMPAR-mediated EPSCs last for hundreds of milliseconds, and it has been proposed that this time course reflects slow diffusion from a complex synaptic space. We show that upon release of glutamate, synaptic AMPARs were desensitized by transmitter by >90%. As glutamate levels subsequently fell, recovery of transmission occurred due to the presence of the AMPAR accessory protein stargazin that enhances the AMPAR response to low levels of transmitter. This gradual increase in receptor activity following desensitization accounted for the majority of synaptic transmission at this synapse. Moreover, the amplitude, duration, and shape of the synaptic response was tightly controlled by plasma membrane glutamate transporters, indicating that clearance of synaptic glutamate during the slow EPSC is dictated by an uptake process.

Corelease of Inhibitory Neurotransmitters in the Mouse Auditory Midbrain.

The central nucleus of the inferior colliculus (ICC) of the auditory midbrain, which integrates most ascending auditory information from lower brainstem regions, receives prominent long-range inhibitory input from the ventral nucleus of the lateral lemniscus (VNLL), a region thought to be important for temporal pattern discrimination. Histological evidence suggests that neurons in the VNLL release both glycine and GABA in the ICC, but functional evidence for their corelease is lacking. We took advantage of the GlyT2-Cre mouse line (both male and female) to target expression of ChR2 to glycinergic afferents in the ICC and made whole-cell recordings in vitro while exciting glycinergic fibers with light. Using this approach, it was clear that a significant fraction of glycinergic boutons corelease GABA in the ICC. Viral injections were used to target ChR2 expression specifically to glycinergic fibers ascending from the VNLL, allowing for activation of fibers from a single source of ascending input in a way that has not been previously possible in the ICC. We then investigated aspects of the glycinergic versus GABAergic current components to probe functional consequences of corelease. Surprisingly, the time course and short-term plasticity of synaptic signaling were nearly identical for the two transmitters. We therefore conclude that the two neurotransmitters may be functionally interchangeable and that multiple receptor subtypes subserving inhibition may offer diverse mechanisms for maintaining inhibitory homeostasis.SIGNIFICANCE STATEMENT Corelease of neurotransmitters is a common feature of the brain. GABA and glycine corelease is particularly common in the spinal cord and brainstem, but its presence in the midbrain is unknown. We show corelease of GABA and glycine for the first time in the central nucleus of the inferior colliculus of the auditory midbrain. Glycine and GABA are both inhibitory neurotransmitters involved in fast synaptic transmission, so we explored differences between the currents to establish a physiological foundation for functional differences in vivo In contrast to the auditory brainstem, coreleased GABAergic and glycinergic currents in the midbrain are strikingly similar. This apparent redundancy may ensure homeostasis if one neurotransmitter system is compromised.