Ventral Cochlear Nucleus Neurons Research Articles

Peripheral Fragile X messenger ribonucleoprotein is required for the timely closure of a critical period for neuronal susceptibility in the ventral cochlear nucleus.

Alterations in neuronal plasticity and critical periods are common across neurodevelopmental diseases, including Fragile X syndrome (FXS), the leading single-gene cause of autism. Characterized with sensory dysfunction, FXS is the result of gene silencing of Fragile X messenger ribonucleoprotein 1 (FMR1) and loss of its product, Fragile X messenger ribonucleoprotein (FMRP). The mechanisms underlying altered critical period and sensory dysfunction in FXS are obscure. Here, we performed genetic and surgical deprivation of peripheral auditory inputs in wildtype and Fmr1 knockout (KO) mice across ages and investigated the effects of global FMRP loss on deafferentation-induced neuronal changes in the ventral cochlear nucleus (VCN) and auditory brainstem responses. The degree of neuronal cell loss during the critical period was unchanged in Fmr1 KO mice. However, the closure of the critical period was delayed. Importantly, this delay was temporally coincidental with reduced hearing sensitivity, implying an association with sensory inputs. Functional analyses further identified early-onset and long-lasting alterations in signal transmission from the spiral ganglion to the VCN, suggesting a peripheral site of FMRP action. Finally, we generated conditional Fmr1 KO (cKO) mice with selective deletion of FMRP in spiral ganglion but not VCN neurons. cKO mice recapitulated the delay in the VCN critical period closure in Fmr1 KO mice, confirming an involvement of cochlear FMRP in shaping the temporal features of neuronal critical periods in the brain. Together, these results identify a novel peripheral mechanism of neurodevelopmental pathogenesis.

NAD+ attenuates bilirubin-induced augmentation of voltage-gated calcium currents in neurons of the ventral cochlear nucleus

Nicotinamide adenine dinucleotide (NAD+) is a ubiquitous molecule with wide-ranging roles in several cell processes, such as regulation of calcium homeostasis and protection against cell injuries. However, the roles of NAD+ in neuroprotection is poorly understood. The main neurons in ventral cochlear nucleus (VCN) are highly susceptible to bilirubin-associated excitotoxicity. We investigated the effects of NAD+ on VCN neurons by whole cell patch-clamp recordings. We found that NAD+ effectively reverses and inhibits bilirubin-mediated enhancement of voltage-gated calcium (VGCC) currents in VCN neurons. Moreover, NAD+ itself did not affect VGCC currents. These results collectively suggest that NAD+ may be neuroprotective by attenuating Ca2+ influx to suppress bilirubin-induced intracellular Ca2+ overloads. Our research provides a basis for evaluation of NAD+ as a promising therapeutic target for bilirubin encephalopathy and excitotoxicity associated with other neurological disorders.

KATP and TRPM2-like channels couple metabolic status to resting membrane potential of octopus neurons in the mouse ventral cochlear nucleus

ATP-sensitive potassium (KATP) channels and transient receptor potential melastatin 2 (TRPM2) channels are commonly expressed both pre- and postsynaptically in the central nervous system (CNS). We hypothesized that KATP and TRPM2 may couple metabolic status to the resting membrane potential of octopus neurons of the mouse ventral cochlear nucleus (VCN).Therefore, we studied the expression of KATP channels and TRPM2 channels in octopus cells by immunohistochemical techniques and their contribution to neuronal electrical properties by the electrophysiological patch clamp technique.In immunohistochemical staining of octopus cells, labelling with Kir6.2 and SUR1 antibodies was strong, and labelling with the SUR2 antibody was moderate, but labelling with Kir6.1 was very weak. Octopus cells had intense staining with TRPM2 antibodies.In patch clamp recordings, bath application of KATP channel agonists H2O2 (880 μM), ATZ (1 mM), cromakalim (50 μM), diazoxide (200 μM), NNC 55-0118 and NN 414 separately resulted in hyperpolarizations of resting potential to different extents. Application of 8-Bro-cADPR (50 μM), a specific antagonist of TRPM2 channels, in the presence of H2O2 (880 μM) resulted in further hyperpolarization by approximately 1 mV. The amplitudes of H2O2-induced outward KATP currents and ADPR-induced inward currents were 206.1 ± 31.5 pA (n = 4) and 136.8 ± 22.4 pA, respectively, at rest. Their respective reversal potentials were -77 ± 2.6 mV (n = 3) and -6.3 ± 2.9 (n = 3) and -6.3 ± 2.9 (n = 3).In conclusion, octopus cells appear to possess both KATP channels and TRPM2-like channels. KATP might largely be constituted by SUR1–Kir6.2 subunits and SUR2–Kir6.2 subunits. Both KATP and TRPM2-like channels might have a modulatory action in setting the membrane potential.

Information Processing by Onset Neurons in the Cat Auditory Brainstem.

Octopus cells in the ventral cochlear nucleus (VCN) have been difficult to study because of the very features that distinguish them from other VCN neurons. We performed in vivo recordings in cats on well-isolated units, some of which were intracellularly labeled and histologically reconstructed. We found that responses to low-frequency tones with frequencies < 1kHz reveal higher levels of neural synchrony and entrainment to the stimulus than the auditory nerve. In responses to higher frequency tones, the neural discharges occur mostly near the stimulus onset. These neurons also respond in a unique way to 100% amplitude-modulated (AM) tones with discharges exhibiting a bandpass tuning. Responses to frequency-modulated sounds (FM) are unusual: Octopus cells react more vigorously during the ascending than the descending parts of the FM stimulus. We examined responses of neurons in the ventral nucleus of the lateral lemniscus (VNLL) whose discharges to tones and AM sounds are similar to octopus cells. Repeated stimulation with short tone pips of VCN and VNLL onset neurons evokes trains of action potentials with gradual shifts toward later times in their first spike latency. This behavior parallels short-term post-synaptic depression observed by other authors in in vitro VCN recordings of octopus cells. VCN and VNLL onset units in cats respond to frozen noise stimuli with gaps as narrow as 1ms with a robust discharge near the stimulus onset following the gap. This finding suggests that VCN and VNLL onset cells play a role in gap detection, which is of great importance to speech perception.

Classification of neurons in the adult mouse cochlear nucleus: Linear discriminant analysis.

The cochlear nucleus (CN) transforms the spike trains of spiral ganglion cells into a set of sensory representations that are essential for auditory discriminations and perception. These transformations require the coordinated activity of different classes of neurons that are embryologically derived from distinct sets of precursors. Decades of investigation have shown that the neurons of the CN are differentiated by their morphology, neurotransmitter receptors, ion channel expression and intrinsic excitability. In the present study we have used linear discriminant analysis (LDA) to perform an unbiased analysis of measures of the responses of CN neurons to current injections to objectively categorize cells on the basis of both morphology and physiology. Recordings were made from cells in brain slices from CBA/CaJ mice and a transgenic mouse line, NF107, crossed against the Ai32 line. For each cell, responses to current injections were analyzed for spike rate, spike shape, input resistance, resting membrane potential, membrane time constant, hyperpolarization-activated sag and time constant. Cells were filled with dye for morphological classification, and visually classified according to published accounts. The different morphological classes of cells were separated with the LDA. Ventral cochlear nucleus (VCN) bushy cells, planar multipolar (T-stellate) cells, and radiate multipolar (D-stellate) cells were in separate clusters and separate from all of the neurons from the dorsal cochlear nucleus (DCN). Within the DCN, the pyramidal cells and tuberculoventral cells were largely separated from a distinct cluster of cartwheel cells. principal axes, whereas VCN cells were in 3 clouds approximately orthogonal to this plane. VCN neurons from the two mouse strains overlapped but were slightly separated, indicating either a strain dependence or differences in slice preparation methods. We conclude that cochlear nucleus neurons can be objectively distinguished based on their intrinsic electrical properties, but such distinctions are still best aided by morphological identification.

Cholinergic responses of acoustically-characterized cochlear nucleus neurons: An in vivo iontophoretic study in Guinea pig

The responses of guinea pig cochlear nucleus neurons to in vivo iontophoretic application of various neurotransmitter agonists were recorded with extracellular multi-barrelled electrodes. Where possible, neurons were physiologically identified using strict criteria. Emphasis was placed on the action of cholinergic agonists in relation to the possible action of olivocochlear collateral innervation. Excitatory responses (increase in action potential firing) to glutamate were confirmed in a number of neuronal response types. Application of acetylcholine (ACh) or the broad spectrum cholinergic agonist carbachol produced reliable excitatory responses in about 47% of neurons (n = 29 out of 61 neurons). The remaining neurons were unresponsive to cholinergic agonists and no inhibitory responses were observed. Cholinergic responses were more common in dorsal cochlear nucleus (DCN) (73% of 30 neurons tested) than in ventral cochlear nucleus (VCN) (23% of 31 neurons). Of the total neuron sample in which cholinergic responses were investigated, 41 neurons were able to be categorized according to established acoustic response features. Excitatory responses to cholinergic agonists were seen in “Pauser-buildup” (Pb) and “Transient chopper” (Ct) response types. Primary-like neurons (PL and Pn) as well as “Onset chopper” (Oc) neurons (n = 6) were unresponsive to either ACh or carbachol. Oc neurons also did not show any effect on their acoustic responses. Robust cholinergic responses were also seen in several VCN and DCN neurons that were either unresponsive to sound, or had acoustic response properties that did not fit standard classification. The results suggest a relatively more robust cholinergic innervation of DCN compared to VCN. The excitatory cholinergic responses of some Ct neurons and the lack of effect on Oc neurons are consistent with previous results in mouse brain slice studies, but are in conflict with reports of medial olivocochlear collateral excitatory responses in onset-type neurons in vivo. The results also indicate that a number of neurons of unknown identity may also receive cholinergic input.

Cholinergic innervation of principal neurons in the cochlear nucleus of the Mongolian gerbil.

Principal neurons in the ventral cochlear nucleus (VCN) receive powerful ascending excitation and pass on the auditory information with exquisite temporal fidelity. Despite being dominated by ascending inputs, the VCN also receives descending cholinergic connections from olivocochlear neurons and from higher regions in the pontomesencephalic tegmentum. In Mongolian gerbils, acetylcholine acts as an excitatory and modulatory neurotransmitter on VCN neurons, but the anatomical structure of cholinergic innervation of gerbil VCN is not well described. We applied fluorescent immunohistochemical staining to elucidate the development and the cellular localization of presynaptic and postsynaptic components of the cholinergic system in the VCN of the Mongolian gerbil. We found that cholinergic fibers (stained with antibodies against the vesicular acetylcholine transporter) were present before hearing onset at P5, but innervation density increased in animals after P10. Early in development cholinergic fibers invaded the VCN from the medial side, spread along the perimeter and finally innervated all parts of the nucleus only after the onset of hearing. Cholinergic fibers ran in a rostro-caudal direction within the nucleus and formed en-passant swellings in the neuropil between principal neurons. Nicotinic and muscarinic receptors were expressed differentially in the VCN, with nicotinic receptors being mostly expressed in dendritic areas while muscarinic receptors were located predominantly in somatic membranes. These anatomical data support physiological indications that cholinergic innervation plays a role in modulating information processing in the cochlear nucleus.

NAD+ Attenuates Bilirubin-Induced Hyperexcitation in the Ventral Cochlear Nucleus by Inhibiting Excitatory Neurotransmission and Neuronal Excitability.

Nicotinamide adenine dinucleotide (NAD+) is an important molecule with extensive biological functions in various cellular processes, including protection against cell injuries. However, little is known regarding the roles of NAD+ in neuronal excitation and excitotoxicity associated with many neurodegenerative disorders and diseases. Using patch-clamp recordings, we studied its potential effects on principal neurons in the ventral cochlear nucleus (VCN), which is particularly vulnerable to bilirubin excitotoxicity. We found that NAD+ effectively decreased the size of evoked excitatory postsynaptic currents (eEPSCs), increased paired-pulse ratio (PPR) and reversed the effect of bilirubin on eEPSCs, implicating its inhibitory effects on the presynaptic release probability (Pr). Moreover, NAD+ not only decreased the basal frequency of miniature EPSCs (mEPSCs), but also reversed bilirubin-induced increases in the frequency of mEPSCs without affecting their amplitude under either condition. Furthermore, we found that NAD+ decreased the frequency of spontaneous firing of VCN neurons as well as bilirubin-induced increases in firing frequency. Whole-cell current-clamp recordings showed that NAD+ could directly decrease the intrinsic excitability of VCN neurons in the presence of synaptic blockers, suggesting NAD+ exerts its actions in both presynaptic and postsynaptic loci. Consistent with these observations, we found that the latency of the first postsynaptic spike triggered by high-frequency train stimulation of presynaptic afferents (i.e., the auditory nerve) was prolonged by NAD+. These results collectively indicate that NAD+ suppresses presynaptic transmitter release and postsynaptic excitability, jointly weakening excitatory neurotransmission. Our findings provide a basis for the exploration of NAD+ for the prevention and treatment of bilirubin encephalopathy and excitotoxicity associated with other neurological disorders.

Ephrin-A2 and ephrin-A5 guide contralateral targeting but not topographic mapping of ventral cochlear nucleus axons.

BackgroundIn the auditory brainstem, ventral cochlear nucleus (VCN) axons project to the contralateral, but not ipsilateral, medial nucleus of trapezoid body (MNTB), terminating in the calyx of Held. Dorsal VCN neurons, representing high frequencies, synapse with medial MNTB neurons, while low frequency-coding ventral VCN neurons synapse with lateral MNTB neurons, reflecting tonotopic organization. The mechanisms that ensure strictly contralateral targeting and topographic ordering are incompletely understood. Here we examined the roles of ephrin-A signaling in both types of targeting.ResultsEphrin-A2 and ephrin-A5 are expressed in VCN cells during late embryonic and early postnatal development. At these ages ephrin-A2 is expressed in axons surrounding MNTB and ephrin-A5 is expressed in MNTB principal neurons. Ephrin-A2/A5 double knockout mice displayed axon targeting errors in which VCN axons project to MNTB on both sides of the brainstem, where they terminate in calyceal endings. Ephrin-A2 and ephrin-A5 single knockout mice showed a similar phenotype. In contrast to effects on contralateral targeting, ephrin-A2/A5 double knockout mice showed no defects in formation of tonotopically ordered projections from VCN to MNTB.ConclusionsThese findings demonstrate that distinct mechanisms regulate targeting of VCN axons to the contralateral MNTB and targeting to appropriate tonotopic locations. Ephrin-A signaling plays a similar role to ephrin-B signaling in the VCN-MNTB pathway, where both classes normally prevent formation of calyceal projections to ipsilateral MNTB. These classes may rely in part on common signaling pathways.

Heterogeneous Calretinin Expression in the Avian Cochlear Nucleus Angularis

Multiple calcium-binding proteins (CaBPs) are expressed at high levels and in complementary patterns in the auditory pathways of birds, mammals, and other vertebrates, but whether specific members of the CaBP family can be used to identify neuronal subpopulations is unclear. We used double immunofluorescence labeling of calretinin (CR) in combination with neuronal markers to investigate the distribution of CR-expressing neurons in brainstem sections of the cochlear nucleus in the chicken (Gallus gallus domesticus). While CR was homogeneously expressed in cochlear nucleus magnocellularis, CR expression was highly heterogeneous in cochlear nucleus angularis (NA), a nucleus with diverse cell types analogous in function to neurons in the mammalian ventral cochlear nucleus. To quantify the distribution of CR in the total NA cell population, we used antibodies against neuronal nuclear protein (NeuN), a postmitotic neuron-specific nuclear marker. In NA neurons, NeuN label was variably localized to the cell nucleus and the cytoplasm, and the intensity of NeuN immunoreactivity was inversely correlated with the intensity of CR immunoreactivity. The percentage of CR + neurons in NA increased from 31% in embryonic (E)17/18 chicks, to 44% around hatching (E21), to 51% in postnatal day (P) 8 chicks. By P8, the distribution of CR + neurons was uniform, both rostrocaudal and in the tonotopic (dorsoventral) axis. Immunoreactivity for the voltage-gated potassium ion channel Kv1.1, used as a marker for physiological type, showed broad and heterogeneous postsynaptic expression in NA, but did not correlate with CR expression. These results suggest that CR may define a subpopulation of neurons within nucleus angularis.

Minocycline cannot protect neurons against bilirubin-induced hyperexcitation in the ventral cochlear nucleus

Excitotoxicity has been suggested to play an important role in many central nervous system diseases, particularly in bilirubin encephalopathy. Minocycline treatment has been proposed to be one of the most promising potential therapies for excitotoxicity-induced neurological disorders. However, some key questions, such as the electrophysiological effect of minocycline on neuronal excitability and hyperexcitation in pathological conditions, require clarification. In this study, using patch-clamp techniques, we showed that bilirubin increased the frequency of both spontaneous excitatory postsynaptic currents (sEPSCs) and neuronal firing in isolated ventral cochlear nucleus (VCN) neurons at postnatal days 11–14 (P11–14) in rats but it did not affect the amplitude of sEPSCs or glutamate-activated (IGlu) currents. However, minocycline had no effect on sEPSC frequency or IGlu amplitude. Furthermore, minocycline pretreatment did not abolish bilirubin-induced sEPSC potentiation or neuron firing. These data suggest that minocycline does not affect excitatory synaptic transmission or hyperexcitation induced by bilirubin in VCN neurons. From these results, we propose that the neuroprotective efficacy of minocycline, if it can protect neurons against neurotoxicity induced by substances like bilirubin, is mediated by either an alternative mechanism or downstream events post neuronal hyperexcitation. Certainly, additional investigation of the neuroprotective effects of minocycline is required before embarking on further clinical trials.

Embryonic assembly of auditory circuits: spiral ganglion and brainstem

During early development, peripheral sensory systems generate physiological activity prior to exposure to normal environmental stimuli. This activity is thought to facilitate maturation of these neurons and their connections, perhaps even promoting efficacy or modifying downstream circuitry. In the mammalian auditory system, initial connections form at embryonic ages, but the functional characteristics of these early neural connections have not been assayed. We investigated processes of embryonic auditory development using a whole-head slice preparation that preserved connectivity between peripheral and brainstem stations of the auditory pathway. Transgenic mice expressing fluorescent protein provided observation of spiral ganglion and cochlear nucleus neurons to facilitate targeted electrophysiological recording. Here we demonstrate an apparent peripheral-to-central order for circuit maturation. Spiral ganglion cells acquire action potential-generating capacity at embryonic day 14 (E14), the earliest age tested, and action potential waveforms begin to mature in advance of comparable states for neurons of the ventral cochlear nucleus (VCN) and medial nucleus of the trapezoid body (MNTB). In accordance, auditory nerve synapses in the VCN are functional at E15, prior to VCN connectivity with the MNTB, which occurs at least 1 day later. Spiral ganglion neurons exhibit spontaneous activity at least by E14 and are able to drive third-order auditory brainstem neurons by E17. This activity precedes cochlear-generated wave activity by 4 days and ear canal opening by at least 2 weeks. Together, these findings reveal a previously unknown initial developmental phase for auditory maturation, and further implicate the spiral ganglion as a potential controlling centre in this process.

Relationship between noise-induced hearing-loss, persistent tinnitus and growth-associated protein-43 expression in the rat cochlear nucleus: does synaptic plasticity in ventral cochlear nucleus suppress tinnitus?

Aberrant, lesion-induced neuroplastic changes in the auditory pathway are believed to give rise to the phantom sound of tinnitus. Noise-induced cochlear damage can induce extensive fiber growth and synaptogenesis in the cochlear nucleus, but it is currently unclear if these changes are linked to tinnitus. To address this issue, we unilaterally exposed nine rats to narrow-band noise centered at 12 kHz at 126 dB sound pressure level (SPL) for 2 h and sacrificed them 10 weeks later for evaluation of synaptic plasticity (growth-associated protein 43 [GAP-43] expression) in the cochlear nucleus. Noise-exposed rats along with three age-matched controls were screened for tinnitus-like behavior with gap prepulse inhibition of the acoustic startle (GPIAS) before, 1-10 days after, and 8-10 weeks after the noise exposure. All nine noise-exposed rats showed similar patterns of severe hair cell loss at high- and mid-frequency regions in the exposed ear. Eight of the nine showed strong up-regulation of GAP-43 in auditory nerve fibers and pronounced shrinkage of the ventral cochlear nucleus (VCN) on the noise-exposed side, and strong up-regulation of GAP-43 in the medial ventral VCN, but not in the lateral VCN or the dorsal cochlear nucleus. GAP-43 up-regulation in VCN was significantly greater in Noise-No-Tinnitus rats than in Noise-Tinnitus rats. One Noise-No-Tinnitus rat showed no up-regulation of GAP-43 in auditory nerve fibers and only little VCN shrinkage, suggesting that auditory nerve degeneration plays a role in tinnitus generation. Our results suggest that noise-induced tinnitus is suppressed by strong up-regulation of GAP-43 in the medial VCN. GAP-43 up-regulation most likely originates from medial olivocochlear neurons. Their increased excitatory input on inhibitory neurons in VCN may possibly reduce central hyperactivity and tinnitus.

Hyperactivity in the Ventral Cochlear Nucleus after Cochlear Trauma

The emergence of hyperactivity in the form of elevated spontaneous firing rates after cochlear trauma has been well documented in a number of central auditory structures, including the auditory cortex, inferior colliculus, and dorsal subdivision of the cochlear nucleus. This hyperactivity is of interest as a possible neural substrate of tinnitus. Whether the ventral subdivision of the cochlear nucleus shows hyperactivity has never been investigated despite the fact that, like the dorsal division, it also receives direct input from the damaged cochlea and supplies major ascending inputs to brainstem and midbrain auditory centers. We investigated spontaneous neuronal firing rates in the ventral cochlear nucleus in a guinea pig model of cochlear trauma in which we have shown that hyperactivity consistently develops in the inferior colliculus (Mulders and Robertson, 2009). The mean spontaneous firing rates of ventral cochlear nucleus neurons was significantly elevated compared to sham controls. This hyperactivity was more evident in primary-like and onset categories of neurons. Hyperactivity in the ventral subdivision of cochlear nucleus therefore needs to be considered in relation to neural models of the genesis of tinnitus.

Bilirubin facilitates depolarizing GABA/glycinergic synaptic transmission in the ventral cochlear nucleus of rats

Excitotoxicity contributes to bilirubin-induced central nervous system injury; however, the mechanisms involved remain controversial. Previous studies from our lab have demonstrated that in juvenile rats bilirubin faciliates γ-aminobutyric acid (GABA)/glycinergic synaptic transmission through activation of presynaptic protein kinase A (PKA) in isolated neurons of the ventral cochlear nucleus (VCN). However, the descending mechanism and physiological effects of bilirubin-induced potentiation remain unclear. Here, whole-cell recordings show that 3×10−6M bilirubin increased the frequency of both spontaneous (sPSCs) and miniature (mPSCs) GABA/glycinergic postsynaptic currents in VCN neurons of postnatal day 12–14 (P12–14) rats. This action was dependent on the concentration and duration of exposure to bilirubin and was only partially suppressed by 10−5M bicuculline. The potentiation effect on mPSCs persisted in a Ca2+-free solution, but was fully occluded by pretreatment with 1,2 bis-(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM), an intracellular Ca2+ chelator. Following pretreatment of the neurons with BAPTA-AM, forskolin, a PKA activator, had no effect on the frequency or amplitude of mPSCs. This suggests that Ca2+ release from presynaptic stores is part of the descending pathway of PKA activation and is responsible for biliurbin-induced potentiation of cell activity. Using gramicidin-perforated patch recordings, the reversal potential of GABA-evoked currents (EGABA) was also investigated. The GABA response resulted in depolarization of 12 of 20 recorded VCN neurons from P12–14 rats. Therefore, potentiation of depolarizing GABA/glycinergic transmission by bilirubin may underlie bilirubin excitotoxicity, which may play a role in the hearing impairment observed among hyperbilirubinemic neonates.

Low-voltage activated Kv1.1 subunits are crucial for the processing of sound source location in the lateral superior olive in mice

Voltage-gated potassium (Kv) channels containing Kv1.1 subunits are strongly expressed in neurons that fire temporally precise action potentials (APs). In the auditory system, AP timing is used to localize sound sources by integrating interaural differences in time (ITD) and intensity (IID) using sound arriving at both cochleae. In mammals, the first nucleus to encode IIDs is the lateral superior olive (LSO), which integrates excitation from the ipsilateral ventral cochlear nucleus and contralateral inhibition mediated via the medial nucleus of the trapezoid body. Previously we reported that neurons in this pathway show reduced firing rates, longer latencies and increased jitter in Kv1.1 knockout (Kcna1−/−) mice. Here, we investigate whether these differences have direct impact on IID processing by LSO neurons. Single-unit recordings were made from LSO neurons of wild-type (Kcna1+/+) and from Kcna1−/− mice. IID functions were measured to evaluate genotype-specific changes in integrating excitatory and inhibitory inputs. In Kcna1+/+ mice, IID sensitivity ranged from +27 dB (excitatory ear more intense) to −20 dB (inhibitory ear more intense), thus covering the physiologically relevant range of IIDs. However, the distribution of IID functions in Kcna1−/− mice was skewed towards positive IIDs, favouring ipsilateral sound positions. Our computational model revealed that the reduced performance of IID encoding in the LSO of Kcna1−/− mice is mainly caused by a decrease in temporal fidelity along the inhibitory pathway. These results imply a fundamental role for Kv1.1 in temporal integration of excitation and inhibition during sound source localization.

Vesicular glutamate transporters: spatio-temporal plasticity following hearing loss

An immunocytochemical comparison of vGluT1 and vGluT3 in the cochlear nucleus (CN) of deafened versus normal hearing rats showed the first example of vGluT3 immunostaining in the dorsal and ventral CN and revealed temporal and spatial changes in vGluT1 localization in the CN after cochlear injury. In normal hearing rats vGluT1 immunostaining was restricted to terminals on CN neurons while vGluT3 immunolabeled the somata of the neurons. This changed in the ventral cochlear nucleus (VCN) 3 days following deafness, where vGluT1 immunostaining was no longer seen in large auditory nerve terminals but was instead found in somata of VCN neurons. In the dorsal cochlear nucleus (DCN), while vGluT1 labeling of terminals decreased, there was no labeling of neuronal somata. Therefore, loss of peripheral excitatory input results in co-localization of vGluT1 and vGluT3 in VCN neuronal somata. Postsynaptic glutamatergic neurons can use retrograde signaling to control their presynaptic inputs and these results suggest vGluTs could play a role in regulating retrograde signaling in the CN under different conditions of excitatory input. Changes in vGluT gene expression in CN neurons were found 3 weeks following deafness using qRT-PCR with significant increases in vGluT1 gene expression in both ventral and dorsal CN while vGluT3 gene expression decreased in VCN but increased in DCN.

Protein kinase A and C signaling induces bilirubin potentiation of GABA/glycinergic synaptic transmission in rat ventral cochlear nucleus neurons

Previous studies have suggested that bilirubin can potentiate GABA/glycinergic synaptic transmission in lateral superior olivary nucleus neurons, but the cellular mechanism has not been defined. The present study evaluated the possible roles of protein kinase A (PKA) and C (PKC) in bilirubin potentiation of GABA/glycinergic synaptic transmission in rat ventral cochlear nucleus (VCN) neurons. VCN neurons were acutely isolated from postnatal 10-12-day-old (P10-12) rats and were voltage-clamped in whole-cell mode. Miniature inhibitory postsynaptic currents (mIPSC) frequencies, but not amplitude, were increased by bilirubin. Forskolin (PKA activator) and H-89 (PKA inhibitor) also individually increased mIPSCs frequency, with an additional increase induced by co-incubation with bilirubin and H-89. Pretreatment with forskolin blocked bilirubin potentiation. mIPSC frequency was not altered by phorbol 12,13-diacetate (PKC activator), but mIPSC frequency was increased following co-application of bilirubin. The mIPSC frequency was increased by chelerythrine (PKC inhibitor), and then further increased after the addition of bilirubin. Neither H-89, forskolin, nor PDA, nor their co-application with bilirubin affected mIPSC amplitudes of GABA-activated (I GABA)/glycine-activated (I gly) currents, suggesting a presynaptic locus of activity. Chelerythrine decreased the mIPSC amplitudes and I GABA/I gly, suggesting a postsynaptic locus of activity. These data suggest that both PKA and PKC can modulate GABA and glycine release in rat VCN neurons. Bilirubin facilitates transmitter release via presynaptic PKA activation, which might provide insight into the cellular mechanism underlying bilirubin-induced hearing dysfunction.

Differences in glutamate-mediated calcium responses in the ventral cochlear nucleus and inferior colliculus of the developing rat

Using optical recordings of neuronal [Ca2+]i in brain slices from young rats (P9-P11), we report distinct regulation of Ca2+ signalling mediated by the activity of glutamate receptors in the ventral cochlear nucleus (VCN) and the central nucleus of the inferior colliculus (CIC) in the midbrain. [Ca2+]i increases were recorded after bath-stimulation of slices with glutamate agonists of both ionotropic (AMPA/kainate or NMDA) and group I metabotropic glutamate receptors (mGluRs).NMDA-induced [Ca2+]i responses were similar in both auditory nuclei. Kainate-induced [Ca2+]i increases recorded in the VCN were over two-fold larger than those in the CIC. Blockade of kainate-induced [Ca2+]i responses in VCN neurons with 1-naphtylacetyl spermine (NAS) demonstrated that Ca2+-permeable AMPA receptors predominated in the VCN. In contrast, abundant Ca2+-impermeable AMPA receptors were found in the CIC.Both mGluR1 and mGluR5 subtypes of group I mGluRs were present in the CIC and the VCN. However, Group I mGluR [Ca2+]i responses elicited by DHPG were two fold higher in CIC than in VCN neurons. Therefore, our findings suggest that Ca2+ signalling in auditory neurons may be differentially regulated at different levels of the auditory pathway through preferential activation of different classes of glutamate receptors, which may have implications for hierarchical auditory signal processing and plasticity, at least during the early developmental stages of hearing.

Deficiency of neural recognition molecule NB-2 affects the development of glutamatergic auditory pathways from the ventral cochlear nucleus to the superior olivary complex in mouse

Neural recognition molecule NB-2/contactin 5 is expressed transiently during the first postnatal week in glutamatergic neurons of the central auditory system. Here, we investigated the effect of NB-2 deficiency on the auditory brainstem in mouse. While almost all principal neurons are wrapped with the calyces of Held in the medial nucleus of the trapezoid body (MNTB) in wild type, 8% of principal neurons in NB-2 knockout (KO) mice lack the calyces of Held at postnatal day (P) 6. At P10 and P15, apoptotic principal neurons were detected in NB-2 KO mice, but not in wild type. Apoptotic cells were also increased in the ventral cochlear nucleus (VCN) of NB-2 KO mice, which contains bushy neurons projecting to the MNTB and the lateral superior olive (LSO). At the age of 1 month, the number of principal neurons in the MNTB and of glutamatergic synapses in the LSO was reduced in NB-2 KO mice. Finally, interpeak latencies for auditory brainstem response waves II-III and III-IV were significantly increased in NB-2 KO mice. Together, these findings suggest that NB-2 deficiency causes a deficit in synapse formation and then induces apoptosis in MNTB and VCN neurons, affecting auditory brainstem function.