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Globular bushy cells (GBCs) of the cochlear nucleus play central roles in the temporal processing of sound. Despite investigation over many decades, fundamental questions remain about their dendrite structure, afferent innervation, and integration of synaptic inputs. Here, we use volume electron microscopy (EM) of the mouse cochlear nucleus to construct synaptic maps that precisely specify convergence ratios and synaptic weights for auditory- nerve innervation and accurate surface areas of all postsynaptic compartments. Detailed biophysically-based compartmental models can help develop hypotheses regarding how GBCs integrate inputs to yield their recorded responses to sound. We established a pipeline to export a precise reconstruction of auditory nerve axons and their endbulb terminals together with high-resolution dendrite, soma, and axon reconstructions into biophysically-detailed compartmental models that could be activated by a standard cochlear transduction model. With these constraints, the models predict auditory nerve input profiles whereby all endbulbs onto a GBC are subthreshold (coincidence detection mode), or one or two inputs are suprathreshold (mixed mode). The models also predict the relative importance of dendrite geometry, soma size, and axon initial segment length in setting action potential threshold and generating heterogeneity in sound-evoked responses, and thereby propose mechanisms by which GBCs may homeostatically adjust their excitability. Volume EM also reveals new dendritic structures and dendrites that lack innervation. This framework defines a pathway from subcellular morphology to synaptic connectivity, and facilitates investigation into the roles of specific cellular features in sound encoding. We also clarify the need for new experimental measurements to provide missing cellular parameters, and predict responses to sound for further in vivo studies, thereby serving as a template for investigation of other neuron classes.

Dendritic spines in the developing mammalian neocortex are initially overproduced and then eliminated during adolescence to achieve appropriate levels of excitation in mature networks. We show here that the L1 family cell adhesion molecule Close Homolog of L1 (CHL1) and secreted repellent ligand Semaphorin 3B (Sema3B) function together to induce dendritic spine pruning in developing cortical pyramidal neurons. Loss of CHL1 in null mutant mice in both genders resulted in increased spine density and a greater proportion of immature spines on apical dendrites in the prefrontal and visual cortex. Electron microscopy showed that excitatory spine synapses with postsynaptic densities were increased in the CHL1-null cortex, and electrophysiological recording in prefrontal slices from mutant mice revealed deficiencies in excitatory synaptic transmission. Mechanistically, Sema3B protein induced elimination of spines on apical dendrites of cortical neurons cultured from wild-type but not CHL1-null embryos. Sema3B was secreted by the cortical neuron cultures, and its levels increased when cells were treated with the GABA antagonist gabazine. In vivo CHL1 was coexpressed with Sema3B in pyramidal neuron subpopulations and formed a complex with Sema3B receptor subunits Neuropilin-2 and PlexinA4. CHL1 and NrCAM, a closely related L1 adhesion molecule, localized primarily to distinct spines and promoted spine elimination to Sema3B or Sema3F, respectively. These results support a new concept in which selective spine elimination is achieved through different secreted semaphorins and L1 family adhesion molecules to sculpt functional neural circuits during postnatal maturation.SIGNIFICANCE STATEMENT Dendritic spines in the mammalian neocortex are initially overproduced and then pruned in adolescent life through unclear mechanisms to sculpt maturing cortical circuits. Here, we show that spine and excitatory synapse density of pyramidal neurons in the developing neocortex is regulated by the L1 adhesion molecule, Close Homolog of L1 (CHL1). CHL1 mediated spine pruning in response to the secreted repellent ligand Semaphorin 3B and associated with receptor subunits Neuropilin-2 and PlexinA4. CHL1 and related L1 adhesion molecule NrCAM localized to distinct spines, and promoted spine elimination to Semaphorin 3B and -3F, respectively. These results support a new concept in which selective elimination of individual spines and nascent synapses can be achieved through the action of distinct secreted semaphorins and L1 adhesion molecules.

Parvalbumin (PV)-expressing basket interneurons in the prefrontal cortex (PFC) regulate pyramidal cell firing, synchrony, and network oscillations. Yet, it is unclear how their perisomatic inputs to pyramidal neurons are integrated into neural circuitry and adjusted postnatally. Neural cell adhesion molecule NCAM is expressed in a variety of cells in the PFC and cooperates with EphrinA/EphAs to regulate inhibitory synapse density. Here, analysis of a novel parvalbumin (PV)-Cre: NCAM F/F mouse mutant revealed that NCAM functions presynaptically in PV+ basket interneurons to regulate postnatal elimination of perisomatic synapses. Mutant mice exhibited an increased density of PV+ perisomatic puncta in PFC layer 2/3, while live imaging in mutant brain slices revealed fewer puncta that were dynamically eliminated. Furthermore, EphrinA5-induced growth cone collapse in PV+ interneurons in culture depended on NCAM expression. Electrophysiological recording from layer 2/3 pyramidal cells in mutant PFC slices showed a slower rise time of inhibitory synaptic currents. PV-Cre: NCAM F/F mice exhibited impairments in working memory and social behavior that may be impacted by altered PFC circuitry. These findings suggest that the density of perisomatic synapses of PV+ basket interneurons is regulated postnatally by NCAM, likely through EphrinA-dependent elimination, which is important for appropriate PFC network function and behavior.

Background In the cochlea, auditory development depends on precise patterns of innervation by afferent and efferent nerve fibers, as well as a stereotyped arrangement of hair and supporting cells. Neuronal cell adhesion molecule (NrCAM) is a homophilic cell adhesion molecule that controls diverse aspects of nervous system development, but the function of NrCAM in cochlear development is not well understood. Results Throughout cochlear innervation, NrCAM is detectable on spiral ganglion neuron (SGN) afferent and olivocochlear efferent fibers, and on the membranes of developing hair and supporting cells. Neonatal Nrcam-null cochleae show errors in type II SGN fasciculation, reduced efferent innervation, and defects in the stereotyped packing of hair and supporting cells. Nrcam loss also leads to dramatic changes in the profiles of presynaptic afferent and efferent synaptic markers at the time of hearing onset. Despite these numerous developmental defects, Nrcam-null adults do not show defects in auditory acuity, and by postnatal day 21, the developmental deficits in ribbon synapse distribution and sensory domain structure appear to have been corrected. Conclusions NrCAM is expressed by several neural and sensory epithelial subtypes within the developing cochlea, and the loss of Nrcam confers numerous, but nonpermanent, developmental defects in innervation and sensory domain patterning. Developmental Dynamics 247:934-950, 2018. © 2018 Wiley Periodicals, Inc.

Models of the auditory brainstem have been an invaluable tool for testing hypotheses about auditory information processing and for highlighting the most important gaps in the experimental literature. Due to the complexity of the auditory brainstem, and indeed most brain circuits, the dynamic behavior of the system may be difficult to predict without a detailed, biologically realistic computational model. Despite the sensitivity of models to their exact construction and parameters, most prior models of the cochlear nucleus have incorporated only a small subset of the known biological properties. This confounds the interpretation of modelling results and also limits the potential future uses of these models, which require a large effort to develop. To address these issues, we have developed a general purpose, biophysically detailed model of the cochlear nucleus for use both in testing hypotheses about cochlear nucleus function and also as an input to models of downstream auditory nuclei. The model implements conductance-based Hodgkin-Huxley representations of cells using a Python-based interface to the NEURON simulator. Our model incorporates most of the quantitatively characterized intrinsic cell properties, synaptic properties, and connectivity available in the literature, and also aims to reproduce the known response properties of the canonical cochlear nucleus cell types. Although we currently lack the empirical data to completely constrain this model, our intent is for the model to continue to incorporate new experimental results as they become available.

Cholinergic systems contribute to the refinement of auditory cortical receptive fields by activating muscarinic acetylcholine receptors (mAChRs). However, the specific cellular and synaptic mechanisms underlying acetylcholine’s effects on cortical circuits are not fully understood. In this study, we investigate the effects of muscarinic receptor modulation on spike-timing dependent plasticity (STDP) at synapses onto layer 2/3 pyramidal neurons in mouse auditory cortex (AC). Synapses onto layer 2/3 pyramidal neurons exhibit a STDP rule for pairing of postsynaptic spike bursts with single presynaptic stimuli. Pre-before-post pairing at +10 ms results in a timing-dependent long-term potentiation (tLTP), whereas pre-before-post pairing at +50 ms intervals, and post-before-pre pairing at -10 to -20 ms produce a timing-dependent long-term depression. We also characterize how mAChR activation affects plasticity at these synapses, focusing on the induction of tLTP. During pre-before-post pairing at +10 ms, mAChR activation by either carbachol or oxotremorine-M suppresses tLTP. mAChR activation also reduces the NMDA-receptor dependent synaptically evoked increase in calcium in dendrites, apparently without affecting presynaptic transmitter release. Pharmacological experiments suggest that M1 and M3 receptors are not involved in the mAChR-mediated suppression of tLTP. Taken together, these results suggest activating mAChRs in layer 2/3 intracortical circuits can modify the circuit dynamics of AC by depressing tLTP mediated by NMDA receptors, and depressing calcium influx at excitatory synapses onto layer 2/3 pyramidal cells.

Key points Synaptic transmission at the endbulb of Held was assessed by whole-cell patch clamp recordings from auditory neurons in mature (2-4 months) and aged (20-26 months) mice. Synaptic transmission is degraded in aged mice, which may contribute to the decline in neural processing of the central auditory system during age-related hearing loss. The changes in synaptic transmission in aged mice can be partially rescued by improving calcium buffering, or decreasing action potential-evoked calcium influx. These experiments suggest potential mechanisms, such as regulating intraterminal calcium, that could be manipulated to improve the fidelity of transmission at the aged endbulb of Held. Abstract Age-related hearing loss (ARHL) is associated with changes to the auditory periphery that raise sensory thresholds and alter coding, and is accompanied by alterations in excitatory and inhibitory synaptic transmission, and intrinsic excitability in the circuits of the central auditory system. However, it remains unclear how synaptic transmission changes at the first central auditory synapses during ARHL. Using mature (2-4 months) and old (20-26 months) CBA/CaJ mice, we studied synaptic transmission at the endbulb of Held. Mature and old mice showed no difference in either spontaneous quantal synaptic transmission or low frequency evoked synaptic transmission at the endbulb of Held. However, when challenged with sustained high frequency stimulation, synapses in old mice exhibited increased asynchronous transmitter release and reduced synchronous release. This suggests that the transmission of temporally precise information is degraded at the endbulb during ARHL. Increasing intraterminal calcium buffering with EGTA-AM or decreasing calcium influx with ω-agatoxin IVA decreased the amount of asynchronous release and restored synchronous release in old mice. In addition, recovery from depression following high frequency trains was faster in old mice, but was restored to a normal time course by EGTA-AM treatment. These results suggest that intraterminal calcium in old endbulbs may rise to abnormally high levels during high rates of auditory nerve firing, or that calcium-dependent processes involved in release are altered with age. These observations suggest that ARHL is associated with a decrease in temporal precision of synaptic release at the first central auditory synapse, which may contribute to perceptual deficits in hearing.

Hearing loss generally occurs in the auditory periphery but leads to changes in the central auditory system. Noise-induced hearing loss (NIHL) and age-related hearing loss (ARHL) affect neurons in the ventral cochlear nucleus (VCN) at both the cellular and systems levels. In response to a decrease in auditory nerve activity associated with hearing loss, the large synaptic endings of the auditory nerve, the endbulbs of Held, undergo simplification of their structure and the volume of the postsynaptic bushy neurons decreases. A major functional change shared by NIHL and ARHL is the development of asynchronous transmitter release at endbulb synapses during periods of high afferent firing. Compensatory adjustements in transmitter release, including changes in release probability and quantal content, have also been reported. The excitability of the bushy cells undergoes subtle changes in the long-term, although short-term, reversible changes in excitability may also occur. These changes are not consistently observed across all models of hearing loss, suggesting that the time course of hearing loss, and potential developmental effects, may influence endbulb transmission in multiple ways. NIHL can alter the representation of the loudness of tonal stimuli by VCN neurons and is accompanied by changes in spontaneous activity in VCN neurons. However, little is known about the representation of more complex stimuli. The relationship between mechanistic changes in VCN neurons with noise-induced or age-related hearing loss, the accompanying change in sensory coding, and the reversibility of changes with the reintroduction of auditory nerve activity are areas that deserve further thoughtful exploration.

In the cochlea, auditory development depends on precise patterns of innervation by afferent and efferent nerve fibers, as well as a stereotyped arrangement of hair and supporting cells. Neuronal cell adhesion molecule (NrCAM) is a homophilic cell adhesion molecule that controls diverse aspects of nervous system development, but the function of NrCAM in cochlear development is not well understood.Throughout cochlear innervation, NrCAM is detectable on spiral ganglion neuron (SGN) afferent and olivocochlear efferent fibers, and on the membranes of developing hair and supporting cells. Neonatal Nrcam-null cochleae show errors in type II SGN fasciculation, reduced efferent innervation, and defects in the stereotyped packing of hair and supporting cells. Nrcam loss also leads to dramatic changes in the profiles of presynaptic afferent and efferent synaptic markers at the time of hearing onset. Despite these numerous developmental defects, Nrcam-null adults do not show defects in auditory acuity, and by postnatal day 21, the developmental deficits in ribbon synapse distribution and sensory domain structure appear to have been corrected.NrCAM is expressed by several neural and sensory epithelial subtypes within the developing cochlea, and the loss of Nrcam confers numerous, but nonpermanent, developmental defects in innervation and sensory domain patterning. Developmental Dynamics 247:934-950, 2018. © 2018 Wiley Periodicals, Inc.

The cochlear nucleus (CN) transforms the spike trains of spiral ganglion cells into a new 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 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 mathematically separate cells on the basis of both morphology and physiology. Recordings were made from cells in brain slices from CBA 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 (action potential height, afterhyperpolarization depth, first spike half-width), 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 were also separated 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 clusters of cartwheel cells. DCN cells fell largely within a plane in the first 3 principal axes, whereas VCN cells were in 3 clouds approximately orthogonal to this plane. VCN neurons from the two mouse strains were slightly separated, indicating either a strain dependence or the differences in slice preparation methods. We conclude that cochlear nucleus neurons can be objectively distinguished based on their intrinsic electrical properties, but that such distinctions are still best aided by morphological identification.

The neural cell adhesion molecule (NCAM), has been shown to be an obligate regulator of synaptic stability and pruning during critical periods of cortical maturation. However, the functional consequences of NCAM deletion on the organization of inhibitory circuits in cortex are not known. In vesicular gamma-amino butyric acid (GABA) transporter (VGAT)-channelrhodopsin2 (ChR2)-enhanced yellow fluorescent protein (EYFP) transgenic mice, NCAM is expressed postnatally at perisomatic synaptic puncta of EYFP-labeled parvalbumin, somatostatin and calretinin-positive interneurons, and in the neuropil in the anterior cingulate cortex (ACC). To investigate how NCAM deletion affects the spatial organization of inhibitory inputs to pyramidal cells, we used laser scanning photostimulation in brain slices of VGAT-ChR2-EYFP transgenic mice crossed to either NCAM-null or wild type (WT) mice. Laser scanning photostimulation revealed that NCAM deletion increased the strength of close-in inhibitory connections to layer 2/3 pyramidal cells of the ACC. In addition, in NCAM-null mice, the intrinsic excitability of pyramidal cells increased, whereas the intrinsic excitability of GABAergic interneurons did not change. The increase in inhibitory tone onto pyramidal cells, and the increased pyramidal cell excitability in NCAM-null mice will alter the delicate coordination of excitation and inhibition (E/I coordination) in the ACC, and may be a factor contributing to circuit dysfunction in diseases such as schizophrenia and bipolar disorder, in which NCAM has been implicated.

In addition to auditory inputs, dorsal cochlear nucleus (DCN) pyramidal cells in the guinea pig receive and respond to somatosensory inputs and perform multisensory integration. DCN pyramidal cells respond to sounds with characteristic spike-timing patterns that are partially controlled by rapidly inactivating potassium conductances. Deactivating these conductances can modify both spike rate and spike timing of responses to sound. Somatosensory pathways are known to modify response rates to subsequent acoustic stimuli, but their effect on spike timing is unknown. Here, we demonstrate that preceding tonal stimulation with spinal trigeminal nucleus (Sp5) stimulation significantly alters the first spike latency, the first interspike interval and the average discharge regularity of firing evoked by the tone. These effects occur whether the neuron is excited or inhibited by Sp5 stimulation alone. Our results demonstrate that multisensory integration in DCN alters spike-timing representations of acoustic stimuli in pyramidal cells. These changes likely occur through synaptic modulation of intrinsic excitability or synaptic inhibition.

The majority of auditory nerve fibers exhibit prominent spontaneous activity in the absence of sound. More than half of all auditory nerve fibers in CBA mice have spontaneous firing rates higher than 20 spikes/s, with some fibers exceeding 100 spikes/s. We tested whether and to what extent endbulb synapses are depressed by activity between 10 and 100 Hz, within the spontaneous firing rates of auditory nerve fibers. In contrast to rate-dependent depression seen at rates >100 Hz, we found that the extent of depression was essentially rate-independent (∼35%) between 10 and 100 Hz. Neither cyclothiazide nor γ-d-glutamylglycine altered the rate-independent depression, arguing against receptor desensitization and/or vesicle depletion as major contributors for the depression. When endbulb synaptic transmission was more than half-blocked with the P/Q Ca(2+) channel blocker ω-agatoxin IVA, depression during 25 and 100 Hz trains was significantly attenuated, indicating P/Q Ca(2+) channel inactivation may contribute to low frequency synaptic depression. Following conditioning with a 100 Hz Poisson train, the EPSC paired-pulse ratio was increased, suggesting a reduced release probability. This in turn should reduce subsequent depletion-based synaptic depression at higher activation rates. To probe whether this conditioning of the synapse improves the reliability of postsynaptic responses, we tested the firing reliability of bushy neurons to 200 Hz stimulation after conditioning the endbulb with a 25 Hz or 100 Hz stimulus train. Although immediately following the conditioning train, bushy cells responded to minimal suprathreshold stimulation less reliably, the firing reliability eventually settled to the same level (

Sensorineural hearing loss during early childhood alters auditory cortical evoked potentials in humans and profoundly changes auditory processing in hearing-impaired animals. Multiple mechanisms underlie the early postnatal establishment of cortical circuits, but one important set of developmental mechanisms relies on the neuromodulator serotonin (5-hydroxytryptamine [5-HT]). On the other hand, early sensory activity may also regulate the establishment of adultlike 5-HT receptor expression and function. We examined the role of 5-HT in auditory cortex by first investigating how 5-HT neurotransmission and 5-HT2 receptors influence the intrinsic excitability of layer II/III pyramidal neurons in brain slices of primary auditory cortex (A1). A brief application of 5-HT (50 μM) transiently and reversibly decreased firing rates, input resistance, and spike rate adaptation in normal postnatal day 12 (P12) to P21 rats. Compared with sham-operated animals, cochlear ablation increased excitability at P12–P21, but all the effects of 5-HT, except for the decrease in adaptation, were eliminated in both sham-operated and cochlear-ablated rats. At P30–P35, cochlear ablation did not increase intrinsic excitability compared with shams, but it did prevent a pronounced decrease in excitability that appeared 10 min after 5-HT application. We also tested whether the effects on excitability were mediated by 5-HT2 receptors. In the presence of the 5-HT2-receptor antagonist, ketanserin, 5-HT significantly decreased excitability compared with 5-HT or ketanserin alone in both sham-operated and cochlear-ablated P12–P21 rats. However, at P30–P35, ketanserin had no effect in sham-operated and only a modest effect cochlear-ablated animals. The 5-HT2-specific agonist 5-methoxy- N, N-dimethyltryptamine also had no effect at P12–P21. These results suggest that 5-HT likely regulates pyramidal cell excitability via multiple receptor subtypes with opposing effects. These data also show that early sensorineural hearing loss affects the ability of 5-HT receptor activation to modulate A1 pyramidal cell excitability.

Objectives/Hypothesis: Auditory nerve synapses in ventral cochlear nucleus end on two principal cell types, bushy and stellate cells. Although the effects of hearing loss on bushy cells have been well studied, little is known about the effects of hearing loss on synaptic input to the stellate cells. Based on prior observations in bushy cells, we hypothesized that noise-induced hearing loss (NIHL) would decrease quantal release onto stellate cells. Study Design: Prospective, randomized animal study. Methods: CBA/CaJ mice were exposed for 2 hours to 98 dB sound pressure level (SPL) 8- to 16-kHz noise to produce a temporary threshold shift (TTS) or 114 dB SPL to produce a permanent threshold shift (PTS). Spontaneous miniature excitatory postsynaptic currents (mEPSCs) were then measured in stellate cells in brain slices of the cochlear nucleus. Results: Click auditory brainstem evoked response thresholds were elevated by 35 dB in both TTS and PTS mice. Spontaneous mEPSC frequency was found to be five-fold higher than normal in stellate cells of TTS mice and three-fold higher in PTS mice. The mEPSC amplitude was also larger in PTS mice. The mEPSC time course was not different between experimental and control groups. Conclusions: The dramatic increase in mEPSC frequency after NIHL was not expected. The increase in mEPSC amplitude in PTS mice suggests a postsynaptic remodeling process. Both of these effects could contribute to increased spontaneous firing in the cochlear nucleus in the absence of sound. Our results also suggest that hearing loss may have different effects at auditory nerve synapses on bushy and stellate cells in the VCN. Laryngoscope, 2010

Individual neurons have been shown to exhibit target cell-specific synaptic function in several brain areas. The time course of the postsynaptic conductances (PSCs) strongly influences the dynamics of local neural networks. Cartwheel cells (CWCs) are the most numerous inhibitory interneurons in the dorsal cochlear nucleus (DCN). They are excited by parallel fiber synapses, which carry polysensory information, and in turn inhibit other CWCs and the main projection neurons of the DCN, pyramidal cells (PCs). CWCs have been implicated in “context-dependent” inhibition, producing either depolarizing (other CWCs) or hyperpolarizing (PCs) post synaptic potentials. In the present study, we used paired whole cell recordings to examine target-dependent inhibition from CWCs in neonatal rat DCN slices. We found that CWC inhibitory postsynaptic potentials (IPSPs) onto PCs are large (1.3 mV) and brief (half-width = 11.8 ms), whereas CWC IPSPs onto other CWCs are small (0.2 mV) and slow (half-width = 36.8 ms). Evoked IPSPs between CWCs exhibit paired-pulse facilitation, while CWC IPSPs onto PCs exhibit paired-pulse depression. Perforated-patch recordings showed that spontaneous IPSPs in CWCs are hyperpolarizing at rest with a mean estimated reversal potential of −67 mV. Spontaneous IPSCs were smaller and lasted longer in CWCs than in PCs, suggesting that the kinetics of the receptors are different in the two cell types. These results reveal that CWCs play a dual role in the DCN. The CWC-CWC network interactions are slow and sensitive to the average rate of CWC firing, whereas the CWC-PC network is fast and sensitive to transient changes in CWC firing.

The endbulb of Held synapses between the auditory nerve fibers (ANF) and cochlear nucleus bushy neurons convey fine temporal information embedded in the incoming acoustic signal. The dynamics of synaptic depression and recovery is a key in regulating synaptic transmission at the endbulb synapse. We studied short-term synaptic depression and recovery in mature (P22-38) CBA mice with stimulation rates that were comparable to sound-driven activities recorded in vivo. Synaptic depression in mature mice is less severe (∼40% at 100 Hz) than reported for immature animals and the depression is predominately due to depletion of releasable vesicles. Recovery from depression depends on the rate of activity and accumulation of intracellular Ca2+ at the presynaptic terminal. With a regular stimulus train at 100 Hz in 2 mM external [Ca2+], the recovery from depletion was slow (τslow, ∼2 s). In contrast, a fast (τfast, ∼25 ms), Ca2+-dependent recovery followed by a slower recovery (τslow, ∼2 s) was seen when stimulus rates or external [Ca2+] increased. In normal [Ca2+], recovery from a 100-Hz Poisson-like train is rapid, suggesting that Poisson-like trains produce a higher internal [Ca2+] than regular trains. Moreover, the fast recovery was slowed by approximately twofold in the presence of calmidazolium, a Ca2+/calmodulin inhibitor. Our results suggest that endbulb synapses from high spontaneous firing rate auditory nerve fibers normally operate in a depressed state. The accelerated synaptic recovery during high rates of activity is likely to ensure that reliable synaptic transmission can be achieved at the endbulb synapse.

Layer 4 (L4) of primary auditory cortex (A1) receives a tonotopically organized projection from the medial geniculate nucleus of the thalamus. However, individual neurons in A1 respond to a wider range of sound frequencies than would be predicted by their thalamic input, which suggests the existence of cross-frequency intracortical networks. We used laser scanning photostimulation and uncaging of glutamate in brain slices of mouse A1 to characterize the spatial organization of intracortical inputs to L4 neurons. Slices were prepared to include the entire tonotopic extent of A1. We find that L4 neurons receive local vertically organized (columnar) excitation from layers 2 through 6 (L6) and horizontally organized excitation primarily from L4 and L6 neurons in regions centered ~300-500 μm caudal and/or rostral to the cell. Excitatory horizontal synaptic connections from layers 2 and 3 were sparse. The origins of horizontal projections from L4 and L6 correspond to regions in the tonotopic map that are approximately an octave away from the target cell location. Such spatially organized lateral connections may contribute to the detection and processing of auditory objects with specific spectral structures.

The bushy cells of the anterior ventral cochlear nucleus (AVCN) preserve or improve the temporal coding of sound information arriving from auditory nerve fibers (ANF). The critical cellular mechanisms entailed in this process include the specialized nerve terminals, the endbulbs of Held, and the membrane conductance configuration of the bushy cell. In one strain of mice (DBA/2J), an early-onset hearing loss can cause a reduction in neurotransmitter release probability, and a smaller and slower spontaneous miniature excitatory postsynaptic current (EPSC) at the endbulb synapse. In the present study, by using a brain slice preparation, we tested the hypothesis that these changes in synaptic transmission would degrade the transmission of timing information from the ANF to the AVCN bushy neuron. We show that the electrical excitability of bushy cells in hearing-impaired old DBA mice was different from that in young, normal-hearing DBA mice. We found an increase in the action potential (AP) firing threshold with current injection; a larger AP afterhyperpolarization; and an increase in the number of spikes produced by large depolarizing currents. We also tested the temporal precision of bushy cell responses to high-frequency stimulation of the ANF. The standard deviation of spikes (spike jitter) produced by ANF-evoked excitatory postsynaptic potentials (EPSPs) was largely unaffected in old DBA mice. However, spike entrainment during a 100-Hz volley of EPSPs was significantly reduced. This was not a limitation of the ability of bushy cells to fire APs at this stimulus frequency, because entrainment to trains of current pulses was unaffected. Moreover, the decrease in entrainment is not attributable to increased synaptic depression. Surprisingly, the spike latency was 0.46 ms shorter in old DBA mice, and was apparently attributable to a faster conduction velocity, since the evoked excitatory postsynaptic current (EPSC) latency was shorter in old DBA mice as well. We also tested the contribution of the low-voltage-activated K+ conductance (g KLV) on the spike latency by using dynamic clamp. Alteration in g KLV had little effect on the spike latency. To test whether these changes in DBA mice were simply a result of continued postnatal maturation, we repeated the experiments in CBA mice, a strain that shows normal hearing thresholds through this age range. CBA mice exhibited no reduction in entrainment or increased spike jitter with age. We conclude that the ability of AVCN bushy neurons to reliably follow ANF EPSPs is compromised in a frequency-dependent fashion in hearing-impaired mice. This effect can be best explained by an increase in spike threshold.

Voltage gated potassium channels play critical roles in determining the responses of auditory brainstem neurons to acoustic stimuli. In the present study, we examined the developmental expression of potassium channels in rat cochlear nucleus. Quantitative RT-PCR revealed that Kv1.1 , Kv1.2 and Kv3.1 showed a monotonic increase in mRNA levels from postnatal days 3-28 (P3-P28), after which mRNA level was relatively constant until P56. In contrast, Kv4.2 mRNA levels were lower on average by a factor of 2 after P28 than before P28. Relative to Kv1.1, Kv3.1 and Kv1.2 mRNA were more abundant before P10 and less abundant thereafter. To address the relationship between message and protein levels, we performed semi-quantitative Western blotting for Kv1.2. The message for Kv1.2 increased earlier in development than the protein levels. Immunocytochemistry revealed a broad expression of Kv1.1 and Kv1.2 in the VCN. Staining intensity increased from 7-28 days postnatal. Kv1.2 immunostaining was less variable across cells than Kv1.1 staining. We conclude that maturation of potassium channel expression in the rat cochlear nucleus continues until at least 4 weeks postnatal.

Many neuronal systems represent information by the timing of individual spikes, and it is generally assumed that spike timing predominantly encodes excitatory inputs. We show here that the timing of inhibition can also be explicitly encoded in spike times using time-dependent and voltage-dependent properties of a rapidly inactivating potassium channel ( IKIF). In vitro recordings in rat dorsal cochlear nucleus show that the effects of inhibition on spike timing can long outlast the duration of the inhibitory potential and that this depends only on the membrane voltage change during the inhibitory postsynaptic potential. Modeling results show that small neuronal populations with a heterogeneous distribution of IKIF voltage dependence can robustly encode intervals of >100 ms between inhibition and excitation. Thus neuronal systems can detect and represent the precise timing of inhibition, suggesting the importance of inhibition in information encoding.

During intracellular recordings in rodent brainstem slice preparations, dorsal cochlear nucleus (DCN) pyramidal cells (PCs) exhibit characteristic discharge patterns to depolarizing current injection that depend on the membrane potential from which the responses are evoked. When depolarized from hyperpolarized potentials, PCs can respond with a short-latency action potential followed by a long silent interval (pauser) or a train of action potentials with a long latency (buildup). During the silent intervals in a pauser or a buildup response, the membrane potential slowly depolarizes towards spike threshold, often exhibiting distinct voltage oscillations of 1-2 mV before the first spike. The subthreshold voltage oscillations were investigated using whole cell recordings from DCN PCs in rat pup (P10-14) brainstem slices. The oscillations were unaffected by excitatory and inhibitory neurotransmitter antagonists, and were not temporally locked to the onset of the depolarization. The oscillations typically became larger as spike threshold was approached, and had a characteristic frequency between 40 and 100 Hz. In the presence of tetrodotoxin (TTX, 500 nM), the oscillations were significantly suppressed, and could not be evoked at any voltage below or above spike threshold. The oscillations were not blocked by phenytoin or Cd2+, but they were affected by prior activity in the neuron for approximately 1 s. We conclude that voltage-gated Na+ channels are required to generate membrane oscillations during the buildup phase. We suggest that the subthreshold oscillations play a role in controlling spike timing in PCs when the membrane potential slowly approaches, or hovers near, spike threshold.

Simultaneous fluorescence imaging and electrophysiologic recordings were used to investigate the Ca2+influx initiated by action potentials (APs) into dorsal cochlear nucleus (DCN) pyramidal cell (PC) and cartwheel cell (CWC) dendrites. Local application of Cd2+blocked Ca2+transients in PC and CWC dendrites, demonstrating that the Ca2+influx was initiated by dendritic Ca2+channels. In PCs, TTX eliminated the dendritic Ca2+transients when APs were completely blocked. However, the Ca2+influx could be partially recovered during an incomplete block of APs or when a large depolarization was substituted for the blocked APs. In CWCs, dendritic Ca2+transients evoked by individual APs, or simple spikes, were blocked by TTX and could be recovered during an incomplete block of APs or by a large depolarization. In contrast, dendritic Ca2+transients evoked by complex spikes, a burst of APs superimposed on a slow depolarization, were not blocked by TTX, despite eliminating the APs superimposed on the slow depolarization. These results suggest two different mechanisms for the retrograde activation of dendritic Ca2+channels: the first requires fast Na+channel-mediated APs or a large somatic depolarization, whereas the second is independent of Na+channel activation, requiring only the slow depolarization underlying complex spikes.

Many cells in the outer two layers of the dorsal cochlear nucleus (DCN) express high levels of the phospholipid-activated, calcium dependent kinase, protein kinase C (PKC), an enzyme that can phosphorylate numerous proteins involved in neurotransmission and postsynaptic signaling. We investigated the effects of stimulating PKC with phorbol esters (phorbol 12-13 diacetate; PDAc) on parallel fiber synaptic transmission in brain slices of the guinea pig DCN. Phorbol esters increased the amplitude of the postsynaptic components of the field potential, including the excitatory post-synaptic field potential (fEPSP) and the population spike following electric stimulation of parallel fibers. Phorbol esters simultaneously decreased paired-pulse facilitation, suggesting that transmitter release mechanisms were affected. Potentiation of synaptic transmission and diminished paired-pulse potentiation were also observed in intracellular recordings of DCN neurons. The effects of phorbol esters were antagonized by the specific PKC blockers bisindolylmaleimide and calphostin C. Although modulation of the synaptic potentials appears to be mediated by presynaptic PKC, the differential effects of PDAc on the fEPSP and the population spike also suggest the involvement of postsynaptic PKC and postsynaptic targets. These experiments demonstrate that protein kinase C is capable of profoundly modulating synaptic transmission at parallel fiber synapses in the DCN.

Both glycine and GABA mediate inhibitory synaptic transmission in the ventral cochlear nucleus (VCN). In mice, the time course of glycinergic inhibition is slow in bushy cells and fast in multipolar (stellate) cells, and is proposed to contribute to the processing of temporal cues in both cell types. Much less is known about GABAergic synaptic transmission in this circuit. Electrical stimulation of the auditory nerve or the tuberculoventral pathway evokes little GABAergic synaptic current in brain slice preparations, and spontaneous GABAergic miniature synaptic currents occur infrequently. To investigate synaptic currents carried by GABA receptors in bushy and multipolar cells, we used transgenic mice in which channelrhodopsin-2 and EYFP is driven by the vesicular GABA transporter (VGAT-ChR2-EYFP) and is expressed in both GABAergic and glycinergic neurons. Light stimulation evoked action potentials in EYFP-expressing presynaptic cells, and evoked inhibitory postsynaptic potentials (IPSPs) in non-expressing bushy and planar multipolar cells. Less than 10% of the IPSP amplitude in bushy cells arose from GABAergic synapses, whereas 40% of the IPSP in multipolar neurons was GABAergic. In voltage clamp, glycinergic IPSCs were significantly slower in bushy neurons than in multipolar neurons, whereas there was little difference in the kinetics of the GABAergic IPSCs between two cell types. During prolonged stimulation, the ratio of steady state versus peak IPSC amplitude was significantly lower for glycinergic IPSCS. Surprisingly, the reversal potentials of GABAergic IPSCs were negative to those of glycinergic IPSCs in both bushy and multipolar neurons. In the absence of receptor blockers, repetitive light stimulation was only able to effectively evoke IPSCs up to 20 Hz in both bushy and multipolar neurons. We conclude that local GABAergic release within the VCN can differentially influence bushy and multipolar cells.

The processes of neuronal differentiation and survival are key questions in neurobiology. The olfactory system possesses unique regenerative capacity, as its neurons are continually replaced throughout adulthood from a maintained population of precursor cells. Primary cultures of olfactory epithelium enriched in olfactory neurons would provide a useful model to study the processes of neurogenesis, differentiation and senescence. To determine whether immature olfactory neurons could be isolated in primary culture and to investigate the mechanisms underlying these processes, culture conditions which selectively favored the presence of immature olfactory neurons were optimized. Using low plating densities, a population of cells was identified which, by reverse transcription–polymerase chain reaction, demonstrated messages for olfactory neuronal markers, including G olf , olfactory cyclic nucleotide-gated channel and olfactory marker protein, as well as the p75 low-affinity nerve growth factor receptor. Immunocytochemical analysis showed that these putative immature olfactory neurons possessed immunoreactivity to G olf , neuron-specific tubulin, neural cell adhesion molecule, synaptophysin and neurofilament. These neurons were defined as olfactory receptor neuron-1 cells. Under these conditions, a separate class of rarely occurring cells with different morphology demonstrated immunoreactivity to mature markers, such as adenylyl cyclase III and olfactory marker protein. Electrophysiologically, these cells displayed properties consistent with those of acutely dissociated olfactory receptor neurons. Another class of rarer cells which represented less than 2% of cells in culture demonstrated immunoreactivity to glial fibrillary acidic protein. These cultures can serve as a model for in vitro analysis of olfactory receptor neuronal development and maintenance, and provide a potential substrate for the development of cell lines.

Pyramidal cells in the dorsal cochlear nucleus (DCN) show three distinct temporal discharge patterns in response to sound: “pauser,” “buildup,” and “chopper.” Similar discharge patterns are seenin vitroand depend on the voltage from which the cell is depolarized. It has been proposed that an inactivating A-type K+current (IKI) might play a critical role in generating the three different patterns. In this study we examined the characteristics of transient currents in DCN pyramidal cells to evaluate this hypothesis. Morphologically identified pyramidal cells in rat brain slices (P11–P17) exhibited the three voltage-dependent discharge patterns. Two inactivating currents were present in outside-out patches from pyramidal cells: a rapidly inactivating (IKIF, τ ∼11 msec) current insensitive to block by tetraethylammonium (TEA) and variably blocked by 4-aminopyridine (4-AP) with half-inactivation near −85 mV, and a slowly inactivating TEA- and 4-AP-sensitive current (IKIS, τ ∼145 msec) with half-inactivation near −35 mV. Recovery from inactivation at 34°C was described by a single exponential with a time constant of 10–30 msec, similar to the rate at which first spike latency increases with the duration of a hyperpolarizing prepulse. Acutely isolated cells also possessed a rapidly activating (IKIFwas correlated with the discharge patterns. Overall, the properties of the fast inactivating K+current were consistent with their proposed role in shaping the discharge pattern of DCN pyramidal cells.

Voltage-gated Ca2+ conductances in acutely isolated guinea pig dorsal cochlear nucleus neurons. Although it is known that voltage-gated Ca2+ conductances (VGCCs) contribute to the responses of dorsal cochlear nucleus (DCN) neurons, little is known about the properties of VGCCs in the DCN. In this study, the whole cell voltage-clamp technique was used to examine the pharmacology and voltage dependence of VGCCs in unidentified DCN neurons acutely isolated from guinea pig brain stem. The majority of cells responded to depolarization with sustained inward currents that were enhanced when Ca2+ was replaced by Ba2+, were blocked partially by Ni2+ (100 μM), and were blocked almost completely by Cd2+ (50 μM). Experiments using nifedipine (10 μM), ωAga IVA (100 nM) and ωCTX GVIA (500 nM) demonstrated that a variety of VGCC subtypes contributed to the Ba2+current in most cells, including the L, N, and P/Q types and antagonist-insensitive R type. Although a large depolarization from rest was required to activate VGCCs in DCN neurons, VGCC activation was rapid at depolarized levels, having time constants 2+ currents were carried by high-voltage activated VGCCs. The VGCC subtypes contributing to the overall Ba2+ current had similar voltage-dependent properties, with the exception of the antagonist-insensitive R-type component, which had a slower activation and a more pronounced inactivation than the other components. These data suggest that a variety of VGCCs is present in DCN neurons, and these conductances generate a rapid Ca2+influx in response to depolarizing stimuli.

The cochlear nuclei are the first central processors of auditory information and provide inputs to all the major brainstem and midbrain auditory nuclei. Although the local circuits within the cochlear nuclei are understood at a cellular level, the spatial patterns of connectivity and the connection strengths in these circuits have been less well characterized. We have applied a novel, quantitative approach to mapping local circuits projecting to cells in the mouse anteroventral cochlear nucleus (AVCN) using laser-scanning photostimulation and glutamate uncaging. The amplitude and kinetics of individual evoked synaptic events were measured to reveal the patterns and strengths of synaptic connections. We found that the two major excitatory projection cell classes, the bushy and T-stellate cells, receive a spatially broad inhibition from D-stellate cells in the AVCN, and a spatially confined inhibition from the tuberculoventral cells of the dorsal cochlear nucleus. Furthermore, T-stellate cells integrate D-stellate inhibition from an area that spans twice the frequency range of that integrated by bushy cells. A subset of both bushy and T-stellate cells receives inhibition from an unidentified cell population at the dorsal–medial boundary of the AVCN. A smaller subset of cells receives local excitation from within the AVCN. Our results show that inhibitory circuits can have target-specific patterns of spatial convergence, synaptic strength, and receptor kinetics, resulting in different spectral and temporal processing capabilities.

The complexity of modern neurophysiology experiments requires specialized software to coordinate multiple acquisition devices and analyze the collected data. We have developed ACQ4, an open-source software platform for performing data acquisition and analysis in experimental neurophysiology. This software integrates the tasks of acquiring, managing, and analyzing experimental data. ACQ4 has been used primarily for standard patch-clamp electrophysiology, laser scanning photostimulation, multiphoton microscopy, intrinsic imaging, and calcium imaging. The system is highly modular, which facilitates the addition of new devices and functionality. The modules included with ACQ4 provide for rapid construction of acquisition protocols, live video display, and customizable analysis tools. Position-aware data collection allows automated construction of image mosaics and registration of images with 3-dimensional anatomical atlases. ACQ4 uses free and open-source tools including Python, NumPy/SciPy for numerical computation, PyQt for the user interface, and PyQtGraph for scientific graphics. Supported hardware includes cameras, patch clamp amplifiers, scanning mirrors, lasers, shutters, Pockels cells, motorized stages, and more. ACQ4 is available for download at http://www.acq4.org.

The ability of mammals to discriminate which ear is the first to receive acoustic stimuli with a resolution of approximately 10 μS is remarkable. It requires precise detection of the acoustic signal by the inner ear and equally precise communication and refinement of the information through at least three synapses prior to dichotic integration in the superior olive. The Jeffress place theory for sound localization was introduced in 1948 and remains a touchstone for research in the area. At its heart are assumptions about the ability of the auditory system to detect, transmit, and process temporal information. More than a half century of research has established that rapid processing of acoustic information in hair cells and brain stem auditory neurons is achieved with surprisingly similar mechanisms having high metabolic demands. Short electrical time constants are required for rapid temporal processing. Hair cells and auditory brain stem neurons have a high conductance at their resting potential that contributes to short time constants, but at the same time leads to large resting currents that have high metabolic demands. The membrane proteins that modulate these currents have a shallow, nonuniform, voltage dependence that results in a broad dynamic range, and further increases metabolic demand. High-speed performance is energetically costly, as is the case with automobiles, and provides the rationale for the high metabolic activity associated with auditory structures. Energetic considerations provide a conceptual bridge between the peripheral and brain stem auditory system.

One of the major contributors to the response profile of neurons in the auditory pathways is the I h current. Its properties such as magnitude, activation, and kinetics not only vary among different types of neurons (Banks et al., J Neurophysiol 70:1420-1432, 1993; Fu et al., J Neurophysiol 78:2235-2245, 1997; Bal and Oertel, J Neurophysiol 84:806-817, 2000; Cao and Oertel, J Neurophysiol 94:821-832, 2005; Rodrigues and Oertel, J Neurophysiol 95:76-87, 2006; Yi et al., J Neurophysiol 103:2532-2543, 2010), but they also display notable diversity in a single population of spiral ganglion neurons (Mo and Davis, J Neurophysiol 78:3019-3027, 1997), the first neural element in the auditory periphery. In this study, we found from somatic recordings that part of the heterogeneity can be attributed to variation along the tonotopic axis because I h in the apical neurons have more positive half-activation voltage levels than basal neurons. Even within a single cochlear region, however, I h current properties are not uniform. To account for this heterogeneity, we provide immunocytochemical evidence for variance in the intracellular density of the hyperpolarization-activated cyclic nucleotide-gated channel α-subunit 1 (HCN1), which mediates I h current. We also observed different combinations of HCN1 and HCN4 α-subunits from cell to cell. Lastly, based on the physiological data, we performed kinetic analysis for the I h current and generated a mathematical model to better understand varied I h on spiral ganglion function. Regardless of whether I h currents are recorded at the nerve terminals (Yi et al., J Neurophysiol 103:2532-2543, 2010) or at the somata of spiral ganglion neurons, they have comparable mean half-activation voltage and induce similar resting membrane potential changes, and thus our model may also provide insights into the impact of I h on synaptic physiology.

The principal inhibitory neurotransmitter in the mammalian cochlear nucleus (CN) is glycine. During age-related hearing loss (AHL), glycinergic inhibition becomes weaker in CN. However, it is unclear what aspects of glycinergic transmission are responsible for weaker inhibition with AHL. We examined glycinergic transmission onto bushy cells of the anteroventral CN in normal-hearing CBA/CaJ mice and in DBA/2J mice, a strain that exhibits an early onset AHL. Glycinergic synaptic transmission was examined in brain slices of mice at 10–15 postnatal days old, 20–35 days old, and at 6–7 mo old. Spontaneous inhibitory postsynaptic current (sIPSC) event frequency and amplitude were the same among all three ages in both strains of mice. However, the amplitudes of IPSCs evoked (eIPSC) from stimulating the dorsal CN were smaller, and the failure rate was higher, with increasing age due to decreased quantal content in both mouse strains, independent of hearing status. The coefficient of variation of the eIPSC amplitude also increased with age. The decay time constant (τ) of sIPSCs and eIPSCs were constant in CBA/CaJ mice at all ages, but were significantly slower in DBA/2J mice at postnatal days 20–35, following the onset of AHL, and not at earlier or later ages. Our results suggest that glycinergic inhibition at the synapses onto bushy cells becomes weaker and less reliable with age through changes in release. However, the hearing loss in DBA/2J mice is accompanied by a transiently enhanced inhibition, which could disrupt the balance of excitation and inhibition.

1. N-methyl-D-aspartate (NMDA) binding and NMDA-receptors immunolocalization experiments have revealed an enhanced expression of these receptors in the outer two layers of the dorsal cochlear nucleus (DCN). The distribution of the receptors is congruent with the distribution of synapses produced by the granule cell-parallel fiber system. To determine the functional distribution and contribution of NMDA receptors at parallel fiber synapses, synaptic responses to parallel fiber stimulation were studied in in vitro brain slice preparations of the guinea pig and rat dorsal cochlear nucleus. 2. The field potential response to parallel fiber stimulation in guinea pigs includes three postsynaptic components. The short latency components (the P3(2) and N2(2)) are blocked by general excitatory receptor antagonists, including the non-NMDA-receptor blockers 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but are insensitive to NMDA-receptor antagonists. 3. A slower component (P4(2)) is revealed when the slices are washed with a low magnesium solution to eliminate the magnesium block of currents through NMDA receptors. This slow component is reduced by D- or DL-2-amino-5-phosphonovaleric acid (D-APV, DL-APV) and 3-[(+/-)-2-carboxypiperazine-4-yl] propyl-1-phosphonate, but is not blocked by DNQX or CNQX. Eliminating the voltage dependence of the NMDA receptors also results in a complex oscillatory response in some slices. This response exhibits the same pharmacological sensitivity as the slow potential. The pharmacologic sensitivity to NMDA-receptor antagonists suggest that the slow component (P4(2)) and the associated oscillatory response are mediated through activation of NMDA receptors. 4. Current source-density analysis of the parallel fiber-evoked field potentials was carried out to determine the relative spatial distributions of the fast and slow synaptic currents. Both synaptic components were associated with a superficial current sink and a deeper current source, localized within the superficial 250 microM of the nucleus. The slow (APV-sensitive) current was slightly shifted in depth relative to the fast (DNQX-sensitive) current in three of five slices with the maximum current sink and source occurring approximately 16 microns further from the surface of the DCN. These data suggest that either the NMDA receptors are not present at all of the synapses that generate the fast non-NMDA currents or that postsynaptic cells with different dendritic distributions have different densities of NMDA receptors. 5. The types of cells in layers 1 and 2 exhibiting NMDA-receptor-mediated synaptic potentials were investigated. Intracellular recordings with sharp electrodes in guinea pig slices showed that eliminating the voltage dependence of the NMDA receptors in low magnesium revealed a slow excitatory postsynaptic potential (EPSP) in both simple and complex spiking cells. The late phase of the EPSP could be reduced by APV in both cell types. These results could be explained by NMDA receptors on the postsynaptic cells or by NMDA receptors on excitatory interneurons. Attempts to demonstrate an appropriate voltage dependence of the parallel fiber synaptic response in normal magnesium medium under current clamp were confounded by the intrinsic voltage-dependent conductances of the cells. 6. To determine whether NMDA receptors were present on postsynaptic cells, the direct sensitivity of DCN cells to NMDA application was examined during intracellular recording. Both simple spiking and complex spiking cells responded to NMDA with depolarization. The response to NMDA persisted when non-NMDA receptors were blocked with CNQX or DNQX. However in all cells tested, the response to NMDA was blocked by APV. These experiments further support the postsynaptic localization of NMDA receptors on both simple and complex spiking cells. (ABSTRACT TRUNCATED)

1. Glycine was applied to acutely dissociated neurons of the guinea pig ventral cochlear nucleus (VCN) with the use of iontophoresis. With approximately equal chloride concentrations in the extra- and intracellular solutions (i.e., chloride equilibrium potential = 0 mV), cells held at -60 mV responded with inward currents that were 1-10 nA in amplitude, had rise times of approximately 50 ms, and decayed to half of the peak amplitude in 50-600 ms. More than 95% of cells with diameters > 12 microns responded to glycine. Response amplitude and area increased with increasing duration of the iontophoretic pulse. Response amplitude saturated at pulse durations of 60-80 ms, whereas response area did not exhibit saturation for pulse durations of 10-100 ms. 2. The glycine antagonist strychnine was added to the extracellular solution at concentrations of 0.5-500 nM to evaluate its effect on glycine-evoked responses. Strychnine produced a 50% reduction in the response at a concentration of 12 nM and the dose-response function had a limiting slope (Hill coefficient) of 1.4. 3. Changes in glycine-evoked currents as a function of cell membrane potential were examined in the presence of tetrodotoxin, tetraethylammonium chloride, and 4-aminopyridine, which block sodium and potassium conductances activated by depolarization. Both the amplitude and the decay of glycine-evoked currents displayed a voltage dependence. Under conditions where the glycine currents reversed at -35 mV, the amplitudes of responses evoked at membrane potentials of 0 mV were 2.3 times larger than those of responses evoked at -70 mV. The decay time constant at 0 mV was 1.49 times longer than that at -70 mV. 4. Acutely dissociated neurons of the VCN previously have been classified on the basis of the absence (type I) or presence (type II) of a low-threshold outward current. Type I cells fire repetitively in response to current pulses, whereas type II cells fire transiently. Glycine-evoked responses were compared in cells identified electrophysiologically as type I or type II on the basis of previously established criteria under voltage clamp. The average amplitudes of responses recorded at a membrane potential of -70 mV were 1.1 and 1.3 nA for type I and type II cells, respectively. The rise time of the glycine current for the two groups of cells was similar (52 ms for type I and 57 ms for type II), but the decay of currents to half-maximum amplitude following the offset of the iontophoretic pulse was longer in type II cells (340 ms) than in type I cells (173 ms). No differences between the two groups were noted with regard to the outward rectification of peak currents or the voltage dependence of current decay. 5. The reversal potential of glycine-evoked responses was determined in extracellular solutions with varying chloride concentrations. The change in the glycine reversal potential (54 mV) for a 10-fold change in chloride concentration was similar to the change in the chloride equilibrium potential (58 mV) over the same range of extracellular chloride concentrations. A similar result was obtained by maintaining the extracellular chloride concentration constant and varying the chloride concentration in the intracellular solution. Glycine-evoked responses were not affected by changes in the potassium or sodium equilibrium potentials. The glycine receptors are therefore principally permeable to chloride. 6. In the VCN, glycine-mediated currents are readily evoked from the majority of larger neurons, indicating an abundance of glycine receptors on the somata and proximal processes of these neurons. The properties of glycine receptors in VCN and other areas of the nervous system are generally similar. The voltage dependence of glycine-evoked currents implies that the inhibitory effectiveness of glycine receptors in VCN increases nonlinearly with depolarization.

The acoustic environment contains biologically relevant information on timescales from microseconds to tens of seconds. The auditory brainstem nuclei process this temporal information through parallel pathways that originate in the cochlear nucleus from different classes of cells. Although the roles of ion channels and excitatory synapses in temporal processing have been well studied, the contribution of inhibition is less well understood. Here, we show in CBA/CaJ mice that the two major projection neurons of the ventral cochlear nucleus, the bushy and T-stellate cells, receive glycinergic inhibition with different synaptic conductance time courses. Bushy cells, which provide precisely timed spike trains used in sound localization and pitch identification, receive slow inhibitory inputs. In contrast, T-stellate cells, which encode slower envelope information, receive inhibition that is eightfold faster. Both types of inhibition improved the precision of spike timing but engage different cellular mechanisms and operate on different timescales. Computer models reveal that slow IPSCs in bushy cells can improve spike timing on the scale of tens of microseconds. Although fast and slow IPSCs in T-stellate cells improve spike timing on the scale of milliseconds, only fast IPSCs can enhance the detection of narrowband acoustic signals in a complex background. Our results suggest that target-specific IPSC kinetics are critical for the segregated parallel processing of temporal information from the sensory environment.

A novel function for the neural cell adhesion molecule (NCAM) was identified in ephrinA/EphA-mediated repulsion as an important regulatory mechanism for development of GABAergic inhibitory synaptic connections in mouse prefrontal cortex. Deletion of NCAM, EphA3, or ephrinA2/3/5 in null mutant mice increased the numbers and size of perisomatic synapses between GABAergic basket interneurons and pyramidal cells in the developing cingulate cortex (layers II/III). A functional consequence of NCAM loss was increased amplitudes and faster kinetics of miniature inhibitory postsynaptic currents in NCAM null cingulate cortex. NCAM and EphA3 formed a molecular complex and colocalized with the inhibitory presynaptic marker vesicular GABA transporter (VGAT) in perisomatic puncta and neuropil in the cingulate cortex. EphrinA5 treatment promoted axon remodeling of enhanced green fluorescent protein-labeled basket interneurons in cortical slice cultures and induced growth cone collapse in wild-type but not NCAM null mutant neurons. NCAM modified with polysialic acid (PSA) was required to promote ephrinA5-induced axon remodeling of basket interneurons in cortical slices, likely by providing a permissive environment for ephrinA5/EphA3 signaling. These results reveal a new mechanism in which NCAM and ephrinAs/EphA3 coordinate to constrain GABAergic interneuronal arborization and perisomatic innervation, potentially contributing to excitatory/inhibitory balance in prefrontal cortical circuitry. © 2012 The Author.

Intracellular recordings from the dorsal cochlear nucleus have identified cells with both simple and complex action potential waveforms. We investigated the hypothesis that cartwheel cells are a specific cell type that generates complex action potentials, based on their analogous anatomical, developmental, and biochemical similarities to cerebellar Purkinje cells, which are known to discharge complex action potentials. Intracellular recordings were made from a brain slice preparation of the guinea pig dorsal cochlear nucleus. A subpopulation of cells discharged a series of two or three action potentials riding on a slow depolarization as an all-or-none event; this discharge pattern is called a complex spike or burst. These cells also exhibited anodal break bursts, anomalous rectification, subthreshold inward rectification, and frequent inhibitory postsynaptic potentials (IPSPs). Seven complex-spiking cells were stained with intracellular dyes and subsequently identified as cartwheel neurons. In contrast, six identified simple-spiking cells recorded in concurrent experiments were pyramidal cells. The cartwheel cell bodies reside in the lower part of layer 1 and the upper part of layer 2 of the nucleus. The cells are characterized by spiny dendrites penetrating the molecular layer, a lack of basal dendritic processes, and an axonal plexus invading layers 2 and 3, and the inner regions of layer 1. The cartwheel cell axons made putative synaptic contacts at the light microscopic level with pyramidal cells and small cells, including stellate cells, granule cells, and other cartwheel cells in layers 1 and 2. The axonal plexus of individual cartwheel cells suggests that they can inhibit cells receiving input from either the same or adjacent parallel fibers and that this inhibition is distributed along the isofrequency contours of the nucleus.

1. Intracellular recordings were obtained from neurons in parasagittal brain slices of the guinea pig ventral cochlear nucleus (VCN). The principal neurons of the VCN can be parceled into two categories. Regular-spiking (Type I) neurons have a linear current-voltage (I–V) relationship over a large range of intracellularly injected currents and fire tonically in response to suprathreshold depolarizing currents. Phasically spiking (Type II) neurons have a nonlinear I–V relationship and fire only phasically at the onset of a depolarizing current or offset of a hyperpolarizing current. Regular-spiking neurons have been shown to be of the stellate morphological type, whereas phasically spiking neurons have been shown to be bushy cells. 2. The electrotonic structure of regular-spiking neurons was studied by applying previously developed modeling techniques based on the somatic shunt model. In these techniques, physiological data are used to determine the set of parameters best describing the neuron. As predicted from previous theoretical investigations, the use of an anatomic constraint (somatic surface area) reduces the variance in estimates of model parameters, especially for the dendritic membrane time constant tau D. 3. Model representations of regular-spiking cells fall into two categories: those with (passive) somatic membrane properties that are nearly identical to those of the dendrite (8/15 cases), and those with a significant amount of somatic shunt (7/15). Estimates of tau D (mean = 7.7 ms) are lower than those often described in the literature. We argue that this low value of tau D may be related to the need of neurons in the auditory brainstem to operate at high firing rates and/or to encode audio-frequency temporal fluctuations. 4. Dendritic transfer functions were calculated as functions of synaptic location using somatic shunt representations of regular-spiking neurons. These transfer functions allow us to predict that mid-range auditory frequencies (approximately 1 kHz) are greatly attenuated, even for synapses near the soma. Thus it is suggested that the electrotonic architecture of regular-spiking cells creates sufficient low-pass filtering of synaptic inputs to reduce the synchronization of firing of these neurons to mid-frequency auditory stimuli.

A remarkable nerve terminal is present at the endings of the auditory nerve fibers (ANFs) in the anterior ventral cochlear nucleus (AVCN), called the “endbulb of Held.” The endbulbs are complex synaptic endings that provide a coordinated release from multiple presynaptic sites of neurotransmitter onto their target postsynaptic cells, the globular and spherical bushy cells of the cochlear nucleus. These synapses play a key role in bringing a precisely timed representation of sound into the central auditory system. Traditionally, the endbulbs, owing to their large size and the presence of multiple presynaptic release zones, were thought to provide a “secure” synapse between auditory nerve fibers and the target neurons, the globular and spherical bushy cells. However, this view has been strongly challenged by several recent observations. While the endbulbs are indeed a particularly strong synapse, they are subject to dynamic regulation of transmitter release probability and receptor sensitivity, and their ability to initiate action potentials in the postsynaptic cell is not immune to postsynaptic inhibition. Integration by convergence of endbulb synapses onto target cells is an important part of central auditory processing. In particular, cells postsynaptic to the endbulbs can fire more precisely to specific temporal features of acoustic stimuli than their individual auditory nerve fiber inputs. The endbulb synapses are found widely in mammals including humans (Adams 1986), as well as in birds (Carr and Boudreau 1991; Koppl 1994) and reptiles (Browner and Marbey 1988; Szpir et al. 1990), but their presence in amphibians is less clear (Lewis et al. 1980; Feng and Lin 1996).

In addition to auditory inputs, dorsal cochlear nucleus (DCN) pyramidal cells in the guinea pig receive and respond to somatosensory inputs and perform multisensory integration. DCN pyramidal cells respond to sounds with characteristic spike-timing patterns that are partially controlled by rapidly inactivating potassium conductances. Deactivating these conductances can modify both spike rate and spike timing of responses to sound. Somatosensory pathways are known to modify response rates to subsequent acoustic stimuli, but their effect on spike timing is unknown. Here, we demonstrate that preceding tonal stimulation with spinal trigeminal nucleus (Sp5) stimulation significantly alters the first spike latency, the first interspike interval, and the average discharge regularity of firing evoked by the tone. These effects occur whether the neuron is excited or inhibited by Sp5 stimulation alone. Our results demonstrate that multisensory integration in DCN alters spike-timing representations of acoustic stimuli in pyramidal cells. These changes likely occur through synaptic modulation of intrinsic excitability or synaptic inhibition.

The dynamics of synaptic transmission between neurons plays a major role in neural information processing. In the cochlear nucleus, auditory nerve synapses have a relatively high release probability and show pronounced synaptic depression that, in conjunction with the variability of interspike intervals, shapes the information transmitted to the postsynaptic cells. Cellular mechanisms have been best analyzed at the endbulb synapses, revealing that the recent history of presynaptic activity plays a complex, non-linear, role in regulating release. Emerging evidence suggests that the dynamics of synaptic function differs according to the target neuron within the cochlear nucleus. One consequence of hearing loss is changes in evoked release at surviving auditory nerve synapses, and in some situations spontaneous release is greatly enhanced. In contrast, even with cochlear ablation, postsynaptic excitability is less affected. The existing evidence suggests that different modes of hearing loss can result in different dynamic patterns of synaptic transmission between the auditory nerve and postsynaptic neurons. These changes in dynamics in turn will affect the efficacy with which different kinds of information about the acoustic environment can be processed by the parallel pathways in the cochlear nucleus.