A GABAergic Maf-expressing interneuron subset regulates the speed of locomotion in Drosophila.

Interneurons (INs) coordinate motoneuron activity to generate appropriate patterns of muscle contractions, providing animals with the ability to adjust their body posture and to move over a range of speeds. In Drosophila larvae several IN subtypes have been morphologically described and their function well documented. However, the general lack of molecular characterization of those INs prevents the identification of evolutionary counterparts in other animals, limiting our understanding of the principles underlying neuronal circuit organization and function. Here we characterize a restricted subset of neurons in the nerve cord expressing the Maf transcription factor Traffic Jam (TJ). We found that TJ+ neurons are highly diverse and selective activation of these different subtypes disrupts larval body posture and induces specific locomotor behaviors. Finally, we show that a small subset of TJ+ GABAergic INs, singled out by the expression of a unique transcription factors code, controls larval crawling speed.

Alpha7-acetylcholine receptor antibodies in two patients with Rasmussen encephalitis.

Rasmussen encephalitis (RE) sera were screened for antibodies to human alpha7 nicotinic acetylcholine receptors (nAChRs) using electrophysiology, calcium imaging, and ligand binding assays. Sera from two of nine patients with RE blocked ACh-induced currents through alpha7 nAChRs and the ACh-induced rise in intracellular free calcium ([Ca2+]i) and inhibited (125)I-alpha-bungarotoxin binding in cells expressing alpha7 nAChRs. Thus, the alpha7 nAChR is a potential target for pathogenic antibodies in patients with RE.

Molecular diversity of voltage-gated sodium channel alpha subunits expressed in neuronal and non-neuronal excitable cells.

In order to investigate the role of molecular diversity of voltage-activated sodium channel alpha-subunits in excitability of neuronal and non-neuronal cells, we carried out patch-clamp recordings and single-cell RT-PCR on two different types of mammalian excitable cells i.e. hippocampal neurons and non-neuronal utricular epithelial hair cells. In each cell type, multiple different combinations of sodium channel alpha-subunits exist from cell to cell despite similar sodium current properties. The mRNA isoforms, Nav1.2 and Nav1.6, are the most frequently detected by single cell analysis in the two cell types while Nav1.3 and Nav1.7 are also moderately expressed in embryonic hippocampal neurons and in neonatal utricular hair cells respectively. By investigating the particular alternate splice isoforms of Nav1.6 occurring at the exon 18 of the mouse orthologue SCN8A, we revealed that this subunit co-exist in the two cell types under different alternative spliced isoforms. The expression of non-functional isoforms of Nav1.6 in utricular epithelial hair cells excludes the involvement of this subunit in supporting their excitability. Thus, from a functional point of view, the present results suggest that, at the single cell level, both neuronal and non-neuronal excitable cells expressed different and complex patterns of sodium channel gene transcripts but this diversity alone cannot explain the sodium current properties of these cell types.

Absence of antibodies to glutamate receptor type 3 (GluR3) in Rasmussen encephalitis.

OBJECTIVE: To determine the prevalence of serum antibodies to the ionotropic glutamate receptor 3 (GluR3) in patients with Rasmussen encephalitis (RE), a severe epileptic disorder, and to compare with serum from control subjects and patients with intractable epilepsy (IE). METHODS: The authors looked for serum immunoglobulin (Ig) G antibodies to GluR3 in 30 patients with RE, including two patients who had plasma exchange and 12 who had been treated with IV Igs with varying results, and 49 patients with IE and 23 healthy individuals, using ELISA with GluR3B peptide, Western blot analysis of recombinant full-length GluR3, immunoprecipitation of [35S]- and [125I]-labeled GluR3 extracellular domains, immunohistochemistry on rat brain sections, and electrophysiology of GluR3 expressed in Xenopus oocytes. RESULTS: Low levels of antibodies to the GluR3B peptide were detected using ELISA in only 4 of the 79 patients with epilepsy (2 with RE and 2 with IE); binding to GluR3B in other sera was shown to be nonspecific. One other patient with IE had antibodies to recombinant GluR3 on Western blot analysis. However, none of the sera tested precipitated either the [35S]- or the [125I]-labeled GluR3 domains; none bound to rat brain sections in a manner similar to rabbit antibodies to GluR3; and none of the nine sera tested affected the electrophysiologic function of GluR3. CONCLUSIONS: GluR3 antibodies were only infrequently found in Rasmussen encephalitis or intractable epilepsy.

The alpha(1A) subunits of rat brain calcium channels are developmentally regulated by alternative RNA splicing.

Calcium influx through voltage-gated calcium channels governs important aspects of CNS development. Multiple alternative splicings of the pore-forming alpha(1) subunits have been evidenced in adult brain but little information about their expression during ontogenesis is presently available. The aim of this study was to focus on the expression of three rat voltage-gated calcium channel alpha(1A) splice variants (alpha(1A-a), alpha(1A-b) and alpha(1A-EFe)) during brain ontogenesis in vivo. Using a reverse transcription-polymerase chain reaction strategy, we found that the three isoforms have different timings of development throughout the brain: alpha(1A-b) is expressed from embryonic to the adult stage, alpha(1A--EFe) is restricted to the embryonic period whereas alpha(1A-a) is expressed only postnatally. In situ hybridization indicated that alpha(1A-a) and alpha(1A-b) isoforms develop with different regional and cellular patterns. In hippocampus and cerebellum, alpha(1A-b) represented the predominant isoform at all developmental stages. Taken together, these data reveal that alternative RNA splicing may modulate the alpha(1A) calcium channel properties during development.

An activity-dependent neurotrophin-3 autocrine loop regulates the phenotype of developing hippocampal pyramidal neurons before target contact.

Neurotrophin-3 (NT-3), its cognate receptor trkC, and voltage-gated calcium channels are coexpressed by embryonic pyramidal neurons before target contact, but their functions at this stage of development are still unclear. We show here that, in vitro, anti-NT-3 and anti-trkC antibodies blocked the increase, and NT-3 reversed the decrease in the number of calbindin-D(28k)-positive pyramidal neurons induced by, respectively, calcium channel activations and blockades. Similar results were obtained with single-neuron microcultures. In addition, voltage-gated calcium channel inhibition downregulates the extracellular levels of NT-3 in high-density cultures. Moreover, electrophysiological experiments in single-cell cultures reveal a tetrodotoxin-sensitive spontaneous electrical activity allowing voltage-gated calcium channel activation. The mouse NT-3 (-/-) mutation decreases by 40% the number of developing calbindin-D(28k)-positive pyramidal neurons, without affecting neuronal survival, both in vitro and in vivo. Thus, present results strongly support that an activity-dependent autocrine NT-3 loop provides a local, intrinsic mechanism by which, before target contact, hippocampal pyramidal-like neurons may regulate their own differentiation, a role that may be important during early CNS differentiation or after adult target disruption.

Hyperpolarization-activated (Ih) current in mouse vestibular primary neurons.

The presence of a hyperpolarization-activated inward current (Ih) was investigated in mouse vestibular primary neurons using the whole-cell patch-clamp technique. In current-clamp configuration, injection of hyperpolarizing currents induced variations of membrane voltage with prominent time-dependent rectification increasing with current amplitudes. This effect was abolished by 2 mM Cs+ or 100 microM ZD7288. In voltage-clamp configuration, hyperpolarization pulses from -60 mV to -140 mV triggered a slow activating and non inactivating inward current that was sensitive to the two blockers, but insensitive to 5 mM Ba2+. Changing Na+ and K+ concentrations demonstrated that Ih current is carried by both these monovalent cations. This is the first demonstration of a Ih current in vestibular primary neurons.

Q- and L-type calcium channels control the development of calbindin phenotype in hippocampal pyramidal neurons in vitro.

Cultured immature hippocampal neurons from embryonic 17-day-old rats were used to explore activity-dependent regulation of neuronal phenotype differentiation in the developing hippocampus. The calbindin-D28k phenotype of the pyramidal neurons appeared during the first 6 days in culture, and was expressed by 12% of the cells on day 6. Daily stimulation with 50 mM KCl during the first 5 days in vitro increased the number of calbindin-D28k-positive pyramidal neurons without affecting neuronal survival. This effect was prevented by buffering extracellular Ca2+. Omega-agatoxin-IVA-sensitive Q-type and nitrendipine-sensitive L-type voltage-gated Ca2+ channels (VGCCs) carried Ca2+ currents and Ca2+ influx in immature pyramidal neurons at somata level. Blockade of these channels inhibited calbindin-D28k phenotype induced by 50 mM KCl. Conversely, glutamate-activated Ca2+ channel antagonists did not affect the KCl-induced calbindin-D28k phenotype. Chronic blockade of Q- and/or L-type VGCCs downregulated the normal calbindin-D28k development of immature pyramidal neurons without affecting neuronal survival, the somatic area of pyramidal neurons or the number of GABAergic-positive (gamma-aminobutyric acid) interneurons. However, at later developmental stages, Q-type VGCCs lost their ability to control Ca2+ influx at somata level, and both Q- and L-type VGCCs failed to regulate calbindin-D28k phenotype. These results suggest that Q-type channels, which have been predominantly associated with neurotransmitter release in adult brain, transiently act in synergy with L-type VGCCs to direct early neuronal differentiation of hippocampal pyramidal neurons before the establishment of their synaptic circuits.

Sarco-endoplasmic ATPase blocker 2,5-Di(tert-butyl)-1, 4-benzohydroquinone inhibits N-, P-, and Q- but not T-, L-, or R-type calcium currents in central and peripheral neurons.

The effects of 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBHQ), a synthetic phenolic antioxidant and a blocker of the sarco-endoplasmic ATPase, were evaluated on low and high voltage-activated Ca(2+) currents (ICas) with rodent dorsal root ganglion, hippocampal, and motor neurons. In all cell types tested, tBHQ (IC(50) = 35 microM) blocked ICa at concentrations used to inhibit sarco-endoplasmic ATPase. This effect was specific to tBHQ because the other sarco-endoplasmic reticulum calcium ATPase pump inhibitors (thapsigargin and cyclopiazonic acid) had no effect. Selective blockade of the N-type current with omega-conotoxin GVIA and of P- (motoneuron) or Q-type currents (hippocampal neuron) with omega-agatoxin IVA indicated that tBHQ inhibited N, P, and Q types of ICa. tBHQ had no effect on nitrendipine-sensitive (L-type) and residual drug-resistant (R-type) ICa, nor on the low voltage-activated T-type ICa. Contrary to neuronal cells, the L-type ICa was inhibited by tBHQ in a differentiated mouse neuroblastoma and rat glioma hybrid cell line. Injection of cDNAs encoding the alpha1A, alpha1B, alpha1C, and alpha1E subunits into oocytes showed that tBHQ blocked ICas at the level of the pore-forming protein. This effect of tBHQ on ICa should be considered when interpreting results obtained with tBHQ used on neuronal preparations. It also may be useful for developing new strategies for the generation of more potent intracellular calcium transient inhibitors.

Regulation of calcium channel alpha(1A) subunit splice variant mRNAs in kainate-induced temporal lobe epilepsy.

P/Q-type voltage-gated Ca(2+) channels (VGCC) regulate neurotransmitter release in the hippocampus and molecular alterations of their alpha(1A) pore-forming subunits are involved in various animal and human CNS diseases. We evaluated, using RT-PCR and in situ hybridization, the spatio-temporal activation of two alpha(1A) subunits splice variants (alpha(1A-a) and alpha(1A-b)) in control and kainic acid (KA)-treated rats. Six hours after KA treatment, alpha(1A-a) and alpha(1A-b) mRNAs increased, decreased or remained unchanged with area specific patterns. These changes were evidenced in the hippocampus and the dentatus gyrus and absent in the cerebellum. The alpha(1A) mRNA upregulation lasted for at least 7 days after KA treatment. Altogether, these results indicate that alpha(1A-a) and alpha(1A-b) mRNAs following seizure onset exhibit a complex and specific spatio-temporal pattern. The long-lasting changes in alpha(1A) subunit mRNA contents suggests that VGCC may be involved in the mechanisms generating chronic focal hyperexcitability and/or cellular damage in temporal lobe epilepsy.

Calcium channel subtypes responsible for voltage-gated intracellular calcium elevations in embryonic rat motoneurons.

The central role of electrical activity and Ca2+ influx in motoneuron development raises important questions about the regulation of Ca2+ signalling induced by voltage-dependent Ca2+ influx. In the purified embryonic rat motoneuron preparation, we recorded barium currents through voltage-activated Ca2+ channels using the whole-cell configuration of the patch-clamp technique. We found that motoneurons express at least four types of high-voltage-activated Ca2+ channels, based on their kinetics, voltage-dependences and pharmacological properties. Of the sustained Ca2+ current activated at 0 mV from a holding potential of -100 mV, approximately 45% was omega-conotoxin-GVIA (1 microM) sensitive, 25% was omega-agatoxin-IVA (30 nM) sensitive and 20% was nitrendipine (250 nM) sensitive. The residual current, after applying these three antagonists, was an inactivating current that differs from classical T-type Ca2+ currents. Based on this pharmacology, changes in intracellular free Ca2+ concentrations were then monitored by Fura 2 digital imaging microspectrofluorimetry. Upon K+ depolarization, the intracellular Ca2+ transient induced by the activation of each type of Ca2+ channel appeared to be quantitatively proportional to their Ca2+ influx. The existence of a calcium-induced calcium release mechanism through activation of caffeine-, ryanodine-sensitive intracellular stores was then investigated. High doses of caffeine and low doses of ryanodine failed to increase intracellular free calcium concentrations and low concentrations of caffeine and high concentrations of ryanodine did not affect K+-induced intracellular free calcium concentration transients indicating both the absence of Ca2+-gated Ca2+-release channels and of a Ca2+-induced Ca2+ release mechanism. Together, these data provide evidence that embryonic motoneurons express multiple Ca2+ channels that function as important regulators of intracellular Ca2+ signalling and may be involved in their development.

Developmental regulation of T-, N- and L-type calcium currents in mouse embryonic sensory neurones.

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.

Rat embryonic hippocampal neurons express a new class A calcium channel variant.

The aim of this study was to investigate, using a RT-PCR strategy, rat voltage-gated class A calcium channel (alpha1A) splice variants during rat hippocampus development. Results demonstrate the presence of multiple alpha1A mRNAs with the hippocampus formation and revealed a new variant of the rat alpha1A subunit (alpha1A-EFe) that diverges from alpha1A-a in the EF-hand domain. alpha1A-EFe expression in hippocampal neurons is restricted to the embryonic period. This in vivo developmental program is recapitulated in dissociated cultures of E17 embryonic hippocampal neurons. These data demonstrate that rat hippocampus neurons express a unique alpha1A splice variant during the embryonic period and suggest that alternative RNA splicing may modulate neuronal calcium channel properties during development.

Toxin-resistant calcium currents in embryonic mouse sensory neurons.

We characterized toxin-insensitive calcium currents expressed by acutely dissociated embryonic dorsal root ganglion neurons. In the presence of 3 microM omega-conotoxin-GVIA, 3 microM nitrendipine and either 500 nM omega-agatoxin-IVA or 500 nM omega-conotoxin-MVIIC to inhibit N-, L- and P/Q-type currents, respectively, all neurons expressed two residual currents: a T-type and another which we referred to as toxin-resistant current. The toxin-resistant current (i) consisted of an inactivating and a sustained components, (ii) had a threshold of activation and a steady-state inactivation comprised between that of the T-type current and that of the other high-voltage-activated currents, (iii) had the same permeability for barium and calcium used as charge carriers, (iv) was highly sensitive to both cadmium and nickel; and (v) was insensitive to 500 microM amiloride which abolished the T-type at this concentration. The properties of the toxin-resistant current are very similar to those of the currents expressed in oocytes following injection of alpha(1E) subunits which we demonstrated to be present in these neurons. Therefore a component of the toxin-resistant current calcium channels in sensory neurons may be closely related to those calcium channels formed by alpha(1E) subunits.

Multiple voltage-dependent calcium currents in acutely isolated mouse vestibular neurons.

We investigated the presence of voltage-gated calcium currents in vestibular neurons acutely isolated from postnatal mice vestibular ganglions using the whole-cell patch-clamp technique. The neuronal origin of the recorded cells was confirmed by immunohistochemical detection of neurofilaments and calretinin. High and low voltage-activated calcium currents were recorded. High voltage-activated currents were present in all investigated neurons. Low voltage-activated currents were recorded in only a few large vestibular neurons. High and low voltage-activated currents were distinguished by their thresholds of activation and their ability to run-up during early recordings. Among high voltage-activated currents. L-, N- and P-type currents were identified by their sensitivity to, respectively, the dihydropyridines agonist Bay K 8644 (3 microM) and antagonist nitrendipine (3 microM), the co-conotoxin GVIA (3 microM) and the omega-agatoxin IVA at low concentration (50 nM). An inactivating current sensitive to 1 microM omega-agatoxin IVA with characteristics similar to those of the Q-type current was also recorded in vestibular neurons. When L-, N-, P-, Q-type barium currents were blocked, a residual high voltage-activated current defined by its resistance to saturating concentrations of all above blockers was detected. This residual current was completely blocked by 0.5 mM nickel and cadmium. Our results reveal that primary vestibular neurons express a variety of voltage-activated calcium currents with distinct physiological and pharmacological properties. This diversity could be related both with their functional synaptic characteristic, and with the intrinsic physiological properties of each class of vestibular afferents.

Voltage-activated sodium currents in acutely isolated mouse vestibular ganglion neurones.

Voltage-activated sodium currents (INa) in vestibular ganglion neurones acutely isolated from postnatal mice were investigated using the whole-cell configuration of the patch-clamp technique. Under recording conditions designed to allow the complete isolation of INa depolarizations from a holding potential of -80 mV revealed a fast inactivating inward current which was activated around -60 mV and exhibited maximum peak current around -30 mV. This current was eliminated when the cells were perifused with a Na(+)-free solution and almost totally blocked by application of 100 nM tetrodotoxin (TTX). These properties identify this inward current as TTX-sensitive INa. The half-maximum activation potential of INa was -46 mV and its half-maximum inactivation potential was -69 mV. This is the first report of voltage-activated sodium currents in vestibular primary neurones.

Opposite developmental regulation of P- and Q-type calcium currents during ontogenesis of large diameter mouse sensory neurons.

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.

Early postnatal muscle contractile activity regulates the carbonic anhydrase phenotype of proprioceptive neurons in young and mature mice: evidence for a critical period in development.

Carbonic anhydrase activity, a marker of mouse proprioceptive neurons in adult dorsal root ganglia, is first detectable in the perinatal period, increases until postnatal day 60 and remains stable in adulthood. The onset of carbonic anhydrase staining begins after the neurons have made connections with their targets suggesting that neuron-target interactions regulate carbonic anhydrase phenotype development. To examine this possibility, we first analysed carbonic anhydrase expression in mdx mice which are characterized by a massive but reversible degeneration of skeletal muscle concomitant with the carbonic anhydrase ontogenesis. Neuronal carbonic anhydrase expression in mdx mice stopped developing when the period of muscular degeneration-regeneration began. Furthermore this alteration persisted during adulthood. We then analysed carbonic anhydrase expression in fifth lumbar dorsal root ganglion of developing control mice before and after surgical procedures that might interfere with central and peripheral target influences on dorsal root ganglion neurons. Central disconnection (dorsal rhizotomy) did not affect the development of carbonic anhydrase activity. Disrupting neuron-peripheral target interactions by sciatic nerve transection or blocking muscle contraction by tenotomy stopped the development of neuronal carbonic anhydrase content. Finally, recovery was monitored following sciatic nerve crush. In adults, recovery of carbonic anhydrase activity was obtained after functional recuperation; similar manipulations during the first month of life induced irreversible alteration of the carbonic anhydrase phenotype. These results show that the development of carbonic anhydrase activity in proprioceptive neurons is regulated by neuron-muscle interactions (i.e. muscle contraction). They also provide evidence for a critical period in the development of the carbonic anhydrase phenotype. We suggest that these two mechanisms are responsible for the altered carbonic anhydrase phenotype of the dorsal root ganglion neurons in mdx mice, a model of human muscular dystrophy.

Diversity of voltage-gated calcium currents in large diameter embryonic mouse sensory neurons.

Voltage-gated Ca2+ currents were investigated in a subpopulation of dorsal root ganglion neurons (large diameter, neurofilament-positive) acutely isolated from 13-day-old mouse embryos and recorded using the whole-cell patch-clamp technique. Low- and high-voltage-activated calcium currents were recorded. These currents could be identified and separated by their distinct (i) threshold of activation, (ii) ability to run-up during the early phase of recording and (iii) decay kinetics using Ba2+ instead of Ca2+ as the charge carrier. Among high-voltage-activated currents, L-, N- and P-type Ca2+ currents were identified by their sensitivity to, respectively, the dihydropyridine agonist Bay K 8644 (5 microM) and antagonist nitrendipine (3 microM), omega-conotoxin GVIA (3 microM) and omega-agatoxin IVA (30 nM). In the combined presence of nitrendipine (3 microM), omega-conotoxin GVIA (3 microM) and omega-agatoxin IVA (30 nM), two additional high-voltage-activated components were detected. One, blocked by 500 nM omega-conotoxin MVIIC and 1 microM omega-agatoxin IVA, had properties similar to those of the Q-type Ca2+ current first reported in cerebellar granule cells. The other, defined by its resistance to saturating concentrations of all the blockers mentioned above applied in combination, resembles the R-type Ca2+ current also described in cerebellar granule cells. In conclusion, embryonic sensory neurons appear to express a large repertoire of voltage-activated Ca2+ currents with distinct pharmacological properties. This diversity suggests a great variety of pathways for Ca2+ signaling which may support different functions during development.

Dihydropyridines, phenylalkylamines and benzothiazepines block N-, P/Q- and R-type calcium currents.

We compared the effects of representative members of three major classes of cardiac L-type channel antagonists, i.e. dihydropyridines (DHPs), phenylalkylamines (PAAs) and benzothiazepines (BTZs) on high-voltage-activated (HVA) Ca2+ channel currents recorded from a holding potential of -100 mV in rat ventricular cells, mouse sensory neurons and rat motoneurons. Nimodipine (DHP), verapamil (PAA) and diltiazem (BTZ) block the cardiac L-type Ca2+ channel current (EC50: 1 microM, 4 microM and 40 microM, respectively). At these concentrations, the drugs could also inhibit HVA Ca2+ channel currents in both sensory and motor neurons. Large blocking effects (> 50%) could be observed at 2-10 times these concentrations. The omega -conotoxin-GVIA-sensitive (omega -CTx-GVIA, N-type), omega -agatoxin-IVA-sensitive (omega -Aga-IVA, P- and Q-types) and non-L-type omega -CTx-GVIA-, omega -Aga-IVA-insensitive (R-types) currents accounted for more than 90% of the global current. Furthermore, our data showed that omega -CTx-GVIA and omega -Aga-IVA spare L-type currents and have only additive blocking effects on neuronal HVA currents. We conclude that DHPs, PAAs and BTZs have substantial inhibitory effects on neuronal non-L-type Ca2+ channels. Inhibitions occur at concentrations that are not maximally active on cardiac L-type Ca2+ channels.