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MS 9123 Received 5 January 1999; accepted after revision 30 March 1999.
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ABSTRACT |
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-conotoxin-GVIA (N-type), and high concentrations of -agatoxin-IVA (Q-type). The P-type current, sensitive to low concentrations of -agatoxin-IVA, transiently increased between E15 and E17, and then both current density and its proportion of the global current decreased.
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INTRODUCTION |
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The development of mouse vestibular receptors has been extensively studied, including the various steps in the differentiation and maturation of primary neurons and their related synaptogenesis from the undifferentiated stage to adult (Anniko, 1983). In mouse vestibular sensory epithelia, the main period of neuronal growth occurs around embryonic day 17 (E17) (Nordemar, 1983; Anniko, 1983). Furthermore, between E15 and birth there is a substantial development of synaptic contacts between primary vestibular afferents and sensory hair cells (Nordemar, 1983; Mbiene et al. 1988). Conversely, the development of vestibular function in mammals has been the subject of few studies dealing with the development of physiological activity (Desmadryl et al. 1986) and the increase of the postnatal sensitivity of primary afferents (Desmadryl, 1991). This functional development of afferents could be related to changes in ionic channel expression in the primary neurons (Desmadryl, 1991). The principal conclusion of these studies is that morphological and physiological properties of the primary afferents are mainly determined during embryonic ontogenesis. In view of these results, we decided to investigate the embryonic development of voltage-dependent calcium channel currents in vestibular neurons, since intracellular calcium regulation is known to play a major regulatory role in ontogenesis.
In neurons, calcium is implicated in various developmental events such as neurite outgrowth, synapse formation and elimination, phenotypic differentiation (Larmet et al. 1992; Desarmenien et al. 1993; Spitzer, 1994; Gu & Spitzer, 1995) and physiological maturation (D'Angelo et al. 1997). Also, calcium acts as a second messenger for gene expression (Bading et al. 1993). We have previously demonstrated the presence of L-, N-, P- and Q-type high voltage-activated (HVA) calcium currents in postnatal mouse primary vestibular neurons. In comparison, low voltage-activated (LVA) current was only found in some large diameter neurons (Desmadryl et al. 1997). These different conductances have been associated with specific functions (McCobb & Beam, 1991; Murphy et al. 1991; Wheeler et al. 1994; Mintz et al. 1996).
The present study investigates, for the first time, changes in calcium currents recorded in cell bodies of mouse vestibular ganglion neurons during development and maturation, using the whole-cell patch-clamp technique. This work focused on the embryonic period when the developmental processes of neuritic growth, target innervation, synaptogenesis and increase in postsynaptic sensitivity occur.
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METHODS |
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Cell culture
Calcium channel currents were studied in neurons isolated from the superior branch of the vestibular nerve innervating the utricular macula and the horizontal and lateral cristae. Ganglia were aseptically dissected from Swiss mice (CERJ, Le Genest, France). Embryos were obtained from pregnant mice killed by inhalation of CO2 followed by cervical dislocation, in accordance with national guidelines. Embryos and new-born mice were rapidly killed by decapitation. Embryonic age was calculated from the day on which the vaginal plug was detected (day 1), and mice were born after 20 days of gestation (E20 = P0). About 20 ganglia for each experiment were collected in phosphate-buffered saline (PBS) (Gibco) and then incubated at 37°C in PBS containing EDTA- trypsin (Gibco). We tested different incubation times and trypsin concentrations at each stage without any difference in the amplitude or the shape of the recorded currents. We settled on protocols employing trypsin at 0·25 % for 5 min (E14), 6 min (E15), 8 min (E17) and 10 min (P0). Ganglia were triturated with fire-polished Pasteur pipettes of two decreasing diameters in Neurobasal medium (Gibco) containing 10 % B27 (Gibco), 25 µM glutamate and 0·25 mM glutamine. Neurons were plated onto 35 mm culture dishes (Nunc) coated with 10 µg ml-1 poly-D-ornithine (Sigma). Cells were used between 1 and 6 h after dissociation. Under phase contrast microscopy, dissociated neurons had spherical refringent cytoplasm as previously reported (Desmadryl et al. 1997). The diameter of the recorded cells ranged between 12·5 and 25 µm, and we used neurons without processes in order to avoid space clamp artefacts.
Electrophysiological recordings
Whole-cell recordings of voltage-activated barium currents (IBa) through calcium channels were obtained at room temperature under conditions optimized to ensure their complete isolation from other voltage-activated currents. Tetrodotoxin (1 µM, Sigma) was used to block Na+ currents, and extracellular Na+ was replaced by the non-permeant ion tetraethylammonium (TEA+). Potassium currents were blocked by replacing internal K+ with Cs+ ions and by using extracellular TEA+. The extracellular solution contained (mM): TEACl, 120; BaCl2, 5; Hepes, 10; and glucose, 10. The pH was adjusted to 7·35 with TEAOH and the osmolarity was set at 310 mosmol l-1 with TEACl. Recording pipettes were pulled from haematocrit tubes (Modulohm I/S, Herlev, Denmark) and filled with the following intracellular solution (mM): CsCl, 130; EGTA, 10; Hepes, 25; MgATP, 3; NaGTP, 1; and glucose, 10. The pH was adjusted to 7·35 with CsOH and the osmolarity set at 310 mosmol l-1 with CsCl. The resistance of the filled pipettes was between 2 and 3 M
. Whole-cell currents were recorded using a Biologic RK400 patch-clamp amplifier. After seal formation and membrane disruption, cell capacitance and series resistance were estimated from the decay of the capacitance transient induced by a ±10 mV pulse from a holding potential of -100 mV. The series resistance was in the range of 4-8 M and the membrane capacitance could be charged with a time constant around 100 µs. Series resistance was compensated for 60 % when necessary, after cancellation of the capacitive transients. Neurons were discarded when the residual series resistance gave a voltage error greater than 5 mV for the peak current. The liquid junction potential between the internal and the extracellular solution, measured according to Neher (1992), was -5·4 mV at 20°C (data presented were not corrected for junction potential). For pharmacological experiments using calcium current antagonists, current-voltage relationships were monitored before and after drug delivery, and cells were rejected for analysis if there was a displacement of the peak current after drug delivery. Neurons were held at a holding potential of -100 mV and currents were recorded in response either to 300 ms pulses at various voltages from -100 to +50 mV, or to a voltage ramp of 500 ms duration from -100 to +50 mV. Current signals were filtered at 3 kHz, digitized and stored. The linear component of the leakage current, though usually small, was calculated from an appropriately scaled current elicited by a 10 mV pulse, and digitally subtracted from the current traces. Rundown of barium currents in the vestibular neurons was very small even during prolonged recording.
Drugs
Nitrendipine (Sandoz) and Bay K 8644 (Bayer AG) were dissolved in dimethylsulfoxide to make a stock solution (10 mM) which was then added to the extracellular medium. -Conotoxin-GVIA (-CTX-GVIA, Sigma) and -agatoxin-IVA (-Aga-IVA, Pfizer) were dissolved in bidistilled water and stored at -80°C. Cytochrome c (Sigma) (1 mg ml-1) was added to -Aga-IVA solutions to prevent adsorption onto the walls of the container. A pressure syringe pump (Microlab 500, Hamilton) was used to deliver drugs and toxins. This system made it possible to use small volumes of peptides (typically 50 µl) in a broken tip microelectrode (
20 µm). The solutions were delivered close to the cell over a 60-120 s period.
Analysis
All experimental parameters, such as holding potential and test potential, were controlled with an IBM PC equipped with a Tecmar Labmaster analog interface (Axon Instruments). Cell stimulation, data acquisition, and analysis were performed using pCLAMP software (version 5.5, Axon Instruments). From E14 to E17, all neurons tested had LVA and HVA IBa. These two classes of current could not be completely separated by voltage protocols or pharmacological tools. However, the LVA IBa was transient with a fast decay and did not contribute to the global IBa measured after 200 ms of depolarization. Therefore, the amplitude of the HVA IBa was systematically measured 250 ms after the beginning of the depolarizing pulse. LVA IBa decay, measured at a -40 mV test pulse, was fitted (pCLAMP software, Axon Instruments) with a single exponential according to the equation I(t) = Is + IBaexp(-t/
), where Is represents the sustained current, IBa is the amplitude, and is the time constant of current decay. Data are given as means ± standard deviation. Statistical significance was tested as appropriate, either by Student's t test or by one-way analysis of variance (ANOVA) followed, when necessary, by Fisher's post hoc test.
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RESULTS |
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The distribution and characteristics of voltage-gated IBa were studied in 13 (E14), 53 (E15), 43 (E17) and 68 (P0) acutely dissociated vestibular neurons under conditions designed to eliminate other voltage-activated currents. The average cell capacitances were 13·3 ± 4·5 pF (n = 10), 14·4 ± 4·1 pF (n = 37), 16·4 ± 3·8 pF (n = 23) and 15·4 ± 5·2 pF (n = 29), at respectively E14, E15, E17 and P0. The cell capacitance did not significantly change during development (P > 0·2). Immediately after rupture of the membrane, peak and transient inward currents generated by test pulses from a holding potential of -100 mV to 0 mV increased in amplitude (run-up) reflecting the increase of HVA currents, as previously described in dorsal root ganglion neurons (Diochot et al. 1995) and in our preparation (Desmadryl et al. 1997). These currents reached a steady state in 1-2 min, after which period, physiological and pharmacological experiments were commenced. Addition of inorganic calcium channel blockers to the external solution (Ni2+ and Cd2+ applied together at 0·5 mM) totally eliminated these currents.
Global barium currents
Representative waveforms of voltage-activated inward IBa generated at three different depolarizations from a holding potential of -100 mV are shown in Fig. 1Aa-Da. Corresponding current-voltage (I-V ) relationships show the peak and the sustained currents (Fig. 1Ab-Db). From E14 to P0, all vestibular neurons had HVA IBa (Fig. 1A-D). LVA IBa was present in almost all neurons tested at E14, E15 and E17, but only 20 % of the neurons expressed LVA IBa at P0 (Fig. 1D).
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Aa-Da, typical records of IBa currents elicited by 300 ms depolarizations in the presence of 5 mM barium. Current traces correspond to single records at various voltages (as indicated) from a holding potential of -100 mV. The voltage protocol for each panel is shown at the top. At E14 (Aa) and E17 (Ba) the traces elicited by a pulse to -40 mV show LVA IBa whereas HVA IBa was evoked by higher depolarizations. At P0, two neurons illustrate the presence (Da) and lack (Ca) of LVA IBa during a pulse to -40 mV. At this stage, note the difference in the shape of the current waveforms elicited by depolarizations to -40 and 0 mV between the two cells. Ab-Db, corresponding current-voltage (I-V ) relationships evoked by 10 mV incrementing steps from a holding potential of -100 mV. Each plot shows IBa peak ( | ||
Low voltage-activated current
At each stage of development, LVA IBa had a threshold at around -60 mV from a holding potential of -100 mV, and a maximum peak amplitude at -40 mV (Fig. 1Aa, Ba and Da). This current did not express a run-up and reached its maximum amplitude immediately after disruption of the membrane. The LVA IBa decayed rapidly and was almost totally inactivated after 100 ms (Fig. 1Aa, Ba and Da, -40 mV traces). We studied the characteristics of inactivation of LVA IBa at a depolarization of -40 mV from a holding potential of -100 mV. The time constants of the current decay were 24·2 ± 4·5 ms at E14 (n = 12), 24·6 ± 5·9 ms at E15 (n = 44), 22·0 ± 4·9 ms at E17 (n = 35) and 22·4 ± 4·4 ms at P0 (n = 11). The differences were not statistically significant (P > 0·2). These time constants were similar to the ones reported in developing visual cortical neurons (Tarasenko et al. 1998). Steady-state inactivation was studied using a protocol in which a test pulse at -40 mV was preceded by a 5 s conditioning pulse at various amplitudes from -110 to -30 mV (Fig. 2Aa, Ba and Ca). Peak IBa responses were normalized with respect to maximum peak IBa and plotted against the prepulse voltage for these neurons (Fig. 2Ab, Bb and Cb). The mean half-maximum inactivation potentials were -67·5 ± 4·5 mV at E14 (n = 7), -65·3 ± 2·8 mV at E15 (n = 7), -67·0 ± 3·7 mV at E17 (n = 8) and -70·2 ± 4·8 mV at P0 (n = 6). Differences between stages were not significant (P > 0·1). The characteristics of inactivation of LVA IBa did not change during development.
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Aa-Ca, representative waveforms of currents evoked by a 400 ms test pulse after 5 s conditioning prepulses at various voltages at E14, E15 and E17. Current traces correspond to single records. The voltage protocol is shown at the top of Ba. Ab-Cb, normalized steady-state inactivation was plotted against conditioning prepulse amplitude. The half-maximum inactivation potentials of LVA IBa were -68 mV (E14), -67 mV (E15) and -66 mV (E17) for these neurons. | ||
The proportion of neurons with LVA IBa decreased drastically between E17 and P4 (P4 data taken from Desmadryl et al. 1997) (Fig. 3A). At P0 and P4, LVA IBa was restricted to large diameter neurons. Based on previously published observation (Desmadryl et al. 1997), we divided the current densities of LVA into two groups of neurons. The first of these (Fig. 3B, filled circles) consisted of cells with LVA IBa densities lower than 50 pA pF-1. From developmental stage E17, a second group included neurons with current densities higher than 50 pA pF-1 (Fig. 3B, open circles). The mean current densities of the first group decreased significantly between E17 and P0 (P < 0·001), whereas that of the second group increased between E17 and P4 (P < 0·05) (Fig. 3C).
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LVA IBa was elicited by depolarization to -40 mV from a holding potential of -100 mV (P4 data taken from Desmadryl et al. 1997). A, relative proportions of neurons with LVA IBa at each stage of development. Note the drastic diminution in the number of neurons with LVA IBa between E17 and P0. B, individual values of current densities during development. The densities of LVA IBa have been separated into two groups: the first ( | ||
High voltage-activated currents
All neurons tested at different stages had HVA IBa. The activation thresholds of global HVA IBa ranged between -40 and -30 mV and the maximal amplitudes occurred between -10 and 0 mV for all stages analysed. The mean amplitudes of HVA IBa, measured 250 ms after the beginning of a pulse to 0 mV from a holding potential of -100 mV, were 740 ± 244 pA (n = 13) and 806 ± 440 pA (n = 53) at E14 and E15, respectively. The amplitudes increased in the older stages to 1221 ± 632 pA (n = 41) at E17 and 1044 ± 558 pA (n = 68) at P0. The mean current amplitudes at E14-E15 were significantly lower than at E17-P0 (P < 0·05). The different components of HVA IBa at E15, E17 and P0 were characterized by classical pharmacological tools.
We tested the presence of the L-type current using the specific dihydropyridine (DHP) agonist Bay K 8644 (Hess et al. 1984) (Fig. 4Aa-c). Application of 3 µM Bay K 8644 enhanced IBa elicited by a pulse to -20 mV from a holding potential of -100 mV by 228 ± 159 % at E15 (n = 4), 218 ± 144 % at E17 (n = 4) and 154 ± 33 % at P0 (n = 3). The I-V curve was shifted towards hyperpolarization (data not shown) as reported previously (Desmadryl et al. 1997). In all neurons tested, we observed the typical effects of Bay K 8644 on the inward tail current where deactivation was prolonged when the potential returned to -50 mV after a pulse to -20 mV (Fig. 4Aa-c). The relative proportion of DHP-sensitive L-type HVA IBa was estimated with a pulse to 0 mV from a holding potential of -100 mV using 1 µM nitrendipine, a specific DHP antagonist (Fig. 4Ba-c). Nitrendipine blocked similar proportions of global HVA IBa at each stage of development: 22·6 ± 10·8 % at E15 (n = 4), 17·3 ± 8·8 % at E17 (n = 4), and 19·9 ± 4·5 % at P0 (n = 8).
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A, effect of 3 µM Bay K 8644 on IBa currents evoked by a pulse to -20 mV from a holding potential of -100 mV on neurons isolated at E15 (a), E17 (b) and P0 (c). The voltage protocols for each panel are shown at the top. Application of Bay K 8644 induced an increase of the current and a prolongation of the tail current when returning to -50 mV, for each stage. Note at E17 a large inactivation of the tail current in the presence of Bay K 8644 due to the typical effect of this DHP, which induces displacement towards hyperpolarization of the I-V curve. Current traces correspond to single records. B, effect of 1 µM nitrendipine on IBa generated by a pulse to 0 mV from a holding potential of -100 mV. For each stage, digital subtraction of trace (1) from trace (2) shows the nitrendipine-sensitive current. Note that the blocked current accounts for a similar proportion of the total current studied at each stage. Current traces correspond to single records. | ||
The contribution of N-, P- and Q-type HVA IBa was assessed over the period from E15 to P0 by successive applications of the specific antagonists (-CTX-GVIA, low and high concentrations of -Aga-IVA, respectively). We used the N-type antagonist -CTX-GVIA at a saturating concentration of 1 µM (Fig. 5A-C). The saturating effect of the toxin occurred rapidly after the onset of application. -CTX-GVIA blocked a similar proportion of IBa at each developmental stage: 34·0 ± 13·9 % at E15 (n = 13), 30·1 ± 9·6 % at E17 (n = 12), and 31·9 ± 12·4 % at P0 (n = 15). The blocking effect of -CTX-GVIA was irreversible (data not shown). Digital subtraction of traces obtained before and after -CTX-GVIA deliveries shows the N-type IBa current (Fig. 5Ab-Cb).
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Aa-Ca, effect of 1 µM | ||
As previously described (Mintz et al. 1992), a low concentration of -Aga-IVA (30 nM) inhibited a sustained IBa. In vestibular neurons, maximum inhibition occurred within 3-5 min with current decreases of 21·5 ± 10·1 % at E15 (n = 13), 31·5 ± 11·7 % at E17 (n = 8), and 15·5 ± 3·2 % at P0 (n = 14). Digital subtraction of traces obtained before and after toxin application revealed that this current was not inactivated (Fig. 5Ac-Cc). The effects of this toxin at low concentration were consistent with the presence of a P-type current at each stage of development.
Over the same period using -Aga-IVA at high concentration (1 µM), we characterized Q-type HVA IBa in conditions inhibiting N- and P-type HVA IBa. Vestibular neurons expressed Q-type IBa at each stage of development (Fig. 5Ad-Cd). The percentage of inhibition was similar at each developmental stage: 25·1 ± 8·2 % at E15 (n = 10), 22·7 ± 14·7 % at E17 (n = 5), and 23·9 ± 7·9 % at P0 (n = 9). The Q-type current was more strongly inactivated than the P-type current during 300 ms test pulses, as shown by digital subtraction of the current after inhibition by a high -Aga-IVA concentration (1 µM) (Fig. 5Ad-Cd).
Results for HVA IBa, together with previously published observations obtained at P4 (see Desmadryl et al. 1997), revealed that the densities of DHP-sensitive L-type current did not change throughout the development period studied, whereas those of N- and Q-type IBa increased steadily between E15 and P4 (Fig. 6). In contrast, our results revealed a transient increase in the P-type current density between E15 and E17, followed by a decrease at later stages (Fig. 6).
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The contribution of each current was evaluated by its specific sensitivity to 1 µM nitrendipine (DHP-sensitive L-type), 1 µM | ||
In all stages studied, an additional current characterized by its resistance to saturating concentrations of DHP, -CTX-GVIA and -Aga-IVA was totally blocked by application of 0·5 mM Ni2+ and Cd2+ (data not shown). We did not study this current in detail. However, based on its previously defined characteristics (Desmadryl et al. 1997), this current was similar to the R-type current described in neurons (Randall & Tsien, 1995; Hilaire et al. 1997).
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DISCUSSION |
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Using the whole-cell patch-clamp technique, we studied the development of different types of calcium channel currents in embryonic mouse primary vestibular neurons. At each stage, LVA and HVA IBa were identified on the basis of their activation thresholds, inactivation decays, and sensitivity to specific pharmacological agents (DHPs, -CTX-GVIA, -Aga-IVA). We report here, for the first time, significant changes in the relative contribution of LVA, and N-, P- and Q-type HVA IBa during the period of embryonic development. Because of their correlation in time with the different stages of neuronal ontogenesis, these events are probably related to the development and the maturation of the primary vestibular afferents. An alternative explanation could be that some of the modifications in the expression of these currents are due to a migration of the channels to distal neuritic process, during neurite elongation. However, our results clearly show that some of the calcium channels remain stable (e.g. DHP-sensitive L-type), or increase continuously during development (N- and Q-type) indicating a quantitative change in the number of channels present in the neuronal membrane.
Development of low voltage-activated current
LVA IBa present in vestibular neurons has electrophysiological properties similar to those reported for mouse sensory neurons (Carbone & Lux, 1987), and more generally, to those of the T-type current present in many cell types (Bean, 1989). The characteristics of activation and inactivation did not change between E14 and birth indicating that LVA calcium channel properties remain constant during development. These properties are similar to those reported in large diameter postnatal vestibular neurons (Desmadryl et al. 1997).
In contrast, the present results demonstrate developmental changes both in the number of neurons with LVA IBa and in the amplitude of the current. During the first developmental stages studied (E14-E17), the entire population of vestibular neurons possesses LVA IBa, while at birth (and P4, see Desmadryl et al. 1997), this current was restricted to about 20 % of large diameter neurons. The density of LVA IBa was also modified in the course of ontogenesis. Between E17 and P0, one group of neurons showed a significant mean current density decrease. A second group of cells acquired a high LVA IBa current density which continued to increase during the same developmental period.
The decrease in the number of vestibular neurons with LVA IBa is consistent with that described in other neuronal preparations including Xenopus spinal neurons (Gu & Spitzer, 1993), sensory neurons (Fedulova et al. 1993) and hippocampal pyramidal cells (Thompson & Wong, 1991). Interestingly, Mynlieff & Beam (1992) reported developmental changes of LVA current in different populations of embryonic motoneurons. In one group of neurons, they reported a decrease in the number of cells with the LVA current concomitant with the diminution of the current amplitude, while a second population still expressed T-type currents at postnatal stages. These authors suggested that the expression of calcium currents plays a role in cell death and synapse elimination (Mynlieff & Beam, 1992). In the vestibular ganglion there is no information to date about the regulation of cell death and elimination of supernumerary synapses.
More unusual is the increase of the LVA IBa density between E17 and P0 in the second population of neurons. Such an increase has been reported during neuronal development in vivo of Xenopus spinal neurons (Desarmenien et al. 1993) and in dorsal root ganglion neurons (Desmadryl et al. 1998). The increase observed in the large diameter vestibular neurons might be related to a specific physiological role of this type of calcium channel, as proposed by McCobb & Beam (1991). Indeed, we have previously suggested that the presence of LVA IBa, in only a few large diameter neurons at postnatal stages (P4-P8), is responsible for the specific physiological properties of a particular class of primary vestibular afferent neurons (Desmadryl et al. 1997). Similarly, in chick embryo cochlear neurons, the T-type calcium current has been found in few cells, only in older stages of development (Jiménez et al. 1997). Another explanation is that the large diameter ganglion neurons with LVA IBa were not completely mature at P0 to P8. Only studies of adult neurons will clarify this point.
The LVA IBa, present in developing embryonic vestibular neurons, could be involved in the physiological regulation of calcium influx during neuron growth, as suggested by Gu & Spitzer (1993). Afferent neurite outgrowth occurs in the peripheral vestibular system at these stages, reaching a maximum around E17 (Nordemar, 1983), when the first synaptic contacts to the sensory cells are observed (Anniko, 1983; Mbiene et al. 1988). The disappearance of LVA IBa from the main population of vestibular neurons at later stages may be associated with the end of a calcium-sensitive period of neuronal differentiation which consequently leads to the stabilization of the neuronal network, as proposed for Xenopus neurons (Gu & Spitzer, 1993).
Development of high voltage-activated currents
Global HVA IBa increased throughout development indicating the augmentation of some component or components. At least four types of HVA calcium channel currents were present throughout the developmental period studied with no obvious variation in their kinetic or pharmacological properties, the L-, N-, P- and Q-type. The DHP-sensitive L-type HVA IBa was present in all stages studied, without any change in its density during embryonic development. This stability is probably due to the function of this class of calcium channel which is mainly involved in enzyme activity, protein phosphorylation and gene expression in neuronal cells (Murphy et al. 1991). The N-type calcium current was evidenced in all stages, by the application of -CTX-GVIA, a selective blocker of this calcium channel type (McCleskey et al. 1987). The density of N-type current increased during development. L- and N-type calcium currents have been described throughout the development span of embryonic chick neurons that innervate the cochlea (Jiménez et al. 1997). Similarly to our findings, a non-inactivating DHP-sensitive current, assumed to be an L-type calcium channel current, remained constant during neuronal development (Jiménez et al. 1997). In contrast to the present study, these authors reported a decrease in N-type calcium current and a total lack of -Aga-IVA-sensitive currents. Since vestibular and cochlear neurons originate from the same population, the discrepancies may be due to the difference between species (avian and mammalian). Two types of -Aga-IVA-sensitive currents were found throughout embryonic development. Low concentrations of -Aga-IVA (30 nM) blocked a sustained HVA IBa identical to the P-type current described previously in neurons (Mintz et al. 1992), while higher concentrations (1 µM) blocked an additional current with a transient component similar to the Q-type current defined in cerebellar granule cells (Randall & Tsien, 1995). N- and Q-type currents accounted for similar proportions of total IBa at all developmental stages, but their densities increased slightly from E15 to P0. Since the cell capacitances did not show significant variations between E15 and P0, this augmentation is the consequence of conductance changes in the vestibular neurons during development.
The increase in the densities of N- and Q-type HVA calcium channel currents, which occurs during the principal period of synaptogenesis in sensory vestibular epithelia (Nordemar, 1983), is similar to that described in other neuronal populations (Mynlieff & Beam, 1992; Hilaire et al. 1996; Desmadryl et al. 1998). This increase was assumed to be the consequence of neuronal growth, differentiation and synaptogenesis. It is interesting to note that changes in calcium current densities take place during periods where vestibular neurons require neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) for survival and neuritogenesis (Montcouquiol et al. 1998). A similar pattern has been described in developing cochlear neurons where Jiménez et al. (1997) showed a significant increase in the densities of calcium currents when neurons grew in the presence of NT-3. The increase of these two HVA calcium channel currents may also be associated with the appearance of the first functional synapses in early target innervation, since a combination of these HVA currents triggers neurotransmitter release in several other neuronal systems (Wheeler et al. 1994; Mintz et al. 1996; Jones et al. 1997). In vestibular neurons, most AMPA and NMDA glutamate receptors appear at E17 and reach a maximum at birth (C. J. Dechesne, personal communication), possibly indicating the existence of neurotransmitter mechanisms at embryonic stages.
The P-type current increases between E15 and E17 and decreases thereafter. A similar pattern leading to the total disappearance of P-type calcium current has been described in dorsal root ganglion neurons (Hilaire et al. 1996). The P-type current does not disappear completely from our vestibular neurons during ontogenesis. This increase is correlated with afferent neurite outgrowth and the beginning of synaptogenesis, suggesting that this calcium channel is involved in the regulation of ontogenesis in these neurons.
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REFERENCES |
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The authors thank G. Lennan for critically reviewing the manuscript. We are grateful to F. Scamps, D. Demèmes and J. Valmier for helpful discussions and comments on the manuscript, P. Bideaux for his contribution in technical preparations, P. Milhaud and R. Assié for statistical assistance. The work was supported by DRET grant 95-061 and CNES grant 793-98.
Corresponding author
G. Desmadryl: INSERM U432 Neurobiologie et Développement du Système Vestibulaire, UM2, cp 089 place E. Bataillon, 34095 Montpellier cedex 5, France.
Email: desmad{at}crit.univ-montp2.fr
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) and sustained (
) current measured after 250 ms depolarization. The threshold of activation of LVA IBa occurred around -60 mV whereas that of HVA IBa was at about -40 mV.
Significantly different from E17 (P < 0·01).