Calcium currents in dissociated cochlear neurons from the chick embryo and their modification by neurotrophin-3

Pergamon

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Neuroscience Vol. 77, No. 3, pp. 673–682, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+0.00 S0306-4522(96)00505-2

CALCIUM CURRENTS IN DISSOCIATED COCHLEAR NEURONS FROM THE CHICK EMBRYO AND THEIR MODIFICATION BY NEUROTROPHIN-3 C. JIME u NEZ,* F. GIRE u LDEZ,* J. REPRESA* and J. F. GARCIuA-DIuAZ,†‡ *Instituto de Biologı´a y Gene´tica Molecular, Universidad de Valladolid, Valladolid, Spain †Department of Physiology, Boston University School of Medicine, Boston, Massachusetts, U.S.A. Abstract––Calcium entry through voltage-dependent channels play a critical role in neuronal development. Using patch-clamp techniques we have identified the components of the macroscopic Ca2+ current in acutely-isolated chick cochlear ganglion neurons and analysed their functional expression throughout embryonic development. With Ba2+ as a charge carrier, the currents exhibited two main components, both with a high activation threshold but differing in their inactivation kinetics. One component showed inactivation with a time constant around 100 ms (transient) whereas the other hardly inactivated (sustained). The currents were sensitive to ù-Conotoxin GVIA and dihydropyridines, blocked by 20 µM Cd2+, but unaffected by ù-Agatoxin IVA. In a few cases, only with Ca2+ as a charge carrier, an additional component with low activation threshold and fast inactivation (time constant of 20 ms), was observed. Currents were first detected at day 7 of embryonic development. Current density (amplitude/cell capacitance) increased through embryonic day 9, when early synaptic contacts are established, and decreased thereafter to lower steady values. The effect of neurotrophin-3, a neurotrophic factor required for survival and differentiation of cochlear ganglion neurons, was also examined. Neurons isolated at embryonic day 7 or day 11 and maintained two days in culture with 2 ng/ml neurotrophin-3 showed a substantial increase in Ca2+ current density, particularly in the transient component. These findings indicate that the expression of neuronal Ca2+ channels is predominant at the time of synapse formation between transducing hair cells and their primary afferents. Besides its effects on survival and neuritogenesis, neurotrophin-3 enhances the expression of Ca2+ channels in cultured neurons. Taken together these results suggest that the functional expression of Ca2+ channels is regulated during embryonic development of cochlear neurons by the release of neurotrophin-3 from the differentiating sensory epithelium of the cochlea. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: cochlear ganglion, developing neurons, calcium currents, neurotrophin-3.

The expression of voltage-gated ion channels, a fundamental aspect of the neuronal phenotype, is tightly regulated during differentiation. Numerous examples indicate that, for a given cell type, the pattern of expression of functional ion channels follows a predetermined sequence. This sequence varies from one cell type to another.41,42 There are, however, certain features common to many cells. For example, a transient Ca2+ current is prominent at early stages of development,17,29,44,52 whereas a fast, transient K+ current (IA) appears late in neuronal development. 10,31,37,38,46 The physiological significance of the expression pattern of ionic ‡To whom correspondence should be addressed. Abbreviations: BDNF, brain-derived neurotrophic factor; CG, cochlear ganglion; DHP, dihydropyridine; E, embryonic day; EDTA, ethylenediaminetetra-acetate; EGTA, 5-ethyleneglycolbis(â-aminoethylether)-N,N,N*,N*-tetraacetate; FCS, fetal calf serum; HEPES, N-2hydroxyethylpiperazine-N*-2-ethanosulphonic acid; HS, horse serum; NGF, nerve growth factor; NT-3, neurotrophin-3; PC12, rat pheochromocytoma cell line; TEA, tetraethylammonium; ù-AgTx, ù-Agatoxin IVA; ù-CgTx, ù-Conotoxin GVIA.

channels is now being recognized.41 In particular, calcium influx via voltage-dependent channels during early stages of neuronal differentiation has been implicated in the regulation of neurite initiation and growth cone motility,2,7,14 neurite elongation,1,27 neuronal migration23 and development of a normal neural phenotype.21 However, the exact mechanisms by which ion fluxes regulate neuronal differentiation and the factors that control the expression of ionic channels during development are still poorly understood. The cochlear ganglion (CG) contains the primary afferent neurons of the vertebrate auditory system. Cochlear neurons are bipolar, with a central process that synapses in the cochlear nucleus of the brainstem and a peripheral process that terminates on the sensory epithelium of the inner ear. The majority of the cochlear neurons (about 95%) are large, type I neurons, that innervate inner hair cells (tall hair cells in the avian cochlea). The development of excitability in chick CG neurons, particularly the sequence of appearance of K+ and Na+ currents and its relation to the innervation pattern of the cochlea, has been described.42 There is no information available,

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however, on the ontogeny of Ca2+ currents on cochlear neurons. The survival and differentiation of neuronal populations, including the endowment of excitability, depend on trophic factors released from their innervation targets. Rat pheochromocytoma (PC12) cells differentiate into sympathetic-like neurons in the presence of nerve growth factor (NGF).18 The differentiation includes an increased level of expression of voltage-dependent Na+ channels,9,26 a process that involves gene regulatory mechanisms.8,26 Recent studies have shown that NGF increases also the expression of voltage-gated Ca2+ channels in neuronal cell lines24,45 and developing neurons.25,43 Other neurotrophins of the NGF family also regulate the differentiation of specific neuronal populations.6 For example, survival and differentiation of CG neurons depend on the presence of the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3).3,34 The physiological relevance of these findings has been confirmed by the analysis of the phenotype of mice carrying null mutations for these neurotrophins or their receptors genes.11,12,40 In particular, these studies have shown that NT-3 is required for survival of type I neurons and the maintenance of their innervation.11,40 The aim of the present study was the identification of the components of the Ca2+ current in acutely dissociated chick CG neurons and the characterization of the expression pattern of these components throughout embryonic development. Furthermore, in order to investigate the possible role of the neurotrophin NT-3 in the regulation of Ca2+ channel expression, we have examined its effect on Ca2+ currents in neurons isolated at specific stages of development. EXPERIMENTAL PROCEDURES

Cell preparation and culture Cochlear ganglia (CG) were isolated from embryonic day (E)6 to E17 chick embryos (Spafas, Preston, CT) following the method described by Represa and Bernd36 (see also Avila et al.3). Briefly, the cephalic region was split by a medial-sagittal incision, the whole membranous labyrinth was isolated and the cochlear duct opened longitudinally to dissect the CG from the otic epithelium of the basilar papillae. The dissected CG were incubated with intermittent agitation for 15–30 min at 37)C in enzyme solutions made in Ca2+- and Mg2+-free Hanks’ salt solutions with 20 mM HEPES and 6 g/l glucose. In initial experiments several digesting enzymes were tried (dispase, collagenase, trypsin, protease) but best results, as judged by the appearance and survival of neurons, were obtained with papain (Sigma P-3125, St. Louis, MO) at a concentration of 12 units/ml, preincubated with cysteine (1 mM) and EDTA (0.5 mM). After 2 min low speed centrifugation the cells were resuspended in culture medium (see below) containing 5% fetal calf serum (FCS) and 5% horse serum (HS) (GIBCO, Grand Island, NY). Dissociated single cells were obtained by gentle and repeated passing through a flame-polished Pasteur pipette. Cell suspensions were filtered through a 70 µm nylon mesh and plated on circular 12 mm glass coverslips (Fisher, Springfield, NJ), previously coated with poly-lysine and either laminin or collagen type I (all chemicals

from Sigma). No differences were found in either neuronal survival or magnitude of the Ca2+ currents between cells plated in laminin or collagen, although cells seemed to adhere better to collagen-coated coverslips. The culture medium employed was M-199 Earles’s salts (GIBCO) supplemented with 6 g/l glucose, 2 mM glutamine, 60 ng/ml progesterone, 16 µg/ml putrescine, 50 units/ml penicillin, 50 µg/ml streptomycin, 5% FCS and 5% HS. All cultures were maintained in 5% CO2 at 37)C in a humidified atmosphere. Experiments were carried out at room temperature on large (12–30 µm), birefringent neurons, that in most cases exhibited a bipolar shape. Currents were recorded within 6 h of cell isolation, except for experiments with NT-3 in which they were recorded after two days in culture. Electrophysiology Coverslips with plated cells were mounted on the recording chamber on the stage of an inverted microscope (Nikon Diaphot) equipped with phase-contrast optics. Cells were initially perfused with a solution containing (mM): NaCl 135, KCl 5, CaCl2 2.5, MgCl2 1, glucose 17 and HEPES/ NaOH 10 (pH 7.4), 322 mOsm. For recording Ca2+ currents, the external solution contained (mM): tetraethylammonium (TEA)–Cl 120, CaCl2 10, MgCl2 1, glucose 17 and HEPES/TEA–OH 10 (pH 7.4), 323 mOsm. Ba2+ currents were measured in the same external solution but with isosmolar substitution of BaCl2 for CaCl2. All solutions were filtered. Tight-seal whole-cell recordings20 were made with fire-polished pipettes fabricated from Corning 7052, 1.5 mm o.d., glass capillaries (Garner Glass Co.) with a horizontal electrode puller (Sutter Instruments). The pipettes were filled with (mM): CsCl 135, MgATP 4, EGTA Na2 2, GTP 0.2, HEPES/CsOH 10 (pH 7.2), 300 mOsm. For pharmacological experiments, 100 µM of the protease inhibitor leupeptine (Sigma) and 1.6 µg/ml of the catalytic subunit of protein kinase A (Sigma or New England Biolabs, Beverly, MA) were included in the pipette solution to reduce spontaneous rundown of the Ca2+ currents. Pipette resistances were 3–6 MÙ. Gigaohm seals were usually formed with the 10 mM Ca external solution, by applying to the pipette gentle suction controlled by lowpressure regulators (Air-Trol, Minuteman Controls, Wakefield, MA). Bath solutions were superfused by gravity at a rate of 0.5–1 ml/min and changed by means of a six-way rotary valve. Drug-containing solutions were applied by pressure ejection from a puffer pipette located near the recorded neuron. The change in solution surrounding the cell was completed within 1 s. With termination of the air pressure pulse, bath superfusion removed the test solution in <10 s. All drugs were stored as 200–1000X stock solutions at "20)C. Nitrendipine and nifedipine (Sigma) stocks solutions were made with dimethylsulfoxide. Working solutions of ù-Conotoxin GVIA (ù-CgTx, Alomone, Israel) and ù-Agatoxin IVA (ù-AgTx, Pfizer, Groton, CT) contained 1 mg/ml lysozyme (Sigma) to block non-specific binding. Working solutions of NT-3 (human recombinant, Austral Biological, San Ramon, CA) contained 0.1 mg/ml of bovine serum albumin. Recordings were made with a patch-clamp amplifier built around a low-noise IV converter (Burr-Brown OPA 101, Tucson, AZ) with a feedback resistor of 100 MÙ. The instrument provided capacitance compensation with three time constants, series resistance compensation and 4-pole Bessel filter. Pulses were applied with a digital stimulator (DAQ Stim6, Ionoptix, Milton, MA). Seal formation was monitored with a "10 mV pulse of 40 ms applied every 200 ms. After formation of a GÙ seal, the fast capacitance transient was compensated. Whole-cell configuration was achieved by slowly increasing the suction applied to the pipette by means of the low vacuum regulator until a large capacitance transient appeared. Analysis of the transient (with the filter set at 10 kHz) showed that it could be fitted

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with a single exponential. Values of cell capacitance obtained by fitting of the exponential decay53 correlated well with those calculated from cell sizes, assuming a specific capacitance of 1 µF/cm2, and with the capacitance compensation read-outs of the patch amplifier. Some of the cells kept in culture with NT-3 presented an extensive dendritic arborization and were difficult to space clamp. These cells were excluded from the analysis. Series resistance and capacitative transients were partially compensated before recording. Whole-cell currents were elicited by pulses of 200 or 250 ms duration every 10 or 15 s, in 10 mV steps, from selected holding voltages. For the analysis of drug actions on Ba2+ currents we used a holding voltage of "80 or "90 mV and test pulses to 0 mV, which usually elicited the maximal inward current. Current and voltages were monitored on a digital storage oscilloscope (Nicolet 310, Madison, WI), filtered (1 or 2 kHz) and digitized (5 kHz) on-line, and stored on disk using and IBM AT computer equipped with DT 2821 AD/DA board (Data Translation, Marlborough, MA). The computer software employed for collection, leak subtraction and analysis of whole-cell currents was developed by Dr E. Nasi of the Dept. of Physiology, Boston University. For comparison among experiments, currents were normalized by cell capacitance. Average values are given as mean&S.E.M. RESULTS

Components of the calcium current We isolated the Ca2+ currents by using either Ba2+ or Ca2+ as charge carrier and blocking all Na+ and K+ currents. Currents through different types of voltage-gated Ca2+ channels can be distinguished by kinetic and pharmacological criteria.4,13 To identify the different components of the current we analysed the kinetics of inactivation and the effects of several blockers on neurons from embryonic stages E10 to E16. With the exception of the fast inactivating component to be described later, the results shown in Figs 1–4 are representative of all neurons from these embryonic stages. Figure 1 (A,B) shows the currents obtained in an E11 neuron, with 10 mM Ba2+ in the bath, after application of depolarizing voltage steps from a holding potential of "90 mV. As shown by the I–V relation (Fig. 1C), inward currents appeared at "40 mV, increasing in amplitude with depolarization to reach a maximum near 0 mV. Partial inactivation of the current during the 200 ms pulse is apparent for test pulses between "10 and 30 mV (Fig. 1A,B). With Ba2+ as a charge carrier, the inactivation decay could always be fitted by a single exponential with time constant of the order of 100 ms (Fig. 2A). The inactivation time constant increased slightly with voltage (Fig. 2B). The steady-state inactivation was analysed with a double-pulse protocol. A prepulse of 1 or 2 s duration to several depolarizing voltages in steps of 10 mV, was followed by a pulse to 0 mV (Fig. 2C, lower panel). Both peak and steady (at end of pulse) currents decreased with less negative prepulse voltages, as shown in Fig. 2C. To analyse the voltage dependence of inactivation of the transient component we plotted the difference between peak and end-of-pulse currents versus the prepulse voltage (Fig. 2D). The sigmoidal relation

Fig. 1. Ba2+ currents recorded in an E11 neuron. A and B) Family of currents obtained from a holding voltage of "90 mV following depolarizing pulses to the values indicated. C) Current–voltage relationship for the peak currents shown in A and B.

was adjusted to a Boltzmann equation (see Fig. 2 legend). For three different experiments, the halfinactivation voltage, V1/2, averaged "45&5 mV and the slope factor was k=15&1 mV. The high activation threshold ("40 mV), the time constant for inactivation (around 100 ms) and the value of V1/2, are all consistent with the presence of a transient current mediated by N-type Ca2+ channels.4,13 Occasionally, in embryos of age E13 and older, and always with Ca2+ as a charge carrier, we observed the presence of more than one inactivating component. Figure 3A shows the currents recorded in one of these experiments. Small, fully-inactivating currents start at "60 mV, slowly increasing in

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Fig. 2. Inactivation of Ba2+ currents. A) Decay of the current obtained in response to a pulse from "80 to 10 mV and fitting to a single exponential (E12 neuron). B) Voltage dependence of the inactivation time constant, n=7. Error bars represent&S.E.M. C) Steady-state inactivation. Currents obtained in response to a pulse to 0 mV from the prepulse voltages indicated (E10 neuron). Lower inset shows the voltage protocol. D) Voltage dependence of steady state inactivation for the transient component of the currents shown in C. Peak minus end of pulse values are represented versus the prepulse voltages. Solid curve shows the fitting, using a Marquardt–Levenberg algorithm, to the equation: Ipeak"Isteady=Imax/ [1+exp[(V"V1/2)/k]], where Imax is the value of Ipeak"Isteady at "100 mV.

amplitude with depolarization. An abrupt increase in amplitude is seen at voltages more positive than "10 mV, reflecting the activation of high threshold currents. The presence of the low threshold current is indicated by a shoulder in the I–V relation (Fig. 3B). Note that the I–V relation is shifted to the right in the presence of Ca2+ (compare with Fig. 1C), indicating a surface charge binding effect of Ca2+. The analysis of the inactivation kinetics showed the presence of two time constants. Figure 3C shows the decay of the current obtained at +10 mV and the fitting obtained by either a single exponential of time constant 132 ms or a double exponential of time constants 20 and 132 ms. The voltage dependence of the slow time constant was similar to that described previously for the inactivation seen with Ba2+, with a slight increase with voltage. The fast time constant, however, showed a decrease with voltage (Fig. 3D). This fast inactivating component of lower activation threshold probably reflects the presence of T-type channels.4,13,44 These channels have also been described in adult cochlear neurons by Yamaguchi and Ohmori.53 The fact that we observed these currents only with

Ca2+ as charge carrier probably reflects the higher permeability of T channels for Ca2+ than for Ba2+.13,53 The total current was higher, however, when Ba2+ was used as charge carrier. In six neurons where the currents were consecutively recorded with both ions the ratio IBa/ICa of maximum peak currents was 2.8&0.1. The use of specific Ca2+ channel blockers has allowed, in certain preparations, the dissection of the different components of the current. Cd2+ is a non-selective blocker of all types of Ca2+ channels, although it is somewhat more effective on N- and L-type channels at concentrations lower than 50 µM.4,13 Figure 4A shows the fast and reversible inhibition of current by 20 µM Cd2+ applied from a puffer pipette. The circles indicate the peak Ba2+ current in response to a voltage pulse from "80 to 0 mV applied every 12 s. The record starts 1 min after the attainment of the whole-cell configuration. Application of 20 µM Cd2+ resulted in the inhibition of 92% of the current. In two other experiments the percentage of current blocked by Cd2+ was 93% and 98%. ù-CgTx, a preferential blocker of N-type Ca2+

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Fig. 3. Ca2+ currents in an E13 neuron. A) Family of currents obtained by depolarizing from "90 mV to the voltages indicated, using 10 mM Ca2+ as charge carrier. B) Current–voltage relationship for the peak currents shown in A. C) Decay of the current in response to a pulse to +10 mV and fitting to either single or double exponentials. D) Voltage dependence of fast (open circles) and slow (filled circles) inactivation time constants for the currents shown in A.

channels, inhibited a fraction of the Ba2+ current when applied at a concentration of 5 µM (Fig. 4B). The inhibition by this toxin was irreversible. On average, 5 µM ù-CgTx blocked 48&7% of the current (n=6). On the other hand, 1 µM ù-AgTx, a blocker of mammalian P- and Q-type Ca2+ channels, did not have any effect on the Ba2+ current (n=4, data not shown). Sensitivity to the dihydropyridines (DHPs) is considered a pharmacological hallmark for the presence of L-type Ca2+ channels, characterized by a high activation threshold and long inactivation kinetics. The DHPs nisoldipine, nitrendipine and nifedipine partially inhibited the Ba2+ current. Figure 4C shows the inhibition by 2 µM nitrendipine. The inhibition was slowly reversible. The fraction blocked by the DHPs exhibited very little inactivation, as shown in Fig. 4D. Inhibition by the DHPs, at concentrations between 1 and 10 µM, ranged between 18 and 44% (n=7) at a holding voltage of "80 mV. Calcium currents during embryonic development The above experiments indicate that chick CG neurons contain mainly two components of Ca2+ currents, both with a high activation threshold. One of them inactivates with a time constant of some

100 ms whereas the other exhibits a much longer inactivation kinetics. The sensitivity to blockers is also consistent with the presence of both, N- and L-type Ca2+ channels. Evidence for low-voltage activated Ca2+ current such as that mediated by T-type channels was found in few of the cells studied and only in neurons from older embryos. Although it is not possible to rule out the presence of additional components (see Discussion) we have analysed the temporal pattern of expression of Ba2+ currents during embryonic development by separating the total current into two components. A sustained, slowly inactivating component was taken as the current remaining at the end of a 250 ms pulse from "90 to 0 mV. The transient or inactivating component was calculated as the difference between the peak of the current and the sustained fraction. Although this is a convenient method to separate the components, it probably overestimates the amplitude of the sustained current due to incomplete inactivation of the transient current during the 250 ms. Using this approximation, we have calculated the current density (amplitude/cell capacitance) of both components throughout development and plotted the results in Fig. 5. Current densities were employed in order to account for the increase in cell area during

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Fig. 4. Block of Ba2+ currents. Circles indicate peak currents elicited by pulses from "80 to 0 mV applied every 12 s. Puffer application of 20 µM Cd2+, E16 neuron (A), 5 µM ù-CgTx, E12 neuron (B) and 2 µM nitrendipine, E13 neuron (C) during the periods marked. Records start 1 min after attainment of the whole-cell configuration. Insets show current traces at the indicated times. D) Ba2+ currents in response to a pulse from "80 to 0 mV in an E15 neuron, before and after application of 10 µM nifedipine (top) and fraction inhibited by nifedipine obtained by digital subtraction (bottom).

E8–9. Afterwards there was a subsequent decline in current density, with a more drastic reduction of the inactivating component (hatched bars), such that Ba2+ currents in mature neurons (E16) exhibited little inactivation. These results indicate that the maximum expression of Ca2+ channels occurs during the period of early synaptogenesis (E8–9),50 reinforcing the notion that the influx of Ca2+ through voltage-activated channels play an important role in this process. Enhancement of calcium currents by neurotrophin-3

Fig. 5. Temporal pattern of expression of Ba2+ currents during embryonic development of chick CG neurons. Open bars indicate the current at the end of a 250 ms pulse to 0 mV. Hatched bars are peak current minus the value at the end of the pulse. Error bars represent the S.E.M.

development (cell capacitances increased from 6& 0.4 pF at E7 to 25&1.8 pF at E16). The earliest day when Ba2+ currents were consistently found was E7. There was an increase in both components of Ba2+ current early in development, reaching a peak at

NT-3 enhances the survival and neuritogenesis of chick CG neurons3 and it is required for the innervation of the mouse inner hair cells.11 To test whether NT-3 regulates also the expression of Ca2+ channels, we have analysed its effects on Ba2+ currents. Neurons isolated from embryos of age E7 or E11 were grown in medium supplemented with 5% FCS and 5% HS and containing 2 ng/ml of NT-3. Control cells were maintained in medium without NT-3. Few neurons survived after two days in the absence of NT-3 and they showed a lack of neurite extension. On the contrary, neurons maintained with NT-3 survived in larger number and exhibited bipolar

Modification of calcium currents in cochlear neurons Table 1. Sustained and transient (inactivating) components of Ba2+ current in isolated chick cochlear ganglion neurons. Effects of neurotrophin-3

E7 E7+d2 (NT-3) E11 E11+d2 (NT-3)

Sustained

Transient

n

20&3 104&16* 95&31 99&37

36&6 85&5* 81&18 208&43*

4 5 7 4

Sustained and transient current components (pA/pF) were calculated as explained in text. Currents from E7 and E11 neurons were recorded within 6 h of isolation. Currents in the presence of NT-3 (2 ng/ml) were recorded after two days in culture. Values shown are means&S.E.M. *Significantly different from the corresponding values measured in acutely-dissociated neurons (P<0.01)

neuritic processes, as previously described.3 Table 1 shows the sustained and transient components of Ba2+ current elicited by a voltage pulse from "90 to 0 mV. In the presence of NT-3 there was a significant 2.5-fold increase of the transient Ba2+ current component in both, E7 and E11 neurons. The sustained component increased in E7 neurons, but it was not affected in E11 neurons. Neurons in the absence of NT-3 did not survive in numbers sufficient to allow methodical measurement of Ba2+ currents. DISCUSSION

The aims of these studies were to identify the components of Ca2+ current in dissociated cochlear neurons at different stages of development and to assess the importance of the neurotrophin NT-3 in the induction of the currents. The experiments have disclosed the presence of several components of Ca2+ current. A component, characterized by a high activation threshold ("40 mV), relatively fast inactivation (time constant ~100 ms) and steady-state inactivation with V1/2="45 mV, is probably mediated by N-type Ca2+ channels. The sensitivity of the total current to ù-CgTx indicates also the presence of N-type channels. Sensitivity to the DHPs uncovered another high-threshold component with slow inactivation (time constant near 1 s), that is probably mediated by L-type channels. These components, at different proportions, were observed throughout the different stages of embryonic development. An additional component with a low activation threshold and fast inactivation (time constant ~20 ms) was observed only in neurons from embryos older than E13. This component was more evident when using Ca2+ as a charge carrier and it is probably mediated by T-type channels. It is difficult to ascertain the presence of additional current types. Assuming that the inhibitions by ù-CgTx (48%) and the DHP’s (30%) are additive, a resistant component of the total Ca2+ current remains. However, the implicit assumption of complete blocking efficacy by the antagonists may not be true.32 On the other hand, ù-CgTx has

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been found to block, in addition to N-type current, L channels in avian sensory and rat sympathetic neurons28 and in rat hippocampal pyramidal cells.44 Thus, it is possible that the fraction blocked by this toxin includes some of the L current. Finally, the Ca2+ current in chick cochlear neurons was insensitive to ù-AgTx, a strong blocker of P- and Q-type currents in mammalian systems. Although this would suggest the absence of these type of channels, the conclusion has to be taken with care since ù-AgTx is a weak blocker of avian Ca2+ channels.32 Based on differences in activation threshold and inactivation kinetics, Yamaguchi and Ohmori53 have described the presence of T- and L-type Ca2+ currents in mature (E16) chick cochlear neurons. Their description agrees with our observation of T currents in neurons from embryos older that E13. They did not report the presence of N-type current, probably due to the low level of expression of this component on acutely dissociated neurons from later stages of embryonic development, as shown in Fig. 5. Based in our results (Fig. 5), we can trace a correspondence between the development of Ca2+ currents in cochlear neurons and the innervation pattern of the cochlea described by Whitehead and Morest.50 On E6–7 the peripheral endings of the cochlear neurons invade the sensory epithelium. A fraction of the cells in the ganglion concomitantly begin to show voltage-dependent Ca2+ currents. Early synaptic contacts are established near E8–9, in association with continued hair cell differentiation. This period is associated with a rapid increase in the amplitude of the Ca2+ currents. Thereafter, during the intermediate stages of synaptogenesis (E10–13), there is a decrease in the amplitude of the Ca2+ currents, more pronounced in the inactivating component. By E14–15 the mature structure of the cochlear receptor develops. Coincident with this period, the non-inactivating component stabilizes, reaching a plateau, the inactivating component is minimally expressed and a fraction of the neurons exhibits a fast inactivating, low-threshold component. It is interesting to note that chick cochlear hair cells express only L-type Ca2+ channels.15 These currents are present from E12, the earliest stage studied, whereas the Ca2+-activated K+ current, responsible for the high frequency electrical tuning of the cochlea, appears abruptly at E19. The prominent expression of Ca2+ channels at the time of synapse formation suggests that they play an important role in this process, as in other aspects of neuronal development.16,42 Of particular interest is the transient expression of the inactivating component (Fig. 5). In other systems, an inactivating Ca2+ current, although of the T-type, is expressed preferentially during early stages of development.17,29,44,52 However, ù-CgTx-sensitive Ca2+ channels are also involved in a variety of early developmental processes such as neurite outgrowth and the acquisition of neurotransmitter phenotype,21 migration of imma-

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ture neurons23 and the release of glutamate in developing neurons.47 The low level of expression of the transient component in later stages of development could reflect a progressively more restricted localization of these channels to distal neuritic processes during development which are then cut off during the cell isolation procedure. In adult CNS neurons, a subtype of an N-type channel subunit is localized predominantly in dendrites,48 although ù-CgTxsensitive channels have been localized on both, soma and dendrites.22,30 Several studies point to an important role of NT-3 in the survival and differentiation of CG neurons. NT-3 promotes the survival and neuritogenesis of chick CG explants and isolated neurons.3 These effects of NT-3 are stage-dependent with a maximum at the time of early synaptogenesis (E7–8). In situ hybridization studies in the rat and mouse have detected the appearance of mRNAs for the neurotrophin receptors TrkB and TrkC in the CG before and during innervation of the hair cells. Similarly, mRNAs for BDNF and NT-3 mRNAs are expressed by the sensory epithelium.33,34,39,49,54 It should be noted, however, that neither NT-3 nor TrkC mRNAs19,51 have been detected in the chick auditory system. On the other hand, quail CG express mRNAs for NT-3 and TrkC.5 Although lack of detection of mRNA could be due to expression levels below the detection limit of the in situ hybridization technique, it is not possible at this time to discard the possibility that the biological effects of NT-3 on chick CG neurons, including those described in the present study, are due to the activation by NT-3 of receptors others than its cognate receptor, TrkC. The physiological significance of NT-3 for the development of the mouse auditory system has been demonstrated by the morphological analysis of mice carrying a null mutation for BDNF and/or NT-3. The results indicate that NT-3 is required for survival of type I cochlear neurons and the maintenance of innervation.11 This requirement has been validated by the analysis of knockouts of the neurotrophin receptors TrkB and TrkC.40 Based on the above findings and the suggested role of voltage-gated Ca2+ channels in several early developmental processes,41 it was clearly

of interest to investigate whether NT-3 has any effect on the expression of Ca2+ channels in cochlear neurons. Our results show that neurons grown in the presence of NT-3 exhibit an increase in Ca2+ current density, particularly in the transient or inactivating component. This finding suggests that the transient expression of Ca2+ currents during embryonic development shown in Fig. 5 may be directly related to the release of NT-3, or a related neurotrophin, by the differentiating hair cells and surrounding supporting cells of the cochlea. The present study is the first instance describing an effect of NT-3 on the expression of ion channels in differentiating neurons. In PC12 cells NGF increases preferentially the inactivating, ù-CgTxsensitive component of the Ca2+ current.45 Both, p21ras-dependent and independent signal transduction pathways are involved in the NGF induction of Ca2+ channels in this cell line.35 CONCLUSION

Embryonic chick CG neurons exhibit two main components of Ca2+ current. Both components activate with a high voltage threshold but differ in the kinetics of inactivation. An additional component, characterized by a low activation threshold and fast inactivation, was observed in mature neurons. Ca2+ current density reached a maximum at day 9 of embryonic development, when early synaptic contacts between the transducing hair cells and their primary afferents are established. NT-3, a neurotrophic factor required for the survival and differentiation of CG neurons, increased the density of Ca2+ current in dissociated neurons grown in culture. These results suggest a role for NT-3, or a related neurotrophin, in the regulation of the functional expression of Ca2+ channels during the embryonic development of cochlear neurons. Acknowledgements—We thank T. G. Cachero and C. Cornwall for critical reading of the manuscript. This work was supported by DGICYT PB92-0261 (F.G.), an Eusko Jaularitza predoctoral fellowship (C.J.) and DGICYT SAB94-0037 (J.F.G.-D.). Additional support from NIH DK40127 is acknowledged.

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