Cochlear zinc signaling dysregulation is associated with noise-induced hearing loss, and zinc chelation enhances cochlear recovery.

Exposure to loud noise triggers sensory organ damage and degeneration that, in turn, leads to hearing loss. Despite the troublesome impact of noise-induced hearing loss (NIHL) in individuals and societies, treatment strategies that protect and restore hearing are few and insufficient. As such, identification and mechanistic understanding of the signaling pathways involved in NIHL are required. Biological zinc is mostly bound to proteins, where it plays major structural or catalytic roles; however, there is also a pool of unbound, mobile (labile) zinc. Labile zinc is mostly found in vesicles in secretory tissues, where it is released and plays a critical signaling role. In the brain, labile zinc fine-tunes neurotransmission and sensory processing. However, injury-induced dysregulation of labile zinc signaling contributes to neurodegeneration. Here, we tested whether zinc dysregulation occurs and contributes to NIHL in mice. We found that ZnT3, the vesicular zinc transporter responsible for loading zinc into vesicles, is expressed in cochlear hair cells and the spiral limbus, with labile zinc also present in the same areas. Soon after noise trauma, ZnT3 and zinc levels are significantly increased, and their subcellular localization is vastly altered. Disruption of zinc signaling, either via ZnT3 deletion or pharmacological zinc chelation, mitigated NIHL, as evidenced by enhanced auditory brainstem responses, distortion product otoacoustic emissions, and number of hair cell synapses. These data reveal that noise-induced zinc dysregulation is associated with cochlear dysfunction and recovery after NIHL, and point to zinc chelation as a potential treatment for mitigating NIHL.

Cortical Zinc Signaling Is Necessary for Changes in Mouse Pupil Diameter That Are Evoked by Background Sounds with Different Contrasts.

Luminance-independent changes in pupil diameter (PD) during wakefulness influence and are influenced by neuromodulatory, neuronal, and behavioral responses. However, it is unclear whether changes in neuromodulatory activity in a specific brain area are necessary for the associated changes in PD or whether some different mechanisms cause parallel fluctuations in both PD and neuromodulation. To answer this question, we simultaneously recorded PD and cortical neuronal activity in male and female mice. Namely, we measured PD and neuronal activity during adaptation to sound contrast, which is a well-described adaptation conserved in many species and brain areas. In the primary auditory cortex (A1), increases in the variability of sound level (contrast) induce a decrease in the slope of the neuronal input-output relationship, neuronal gain, which depends on cortical neuromodulatory zinc signaling. We found a previously unknown modulation of PD by changes in background sensory context: high stimulus contrast sounds evoke larger increases in evoked PD compared with low-contrast sounds. To explore whether these changes in evoked PD are controlled by cortical neuromodulatory zinc signaling, we imaged single-cell neural activity in A1, manipulated zinc signaling in the cortex, and assessed PD in the same awake mouse. We found that cortical synaptic zinc signaling is necessary for increases in PD during high-contrast background sounds compared with low-contrast sounds. This finding advances our knowledge about how cortical neuromodulatory activity affects PD changes and thus advances our understanding of the brain states, circuits, and neuromodulatory mechanisms that can be inferred from pupil size fluctuations.

A Role for KCNQ Channels on Cell Type-Specific Plasticity in Mouse Auditory Cortex after Peripheral Damage.

Damage to sensory organs triggers compensatory plasticity mechanisms in sensory cortices. These plasticity mechanisms result in restored cortical responses, despite reduced peripheral input, and contribute to the remarkable recovery of perceptual detection thresholds to sensory stimuli. Overall, peripheral damage is associated with a reduction of cortical GABAergic inhibition; however, less is known about changes in intrinsic properties and the underlying biophysical mechanisms. To study these mechanisms, we used a model of noise-induced peripheral damage in male and female mice. We uncovered a rapid, cell type-specific reduction in the intrinsic excitability of parvalbumin-expressing neurons (PVs) in layer (L) 2/3 of auditory cortex. No changes in the intrinsic excitability of either L2/3 somatostatin-expressing or L2/3 principal neurons (PNs) were observed. The decrease in L2/3 PV excitability was observed 1, but not 7, d after noise exposure, and was evidenced by a hyperpolarization of the resting membrane potential, depolarization of the action potential threshold, and reduction in firing frequency in response to depolarizing current. To uncover the underlying biophysical mechanisms, we recorded potassium currents. We found an increase in KCNQ potassium channel activity in L2/3 PVs of auditory cortex 1 d after noise exposure, associated with a hyperpolarizing shift in the minimal voltage activation of KCNQ channels. This increase contributes to the decreased intrinsic excitability of PVs. Our results highlight cell-type- and channel-specific mechanisms of plasticity after noise-induced hearing loss and will aid in understanding the pathologic processes involved in hearing loss and hearing loss-related disorders, such as tinnitus and hyperacusis.SIGNIFICANCE STATEMENT Noise-induced damage to the peripheral auditory system triggers central plasticity that compensates for the reduced peripheral input. The mechanisms of this plasticity are not fully understood. In the auditory cortex, this plasticity likely contributes to the recovery of sound-evoked responses and perceptual hearing thresholds. Importantly, other functional aspects of hearing do not recover, and peripheral damage may also lead to maladaptive plasticity-related disorders, such as tinnitus and hyperacusis. Here, after noise-induced peripheral damage, we highlight a rapid, transient, and cell type-specific reduction in the excitability of layer 2/3 parvalbumin-expressing neurons, which is due, at least in part, to increased KCNQ potassium channel activity. These studies may highlight novel strategies for enhancing perceptual recovery after hearing loss and mitigating hyperacusis and tinnitus.

Neuromodulatory Mechanisms Underlying Contrast Gain Control in Mouse Auditory Cortex.

Neural adaptation enables the brain to efficiently process sensory signals despite large changes in background noise. Previous studies have established that recent background spectro- or spatio-temporal statistics scale neural responses to sensory stimuli via a canonical normalization computation, which is conserved among species and sensory domains. In the auditory pathway, one major form of normalization, termed contrast gain control, presents as decreasing instantaneous firing-rate gain, the slope of the neural input-output relationship, with increasing variability of background sound levels (contrast) across time and frequency. Despite this gain rescaling, mean firing-rates in auditory cortex become invariant to sound level contrast, termed contrast invariance. The underlying neuromodulatory mechanisms of these two phenomena remain unknown. To study these mechanisms in male and female mice, we used a 2-photon calcium imaging preparation in layer 2/3 neurons of primary auditory cortex (A1), along with pharmacological and genetic KO approaches. We found that neuromodulatory cortical synaptic zinc signaling is necessary for contrast gain control but not contrast invariance in mouse A1.SIGNIFICANCE STATEMENT When sound levels in the acoustic environment become more variable across time and frequency, the brain decreases response gain to maintain dynamic range and thus stimulus discriminability. This gain adaptation accounts for changes in perceptual judgments in humans and mice; however, the underlying neuromodulatory mechanisms remain poorly understood. Here, we report context-dependent neuromodulatory effects of synaptic zinc that are necessary for contrast gain control in A1. Understanding context-specific neuromodulatory mechanisms, such as contrast gain control, provides insight into A1 cortical mechanisms of adaptation and also into fundamental aspects of perceptual changes that rely on gain modulation, such as attention.

Development of an automated screen for Kv7.2 potassium channels and discovery of a new agonist chemotype.

To identify pore domain ligands on Kv7.2 potassium ion channels, we compared wild-type (WT) and W236L mutant Kv7.2 channels in a series of assays with previously validated and novel agonist chemotypes. Positive controls were retigabine, flupirtine, and RL-81; i.e. Kv7.2 channel activators that significantly shift voltage-dependent activation to more negative potentials (ΔV50) at 5 µM. We identified 6 new compounds that exhibited differential enhancing activity between WT and W236L mutant channels. Whole cell patch-clamp electrophysiology studies were conducted to identify Kv7.2. Kv7.2/3, Kv7.4, and Kv7.5 selectivity. Our results validate the SyncroPatch platform and establish new structure activity relationships (SAR). Specifically, in addition to selective Kv7.2, Kv7.2/3, Kv7.4. and Kv7.5 agonists, we identified a novel chemotype, ZK-21, a 4-aminotetrahydroquinoline that is distinct from any of the previously described Kv7 channel modifiers. Using flexible receptor docking, ZK-21 was predicted to be stabilized by W236 and bind perpendicular to retigabine, burying the benzyl carbamate group into a tunnel reaching the core of the pore domain.

Defining the Kv2.1-syntaxin molecular interaction identifies a first-in-class small molecule neuroprotectant.

The neuronal cell death-promoting loss of cytoplasmic K+ following injury is mediated by an increase in Kv2.1 potassium channels in the plasma membrane. This phenomenon relies on Kv2.1 binding to syntaxin 1A via 9 amino acids within the channel intrinsically disordered C terminus. Preventing this interaction with a cell and blood-brain barrier-permeant peptide is neuroprotective in an in vivo stroke model. Here a rational approach was applied to define the key molecular interactions between syntaxin and Kv2.1, some of which are shared with mammalian uncoordinated-18 (munc18). Armed with this information, we found a small molecule Kv2.1-syntaxin-binding inhibitor (cpd5) that improves cortical neuron survival by suppressing SNARE-dependent enhancement of Kv2.1-mediated currents following excitotoxic injury. We validated that cpd5 selectively displaces Kv2.1-syntaxin-binding peptides from syntaxin and, at higher concentrations, munc18, but without affecting either synaptic or neuronal intrinsic properties in brain tissue slices at neuroprotective concentrations. Collectively, our findings provide insight into the role of syntaxin in neuronal cell death and validate an important target for neuroprotection.

Mechanisms Underlying Long-Term Synaptic Zinc Plasticity at Mouse Dorsal Cochlear Nucleus Glutamatergic Synapses.

In many brain areas, such as the neocortex, limbic structures, and auditory brainstem, synaptic zinc is released from presynaptic terminals to modulate neurotransmission. As such, synaptic zinc signaling modulates sensory processing and enhances acuity for discrimination of different sensory stimuli. Whereas sensory experience causes long-term changes in synaptic zinc signaling, the mechanisms underlying this long-term synaptic zinc plasticity remain unknown. To study these mechanisms in male and female mice, we used in vitro and in vivo models of zinc plasticity observed at the zinc-rich glutamatergic dorsal cochlear nucleus (DCN) parallel fiber synapses onto cartwheel cells. High-frequency stimulation of DCN parallel fiber synapses induced LTD of synaptic zinc signaling (Z-LTD), evidenced by reduced zinc-mediated inhibition of EPSCs. Low-frequency stimulation induced LTP of synaptic zinc signaling (Z-LTP), evidenced by enhanced zinc-mediated inhibition of EPSCs. Pharmacological manipulations of Group 1 metabotropic glutamate receptors (G1 mGluRs) demonstrated that G1 mGluR activation is necessary and sufficient for inducing Z-LTD and Z-LTP. Pharmacological manipulations of Ca2+ dynamics indicated that rises in postsynaptic Ca2+ are necessary and sufficient for Z-LTD induction. Electrophysiological measurements assessing postsynaptic expression mechanisms, and imaging studies with a ratiometric extracellular zinc sensor probing zinc release, supported that Z-LTD is expressed, at least in part, via reductions in presynaptic zinc release. Finally, exposure of mice to loud sound caused G1 mGluR-dependent Z-LTD at DCN parallel fiber synapses, thus validating our in vitro results. Together, our results reveal a novel mechanism underlying activity- and experience-dependent plasticity of synaptic zinc signaling.SIGNIFICANCE STATEMENT In the neocortex, limbic structures, and auditory brainstem, glutamatergic nerve terminals corelease zinc to modulate excitatory neurotransmission and sensory responses. Moreover, sensory experience causes bidirectional, long-term changes in synaptic zinc signaling. However, the mechanisms of this long-term synaptic zinc plasticity remain unknown. Here, we identified a novel Group 1 mGluR-dependent mechanism that causes bidirectional, long-term changes in synaptic zinc signaling. Our results highlight new mechanisms of brain adaptation during sensory processing, and potentially point to mechanisms of disorders associated with pathologic adaptation, such as tinnitus.

Synaptic Zinc Enhances Inhibition Mediated by Somatostatin, but not Parvalbumin, Cells in Mouse Auditory Cortex.

Cortical inhibition is essential for brain activity and behavior. Yet, the mechanisms that modulate cortical inhibition and their impact on sensory processing remain less understood. Synaptically released zinc, a neuromodulator released by cortical glutamatergic synaptic vesicles, has emerged as a powerful modulator of sensory processing and behavior. Despite the puzzling finding that the vesicular zinc transporter (ZnT3) mRNA is expressed in cortical inhibitory interneurons, the actions of synaptic zinc in cortical inhibitory neurotransmission remain unknown. Using in vitro electrophysiology and optogenetics in mouse brain slices containing the layer 2/3 (L2/3) of auditory cortex, we discovered that synaptic zinc increases the quantal size of inhibitory GABAergic neurotransmission mediated by somatostatin (SOM)- but not parvalbumin (PV)-expressing neurons. Using two-photon imaging in awake mice, we showed that synaptic zinc is required for the effects of SOM- but not PV-mediated inhibition on frequency tuning of principal neurons. Thus, cell-specific zinc modulation of cortical inhibition regulates frequency tuning.

Fine Control of Sound Frequency Tuning and Frequency Discrimination Acuity by Synaptic Zinc Signaling in Mouse Auditory Cortex.

Neurons in the auditory cortex are tuned to specific ranges of sound frequencies. Although the cellular and network mechanisms underlying neuronal sound frequency selectivity are well studied and reflect the interplay of thalamocortical and intracortical excitatory inputs and further refinement by cortical inhibition, the precise synaptic signaling mechanisms remain less understood. To gain further understanding on these mechanisms and their effects on sound-driven behavior, we used in vivo imaging as well as behavioral approaches in awake and behaving female and male mice. We discovered that synaptic zinc, a modulator of neurotransmission and responsiveness to sound, sharpened the sound frequency tuning of principal and parvalbumin-expressing neurons and widened the sound frequency tuning of somatostatin-expressing inhibitory neurons in layer 2/3 of the primary auditory cortex. In the absence of cortical synaptic zinc, mice exhibited reduced acuity for detecting changes in sound frequencies. Together, our results reveal that cell-type-specific effects of zinc contribute to cortical sound frequency tuning and enhance acuity for sound frequency discrimination.SIGNIFICANCE STATEMENT Neuronal tuning to specific features of sensory stimuli is a fundamental property of cortical sensory processing that advantageously supports behavior. Despite the established roles of synaptic thalamocortical and intracortical excitation and inhibition in cortical tuning, the precise synaptic signaling mechanisms remain unknown. Here, we investigated these mechanisms in the mouse auditory cortex. We discovered a previously unknown signaling mechanism linking synaptic zinc signaling with cell-specific cortical tuning and enhancement in sound frequency discrimination acuity. Given the abundance of synaptic zinc in all sensory cortices, this newly discovered interaction between synaptic zinc and cortical tuning can provide a general mechanism for modulating neuronal stimulus specificity and sensory-driven behavior.

Targeting a Potassium Channel/Syntaxin Interaction Ameliorates Cell Death in Ischemic Stroke.

The voltage-gated K+ channel Kv2.1 has been intimately linked with neuronal apoptosis. After ischemic, oxidative, or inflammatory insults, Kv2.1 mediates a pronounced, delayed enhancement of K+ efflux, generating an optimal intracellular environment for caspase and nuclease activity, key components of programmed cell death. This apoptosis-enabling mechanism is initiated via Zn2+-dependent dual phosphorylation of Kv2.1, increasing the interaction between the channel's intracellular C-terminus domain and the SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor) protein syntaxin 1A. Subsequently, an upregulation of de novo channel insertion into the plasma membrane leads to the critical enhancement of K+ efflux in damaged neurons. Here, we investigated whether a strategy designed to interfere with the cell death-facilitating properties of Kv2.1, specifically its interaction with syntaxin 1A, could lead to neuroprotection following ischemic injury in vivo The minimal syntaxin 1A-binding sequence of Kv2.1 C terminus (C1aB) was first identified via a far-Western peptide screen and used to create a protherapeutic product by conjugating C1aB to a cell-penetrating domain. The resulting peptide (TAT-C1aB) suppressed enhanced whole-cell K+ currents produced by a mutated form of Kv2.1 mimicking apoptosis in a mammalian expression system, and protected cortical neurons from slow excitotoxic injury in vitro, without influencing NMDA-induced intracellular calcium responses. Importantly, intraperitoneal administration of TAT-C1aB in mice following transient middle cerebral artery occlusion significantly reduced ischemic stroke damage and improved neurological outcome. These results provide strong evidence that targeting the proapoptotic function of Kv2.1 is an effective and highly promising neuroprotective strategy.SIGNIFICANCE STATEMENT Kv2.1 is a critical regulator of apoptosis in central neurons. It has not been determined, however, whether the cell death-enabling function of this K+ channel can be selectively targeted to improve neuronal survival following injury in vivo The experiments presented here demonstrate that the cell death-specific role of Kv2.1 can be uniquely modulated to provide neuroprotection in an animal model of acute ischemic stroke. We thus reveal a novel therapeutic strategy for neurological disorders that are accompanied by Kv2.1-facilitated forms of cell death.

Photoactivatable Sensors for Detecting Mobile Zinc.

Fluorescent sensors for mobile zinc are valuable for studying complex biological systems. Because these sensors typically bind zinc rapidly and tightly, there has been little temporal control over the activity of the probe after its application to a sample. The ability to control the activity of a zinc sensor in vivo during imaging experiments would greatly improve the time resolution of the measurement. Here, we describe photoactivatable zinc sensors that can be triggered with short pulses of UV light. These probes are prepared by functionalizing a zinc sensor with protecting groups that render the probe insensitive to metal ions. Photoinduced removal of the protecting groups restores the binding site, allowing for zinc-responsive changes in fluorescence that can be observed in live cells and tissues.

AMPA receptor inhibition by synaptically released zinc.

The vast amount of fast excitatory neurotransmission in the mammalian central nervous system is mediated by AMPA-subtype glutamate receptors (AMPARs). As a result, AMPAR-mediated synaptic transmission is implicated in nearly all aspects of brain development, function, and plasticity. Despite the central role of AMPARs in neurobiology, the fine-tuning of synaptic AMPA responses by endogenous modulators remains poorly understood. Here we provide evidence that endogenous zinc, released by single presynaptic action potentials, inhibits synaptic AMPA currents in the dorsal cochlear nucleus (DCN) and hippocampus. Exposure to loud sound reduces presynaptic zinc levels in the DCN and abolishes zinc inhibition, implicating zinc in experience-dependent AMPAR synaptic plasticity. Our results establish zinc as an activity-dependent, endogenous modulator of AMPARs that tunes fast excitatory neurotransmission and plasticity in glutamatergic synapses.

Modulation of extrasynaptic NMDA receptors by synaptic and tonic zinc.

Many excitatory synapses contain high levels of mobile zinc within glutamatergic vesicles. Although synaptic zinc and glutamate are coreleased, it is controversial whether zinc diffuses away from the release site or whether it remains bound to presynaptic membranes or proteins after its release. To study zinc transmission and quantify zinc levels, we required a high-affinity rapid zinc chelator as well as an extracellular ratiometric fluorescent zinc sensor. We demonstrate that tricine, considered a preferred chelator for studying the role of synaptic zinc, is unable to efficiently prevent zinc from binding low-nanomolar zinc-binding sites, such as the high-affinity zinc-binding site found in NMDA receptors (NMDARs). Here, we used ZX1, which has a 1 nM zinc dissociation constant and second-order rate constant for binding zinc that is 200-fold higher than those for tricine and CaEDTA. We find that synaptic zinc is phasically released during action potentials. In response to short trains of presynaptic stimulation, synaptic zinc diffuses beyond the synaptic cleft where it inhibits extrasynaptic NMDARs. During higher rates of presynaptic stimulation, released glutamate activates additional extrasynaptic NMDARs that are not reached by synaptically released zinc, but which are inhibited by ambient, tonic levels of nonsynaptic zinc. By performing a ratiometric evaluation of extracellular zinc levels in the dorsal cochlear nucleus, we determined the tonic zinc levels to be low nanomolar. These results demonstrate a physiological role for endogenous synaptic as well as tonic zinc in inhibiting extrasynaptic NMDARs and thereby fine tuning neuronal excitability and signaling.

Cell-Specific Cholinergic Modulation of Excitability of Layer 5B Principal Neurons in Mouse Auditory Cortex.

UNLABELLED: The neuromodulator acetylcholine (ACh) is crucial for several cognitive functions, such as perception, attention, and learning and memory. Whereas, in most cases, the cellular circuits or the specific neurons via which ACh exerts its cognitive effects remain unknown, it is known that auditory cortex (AC) neurons projecting from layer 5B (L5B) to the inferior colliculus, corticocollicular neurons, are required for cholinergic-mediated relearning of sound localization after occlusion of one ear. Therefore, elucidation of the effects of ACh on the excitability of corticocollicular neurons will bridge the cell-specific and cognitive properties of ACh. Because AC L5B contains another class of neurons that project to the contralateral cortex, corticocallosal neurons, to identify the cell-specific mechanisms that enable corticocollicular neurons to participate in sound localization relearning, we investigated the effects of ACh release on both L5B corticocallosal and corticocollicular neurons. Using in vitro electrophysiology and optogenetics in mouse brain slices, we found that ACh generated nicotinic ACh receptor (nAChR)-mediated depolarizing potentials and muscarinic ACh receptor (mAChR)-mediated hyperpolarizing potentials in AC L5B corticocallosal neurons. In corticocollicular neurons, ACh release also generated nAChR-mediated depolarizing potentials. However, in contrast to the mAChR-mediated hyperpolarizing potentials in corticocallosal neurons, ACh generated prolonged mAChR-mediated depolarizing potentials in corticocollicular neurons. These prolonged depolarizing potentials generated persistent firing in corticocollicular neurons, whereas corticocallosal neurons lacking mAChR-mediated depolarizing potentials did not show persistent firing. We propose that ACh-mediated persistent firing in corticocollicular neurons may represent a critical mechanism required for learning-induced plasticity in AC. SIGNIFICANCE STATEMENT: Acetylcholine (ACh) is crucial for cognitive functions. Whereas in most cases the cellular circuits or the specific neurons via which ACh exerts its cognitive effects remain unknown, it is known that auditory cortex (AC) corticocollicular neurons projecting from layer 5B to the inferior colliculus are required for cholinergic-mediated relearning of sound localization after occlusion of one ear. Therefore, elucidation of the effects of ACh on the excitability of corticocollicular neurons will bridge the cell-specific and cognitive properties of ACh. Our results suggest that cell-specific ACh-mediated persistent firing in corticocollicular neurons may represent a critical mechanism required for learning-induced plasticity in AC. Moreover, our results provide synaptic mechanisms via which ACh may mediate its effects on AC receptive fields.

Potent KCNQ2/3-specific channel activator suppresses in vivo epileptic activity and prevents the development of tinnitus.

Voltage-gated Kv7 (KCNQ) channels are voltage-dependent potassium channels that are activated at resting membrane potentials and therefore provide a powerful brake on neuronal excitability. Genetic or experience-dependent reduction of KCNQ2/3 channel activity is linked with disorders that are characterized by neuronal hyperexcitability, such as epilepsy and tinnitus. Retigabine, a small molecule that activates KCNQ2-5 channels by shifting their voltage-dependent opening to more negative voltages, is an US Food and Drug Administration (FDA) approved anti-epileptic drug. However, recently identified side effects have limited its clinical use. As a result, the development of improved KCNQ2/3 channel activators is crucial for the treatment of hyperexcitability-related disorders. By incorporating a fluorine substituent in the 3-position of the tri-aminophenyl ring of retigabine, we synthesized a small-molecule activator (SF0034) with novel properties. Heterologous expression of KCNQ2/3 channels in HEK293T cells showed that SF0034 was five times more potent than retigabine at shifting the voltage dependence of KCNQ2/3 channels to more negative voltages. Moreover, unlike retigabine, SF0034 did not shift the voltage dependence of either KCNQ4 or KCNQ5 homomeric channels. Conditional deletion of Kcnq2 from cerebral cortical pyramidal neurons showed that SF0034 requires the expression of KCNQ2/3 channels for reducing the excitability of CA1 hippocampal neurons. Behavioral studies demonstrated that SF0034 was a more potent and less toxic anticonvulsant than retigabine in rodents. Furthermore, SF0034 prevented the development of tinnitus in mice. We propose that SF0034 provides, not only a powerful tool for investigating ion channel properties, but, most importantly, it provides a clinical candidate for treating epilepsy and preventing tinnitus.

Cell-specific activity-dependent fractionation of layer 2/3→5B excitatory signaling in mouse auditory cortex.

Auditory cortex (AC) layer 5B (L5B) contains both corticocollicular neurons, a type of pyramidal-tract neuron projecting to the inferior colliculus, and corticocallosal neurons, a type of intratelencephalic neuron projecting to contralateral AC. Although it is known that these neuronal types have distinct roles in auditory processing and different response properties to sound, the synaptic and intrinsic mechanisms shaping their input-output functions remain less understood. Here, we recorded in brain slices of mouse AC from retrogradely labeled corticocollicular and neighboring corticocallosal neurons in L5B. Corticocollicular neurons had, on average, lower input resistance, greater hyperpolarization-activated current (Ih), depolarized resting membrane potential, faster action potentials, initial spike doublets, and less spike-frequency adaptation. In paired recordings between single L2/3 and labeled L5B neurons, the probabilities of connection, amplitude, latency, rise time, and decay time constant of the unitary EPSC were not different for L2/3→corticocollicular and L2/3→corticocallosal connections. However, short trains of unitary EPSCs showed no synaptic depression in L2/3→corticocollicular connections, but substantial depression in L2/3→corticocallosal connections. Synaptic potentials in L2/3→corticocollicular connections decayed faster and showed less temporal summation, consistent with increased Ih in corticocollicular neurons, whereas synaptic potentials in L2/3→corticocallosal connections showed more temporal summation. Extracellular L2/3 stimulation at two different rates resulted in spiking in L5B neurons; for corticocallosal neurons the spike rate was frequency dependent, but for corticocollicular neurons it was not. Together, these findings identify cell-specific intrinsic and synaptic mechanisms that divide intracortical synaptic excitation from L2/3 to L5B into two functionally distinct pathways with different input-output functions.

Synthesis and Evaluation of Potent KCNQ2/3-Specific Channel Activators.

KQT-like subfamily (KCNQ) channels are voltage-gated, noninactivating potassium ion channels, and their down-regulation has been implicated in several hyperexcitability-related disorders, including epilepsy, neuropathic pain, and tinnitus. Activators of these channels reduce the excitability of central and peripheral neurons, and, as such, have therapeutic utility. Here, we synthetically modified several moieties of the KCNQ2-5 channel activator retigabine, an anticonvulsant approved by the U.S. Food and Drug Administration. By introducing a CF3-group at the 4-position of the benzylamine moiety, combined with a fluorine atom at the 3-position of the aniline ring, we generated Ethyl (2-amino-3-fluoro-4-((4-(trifluoromethyl)benzyl)amino)phenyl)carbamate (RL648_81), a new KCNQ2/3-specific activator that is >15 times more potent and also more selective than retigabine. We suggest that RL648_81 is a promising clinical candidate for treating or preventing neurologic disorders associated with neuronal hyperexcitability.

Pathogenic plasticity of Kv7.2/3 channel activity is essential for the induction of tinnitus.

Tinnitus, the perception of phantom sound, is often a debilitating condition that affects many millions of people. Little is known, however, about the molecules that participate in the induction of tinnitus. In brain slices containing the dorsal cochlear nucleus, we reveal a tinnitus-specific increase in the spontaneous firing rate of principal neurons (hyperactivity). This hyperactivity is observed only in noise-exposed mice that develop tinnitus and only in the dorsal cochlear nucleus regions that are sensitive to high frequency sounds. We show that a reduction in Kv7.2/3 channel activity is essential for tinnitus induction and for the tinnitus-specific hyperactivity. This reduction is due to a shift in the voltage dependence of Kv7 channel activation to more positive voltages. Our in vivo studies demonstrate that a pharmacological manipulation that shifts the voltage dependence of Kv7 to more negative voltages prevents the development of tinnitus. Together, our studies provide an important link between the biophysical properties of the Kv7 channel and the generation of tinnitus. Moreover, our findings point to previously unknown biological targets for designing therapeutic drugs that may prevent the development of tinnitus in humans.

Tonic zinc inhibits spontaneous firing in dorsal cochlear nucleus principal neurons by enhancing glycinergic neurotransmission.

In many synapses of the CNS, mobile zinc is packaged into glutamatergic vesicles and co-released with glutamate during neurotransmission. Following synaptic release, the mobilized zinc modulates ligand- and voltage-gated channels and receptors, functioning as an inhibitory neuromodulator. However, the origin and role of tonic, as opposed to phasically released, zinc are less well understood. We investigated tonic zinc in the dorsal cochlear nucleus (DCN), a zinc-rich, auditory brainstem nucleus. Our results show that application of a high-affinity, extracellular zinc chelator (ZX1) enhances spontaneous firing in DCN principal neurons (fusiform cells), consistent with inhibition of this neuronal property by tonic zinc. The enhancing effect was prevented by prior application of strychnine, a glycine receptor antagonist, suggesting that ZX1 interferes with zinc-mediated modulation of spontaneous glycinergic inhibition. In particular, ZX1 decreased the amplitude and the frequency of glycinergic miniature inhibitory postsynaptic currents in fusiform cells, from which we conclude that tonic zinc enhances glycinergic inhibitory neurotransmission. The observed zinc-mediated inhibition in spontaneous firing is present in mice lacking the vesicular zinc transporter (ZnT3), indicating that non-vesicular zinc inhibits spontaneous firing. Noise-induced increase in the spontaneous firing of fusiform cells is crucial for the induction of tinnitus. In this context, tonic zinc provides a powerful break of spontaneous firing that may protect against pathological run-up of spontaneous activity in the DCN.

Synaptic Zn2+ inhibits neurotransmitter release by promoting endocannabinoid synthesis.

Although it is well established that many glutamatergic neurons sequester Zn(2+) within their synaptic vesicles, the physiological significance of synaptic Zn(2+) remains poorly understood. In experiments performed in a Zn(2+)-enriched auditory brainstem nucleus--the dorsal cochlear nucleus--we discovered that synaptic Zn(2+) and GPR39, a putative metabotropic Zn(2+)-sensing receptor (mZnR), are necessary for triggering the synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). The postsynaptic production of 2-AG, in turn, inhibits presynaptic probability of neurotransmitter release, thus shaping synaptic strength and short-term synaptic plasticity. Zn(2+)-induced inhibition of transmitter release is absent in mutant mice that lack either vesicular Zn(2+) or the mZnR. Moreover, mass spectrometry measurements of 2-AG levels reveal that Zn(2+)-mediated initiation of 2-AG synthesis is absent in mice lacking the mZnR. We reveal a previously unknown action of synaptic Zn(2+): synaptic Zn(2+) inhibits glutamate release by promoting 2-AG synthesis.