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Ca2+ influx through voltage-gated calcium channels probably influences neuronal ontogenesis. Many developing neurones transiently express T-type/Cav3 calcium channels that contribute to their electrical activity and potentially to their morphological differentiation. Here we have characterized the electrophysiological properties and the functional role of a large T-type calcium current that is present in mouse developing primary vestibular neurones at embryonic day E17. This T-type current showed fast activation and inactivation, as well as slow deactivation kinetics. The overlap of activation and inactivation parameters produced a window current between -65 and -45 mV. Recovery from short-term inactivation was slow suggesting the presence of the Cav3.2 subunit. This T-type current was blocked by micromolar concentrations of Ni2+ and was inhibited by fast perfusion velocities in a similar fashion to recombinant Cav3.2 T-type channels expressed in HEK-293 cells. More importantly, current clamp experiments have revealed that the T-current could elicit afterdepolarization potentials during the repolarization phase of action potentials, and occasionally generate calcium spikes. Taken together, we demonstrate that the Cav3.2 subunit is likely to be the main T-type calcium channel subunit expressed in embryonic vestibular neurones and should play a key role in the excitability of these neurones during the ontogenesis of vestibular afferentation.

Medium sized dorsal root ganglion neurones are involved in tactile sensation and responsible for allodynia following nerve injury. We examined the effects of sciatic nerve injury on the expression of low and high voltage-gated calcium currents in medium sized neurones isolated from lumbar dorsal root ganglia of adult mice. Based on the relative expression of these calcium channel types, three populations of medium sized neurones were identified in controls. Type I, II and III populations were characterised respectively by small, predominant and no low voltage-gated current compared to the high voltage-gated current. Five days after nerve injury, calcium current expression was differentially affected by axotomy in these three subsets of medium neurones. Altogether, these results suggest that calcium channels are heterogeneously distributed among the medium sized neurones. This heterogeneity should provide specificity not only to sensory functions but also to sensory responses following nerve injury., (Copyright 2003 Lippincott Williams & Wilkins)

We investigated the development of a low (T-type) and two high voltage-activated (N- and L-type) calcium channel currents in large diameter dorsal root ganglion neurones acutely isolated from embryonic mice using the whole-cell patch-clamp technique. The low and high voltage-activated barium currents (LVA and HVA) were identified by their distinct threshold of activation and their sensitivity to pharmacological agents, dihydropyridines and omega-conotoxin-GVIA, at embryonic day 13 (E13), E15 and E17-18, respectively, before, during and after synaptogenesis. The amplitude and density of LVA currents, measured during a -40 mV pulse from a holding potential of -100 mV, increased significantly between E13 and E15, and remained constant between E15 and E17-18. The density of global HVA current, elicited by 0 mV pulse, increased between E13 and E15/E17-18. The density of the N-type current studied by the application of omega-conotoxin-GVIA (1 microM) increased significantly between E13 and E15/E17-18. The use of the dihydropyridine nitrendipine (1 microM) revealed that the density of L-type current remained constant at each stage of development. Nevertheless, application of dihydropyridine Bay K 8644 (3 microM) demonstrated a significant slowing of the deactivation tail current between embryonic days 13 and 15, which may reflect a qualitative maturation of this class of calcium channel current. The temporal relationship between the changes in calcium channel pattern and the period of target innervation suggests possible roles of T-, N- and L-type currents during developmental key events such as natural neurone death and onset of synapse formation.

Analysis of neuronal development has emphasized the importance of voltage-activated Ca2+ currents during the initial period of differentiation. We investigated non-N, non-L Ba2+ currents through Ca2+ channels in freshly dissociated large diameter embryonic mouse dorsal root ganglion neurons using the whole-cell patch-clamp technique. Two types of omega-agatoxin IVA-sensitive currents were clearly distinguished at embryonic day 13: a sustained P-type current blocked selectively at 30 nM (IC50 = 3nM) and an inactivating Q-type current blocked in the range 50-500 nM (IC50 = 120nM). The P-type Ca2+ current disappeared at day 15 whereas the Q-type Ca2+ current increased two- to three-fold during the same embryonic period. In contrast, the contribution of the non-L, non-N, omega-agatoxin IVA-resistant current (R-type) was constant during this developmental span. In conclusion, our results clearly show that P- and Q-type Ca2+ currents are differentially expressed during ontogenesis in large diameter dorsal root ganglion neurons. The developmental change, which occurs during the period of target innervation, could be related to specific key events such as natural neuron death and onset of synapse formation.

1. Extracellular recording techniques were used in the chinchilla to study the discharge properties of utricular afferents, including their discharge regularity, background discharge, and responses to both externally applied galvanic currents and centrifugal forces. 2. A normalized coefficient of variation (CV*), independent of discharge rate, was used to classify units as regularly (CV* less than 0.10), intermediate (0.10 less than or equal to CV* less than or equal to 0.20), or irregularly discharging (CV* greater than 0.20). In some circumstances, it was useful to recognize a group of very regularly discharging afferents (CV* less than 0.05). The CV* ranged from less than 0.020 to greater than 0.60. Regular units outnumbered irregular units by an approximate 3:1 ratio. The distribution of CV*s was bimodal: there was a major peak at CV* = 0.03 and a minor peak at CV* = 0.3. 3. Background rates were measured with the head in a horizontal position. Those of regular units usually fell between 40 and 80 spikes/s (mean: 54 spikes/s); those of irregular units were more broadly distributed (mean: 47 spikes/s). 4. Units were categorized in terms of the tilt directions resulting in increased discharge. There is a broad distribution of excitatory tilt directions with some units excited by ipsilateral rolls, others by contralateral rolls, some by nose-up pitches, and still others by nose-down pitches. In the chinchilla, there are almost equal numbers of units excited by ipsilateral or contralateral tilts. This is in contrast to previous findings in the cat and squirrel monkey, where the former units predominant by a 3:1 ratio. The difference can be related to the fact that the medial zone of the macula, where units excited by ipsilateral tilts reside, makes up a smaller proportion of the sensory epithelium in the chinchilla than in the monkey. 5. Galvanic sensitivity (beta *) and discharge regularity (CV*) were related by a power law, beta* = (CV*), with an exponent, b = 0.70. 6. Responses to sinusoidal centrifugal forces in the frequency range, f, between DC and 2 Hz were characterized by their gains (gf) and phases (phi f), taken with respect to peak linear force. Response linearity was studied by varying the amplitude of a 0.1-Hz sinusoid from 0.05 to 0.4 g. Nonlinear distortion was small (approximately 10%), as was the variation of gain (+/- 10%) and phase (+/- 5 degrees) with amplitude. 7. Response dynamics vary with discharge regularity. Very regular units are tonic. Their gains are typically 50 spikes.s-1/g and almost constant (+/- 10%) over the entire frequency range. Phases hover near zero with small (5 degrees) phase leads at low frequencies and slightly larger (10 degrees) phase lags at high frequencies. Irregular units are more phasic.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The relation between the discharge properties of utricular afferents and their peripheral innervation patterns was studied in the chinchilla by the use of intra-axonal labeling techniques. Fifty-three physiologically characterized units were injected with horseradish peroxidase (HRP) or lucifer yellow CH (LY) and their labeled processes were traced to the utricular macula. For most labeled neurons, the discharge regularity, background discharge, and sensitivity to externally applied galvanic currents were determined, as were the gain (g2 Hz) and phase (phi 2 Hz) of the response to 2-Hz sinusoidal linear forces. Terminal fields were reconstructed and fibers were classified as calyx (n = 13) or dimorphic units (n = 40). No bouton units were recovered. Calyx units were confined to the striola. Dimorphic units were located in the striola (n = 8), the juxtastriola (n = 7), or the peripheral extrastriola (n = 25). 2. To determine whether the intra-axonal sample was representative, the physiological properties of labeled utricular units were compared with those of a larger sample of extracellularly recorded units. A comparison was also made between the morphology of intra-axonally labeled units and those labeled by the extracellular injection of HRP into the vestibular nerve. Most of the discrepancies between the intra-axonal and either extracellular sample can be explained by assuming that small-diameter fibers are underrepresented in the former sample. Dimorphic fibers labeled intra-axonally had more bouton endings and larger terminal trees than did those labeled extracellularly. The latter differences may reflect a sampling bias in the extracellular material. 3. Calyx units were irregularly discharging. The discharge regularity of dimorphic units was related to their macular locations. Only 1/8 dimorphic units in the striola was regularly discharging. The ratio increases to 3/7 in the juxtastriola and to 23/25 in the peripheral extrastriola. Among dimorphic units, there is a tendency for irregularly discharging afferents to have fewer bouton endings. The trend is far from perfect because it is possible to pick a subsample of dimorphic units that have similar numbers of boutons and, yet, have discharge patterns that range from regular to irregular. 4. Published morphological polarization maps can be used to predict the excitatory tilt directions of a unit from its macular location. Predictions were confirmed in 39/41 labeled afferents. 5. The galvanic sensitivity (beta *) of an afferent, irrespective of its peripheral innervation pattern or its epithelial location, was strongly correlated with a normalized coefficient of variation (CV*).(ABSTRACT TRUNCATED AT 400 WORDS)

1. The relation between the response properties of semicircular canal afferents and their peripheral innervation patterns was studied by the use of intra-axonal labeling techniques. Fifty physiologically characterized units were injected with horseradish peroxidase (HRP) or Lucifer yellow CH (LY) and their processes were traced to the crista. The resting discharge, discharge regularity, and responses to both externally applied galvanic currents and sinusoidal head rotations were determined for most neurons. Terminal fields were reconstructed and, as in the preceding paper, the fibers were classified as calyx, bouton, or dimorphic units. 2. To determine if the intra-axonal sample was representative, the physiological properties of the labeled units were compared with those of a sample of extracellularly recorded units. A comparison was also made between the morphology of the intra-axonal units and those labeled by extracellular injection of HRP into the vestibular nerve Most of the discrepancies between the intra-axonal and the two extracellular samples can be explained by assuming that small-diameter fibers are underrepresented in the former sample. 3. A normalized coefficient of variation (CV*), independent of discharge rate, was used to classify units as regular, intermediate, or irregular. The CV* ranged from 0.020 to 0.60. Regular units (CV* less than or equal to 0.10) outnumbered irregular units (CV* greater than or equal to 0.20) by an approximately 3:1 ratio and had higher resting discharges. 4. Calyx units were invariably irregular. The one recovered bouton unit was regular. The discharge regularity of dimorphic units was related to their epithelial location, with those found in the periphery of the crista having a more regular discharge than those located more centrally. Dimorphic units, even those with quite similar morphology, can differ in their discharge regularity. Calyx and dimorphic units, which differ in their morphology, can both be irregular. These observations imply that discharge regularity is not determined by the branching pattern of a fiber or the number and types of hair cells it contacts. 5. The galvanic sensitivity (beta*) of an afferent, irrespective of its peripheral innervation pattern, was strongly correlated with CV*. This is consistent with the notion that discharge regularity and galvanic sensitivity are causally related, both being determined by postspike recovery mechanisms of the afferent nerve terminal.(ABSTRACT TRUNCATED AT 400 WORDS)