Cells In Cochlear Nucleus Research Articles

Characterization of side population (SP) cells in murine cochlear nucleus

Conclusion: We characterized side population (SP) cells in the cochlear nucleus (CN). Some genes of stem/progenitor markers in sorted SP cells were identified by microarray analysis and RT-PCR. Furthermore, some cells in the CN also demonstrated self-renewal and clonal expansion activities. These results suggest that tissue stem/ progenitor like cells would be identified and characterized as a slow cycling and immaturity in SP cells of CN. Objectives: SP cells were sorted and characterized as regards their activity in the CN in order to identify the tissue progenitor/stem cells in the auditory nervous system. Methods: Bromodeoxyuridine (BrdU)-injected mice were prepared and the long-term BrdU-retaining cells were detected by flow cytometry. Gene expression of SP and MP cells was analyzed by microarray analysis and RT-PCR. SP cells were cultured in conditioned medium to expand stem/progenitor cells in vitro and to estimate the spheroid-forming activity of stem cells. Results: In all, 1% of cells in the CN were detected as BrdU-positive. SP cells were detected at a frequency of 4.4% and expressed stem/progenitor markers, Abcb1b, Abcg2, Sca1, Notch1, Notch4, Hes1, and Jag1 in microarray analysis. Expression of Abcb1b, Abcg2, Sca1,Oct3/4, and Sox2 as determined by RT-PCR was supported by the microarray data. CN cells also had sphere-forming activity in young mice, but this activity was decreased by aging.

Intrinsic and synaptic properties of vertical cells of the mouse dorsal cochlear nucleus

Multiple classes of inhibitory interneurons shape the activity of principal neurons of the dorsal cochlear nucleus (DCN), a primary target of auditory nerve fibers in the mammalian brain stem. Feedforward inhibition mediated by glycinergic vertical cells (also termed tuberculoventral or corn cells) is thought to contribute importantly to the sound-evoked response properties of principal neurons, but the cellular and synaptic properties that determine how vertical cells function are unclear. We used transgenic mice in which glycinergic neurons express green fluorescent protein (GFP) to target vertical cells for whole cell patch-clamp recordings in acute slices of DCN. We found that vertical cells express diverse intrinsic spiking properties and could fire action potentials at high, sustained spiking rates. Using paired recordings, we directly examined synapses made by vertical cells onto fusiform cells, a primary DCN principal cell type. Vertical cell synapses produced unexpectedly small-amplitude unitary currents in fusiform cells, and additional experiments indicated that multiple vertical cells must be simultaneously active to inhibit fusiform cell spike output. Paired recordings also revealed that a major source of inhibition to vertical cells comes from other vertical cells.

Expression of the gap junction protein connexin-36 in the adult big brown bat cochlear nucleus may be involved in temporally precise biosonar processing

The big brown bat uses biosonar to orient, navigate, and forage. Successful prey capture requires sonar emissions and returning echoes to be encoded and compared in the central auditory system with precision. Behavioral experiments show that E. fuscus can discriminate echo delay, relative timing of harmonics, and echo phase on the order of 3μs or less. The neurobiological specializations underlying this perceptual acuity remain elusive. New evidence shows that principal cells in the bat's cochlear nucleus (CN) express the neuronal gap junction protein connexin-36. During early postnatal development, connexin-36 expression is seen throughout the AVCN and PVCN of both big brown bats and mice. In adult bats, but not mice, cx36 expression is restricted to a specific population of cells in the ventral AVCN. The retention of cx36 in a discrete population of cells in the adult bat CN suggests that electrical transmission could be involved in processing echolocation sounds with temporal accuracy. Support: NSF grant 0843522 and ONR grant N00014-09-1-0691 to James Simmons.

Planar multipolar cells in the cochlear nucleus project to medial olivocochlear neurons in mouse.

Medial olivocochlear (MOC) neurons originate in the superior olivary complex and project to the cochlea, where they act to reduce the effects of noise masking and protect the cochlea from damage. MOC neurons respond to sound via a reflex pathway; however, in this pathway the cochlear nucleus cell type that provides input to MOC neurons is not known. We investigated whether multipolar cells of the ventral cochlear nucleus have projections to MOC neurons by labeling them with injections into the dorsal cochlear nucleus. The projections of one type of labeled multipolar cell, planar neurons, were traced into the ventral nucleus of the trapezoid body, where they were observed terminating on MOC neurons (labeled in some cases by a second cochlear injection of FluoroGold). These terminations formed what appear to be excitatory synapses, i.e., containing small, round vesicles and prominent postsynaptic densities. These data suggest that cochlear nucleus planar multipolar neurons drive the MOC neuron's response to sound.

Differences in developmental changes in GABAergic response between bushy and stellate cells in the rat anteroventral cochlear nucleus

Many mammalian central nervous system neuron responses mediated by GABAA receptors undergo a developmental transition from excitation to inhibition, but little is known about the time of this switch in specific cell types in the developing anteroventral cochlear nucleus (AVCN). In the present study, bushy and stellate cells, two major cell types in the AVCN, were identified according to their morphology and electrophysiology. The equilibrium potential of GABA-evoked currents (EGABA) was examined using the gramicidin-perforated patch-clamp technique. We found that the action of GABA in bushy and stellate cells switched from predominantly depolarizing to predominantly hyperpolarizing with respect to their resting membrane potential (Vrest) at different postnatal ages. Such a switch in the GABA response of bushy cells occurred before the first postnatal week, whereas that in stellate cells happened at the end of the second postnatal week. Furthermore, we discovered that bushy cells had a more depolarized Vrest than did stellate cells before the second postnatal week; however, the EGABA of bushy and stellate cells was not significantly different. Thus, the discrepancy in the timing of the developmental shift from depolarizing to hyperpolarizing GABA responses between bushy and stellate cells may be due to the difference in their Vrest, but not due to EGABA itself. These results suggest that GABAergic inhibition functions earlier in bushy than in stellate cells. In contrast, the longer excitatory action of GABA on stellate cells possibly renders them more vulnerable than bushy cells to excitotoxic substances during early development.

An investigation of dendritic delay in octopus cells of the mammalian cochlear nucleus

Octopus cells, located in the mammalian auditory brainstem, receive their excitatory synaptic input exclusively from auditory nerve fibers (ANFs). They respond with accurately timed spikes but are broadly tuned for sound frequency. Since the representation of information in the auditory nerve is well understood, it is possible to pose a number of questions about the relationship between the intrinsic electrophysiology, dendritic morphology, synaptic connectivity, and the ultimate functional role of octopus cells in the brainstem. This study employed a multi-compartmental Hodgkin-Huxley model to determine whether dendritic delay in octopus cells improves synaptic input coincidence detection in octopus cells by compensating for the cochlear traveling wave delay. The propagation time of post-synaptic potentials from synapse to soma was investigated. We found that the total dendritic delay was approximately 0.275 ms. It was observed that low-threshold potassium channels in the dendrites reduce the amplitude dependence of the dendritic delay of post-synaptic potentials. As our hypothesis predicted, the model was most sensitive to acoustic onset events, such as the glottal pulses in speech when the synaptic inputs were arranged such that the model's dendritic delay compensated for the cochlear traveling wave delay across the ANFs. The range of sound frequency input from ANFs was also investigated. The results suggested that input to octopus cells is dominated by high frequency ANFs.

Auditory neuroanatomy: a sound foundation for sound processing

Auditory neuroanatomy: a sound foundation for sound processing

Sustained Firing of Cartwheel Cells in the Dorsal Cochlear Nucleus Evokes Endocannabinoid Release and Retrograde Suppression of Parallel Fiber Synapses

Neurons in many brain regions release endocannabinoids from their dendrites that act as retrograde signals to transiently suppress neurotransmitter release from presynaptic terminals. Little is known, however, about the physiological mechanisms of short-term endocannabinoid-mediated plasticity under physiological conditions. Here we investigate calcium-dependent endocannabinoid release from cartwheel cells (CWCs) of the mouse dorsal cochlear nucleus (DCN) in the auditory brainstem that provide feedforward inhibition onto DCN principal neurons. We report that sustained action potential firing by CWCs evokes endocannabinoid release in response to submicromolar elevation of dendritic calcium that transiently suppresses their parallel fiber (PF) inputs by >70%. Basal spontaneous CWC firing rates are insufficient to evoke tonic suppression of PF synapses. However, elevating CWC firing rates by stimulating PFs triggers the release of endocannabinoids and heterosynaptic suppression of PF inputs. Spike-evoked suppression by endocannabinoids selectively suppresses excitatory synapses, but glycinergic/GABAergic inputs onto CWCs are not affected. Our findings demonstrate a mechanism of transient plasticity mediated by endocannabinoids that heterosynaptically suppresses subsets of excitatory presynaptic inputs to CWCs that regulates feedforward inhibition of DCN principal neurons and may influence the output of the DCN.

Postnatal development of microcyst in the anteroventral cochlear nucleus of the Mongolian gerbil: a light- and electron microscopic study

We investigated the postnatal formation and origin of the microcyst, which are not fully elucidated at present, in the cochlear nucleus of gerbils. Sixty-six Mongolian gerbils were investigated at the light microscope level, and 35 of them were observed at the electron microscopic level. Foamy structures were evidently found at 2 days of age and remained unchanged through 4-8 days. The first small vacuole, presumably the former microcyst, appeared at 8 days. Myelin sheath bundles first appeared at 13 days. Electron-dense bodies were frequently found in the junction of the superficial layer and the deep layer at 2 days. The medium-sized vacuole was found in close association with the spherical bushy cells in the anteroventral cochlear nucleus (AVCN) as early as 5 weeks. Various large and small vacuoles were presumably coalesced to form a large vacuole at 3 and 6 months. Membranous structures and red blood cells were in the budding-like vacuoles at 6 months. In addition to membranous structures, the microcyst contained distorted mitochondria and parts of myelin sheaths. The vacuole was interposed between spherical bushy cells at age of 10 months. Small vacuoles were mainly located in the flame-shaped neurons at 14 months. An internal detachment and an external protrusion of the myelin sheath into the adjacent microcyst were found. Thus, this study suggests the first appearance of microcysts at 8 days. Also, the microcyst and the blood vessel may exchange their contents through a leakage in the anteroventral cochlear nucleus.

Presynaptic Ca2+ influx and vesicle exocytosis at the mouse endbulb of Held: a comparison of two auditory nerve terminals

The functional properties of mammalian presynaptic nerve endings remain elusive since most terminals of the central nervous system are not accessible to direct electrophysiological recordings. In this study, direct recordings were performed for the first time at endbulb of Held terminals to characterize passive membrane properties, voltage-gated Ca(2+) channels (VGCCs) and Ca(2+)-dependent exocytosis. Endbulb of Held terminals arise from endings of auditory nerve fibres contacting spherical bushy cells (SBCs) in the anterior ventral cochlear nucleus (AVCN). These terminals had a high mean input resistance (1.1 G) and a small mean capacitance (4.3 pF). Presynaptic VGCCs were predominantly of the P/Q type (86%) and expressed at a high density with an estimated average number of 6400 channels per terminal. Presynaptic Ca(2+) currents (I(Ca(V))) activated and deactivated rapidly. Simulations of action potential (AP)-driven gating of VGCCs suggests that endbulb APs trigger brief Ca(2+) influx with a mean half-width of 240 μs and a peak amplitude of 0.45 nA which results from the opening of approximately 2600 channels. Unlike Ca(2+) currents at the calyx of Held, I(Ca(V)) of endbulb terminals showed no inactivation during trains of AP-like presynaptic depolarizations. Endbulb terminals are endowed with a large readily releasable vesicle pool (1064 vesicles) of which only a small fraction (<10%) is consumed during a single AP-like stimulus. Fast presynaptic APs together with rapidly gating VGCCs will generate brief intracellular Ca(2+) transients that favour highly synchronous transmitter release. Collectively these characteristics ensure sustained and precise transmission of timing information from auditory stimuli at the endbulbSBC synapse.

Excitatory Modulation in the Cochlear Nucleus through Group I Metabotropic Glutamate Receptor Activation

Activation of group I metabotropic glutamate receptors (mGluRs) has been suggested to modulate development of auditory neurons. However, the acute effects of mGluR activation on physiological response properties are unclear. To address this, we studied the effects of mGluRs in bushy cells (BCs) of the mammalian anteroventral cochlear nucleus (AVCN). Activation of mGluRs with dihydroxyphenylglycine (DHPG) caused depolarization of BCs in mice as old as P42, but did not affect neurotransmitter release by presynaptic auditory nerve (AN) fibers. Application of mGluR antagonists indicated that mGluRs are tonically active, and are highly sensitive to small elevations in ambient glutamate by the glutamate reuptake blocker threo-β-benzyloxyaspartic acid (TBOA). mGluR-mediated depolarization enhanced the firing probability in response to AN stimulation, and reduced the latency and jitter. Furthermore, excitation through postsynaptic mGluRs can significantly counterbalance the inhibitory effects of presynaptic GABA(B) receptors. Thus, interaction between these two modulatory pathways may provide additional flexibility for fine-tuning the BC relay.

Immunolocalization of Aquaporins in Rat Brain

The aquaporins (AQPs) are a family of homologous water channels expressed in many tissues. In this study, the expression and immunolocalization of different AQP subtypes in rat brains were investigated by RT-PCR, immunohistochemistry and immunofluorescence. The data showed that AQP1 was expressed in the subpial processes of astrocytes, choroid plexus and ependyma. AQP3, AQP5 and AQP8 had similar distribution patterns in piriform cortex, choroid plexus, hippocampus and dorsal thalamus. AQP4 and AQP9 were widely expressed in the rat brain and distributed in the subpial processes of astrocytes, ependyma, dorsal thalamus, hippocampus, white matter, suprachiasmatic nucleus (SCN) and supraoptic nucleus. AQP3, AQP4, AQP5, AQP8 and AQP9 were found in the Bergmann glial cells of cerebellum, cochlear nucleus and trapezoid nuclei. The distinct localization of various AQPs in cerebrum and the similarities of distribution patterns within cerebellum, cochlear nucleus and trapezoid nuclei suggest that AQPs may play an important role in maintaining the specific microenvironments of the brain.

Factors Controlling the Input–Output Relationship of Spherical Bushy Cells in the Gerbil Cochlear Nucleus

Despite the presence of large endbulb inputs, the spherical bushy cells (SBCs) of the rostral anteroventral cochlear nucleus do not function as simple auditory relays. We used the good signal-to-noise ratio of juxtacellular recordings to dissect the intrinsic and network mechanisms controlling the input-output relationship of SBCs in anesthetized gerbils. The SBCs generally operated close to action potential (AP) threshold and showed no evidence for synaptic depression, suggesting that the endbulbs of Held have low release probability in vivo. Analysis of the complex waveforms suggested that in the absence of auditory stimulation, postsynaptic spike depression and stochastic fluctuations in EPSP size were the main factors determining jitter and reliability of the endbulb synapse. During auditory stimulation, progressively larger EPSPs were needed to trigger APs at increasing sound intensities. Simulations suggested hyperpolarizing inhibition could explain the observed decrease in EPSP efficacy. Synaptic inhibition showed a delayed onset and generally had a higher threshold than excitatory inputs, but otherwise inhibition and excitation showed mostly overlapping frequency-response areas. The recruitment of synaptic inhibition caused postsynaptic spikes to be preferentially triggered by well-timed, large EPSPs, resulting in improved phase locking despite more variable EPSP-AP latencies. Our results suggest that the lack of synaptic depression, caused by low release probability, and the apparent absence of sound-evoked synaptic inhibition at low sound intensity maximize sensitivity of SBCs. At higher sound intensities, the recruitment of synaptic inhibition constrains their firing rate and optimizes their temporal precision.

Axonal Branching Patterns as Sources of Delay in the Mammalian Auditory Brainstem: A Re-Examination

In models of temporal processing, time delays incurred by axonal propagation of action potentials play a prominent role. A pre-eminent model of temporal processing in audition is the binaural model of Jeffress (1948), which has dominated theories regarding our acute sensitivity to interaural time differences (ITDs). In Jeffress' model, a binaural cell is maximally active when the ITD is compensated by an internal delay, which brings the inputs from left and right ears in coincidence, and which would arise from axonal branching patterns of monaural input fibers. By arranging these patterns in systematic and opposite ways for the ipsilateral and contralateral inputs, a range of length differences, and thereby of internal delays, is created so that the ITD is transformed into a spatial activation pattern along the binaural nucleus. We reanalyze single, labeled, and physiologically characterized axons of spherical bushy cells of the cat anteroventral cochlear nucleus, which project to binaural coincidence detectors in the medial superior olive (MSO). The reconstructions largely confirm the observations of two previous reports, but several features are observed that are inconsistent with Jeffress' model. We found that ipsilateral projections can also form a caudally directed delay line pattern, which would counteract delays incurred by caudally directed contralateral projections. Comparisons of estimated axonal delays with binaural physiological data indicate that the suggestive anatomical patterns cannot account for the frequency-dependent distribution of best delays in the cat. Surprisingly, the tonotopic distribution of the afferent endings indicate that low characteristic frequencies are under-represented rather than over-represented in the MSO.

Control of firing patterns by two transient potassium currents: leading spike, latency, bistability.

Transient potassium currents distinctively affect firing properties, particularly in regulating the latency before repetitive firing. Pyramidal cells of the dorsal cochlear nucleus (DCN) have two transient potassium currents, I(Kif) and I(Kis), fast and slowly inactivating, respectively, and they exhibit firing patterns with dramatically variable latencies. They show immediate repetitive firing, or only after a long latency with or without a leading spike, the so-called pauser and buildup patterns. We consider a conductance-based, ten-variable, single-compartment model for the DCN pyramidal cells (Kanold and Manis 2001). We develop and analyze a reduced three-variable integrate-and-fire model (KM-LIF) which captures the qualitative firing features. We apply dynamical systems methods to explain the underlying biophysical and mathematical mechanisms for the firing behaviors, including the characteristic firing patterns, the latency phase, the onset of repetitive firing, and some discontinuities in the timing of latency duration (e.i. first spike latency and first inter spike interval). Moreover, we obtain new insights associated with the leading spike by phase plane analysis. We further demonstrate the effects of possible heterogeneity of I(Kis). The latency before repetitive firing can be controlled to cover a large range by tuning of the relative amounts of I(Kif) and I(Kis). Finally, we find for the full system robust bistability when enough I(Kis) is present.

Cuneate and spinal trigeminal nucleus projections to the cochlear nucleus are differentially associated with vesicular glutamate transporter-2

There are distinct distributions and associations with vesicular glutamate transporters (VGLUTs) for auditory nerve and specific somatosensory projections in the cochlear nucleus (CN). Auditory nerve fibers project primarily to the magnocellular areas of the ventral cochlear nucleus and deepest layer of the dorsal cochlear nucleus and predominantly colabel with VGLUT1; whereas the spinal trigeminal nucleus (Sp5) projections terminate primarily in the granule cell domains (GCD) of CN and predominantly colabel with VGLUT2. Here, we demonstrate that the terminals of another somatosensory pathway, originating in the cuneate nucleus (Cu), also colabel with VGLUT2. Cu projections in cochlear nucleus exhibited a bilateral distribution pattern with ipsilateral dominance, with 30% of these classified as putative mossy fibers (MFs) and 70% as small boutons (SBs). Cu anterograde endings had a more prominent distribution in the GCD than Sp5, with a higher percentage of MF terminals throughout the CN and higher MF/SB ratio in GCD. 56% of Cu endings and only 25% of Sp5 endings colabeled with VGLUT2. In both cases these were mostly MFs with only 43% of Cu SBs and 18% of Sp5 SBs colabeled with VGLUT2. The few Cu and Sp5 terminals that colabeled with VGLUT1 (11% vs. 1%), were evenly distributed between MFs and SBs. The high number of VGLUT2-positive Cu MFs predominantly located in the GCD, may reflect a faster-acting pathway that activates primarily dorsal cochlear nucleus cells via granule cell axons. In contrast, the higher percentage of Sp5-labeled SB terminals and a greater number of projections outside the GCD suggest a slower-acting pathway that activates both dorsal and ventral cochlear nucleus principal cells. Both projections, with their associations to VGLUT2 likely play a role in the enhancement of VGLUT2 after unilateral deafness [Zeng C, Nannapaneni N, Zhou J, Hughes LF, Shore S (2009) J Neurosci 29:4210–4217] that may be associated with tinnitus.

Increased BrdU incorporation reflecting DNA repair, neuronal de-differentiation or possible neurogenesis in the adult cochlear nucleus following bilateral cochlear lesions in the rat

Neurogenesis is known to occur in response to injury in the brain, for example, as a result of neurodegenerative diseases. However, there have been few investigations into how the brain responds to damage to peripheral sensory nerves, in other areas such as the brainstem. Here, we report that bilateral surgical lesions of the cochlea result in increased incorporation of the DNA replication marker, bromodeoxyuridine (BrdU), in cells of the brainstem cochlear nucleus (CN) of the adult rat, suggesting either cell proliferation or DNA repair. Some of the BrdU-labelled cells colabelled for the mature neuron marker, NeuN and the GABAergic enzyme GAD-65, suggesting the possibility that neurogenesis might have occurred and resulted in the generation of new neurons with a GABAergic phenotype. However, some of the mature neurons also re-expressed immature neuronal intermediate filament and microtuble-associated proteins, without apoptotic neuronal death, which suggests that the colabelling of BrdU with NeuN and GAD-65 may not be a true reflection of neurogenesis, but injury-stimulated neuronal dedifferentiation. These results suggest the possibility that DNA repair, neuronal de-differentiation or possible neurogenesis occurs in the cochlear nucleus, in response to damage to the peripheral auditory system.

Calcium imaging of auditory nerve fiber terminals in the cochlear nucleus

One important model for understanding neuronal computation is how auditory information is transformed at the synapses made by auditory nerve (AN) fibers on the bushy cells (BCs) in the anteroventral cochlear nucleus (AVCN). This transformation is influenced by synaptic plasticity, the mechanisms of which have been studied primarily using postsynaptic electrophysiology. However, it is also important to make direct measurements of the presynaptic terminal to consider presynaptic mechanisms. Here we introduce a technique for doing that using calcium imaging of presynaptic AN terminals, by injecting dextran-conjugated fluorophores into the cochlea. To measure the calcium transients, we used calcium-sensitive fluorophores, and measured the changes in fluorescence upon stimulation. As an example of the application of this technique, we showed that activation of GABAB receptors reduces presynaptic calcium influx. This technique could be further extended to study the effects of activity- and other neuromodulator-dependent plasticities on AN terminals.

Early dating influences long‐term synaptic partnerships

Early dating influences long‐term synaptic partnerships

Acute genetic perturbation of exocyst function in the rat calyx of Held impedes structural maturation, but spares synaptic transmission

The exocyst is an octameric protein complex mediating polarized secretion by tethering vesicles to target membranes. In non-vertebrate neurons, the exocyst has been associated with constitutive membrane addition at growth cones and nerve terminals, but its function in synaptic vesicle trafficking at mammalian nerve terminals remains unclear. Here, we examined the role of the exocyst complex in immature postnatal day (P)13 and mature P21 rat calyces of Held. Exo70, an exocyst subunit conferring membrane anchoring of the complex, was tagged with green fluorescent protein (GFP) and overexpressed as a full-length subunit or as a dominant-negative C-terminally truncated variant (Exo70ΔC) disrupting membrane targeting. In vivo expression of the Exo70 subunits in the calyx was achieved by stereotaxic adeno-associated virus-mediated gene transfer into globular bushy cells of the rat ventral cochlear nucleus at P2. Overexpression of dominant-negative Exo70ΔC, but not full-length Exo70, decreased the structural complexity and volume of calyces, as assayed by confocal microscopy and three-dimensional reconstructions. The distribution of active zones and synaptic vesicles remained unaffected. Neither perturbation changed the characteristics of spontaneous and evoked neurotransmitter release, short-term depression or recovery from depression. Together, these data suggest that in central mammalian synapses, the exocyst complex mediates the addition of membrane during postnatal presynaptic maturation, but does not function as a tethering complex in local recycling of vesicles within the synaptic vesicle cycle.