6'-sialyllactose ameliorates the ototoxic effects of the aminoglycoside antibiotic neomycin in susceptible mice.

Sialic acids are terminal sugars of the cellular glycocalyx and are highly abundant in the nervous tissue. Sialylation is sensed by the innate immune system and acts as an inhibitory immune checkpoint. Aminoglycoside antibiotics such as neomycin have been shown to activate tissue macrophages and induce ototoxicity. In this study, we investigated the systemic subcutaneous application of the human milk oligosaccharide 6'-sialyllactose (6SL) as a potential therapy for neomycin-induced ototoxicity in postnatal mice. Repeated systemic treatment of mice with 6SL ameliorated neomycin-induced hearing loss and attenuated neomycin-triggered macrophage activation in the cochlear spiral ganglion. In addition, 6SL reversed the neomycin-mediated increase in gene transcription of the pro-inflammatory cytokine interleukin-1ß (Il-1b) and the apoptotic/inflammatory kinase Pik3cd in the inner ear. Interestingly, neomycin application also increased the transcription of desialylating enzyme neuraminidase 3 (Neu3) in the inner ear. In vitro, we confirmed that treatment with 6SL had anti-inflammatory, anti-phagocytic, and neuroprotective effects on cultured lipopolysaccharide-challenged human THP1-macrophages. Thus, our data demonstrated that treatment with 6SL has anti-inflammatory and protective effects against neomycin-mediated macrophage activation and ototoxicity.

Efficacy and safety of ocrelizumab in patients with relapsing-remitting multiple sclerosis with suboptimal response to prior disease-modifying therapies: A primary analysis from the phase 3b CASTING single-arm, open-label trial.

BACKGROUND AND PURPOSE: Using the treatment goal of "no evidence of disease activity" (NEDA) incorporating magnetic resonance imaging (MRI) re-baselining, we aimed to assess the efficacy of ocrelizumab in patients with relapsing-remitting multiple sclerosis with a prior suboptimal response, defined by MRI or relapse criteria, to one or two disease-modifying therapies (DMTs). METHODS: CASTING was a prospective, international, multicenter, single-arm, open-label phase 3 trial (NCT02861014). Patients (Expanded Disability Status Scale [EDSS] score ≤ 4.0, with discontinued prior DMT of ≥6 months duration due to suboptimal disease control) received intravenous ocrelizumab 600 mg every 24 weeks for 96 weeks. The primary endpoint was NEDA (defined as absence of relapses, disability progression, and inflammatory MRI measures, with prespecified MRI re-baselining at Week 8) over 96 weeks. RESULTS: A total of 680 patients were enrolled, 167 (24.6%) based on MRI activity only. At Week 96, 74.8% (95% confidence interval [CI] 71.3-78.0, n/N = 492/658) of patients had NEDA. NEDA was highest among patients enrolled due to MRI activity alone (80.6% [95% CI 68.6-89.6], n/N = 50/62) versus those enrolled for relapse (75.1% [95% CI 69.0-80.6], n/N = 172/229) or for relapse with MRI (70.5% [95% CI 60.0-79.0], n/N = 74/105). NEDA across subgroups was highest in patients with a baseline EDSS score <2.5 (77.2% [95% CI 72.8-81.2], n/N = 315/408). NEDA was higher in patients receiving one prior DMT (77.6% [95% CI 73.2-81.6], n/N = 312/402) versus two prior DMTs (70.3% [95% CI 64.3-75.8], n/N = 180/256). CONCLUSIONS: In patients switching therapy due to suboptimal disease control, treatment with ocrelizumab led to an overall high NEDA rate across a wide range of disease-related and demographic subgroups, regardless of prior treatment background, with no new safety signals detected.

Small dendritic synapses enhance temporal coding in a model of cochlear nucleus bushy cells.

Spherical bushy cells (SBCs) in the anteroventral cochlear nucleus receive a single or very few powerful axosomatic inputs from the auditory nerve. However, SBCs are also contacted by small regular bouton synapses of the auditory nerve, located in their dendritic tree. The function of these small inputs is unknown. It was speculated that the interaction of axosomatic inputs with small dendritic inputs improved temporal precision, but direct evidence for this is missing. In a compartment model of spherical bushy cells with a stylized or realistic three-dimensional (3-D) representation of the bushy dendrite, we explored this hypothesis. Phase-locked dendritic inputs caused both tonic depolarization and a modulation of the model SBC membrane potential at the frequency of the stimulus. For plausible model parameters, dendritic inputs were subthreshold. Instead, the tonic depolarization increased the excitability of the SBC model and the modulation of the membrane potential caused a phase-dependent increase in the efficacy of the main axosomatic input. This improved response rate and entrainment for low-input frequencies and temporal precision of output at and above the characteristic frequency. A careful exploration of morphological and biophysical parameters of the bushy dendrite suggested a functional explanation for the peculiar shape of the bushy dendrite. Our model for the first time directly implied a role for the small excitatory dendritic inputs in auditory processing: they modulate the efficacy of the main input and are thus a plausible mechanism for the improvement of temporal precision and fidelity in these central auditory neurons.NEW & NOTEWORTHY We modeled dendritic inputs from the auditory nerve that spherical bushy cells of the cochlear nucleus receive. Dendritic inputs caused both tonic depolarization and modulation of the membrane potential at the input frequency. This improved the rate, entrainment, and temporal precision of output action potentials. Our simulations suggest a role for small dendritic inputs in auditory processing: they modulate the efficacy of the main input supporting temporal precision and fidelity in these central auditory neurons.

Expression patterns of chloride transporters in the auditory brainstem of developing chicken.

GABAergic transmission changes from depolarization to hyperpolarization in most vertebrate brain regions during development. By contrast, in the auditory brainstem of chicken a depolarizing effect of GABA persists after hatching. Since auditory brainstem neurons that receive GABAergic input have a Cl- reversal potential above resting membrane potential, a specifically tuned activity of Cl- transporters is likely. We here present a developmental study of the expression patterns of several members of the SLC12 family (NKCC1, NKCC2, KCC1, KCC2, KCC4, CCC6, CCC9) and of AE3 at developmental ages E7, E10, E12, E15, E17, and P1 with quantitative RT-PCR. NKCC2 and CCC9 were not detected in auditory brainstem (positive control: kidney). KCC1, CCC6 and AE3 were expressed, but not regulated, while NKCC1, KCC2 and KCC4 were regulated. The expression of the latter transporters increased, with KCC2 exhibiting the strongest expression at all time points. Biochemical analysis of the protein expression of NKCC1, KCC2 and KCC4 corroborated the findings on the mRNA level. All three transporters showed a localization at the outer rim of the cells, with NKCC1 and KCC2 expressed in neurons, and KCC4 predominantly in glia. The comparison of the published chloride reversal potential and expression of transporter proteins suggest strong differences in the efficiency of the three transporters. Further, the strong KCC2 expression could reflect a role in the structural development of auditory brainstem synapses that might lead to changes in the physiological properties.

Muscarinic modulation of M and h currents in gerbil spherical bushy cells.

Descending cholinergic fibers innervate the cochlear nucleus. Spherical bushy cells, principal neurons of the anterior part of the ventral cochlear nucleus, are depolarized by cholinergic agonists on two different time scales. A fast and transient response is mediated by alpha-7 homomeric nicotinic receptors while a slow and long-lasting response is mediated by muscarinic receptors. Spherical bushy cells were shown to express M3 receptors, but the receptor subtypes involved in the slow muscarinic response were not physiologically identified yet. Whole-cell patch clamp recordings combined with pharmacology and immunohistochemistry were performed to identify the muscarinic receptor subtypes and the effector currents involved. Spherical bushy cells also expressed both M1 and M2 receptors. The M1 signal was stronger and mainly somatic while the M2 signal was localized in the neuropil and on the soma of bushy cells. Physiologically, the M-current was observed for the gerbil spherical bushy cells and was inhibited by oxotremorine-M application. Surprisingly, long application of carbachol showed only a transient depolarization. Even though no muscarinic depolarization could be detected, the input resistance increased suggesting a decrease in the cell conductance that matched with the closure of M-channels. The hyperpolarization-activated currents were also affected by muscarinic activation and counteracted the effect of the inactivation of M-current on the membrane potential. We hypothesize that this double muscarinic action might allow adaptation of effects during long durations of cholinergic activation.

Modulatory influences on time-coding neurons in the ventral cochlear nucleus.

Bushy cells of the ventral cochlear nucleus are time-coding neurons. They receive axosomatic synaptic terminals from the auditory nerve, the so-called endbulb of Held synapses and project to sound localization centers in the superior olivary complex. Bushy cells are specialized to maintain and even improve the temporal code contained in the auditory nerve activity. In the present review an overview is given of the dynamic features and convergent inputs that modulate the response of bushy cells to auditory stimuli. The biophysics and synaptic specializations and dynamics of these neurons were studied extensively. These studies will be reviewed briefly in the initial part of this paper. In addition to auditory nerve input, powerful but slow inhibitory inputs act on bushy cells. Studies on these inhibitory inputs to bushy cells are discussed as part of this review. Furthermore, evidence for four classes of additional or secondary inputs that also impinge on the bushy cells will be reviewed: 1) small auditory nerve boutons, 2) commissural connections that are either inhibitory or excitatory, 3) multimodal inputs from somatosensory nuclei and 4) descending modulatory axons employing monoaminergic transmitters all interact with the main auditory nerve input in the bushy cells. The present article aims at reviewing how complex the influences on neuronal processing are, already in this early stage of the auditory pathway. It is concluded that the various modulatory influences help to better adapt bushy cell coding functions to dynamics of the sensory world.

Intrinsic and Synaptic Dynamics Contribute to Adaptation in the Core of the Avian Central Nucleus of the Inferior Colliculus.

The reduction of neuronal responses to repeated stimulus presentation occurs in many sensory neurons, also in the inferior colliculus of birds. The cellular mechanisms that cause response adaptation are not well described. Adaptation must be explicable by changes in the activity of input neurons, short-term synaptic plasticity of the incoming connections, excitability changes of the neuron under consideration or influences of inhibitory or modulatory network connections. Using whole-cell recordings in acute brain slices of the embryonic chicken brain we wanted to understand the intrinsic and synaptic contributions to adaptation in the core of the central nucleus of the inferior colliculus (ICCc). We described two neuron types in the chicken ICCc based on their action potential firing patterns: Phasic/onset neurons showed strong intrinsic adaptation but recovered more rapidly. Tonic/sustained firing neurons had weaker adaptation but often had additional slow components of recovery from adaptation. Morphological analysis suggested two neuron classes, but no physiological parameter aligned with this classification. Chicken ICCc neurons received mostly mixed AMPA- and NMDA-type glutamatergic synaptic inputs. In the majority of ICCc neurons the input synapses underwent short-term depression. With a simulation of the putative population output activity of the chicken ICCc we showed that the different adaptation profiles of the neuron classes could shift the emphasize of stimulus encoding from transients at long intervals to ongoing parts at short intervals. Thus, we report here that description of biophysical and synaptic properties can help to explain adaptive phenomena in central auditory neurons.

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

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

Cell-free artificial implants of electrospun fibres in a three-dimensional gelatin matrix support sciatic nerve regeneration in vivo.

Surgical repair of larger peripheral nerve lesions requires the use of autologous nerve grafts. At present, clinical alternatives to avoid nerve transplantation consist of empty tubes, which are only suitable for the repair over short distances and have limited success. We developed a cell-free, three-dimensional scaffold for axonal guidance in long-distance nerve repair. Sub-micron scale fibres of biodegradable poly-ε-caprolactone (PCL) and collagen/PCL (c/PCL) blends were incorporated in a gelatin matrix and inserted in collagen tubes. The conduits were tested by replacing 15-mm-long segments of rat sciatic nerves in vivo. Biocompatibility of the implants and nerve regeneration were assessed histologically, with electromyography and with behavioural tests for motor functions. Functional repair was achieved in all animals with autologous transplants, in 12 of 13 rats that received artificial implants with an internal structure and in half of the animals with empty nerve conduits. In rats with implants containing c/PCL fibres, the extent of recovery (compound muscle action potentials, motor functions of the hind limbs) was superior to animals that had received empty implants, but not as good as with autologous nerve transplantation. Schwann cell migration and axonal regeneration were observed in all artificial implants, and muscular atrophy was reduced in comparison with animals that had received no implants. The present design represents a significant step towards cell-free, artificial nerve bridges that can replace autologous nerve transplants in the clinic. Copyright © 2017 John Wiley & Sons, Ltd.

Slow Cholinergic Modulation of Spike Probability in Ultra-Fast Time-Coding Sensory Neurons.

Sensory processing in the lower auditory pathway is generally considered to be rigid and thus less subject to modulation than central processing. However, in addition to the powerful bottom-up excitation by auditory nerve fibers, the ventral cochlear nucleus also receives efferent cholinergic innervation from both auditory and nonauditory top-down sources. We thus tested the influence of cholinergic modulation on highly precise time-coding neurons in the cochlear nucleus of the Mongolian gerbil. By combining electrophysiological recordings with pharmacological application in vitro and in vivo, we found 55-72% of spherical bushy cells (SBCs) to be depolarized by carbachol on two time scales, ranging from hundreds of milliseconds to minutes. These effects were mediated by nicotinic and muscarinic acetylcholine receptors, respectively. Pharmacological block of muscarinic receptors hyperpolarized the resting membrane potential, suggesting a novel mechanism of setting the resting membrane potential for SBC. The cholinergic depolarization led to an increase of spike probability in SBCs without compromising the temporal precision of the SBC output in vitro. In vivo, iontophoretic application of carbachol resulted in an increase in spontaneous SBC activity. The inclusion of cholinergic modulation in an SBC model predicted an expansion of the dynamic range of sound responses and increased temporal acuity. Our results thus suggest of a top-down modulatory system mediated by acetylcholine which influences temporally precise information processing in the lower auditory pathway.

Expression of the postsynaptic scaffold PSD-95 and development of synaptic physiology during giant terminal formation in the auditory brainstem of the chicken.

In the avian nucleus magnocellularis (NM) endbulb of Held giant synapses develop from temporary bouton terminals. The molecular regulation of this process is not well understood. Furthermore, it is unknown how the postsynaptic specialization of the endbulb synapses develops. We therefore analysed expression of the postsynaptic scaffold protein PSD-95 during the transition from bouton-to-endbulb synapses. PSD-95 has been implicated in the regulation of the strength of glutamatergic synapses and could accordingly be of functional relevance for giant synapse formation. PSD-95 protein was expressed at synaptic sites in embryonic chicken auditory brainstem and upregulated between embryonic days (E)12 and E16. We applied immunofluorescence staining and confocal microscopy to quantify pre-and postsynaptic protein signals during bouton-to-endbulb transition. Giant terminal formation progressed along the tonotopic axis in NM, but was absent in low-frequency NM. We found a tonotopic gradient of postsynaptic PSD-95 signals in NM. Furthermore, PSD-95 immunosignals showed the greatest increase between E12 and E15, temporally preceding the bouton-to-endbulb transition. We then applied whole-cell electrophysiology to measure synaptic currents elicited by synaptic terminals during bouton-to-endbulb transition. With progressing endbulb formation postsynaptic currents rose more rapidly and synapses were less susceptible to short-term depression, but currents were not different in amplitude or decay-time constant. We conclude that development of presynaptic specializations follows postsynaptic development and speculate that the early PSD-95 increase could play a functional role in endbulb formation.

Inhibitory properties underlying non-monotonic input-output relationship in low-frequency spherical bushy neurons of the gerbil.

Spherical bushy cells (SBCs) of the anteroventral cochlear nucleus (AVCN) receive input from large excitatory auditory nerve (AN) terminals, the endbulbs of Held, and mixed glycinergic/GABAergic inhibitory inputs. The latter have sufficient potency to block action potential firing in vivo and in slice recordings. However, it is not clear how well the data from slice recordings match the inhibition in the intact brain and how it contributes to complex phenomena such as non-monotonic rate-level functions (RLF). Therefore, we determined the input-output relationship of a model SBC with simulated endbulb inputs and a dynamic inhibitory conductance constrained by recordings in brain slice preparations of hearing gerbils. Event arrival times from in vivo single-unit recordings in gerbils, where 70% of SBC showed non-monotonic RLF, were used as input for the model. Model output RLFs systematically changed from monotonic to non-monotonic shape with increasing strength of tonic inhibition. A limited range of inhibitory synaptic properties consistent with the slice data generated a good match between the model and recorded RLF. Moreover, tonic inhibition elevated the action potentials (AP) threshold and improved the temporal precision of output functions in a SBC model with phase-dependent input conductance. We conclude that activity-dependent, summating inhibition contributes to high temporal precision of SBC spiking by filtering out weak and poorly timed EPSP. Moreover, inhibitory parameters determined in slice recordings provide a good estimate of inhibitory mechanisms apparently active in vivo.

Dynamic fidelity control to the central auditory system: synergistic glycine/GABAergic inhibition in the cochlear nucleus.

GABA and glycine are the major inhibitory transmitters that attune neuronal activity in the CNS of mammals. The respective transmitters are mostly spatially separated, that is, synaptic inhibition in the forebrain areas is mediated by GABA, whereas glycine is predominantly used in the brainstem. Accordingly, inhibition in auditory brainstem circuits is largely mediated by glycine, but there are few auditory synapses using both transmitters in maturity. Little is known about physiological advantages of such a two-transmitter inhibitory mechanism. We explored the benefit of engaging both glycine and GABA with inhibition at the endbulb of Held-spherical bushy cell synapse in the auditory brainstem of juvenile Mongolian gerbils. This model synapse enables selective in vivo activation of excitatory and inhibitory neuronal inputs through systemic sound stimulation and precise analysis of the input (endbulb of Held) output (spherical bushy cell) function. The combination of in vivo and slice electrophysiology revealed that the dynamic AP inhibition in spherical bushy cells closely matches the inhibitory conductance profile determined by the glycine-R and GABAA-R. The slow and potent glycinergic component dominates the inhibitory conductance, thereby primarily accounting for its high-pass filter properties. GABAergic transmission enhances the inhibitory strength and shapes its duration in an activity-dependent manner, thus increasing the inhibitory potency to suppress the excitation through the endbulb of Held. Finally, in silico modeling provides a strong link between in vivo and slice data by simulating the interactions between the endbulb- and the synergistic glycine-GABA-conductances during in vivo-like spontaneous and sound evoked activities.

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.

Neuronal cell growth on iridium oxide.

Iridium oxide is an attractive material for the development of novel multi electrode array (MEA) systems that provide electrodes for stimulation as well as recording single neurons. In this study the biocompatibility of pure iridium and different iridium oxides that differ characteristically in their surface roughness was investigated using two different biological test systems, insect and vertebrate neurons. Iridium oxide surfaces were coated with Concanavalin A and poly-(D)-lysine. In detailed investigations (R(a) value determination, contact angle measurement, marker enzyme assay) the surface characteristics of non-modified and coated iridium oxide films were analysed, demonstrating that the materials can be successfully coated. Furthermore, we show that locust neurons grow well on all substrates tested, while chicken neurons need coated surfaces for proper adhesion. Increasing the roughness of iridium oxide films, which in principle could improve cell adhesion, did not improve the neurocompatibility. These results show that in future applications iridium oxide films can be used with surface morphologies previously shown to be optimal for stimulation purposes (cauliflower-like surface structure).

Increase of Kv3.1b expression in avian auditory brainstem neurons correlates with synaptogenesis in vivo and in vitro.

In the auditory system voltage-activated currents mediated by potassium channels Kv1.1 and Kv3.1b and their interaction with sodium inward currents play a crucial role for computational function. However, it is unresolved how these potassium channels are developmentally regulated. We have therefore combined a biochemical investigation of Kv1.1 and Kv3.1b protein expression with electrophysiological recordings of membrane currents to characterize neuronal differentiation in the auditory brain stem of the chick. Differentiation in vitro was compared with cells prepared from corresponding embryonic stages in vivo. Using a computer model based on the empirical data we were then able to predict physiological properties of developing auditory brain stem neurons. In vivo Kv3.1b expression increased strongly between E10 and E14, a time of functional synaptogenesis in the auditory brainstem. We also found this increase of expression in vitro, again coinciding with synaptogenesis in the cultures. Whole-cell patch recordings revealed a corresponding increase of the (Kv3.1-like) high threshold potassium current. In contrast, Kv1.1 protein expression failed to increase in vitro, and changes in (Kv1.1-like) low threshold potassium current with time in culture were not significant. Electrophysiological recordings revealed that sodium inward currents increased with cultivation time. Thus, our data suggest that Kv3.1b expression occurs with the onset of functional synaptogenesis, while a different signal, absent from cultures of dissociated auditory brain stem, is needed for Kv1.1 expression. A biophysical model constructed with parameters from our recordings was used to investigate the functional impact of the currents mediated by these channels. We found that during development both high and low threshold potassium currents need to be increased in a concerted manner with the sodium conductance for the neurons to exhibit fast and phasic action potential firing and a narrow time window of coincidence detection.

Identification of auditory neurons by retrograde labelling for patch-clamp recordings in a mixed culture of chick brainstem.

We present a method to identify specific sub-populations of auditory neurons in a mixed primary cell culture of the chicken brainstem, allowing the study of individual neurons with a known identity in vitro. To label specific afferent cell types, we injected retrograde tracers (dextrans coupled to fluorescent dyes) into either the mid-line or the superior olivary nuclei (SON) of the isolated chicken brainstem in vitro. Mid-line injections resulted in stable labelling of neurons of the nucleus magnocellularis (NM), whereas injections into the SON retrogradely labelled neurons of the nucleus laminaris (NL). The fluorescent label survives the dissociation procedure and is detectable for at least 1 week in vitro. Only about 0.1% of all cells in vitro are pre-labelled. The auditory identity of the pre-labelled neurons was confirmed with calretinin immunocytochemistry and electrophysiological recordings, where the cells had typical firing patterns of auditory brainstem neurons. In the future, this method can be combined with single cell PCR to match nuclear origin, firing patterns and the expression of functional molecules in vitro.

Neuronal differentiation of the early embryonic auditory hindbrain of the chicken in primary culture.

Neurons in the auditory hindbrain pathway of the chicken are physiologically and morphologically highly specialized. It remains unclear to what extent independent differentiation vs. activity-dependent mechanisms determines the development of this system. To address this question we established a primary culture system of the early auditory hindbrain neurons. Primary cultures of neurons from nucleus magnocellularis and nucleus laminaris were prepared from embryonic day 6.5 chicken. These cells developed in culture under serum-free conditions for up to 15 days. Immunocytochemical staining and whole-cell patch recordings were used to characterize the development of the neurons. A stable expression of the calcium-binding protein calretinin, which serves as a characteristic marker of the auditory pathway, was found at all stages. A voltage-gated potassium channel (Kv3.1b) with a specific function in the auditory system was also expressed after about 1 week in culture. Electrophysiological recordings showed a general maturation of the neuronal phenotype as reflected by an increase in the mean resting membrane potential, a decrease in the mean input resistance as well as a maturation of action potential parameters. Four groups of neurons that generate action potentials could be distinguished. One of these showed the phasic firing pattern of auditory brainstem neurons known from slice preparations. In older cultures we demonstrated functional synaptogenesis in vitro by recording postsynaptic activity elicited by extracellular stimulation and styryl dye loading of vesicles. Thus, isolated neurons from the auditory region of the avian brainstem differentiate to specific neuronal subtypes and autonomously develop synaptic connections in vitro.