Biophysical and pharmacological characterization of voltage-sensitive calcium currents in neonatal rat inferior colliculus neurons

Neuroscience Vol. 96, No. 4, pp. 753–765, 2000 753 Copyright q 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00

Calcium currents in neonatal inferior colliculus neurons

Pergamon PII: S0306-4522(00)00006-3 www.elsevier.com/locate/neuroscience

BIOPHYSICAL AND PHARMACOLOGICAL CHARACTERIZATION OF VOLTAGESENSITIVE CALCIUM CURRENTS IN NEONATAL RAT INFERIOR COLLICULUS NEURONS P. N’GOUEMO* and A. R. RITTENHOUSE† Department of Physiology and Program in Neuroscience, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA 01655, U.S.A.

Abstract—Calcium conductances have been found in neonatal inferior colliculus neurons, however the biophysical and pharmacological profiles of the underlying calcium currents have not yet been characterized. In this study, we examined which types of voltage-activated calcium currents comprise the whole-cell inward current of neonatal inferior colliculus neurons (10–22 mm in diameter). On the basis of their voltage-dependence and pharmacological sensitivities, three major components of barium currents were identified. A low threshold voltage-activated current that activated around 270 mV, a mid threshold voltage-activated current that activated near 250 mV, and a high threshold voltage-activated current that activated around 240 mV. Low and mid threshold voltage-activated currents were present in 33% and 41% of the recordings, respectively, whereas high threshold voltage-activated currents were recorded in all inferior colliculus neurons tested. Nickel chloride (50 mM) and U-92032 (1 mM), which both block low threshold voltage-activated currents, reduced the amplitude of low threshold voltage-activated peak currents at a test potential of 260 mV by 72% and 10%, respectively. In addition, 50 mM nickel chloride and 1 mM U-92032 reduced the amplitude of mid threshold voltage-activated peak currents measured at 220 mV by 55% and 21%, respectively. Further pharmacological analysis indicated the presence of multiple types of high threshold voltage-activated currents in neonatal inferior colliculus neurons. The dihydropyridine nimodipine (1 mM), a selective L-type current antagonist, reduced the amplitude of high threshold voltageactivated peak currents by 25%. In addition, FPL 64176 (1 mM), a non-dihydropyridine L-type current agonist caused a dramatic 534% increase in the amplitude of the slow sustained component of the tail current measured at 240 mV. These data indicate that inferior colliculus neurons express L-type channels. v-Conotoxin GVIA (1 mM), a selective blocker of N-type current, inhibited high threshold voltage-activated peak currents by 28% indicating the presence of N-type channels. v-Agatoxin IVA (300 nM), a potent P/Q-type antagonist, reduced high threshold voltage-activated peak currents by 27%, suggesting that inferior colliculus neurons express P/Q-type channels. Concomitant application of nimodipine (1 mM), v-conotoxin GVIA (1 mM) and v-agatoxin IVA (300 nM) onto inferior colliculus neurons decreased the control high threshold voltage-activated peak currents only by 62%. Thus, inferior colliculus neurons may express at least one more type of calcium current in addition to low and mid threshold voltage-activated currents and L-type, N-type and P/Q-type high threshold currents. q 2000 IBRO. Published by Elsevier Science Ltd. Key words: v-agatoxin, calcium channel, v-conotoxin GVIA, dihydropyridine, nickel, U-92032.

The inferior colliculus (IC), in adult rat, is an important nucleus for auditory processing 1 and the integration of auditory information with motor and other sensory information. 45 In the neonatal rat, the IC plays important roles in sensorymotor function, prior to hearing. 40 It has been reported that a tail pinch stimulus in five-day-old rats induces a spectrum of motor behaviors associated with afterdischarge-like activity with multiple spikes in the IC. 40 In addition, electrical stimulation of neonatal IC produces electrographic discharge and motor activity similar to those observed with a tail pinch stimulus. 41 Thus, a sensory stimulus could induce a change

in neuronal excitability in neonatal IC. Ionic conductances including calcium (Ca 21) currents have been implicated in the in vitro generation of spikes in IC neurons. 57 A Ca 21dependent “hump” as well as high- and low-threshold Ca 21 spikes have been observed in intracellular recordings from adult IC slices, 57 suggesting that IC neurons express multiple types of Ca 21 channels. Recent studies reported that voltageactivated Ca 21 channels are present in neonatal IC neurons, 26 however, their currents have not yet been characterized. Therefore, to understand Ca 21-dependent electrical activity in neonatal IC neurons, it is important to determine first which types of Ca 21 currents are present. Currently, Ca 21 channels can be separated based on biophysical, molecular and pharmacological properties. Thus far, at least nine Ca 21 channel transcripts of a1 poreforming subunits (A-I) have been detected in the CNS. 7,14,32,58,61,64,69 Their currents have been classified as low threshold voltage-activated (LVA) currents, mid-threshold voltage-activated (MVA) currents, and high threshold voltage-activated (HVA) currents. 16 Some Ca 21 currents exhibit varied, but overlapping voltage dependence and kinetic properties making it difficult to further define a type of Ca 21 channel only on the basis of the biophysical properties of its currents. 10,37,38,71 Conveniently, whole-cell LVA, MVA and HVA currents exhibit different pharmacological

*Present address: Department of Pharmacology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington, DC 200072194, U.S.A. †To whom correspondence should be addressed. Tel.: 1 1-508-856-3735; fax: 1 1-508-856-5997. E-mail address: [email protected] (A. R. Rittenhouse). Abbreviations: v-Aga IVA, v-agatoxin IVA; v-CTx GVIA, v-conotoxin GVIA; DHP, dihydropyridine; EGTA, ethyleneglycolbis(aminoethylether)tetra-acetate; FPL, FPL 64176; FTX, funnel-web spider toxin; HEPES, N-2-hydroxyethylpiperazine-N 0 -2-ethanesulphonic acid; HVA, high threshold voltage-activated; IC, inferior colliculus; LVA, low threshold voltage-activated; MVA, mid-threshold voltage-activated; NIM, nimodipine; TEA, tetraethylammonium; U-92032, [7-(bis-4fluorophenyl) (methyl)-(1-piperazinyl)-2-(2-hydroxyethylamino)-4-1(1methylethyl)-2,4,6,-cycloheptatrien-1-one]. 753

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sensitivities that are consistent with molecular and biophysical data, confirming the heterogeneity of Ca 21 channel phenotypes in CNS neurons. In particular, HVA currents have been further divided into five phenotypes including L, N, P/Q, and R based on their sensitivity to various Ca 21 channel ligands. 53 Thus, in addition to biophysical characteristics, pharmacological properties can serve as additional tools to characterize Ca 21 current phenotypes in CNS neurons. In the present study, the biophysical characteristics and pharmacological profiles of whole-cell Ca 21 currents, using barium (Ba 21) as the charge carrier, were examined with the aim of identifying which types of Ca 21 currents are present in neonatal rat IC neurons. A preliminary report of some of these data has been presented. 46 EXPERIMENTAL PROCEDURES

Cell preparation The IC from two- to five-day-old Sprague–Dawley rats (either sex) were removed following decapitation and placed in Neurobasal medium (Life Technologies) at 48C. IC tissue was manually dissected into small pieces (less than 1 mm 3) with a scalpel blade, and digested with papain (2 mg/ml) (Boehringer Mannheim) in Neurobasal medium bubbled with a 95% O2/5% CO2 gas mixture at 378C for 30 min. 9 After enzyme treatment, IC tissues were washed with Neurobasal medium containing bovine serum albumin (1 mg/ml) (Sigma) and trypsin inhibitor (1 mg/ml) (Boehringer Mannheim). IC tissues were transferred into Neurobasal medium supplemented with 20 ml/ml of B27 (Life Technologies), 10% fetal bovine serum, 0.5 mM glutamine, and penicillin (100 U/ml)-streptomycin (0.1 mg/ml). IC neurons were dissociated by gentle trituration with a fire-polished Pasteur pipette; the supernatants after each trituration were collected and mixed. Dissociated IC neurons were then plated onto poly-l-lysine-coated glass coverslips in 25 mm 2 dishes and placed at 378C in a CO2 (5%) humidified incubator. IC neurons were allowed to settle and attach to the polyl-lysine for at least 1 h. Differences in the expression of Ca 21 currents have been reported between acutely dissociated cells and neurons maintained in culture. 39 Thus, to avoid possible phenotypic changes of Ba 21 currents due to culture conditions, IC neurons were used within 10 h following dissection. The IC can be separated further into three nuclei including the central nucleus, the external nucleus and the dorsal cortex. 19 However, the technique of neuron isolation used in this study did not allow a further identification of their origin from within the IC. Animals were cared for and killed according to University of Massachusetts Medical School approved protocol in compliance with the scientist-related provisions of Federal Animal Welfare Act, the US Public Health Service “Policy on Human Care and Use of Animals”. All efforts were made to reduce the number of animals used in these studies. Electrophysiology Patch-clamp recordings were performed using standard whole-cell voltage-clamp techniques. 25 A piece of coverslip with IC neurons was placed into a recording chamber that was mounted on the stage of an inverted microscope. IC neurons were voltage clamped with borosilicate glass patch pipettes fire-polished to < 1 mm tip openings, made with the use of a Narishige model PB-7 two-stage electrode puller (Narishige Scientific Instrument, Tokyo, Japan). The pipette resistance ranged from 1.5 to 2.5 MV when filled with internal solution. For some experiments, patch pipettes were coated with Sylgard (Dow Corning 184). Membrane currents were measured with and voltages were controlled by either an Axopatch 200A (Axon Instrument) or Dagan 3900 (Dagan Corporation) patch-clamp amplifier. Currents were filtered at 1–2 kHz, sampled at 5–20 kHz, and stored on a Pentium/ 133 MHz computer for later analysis. Data acquisition and analysis were performed using Patch software (Cambridge Electronic Design). All experiments were performed at room temperature (20–248C). HVA Ba 21 currents were elicited every 4 s for 40 ms by voltage steps from a holding potential of 290 mV to 1 20 mV, and by a repolarization step to an intermediate potential of 240 mV. The rationale for measuring tail currents at 240 mV was to determine whether a slow sustained component of the tail current was present and composed

of L-type current. 29 LVA currents were elicited at a test potential of 260 mV from a holding potential of 290 mV, followed by repolarization to 270 mV. The rationale for stepping back to 270 mV was to reveal the slow deactivation kinetics of the tail current that characterize LVA currents. 4 MVA Ba 21 currents were evoked from a holding potential of 290 mV by stepping to 220 mV. In some experiments, tail currents were compared to those of LVA currents by repolarization to the same intermediary potential of 270 mV. To avoid contamination of LVA currents, only IC neurons without a slow component of the tail current were retained for statistical analysis of MVA currents. The mean capacitance (CM) and series resistance (RS) were determined by measuring the area under current trace (Q ˆ charge) and the amplitude (IPEAK) of the capacitive current transients elicited every second by a 30 ms hyperpolarizing, 10 mV voltage step (VSTEP) from a holding potential of 290 mV. These currents were sampled at 80 kHz. Capacitance and series resistance were calculated as follows: CM ˆ Q/VSTEP; RS ˆ VSTEP/IPEAK. Linear leak and residual capacitance currents were subtracted off-line from all traces prior to analysis by adding scaled up leak currents generated from test potentials to 2100 mV. The amplitudes of Ba 21 currents were measured using a trough seeking function < 10–20 ms after the onset of the test pulse to measure peak current. Tail currents were measured at either 270 mV or 240 mV, 6 or 12 ms, respectively, after the test pulse. Solutions containing Ca 21 channel antagonists and/or toxin blockers were delivered into the recording chamber with the use of a gravitydriven system and siphoned from the bath by suction. Their effects were tested after obtaining 2–5 min of steady state control Ba 21 current recordings. Whole-cell Ba 21 currents decayed 1–5% over time. Thus, only IC neurons that exhibited a change greater than 5% in the amplitude of Ba 21 currents following application of Ca 21 channel ligands were considered sensitive neurons. To evaluate the effects of Ca 21 channel ligands, the percentage of current inhibition was expressed as 100[1 2 (Idrug/Icontrol)] and used for further analysis. Changes in current amplitudes due to the presence of Ca21 channel ligands were analysed using a two-tailed paired t-test. All data are presented as mean ^ S.E.M. Solutions and drugs Whole-cell currents were established in tyrode’s solution which contained (in mM): 145 NaCl, 10 HEPES, 5.4 KCl, and 5 CaCl2 (pH 7.4 with NaOH, 300–305 mOsm). The extracellular recording solution for isolating Ba 21 currents contained (in mM): 125 tetraethylammonium (TEA) chloride, 20 Ba 21-acetate, 10 glucose, 10 HEPES, and 0.001 tetrodotoxin (RBI) (pH 7.4 with TEA-OH, 297–300 mOsm). Where noted, a low Ba 21 solution (5 mM) was used. The pipette solution contained (in mM): 90 cesium methanesulfonate, 10 EGTA, 14 phosphocreatine, 10 HEPES, 10 glucose, 4 ATP, 5 MgCl2, and 0.4 GTP (pH 7.3 with CsOH, 288–294 mOsm). v-Agatoxin IVA (v-Aga IVA) was a generous gift from Dr N. A. Saccomano (Pfizer) while v-conotoxin GIVA (v-CTx GIVA) was purchased from Bachem (Torrance). U-92032 or,[7-(bis-4-fluorophenyl)methyl)-1-piperazinyl)-2-(2-hydroxyethylamino)-4-1(1-methylethyl)-2,4,6,-cycloheptatrien-1-one] was a generous gift from Dr D. Newburry (Pharmacia and Upjohn). The funnel-web spider toxin (FTX) was a gift from Professor R. Llina´s (New York University Medical Center). Nickel chloride (Ni) was purchased from E.M. Science. Toxin blockers and Ni were prepared as concentrated stock solutions in distilled water and stored in aliquots at 2208C. Stock solutions were diluted .1:1000 with the extracellular recording solution before each experiment. Cytochrome C (0.01%) was added to the v-Aga IVA solution to saturate any non-specific binding sites located on the walls of the tubing and chamber. 44 At this concentration cytochrome C alone had no effect on whole-cell Ba21 currents (n ˆ 3, data not shown). Nimodipine (NIM; Miles Pharmaceuticals and RBI) and U-92032, as well as FPL 64176 (FPL; RBI) were solubilized in 100% ethanol as concentrated stock solutions and protected from ambient light (NIM was protected from light at all times) and stored in aliquots at 2208C. The final dilutions resulted in ,0.01% ethanol concentration which had no effect on whole-cell Ba 21 currents in IC neurons when applied alone (n ˆ 3, data not shown). All other chemicals used in this study were purchased from Sigma. RESULTS

Whole-cell barium currents in inferior colliculus neurons Acutely dissociated IC neurons (n ˆ 129) in this study had

Calcium currents in neonatal inferior colliculus neurons

round soma (10–22 mm in diameter) without processes. Such a spherically-shaped population of neurons has been found previously in microscopic studies in the IC. 2,54 The mean cell capacitance recorded in 46 IC neurons was 14 ^ 1 pF (range: 7–34 pF), and the series resistance was 16 ^ 1 MV (range 6–32 MV). Following its measurement, series resistance was minimized and whole-cell currents were characterized. IC neurons exhibited large whole-cell inward Ba 21 currents following a 40 ms voltage step to 1 20 mV from a holding potential of 290 mV (mean of peak current: 21017 ^ 49 pA; range: 330–2053 pA; n ˆ 102). At this test potential, the Ba 21 current in IC neurons slowly decayed with repeated pulses that resembled rundown although ATP, GTP and phosphocreatine were present in the pipette solution. However, exchanging the bath solution often resulted in a temporary increase in the whole-cell Ba 21 currents, while maintaining constant bath flow reduced the decay of the currents and increased the rate of activation (data not shown). Tonic release of neurotransmitters may have been the cause of this observed reversible decrease since the decaying current displayed slowed activation kinetics reminiscent of neurotransmitter-induced inhibition of the Ba 21 currents. 28 Therefore, in most recordings a slow flow rate ( < 0.2 ml/min) was maintained to minimize this whole-cell current decrease. Under these recording conditions, an initial, small decrease in whole-cell Ba 21 currents was observed after breakthrough, but then currents stabilized. In the process of characterizing whole-cell Ba 21 currents in IC neurons biophysically and pharmacologically, it became clear that the expression pattern of different types of Ca 21 currents was heterogeneous from cell to cell giving rise to complex whole-cell currents. At least three categories of currents including HVA, MVA and LVA currents could be identified biophysically based on threshold of activation, current kinetics and tail current characteristics. High threshold Ca 21 currents begin to activate at approximately 240 mV in other CNS neurons. 47,53,71 In IC neurons devoid of nonHVA currents, plots of the peak current as a function of membrane potential (I–V) revealed that as with other CNS neurons HVA Ba 21 currents typically activated around 240 mV (range: 240 to 225 mV), peaked around 0 mV (range: 210 to 1 20 mV), and reversed between 1 50 mV and 1 65 mV (data not shown). While all IC neurons expressed HVA currents, heterogeneity arose in the varied expression of MVA and LVA currents and in the types of HVA currents found in individual cells. Figures 1–3 give examples of this heterogeneity and document the presence of MVA and LVA currents in neonatal IC neurons. An example of whole-cell Ba 21 currents with both HVA and non-HVA currents is illustrated in Fig. 1A. Current was evoked at test potentials at least as negative as 250 mV, indicating the presence of non-HVA currents. In addition, the current jumped in magnitude when changing the test potential from 230 mV to 220 mV, indicating the opening of HVA channels. The complex tail currents gave additional information about the types of currents present in this wholecell recording. A sustained, long-lasting component of the tail current, in addition to the rapidly deactivating component could be observed when the membrane potential was stepped from test potentials positive to 230 mV back to a tail potential of 240 mV. Moreover activation of a current during the tail potential was observed when cells were stepped from test potentials negative to 240 mV to a tail potential of 240 mV

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(Fig. 2A). Once activated this tail current lasted the duration of the tail pulse; it showed no deactivation. In comparison, a long-lasting tail current was not present in the recording shown in Fig. 1B. L-type Ca 21 channels are known to activate at 240 mV. Therefore, the presence of a long-lasting, sustained component of the tail current at a tail potential equal to or greater than 240 mV suggests the presence of L-type Ca 21 channels in some IC neurons. In this first example, it is clear that both HVA and nonHVA currents are present, however it was not obvious what types of currents made up the non-HVA current. Fig. 1B shows that some IC neurons exhibited a transient component of non-HVA current that could be observed at voltages negative to 0 mV (Fig. 1B). We termed these currents “midthreshold, voltage-activated” (MVA) currents to distinguish them from LVA currents that activated around 270 mV (see below). Examination of individual sweeps (Fig. 1B) and the I–V relationship (data not shown) revealed that MVA currents activated around 250 mV (range: 250 mV to 240 mV). As the test potential became more positive, the amplitude of these currents increased and the rate of activation and inactivation became faster (Fig. 1B). The increase in MVA current kinetics appeared to be maximal around 220 mV (range: 240 mV to 210 mV). However, at more positive voltages the MVA current was obscured by the HVA currents, making it impossible to further analyse these currents. When stepped to 220 mV, MVA currents exhibited a relatively slow rate of activation that reached its peak in 9 ^ 1 ms (range: 4–13 ms; n ˆ 11). The time constant for the decay of the transient component of the current measured at 220 mV was 26 ^ 3 ms (range: 18–37 ms; n ˆ 5). When depolarizing from a holding potential of 290 mV to 220 mV, MVA currents were observed in 41% (11 out of 27) of IC neurons tested. The mean amplitude of MVA peak currents measured at a test potential of 220 mV was 2720 ^ 94 pA (range: 344–1 150 pA; n ˆ 8). The tail currents of cells that exhibited MVA currents (Fig. 1B) deactivated relatively rapidly and did not exhibit a slowly deactivating component as observed with the LVA currents (see Fig. 2B). In addition, a slowly activating current was observed during the tail potential following test potentials more negative than 240 mV (Fig. 1B). This activating current reached a peak and then decreased during the tail pulse, similar in kinetics to MVA currents observed during a test pulse to 240 mV. In contrast to the recording in Fig. 1A, there was no sustained component of the tail current or activation of a sustained component during the tail potential, suggesting the absence of a prominent L-type current in this neuron. A lower threshold of current activation than 250 mV in some IC neurons indicated the presence of LVA channels. To isolate LVA from MVA currents, only IC neurons that exhibited a slow deactivation of the tail currents after repolarization to 270 mV in addition to a low activation threshold (around 270 mV) were considered as expressing LVA channels (Fig. 2A and C). The repolarization at 270 mV was also used to isolate LVA currents from L-type current that could be evoked by stepping back to an intermediary potential of 240 mV. Using these criteria, 33% (9 out of 27) of IC neurons tested exhibited LVA currents. The mean amplitude of LVA peak currents, measured at a test potential of 260 mV was 236 ^ 6 pA (range: 17–70 pA; n ˆ 8) and the slow component of the tail current, measured at 270 mV was 229 ^ 15 pA (range: 6–97 pA; n ˆ 8).

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Fig. 1. Examples of typical whole-cell barium currents recorded in neonatal inferior colliculus neurons. (A) An example of whole-cell Ba 21 currents that are non-inactivating. At test potentials greater than 240 mV, in addition to the typical rapidly deactivating tail currents (asterisk) a slow, longlasting component of tail current also was present (vertical arrow). These currents converged to the same amplitude as currents which activated during the tail potential following test potentials less than 240mV. (B) In a second IC recording, currents activated around 250 mV and exhibited transient (T) and sustained (S) components, illustrating the properties of whole-cell currents containing typical mid-threshold voltage-activated (MVA) current. Note that a slowly activating current was observed during the tail potential following test potentials more negative than 240 mV (arrowhead). Dashed lines indicate zero current level. Twenty millimolar Ba 21 was used as the charge carrier in the present and subsequent experiments unless stated otherwise.

Pharmacological characterization of calcium currents in inferior colliculus neurons Because it was difficult to characterize all types of Ba 21 currents present in IC neurons on the basis of their biophysical properties alone, we dissected the Ba 21 currents further using Ca 21 channel ligands. Currents sensitive to nickel and U-92032. To determine whether LVA and MVA currents could be distinguished pharmacologically, we tested the effects of Ni and U-92032 on both currents. Ni is widely used to identify LVA T-type currents 18 and also has been shown to inhibit MVA currents. 59 U-92032 has been reported to inhibit LVA T-type currents in CNS neurons. 5 Whether U-92032 suppresses MVA current is unknown. Ni and U-92032 at relatively high concentrations have some inhibitory effects on HVA currents, however relatively low concentrations (50 mM Ni and 1 mM U-92032)

Fig. 2. Effects of nickel chloride and U-92032 on low threshold voltageactivated barium currents in neonatal inferior colliculus neurons. (A) Examples of a series of LVA Ba 21 currents. Note that a slow deactivation of the tail current (vertical arrow) that characterizes LVA currents was observed with test pulses positive to 260 mV. (B) The presence of Ni (50 mM) suppressed both the peak and tail currents (b) observed under normal conditions (a). (C) Current–voltage (I–V) relationship obtained from the same neuron as in B revealed that Ba 21 currents activated around 270 mV indicating the presence of LVA currents (inset). (D) Examples of current traces in the absence (a) and presence (b) of U-92032 (1 mM). Note that in this recording, U-92032 did not affect either the peak current elicited at 260 mV or the slow component of the tail current recorded at 270 mV. Dotted lines indicate zero current level.

appear to be sufficient to reduce the peak current and suppress the inactivating component of LVA T-type currents in hippocampal neurons. 5,6 We used 50 mM Ni and 1 mM U-92032 to pharmacologically characterize LVA, and MVA currents in IC neurons. Application of 50 mM Ni reduced the amplitude of LVA peak currents measured at 260 mV by 72 ^ 10% (P , 0.05; range: 44–91%, n ˆ 4) and the slow component of the tail currents after stepping back to 270 mV by 69 ^ 10% (P , 0.05; 46–87%, n ˆ 4). An example of the inhibitory effects of Ni on LVA currents is illustrated in Fig. 2B. To examine the effects of 50 mM Ni on MVA currents, cells were

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Fig. 3. Effects of nickel chloride and U-92032 on whole-cell mid-threshold voltage-activated barium currents in neonatal inferior colliculus neurons. (A) Examples of MVA current traces obtained in the absence (a) and presence (b) of Ni (50 mM) showed that this compound reduced the control peak current by 63%. A substantial inhibition of the tail current was also observed. (B) I–V relationships from the same neuron as in A revealed that the Ni-sensitive current activated around 250 mV. (C) Representative current traces obtained at negative voltages in control (CON), and in the presence of U-92032 (1 mM). MVA currents were detectable at 250 mV. Application of U-92032 suppressed the inactivating component of MVA currents, and reduced the amplitude of peak currents at voltages negative to 210 mV. Note that U-92032 also slowed the activation rate and increased the amplitude of the peak current at the end of the pulse at 220 mV. It also eliminated the slow current activation observed in control sweeps (arrowhead) during the tail potential. (D) I–V curves from the same neuron as in C, illustrating the reduction of currents at voltages positive to 250 mV following application of U-92032, as compared to controls. Note that U92032-sensitive current activated around 250 mV. Five millimolar Ba 21 was used as the charge carrier.

used that had no current at 260 mV and no slowly deactivating tail current, measured at 270 mV (compare Fig. 3A to Fig. 2B), ruling out Ni effects on LVA currents. Under these conditions, Ni significantly reduced the amplitude of the inactivating component of MVA currents elicited at 220 mV by 55 ^ 10% (P , 0.05; range: 30–75%). This inhibition was observed in all IC neurons tested (n ˆ 4), an example of which is shown in Fig. 3A. In addition, Ni inhibited the tail currents measured at 270 mV by 78 ^ 9% (P , 0.005; range: 50–92%; n ˆ 4), suggesting that Ni sensitive channels contribute to the tail currents in some neurons. Examination of the I–V relationship revealed that Ni did not shift the voltage at which the maximum inward current occurred (Fig. 3B). The Ni-sensitive currents activated near 250 mV and peaked at 210 mV (Fig. 3B), consistent with an I–V profile of a MVA current. These data indicate that Ni suppresses both LVA and MVA currents in IC neurons. Examination of the LVA currents showed that 1 mM U92032 reduced the amplitude of peak currents measured at 260 mV by the negligible amount of 10 ^ 3% (range: 5– 20%, n ˆ 4). The slow component of the tail current measured at 270 mV was insensitive to U-92032. The lack of effect of U-92032 on LVA currents is illustrated in Fig. 3D. In contrast, in cells that had current first activating at 250 mV, indicating the absence of LVA current and the presence of MVA current, U-92032 significantly (P , 0.05) reduced the amplitude of the peak current by 21 ^ 9% (range: 8–47%; n ˆ 4). Current was

measured 10 ms after the onset of a test pulse to 220 mV. The effects of U-92032 were complex in that it suppressed the inactivating component of MVA currents but increased the amplitude of the peak current at the end of the 220 mV test pulse (Fig. 3C). The loss of the transient component and the increase in the sustained component at 220 mV suggest that U-92032 slowed the activation of MVA currents, and as a consequence, little if any inactivation or decrease in tail current was observed. The I–V relationship showed that U92032 did not cause a shift in the voltage at which the maximum inward whole-cell Ba 21 current occurred (Fig. 3D). As with the effects of Ni on whole-cell currents that contained MVA currents, the U-92032 sensitive current activated at 250 mV and peaked at 210 mV, consistent with it affecting MVA currents (Fig. 3D). Together, these findings suggest that U-92032 alters MVA currents, but has little effect on LVA currents in IC neurons under the conditions used here. In another set of experiments, we examined the effects of 50 mM Ni (n ˆ 4) and 1 mM U-92032 (n ˆ 4) at 1 20 mV in IC neurons that exhibited MVA currents. Ni and U-92032 consistently caused a 31 ^ 6% (P , 0.05; range: 18–45%, n ˆ 4) and 24 ^ 3% (P , 0.05; range: 17–27%, n ˆ 4) reduction of the peak current at a test potential of 1 20 mV, respectively. When present, the long-lasting component of the tail current, measured after stepping back to 240 mV, was insensitive to U-92032. Examples of the reduction of the peak currents following application of U-92032 and Ni are

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Fig. 4. Effects of nimodipine on high threshold voltage-activated barium currents in neonatal inferior colliculus neurons. (A) An example of the time-course of the reversible action of 1 mM NIM on peak currents measured at 1 20 mV. (B) Representative traces (upper traces) from another IC neuron of the control current (a) and in the presence of NIM (b). In this recording, NIM reduced the amplitude of peak currents by 23.3%. The NIM-sensitive current (lower trace), obtained by subtracting the current in the presence of NIM from the one elicited under control conditions, was noninactivating. (C) Examples of I–V relationships in control and in the presence of NIM showed that NIM reduced the amplitude of peak inward currents at voltages positive to 240 mV. The I–V relationship of the NIM-sensitive current was obtained as described in B. (D) Comparison of the mean ^ S.E.M. (n ˆ 13) amplitude of the peak and the slow, sustained component of the tail current in the absence (CON) and presence of NIM (*P , 0.05, ***P , 0.001). In the present and subsequent experiments, the amplitudes of the peak current and the slow sustained component of the tail current were measured at 1 20 mV and 240 mV, respectively.

illustrated in I–V relationships (Fig. 3B, D). These data suggest that both Ni and U-92032 suppress a component of Ca 21 currents at a test potential of 1 20 mV in IC neurons. At least, a fraction of the sensitive component at 1 20 mV may be MVA current contaminating the HVA current. Currents sensitive to nimodipine. NIM, a dihydropyridine (DHP) L-type channel antagonist, has been used to identify Ltype currents in various CNS neurons. 17,53,70 It has been reported that the reduction of the whole-cell current amplitude by a relatively high concentration (10 mM) of NIM was not significantly different from that elicited by a lower concentration (1 mM) in cerebellar granule neurons. 3 Thus, we avoided using higher concentrations of NIM that can result in nonspecific effects in peripheral neurons. 29 NIM (1 mM) rapidly and reversibly reduced by 25 ^ 3% (range: 8–44%, n ˆ 13) the mean amplitude of control peak Ba 21 currents measured at a test potential of 1 20 mV. The inhibitory effect of NIM was observed in 13 out of 16 IC neurons tested; the remaining IC neurons were NIM-insensitive. The inhibitory action of NIM can be observed in a plot of the peak current vs time (Fig. 4A), in individual traces (Fig. 4B), and on the mean amplitude of the peak current (Fig. 4D). The NIM-sensitive current obtained by subtracting the current in the presence of NIM from the control, was noninactivating (Fig. 4B). An example of the I–V relationship showed that NIM-sensitive current activated near 240 mV, and showed

no change in the voltage at which the maximum inward current occurred (Fig. 4C). Examination of the tail current, measured by stepping back from 1 20 mV to 240 mV (Fig. 4B), revealed that NIM also significantly (P , 0.05) reduced the mean amplitude of the long-lasting component of the tail current by 22 ^ 7% (range: 5–76%, n ˆ 13), compared to controls (Fig. 4D). To further investigate the presence of L-type channels in IC neurons, we examined the effects of the benzoyl pyrrole FPL 64176 (FPL), a non-DHP L-type channel agonist, that has been shown to increase Ba 21 currents in a number of cell preparations including cerebellar granule neurons. 53 Application of 1 mM FPL significantly (P , 0.01) increased the mean amplitude of control peak currents at 210 mV (n ˆ 7), but not at 1 20 mV (n ˆ 7; Fig. 5, Table 1). The mean amplitude of the long-lasting component of the tail current, measured at 240 mV, was significantly increased in the presence of 1 mM FPL as compared to controls (Table 1), whether IC neurons were stepped to 210 mV (P , 0.05; n ˆ 7) or 1 20 mV (P , 0.001; n ˆ 7). The degree of increase in the amplitude of the long-lasting component of the tail current was variable (mean: 534 ^ 101% of control levels; range: 11–1071%; n ˆ 12). The action of FPL had a slow onset in most IC neurons tested (Fig. 5A). Like the NIM-sensitive current, the FPL-sensitive current was non-inactivating (Fig. 5B). Examination of the I–V relationship revealed that FPL caused a significant (P , 0.001) voltage shift of the maximum value

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759

Fig. 5. Effects of 1 mM FPL 64176 on high threshold voltage-activated currents in neonatal inferior colliculus neurons. (A) Example of a time-course of the actions of FPL on the peak and the slow, sustained component of the tail currents. (B) Representative traces (upper traces) of control (a) and in the presence of FPL (b) in another IC neuron. In this example, FPL caused a 560% increase in the amplitude of the slow sustained component of the tail current. Only a modest increase of the peak current was observed. FPL-sensitive current (obtained as described in Fig. 4B) was noninactivating. Current–voltage (I–V) relationships of the peak (C) and the slow sustained component of the tail (D) current from a third IC neuron. I-V relationships were generated by stepping the test potential in 10 mV increments while keeping the tail potential at 240 mV. FPL-sensitive currents were obtained as described in Fig. 4B, D and activated at 240 mV.

neurons. 24,51,55 Application of 1 mM v-CTx GVIA irreversibly reduced by 28 ^ 4% (range: 8–60%) the amplitude of peak currents in 17 out of 19 IC neurons tested; the remaining IC neurons were insensitive to v-CTx GVIA. The inhibitory effects of 1 mM v-CTx GVIA during a step to 1 20 mV is illustrated in Fig. 6A–D. Examples of I–V relationships and individual traces revealed that v-CTx GVIA-sensitive currents activated near 220 mV (Fig. 6C), and appeared non-inactivating (Fig. 6B). v-CTx GVIA did not change the voltage at which the maximum peak inward current occurred (Fig. 6C). As expected, v-CTx GVIA did not affect the amplitude of the long-lasting component of the tail current (Fig. 6B, D). These data strongly suggest that N-type currents are a component of HVA currents in neonatal IC neurons.

of the inward current towards more negative voltages (control: 2 ^ 2 mV, FPL 64176: 27 ^ 1 mV, n ˆ 9). An example of the I–V relationship showed that FPL-sensitive currents activated around 240 mV (Fig. 5C–D) suggesting that these currents may be contaminating some of the MVA currents in IC neurons, as discussed earlier. These results indicate that L-type current contributes to HVA whole-cell currents in neonatal IC neurons and that at least a part of the long-lasting component of the tail current at 240 mV is made up of L-type current. Currents sensitive to v -conotoxin GVIA. We also examined the effects of v-CTx GVIA, a peptide toxin originally isolated from the venom of the marine snail Conus geographus that potently inhibits N-type current in many CNS

Table 1. Comparison of the amplitude of the peak and the long-lasting component of the tail current in the absence and presence of FPL 64176 Current at 1 20 mV Peak Tail CON FPL

860.1 ^ 160.8 862.5 ^ 47.9

176.8 ^ 136.1 936.6 ^ 184.8***

Current at 210 mV Peak Tail

n 7 7

720.8 ^ 212.0 1141.9 ^ 265.1**

All data are mean current amplitude ^ S.E.M. n, number of recordings. *P , 0.05, 1 mM FPL 64176 (FPL) vs Control (CON). **P , 0.01, FPL vs CON. ***P , 0.001, FPL vs CON.

112.1 ^ 39.4 846.6 ^ 238.4*

n 7 7

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Fig. 6. Effects of v-conotoxin GVIA on high threshold voltage-activated currents in neonatal inferior colliculus neurons. (A) An example of the time-course of the irreversible action of 1 mM v-CTx GVIA. (B) Representative current traces in the absence (a) and presence (b) of v-CTx GVIA taken from the same neuron as in A. In this experiment v-CTx GVIA caused a 29% reduction in the amplitude of peak HVA currents, as compared to control. v-CTx GVIA-sensitive current (obtained as described in Fig. 4B) was non-inactivating. (C) Examples of I–V relationships in the absence (a) or presence (b) of v-CTx GVIA, and the v-CTx GVIA-sensitive I–V curve (obtained as described in Fig. 4C). (D) Comparison of mean ^ S.E.M. (n ˆ 17) amplitude of the peak current and the slow sustained component of the tail current in the absence (CON) and presence of v-CTx GVIA. v-CTx GVIA significantly (***P , 0.001) reduced the amplitude of peak currents at 1 20 mV, as compared to controls (CON).

Currents sensitive to funnel-web spider toxin and v agatoxin IVA. Concomitant application of 1 mM v-CTx GVIA and 1 mM NIM caused a 53 ^ 1% (range: 51–65%, n ˆ 5) reduction in control peak currents at 1 20 mV (data not shown), but did not completely suppress this current. These findings raised the possibility that the activity of additional types of HVA Ca 21 channels contributes to whole-cell Ba 21 currents in IC neurons. Therefore, IC neurons were next tested for the presence of P/Q-type currents. To test whether IC neurons exhibit P/Q-type currents, we initially examined the effects of FTX, a partially purified polyamine isolated from the venom of Agelenopsis aperta, a funnel-web spider. 35 It is a reported blocker of P/Q-type channels in cerebellar Purkinje neurons in which P-type current dominates. 36,43,67 Application of FTX at a dilution of 1:1000 induced a 10% and 50% reduction of the total peak HVA currents in two IC neurons tested (data not shown), as compared to controls. However, the reported non-selectivity of FTX-induced blockade of the Ca 21 currents in neocortical pyramidal neurons led to further evaluation of the presence of P/Q-type Ca 21 channels in IC neurons by using v-Aga IVA, a peptide toxin also isolated from Agelenopsis aperta. 11,49 Because of the controversy in the literature as to what concentration of v-Aga IVA selectively blocks P-type versus Q-type channels, we examined the effects of a relatively high concentration (300 nM) of v-Aga IVA which should block both Pand Q-type Ca 21 channel activity. 49,53,55 300 nM v-Aga IVA

irreversibly reduced the amplitude of peak HVA currents by 26 ^ 2% (range: 16–35%, n ˆ 10) in 10 out of 14 IC neurons tested; the remaining IC neurons were v-Aga IVA-insensitive (Fig. 7D). The inhibitory effect of v-Aga IVA on the peak current was maximal 4–10 min after adding it to the bath (Fig. 7A). However, it did not affect the amplitude of the long-lasting component of the tail current suggesting that P/Q-type channels do not contribute to this current (Fig. 7B). Examination of the I–V relationship revealed that v-Aga IVA did not significantly shift the voltage at which peak inward currents occurred (Fig. 7C). Examples of the I–V relationship and current traces revealed that v-Aga IVA-sensitive currents activated around 230 mV (Fig. 7C) and exhibited some inactivation (Fig. 7B). These results indicate that P/Q-type currents also contribute to whole-cell Ba 21 currents in neonatal IC neurons. We next tested whether antagonists of HVA currents could inhibit MVA currents. We expected this current to be insensitive to NIM, v-CTx GVIA and v-Aga IVA. Indeed, in most IC neurons tested the inactivating component of MVA current, measured at 220 mV, was insensitive to 1 mM NIM (four out of five tested neurons, data not shown) and to 1 mM v-CTx GVIA (three out of four tested neurons, data not shown). However, MVA currents appeared to be sensitive to 300 nM v-Aga IVA (two out of three tested neurons, data not shown). These results suggest that MVA currents recorded at 220 mV might have been

Calcium currents in neonatal inferior colliculus neurons

761

Fig. 7. Effects of v-Agatoxin IVA on high threshold voltage-activated barium currents in neonatal inferior colliculus neurons. (A) An example of the timecourse of the actions of 300 nM v-Aga IVA on peak Ba 21 currents. (B) Representative traces in the absence (a) and presence (b) of v-Aga IVA in another IC neuron. v-Aga IVA reduced the amplitude of the control peak current by 31% (upper traces). The v-Aga IVA-sensitive current (obtained as described in Fig. 4B) exhibited some inactivation (lower trace). (C) I–V relationships in control and in the presence of v-Aga IVA demonstrated that the toxin blocker reduced the amplitude of the peak current at voltages positive to 240 mV. The v-Aga IVA-sensitive I–V relationship was obtained as described in Fig. 4D. Comparison of mean ^ S.E.M. (n ˆ 10) amplitude of the peak current and the slow sustained component of the tail current in the absence and presence of v-Aga IVA. The toxin significantly (***P , 0.001) reduced the amplitude of the peak current, but did not affect the slow component of the tail current. 5 mM Ba 21 was used as the charge carrier.

contaminated by some HVA currents, or that these channels show a sensitivity to v-Aga IVA, as has been reported previously. 59 Current insensitive to a combination of nimodipine, v conotoxin GIVA and v -agatoxin GIVA. We also examined whether IC neurons exhibit a component of the HVA current that is insensitive to a combination of Ca 21 channel blockers. Concomitant application of 300 nM v-Aga IVA, 1 mM NIM and 1 mM v-CTx GVIA decreased the control peak currents by 62 ^ 3% (range: 54–65%; n ˆ 4) but did not completely suppress these currents (Fig. 8A, B). Resistant currents following application of 300 nM v-Aga IVA, 1 mM NIM and 1 mM v-CTx GVIA activated near 250 mV (range: 250 to 240 mV), peaked near 210 mV and exhibited partial inactivation at a test potential of 1 20 mV (Fig. 8B, C). Partial inactivation was also observed at test potentials negative to 210 mV, suggesting that currents, which activated at “mid-range” voltages, may contribute to the resistant currents measured at 1 20 mV. Consistent with this idea, application of 1 mM U-92032 in the continued presence of 300 nM v-Aga IVA, 1 mM NIM and 1 mM v-CTx GVIA caused a further 45% reduction in resistant currents in a neuron that also exhibited MVA currents (data not shown). The persistent

residual currents following application of 300 nM v-Aga IVA, 1 mM NIM, 1 mM v-CTx GVIA and 1 mM U-92032 exhibited partial inactivation and were completely suppressed by 50 mM cadmium (data not shown), indicating that the residual currents were Ba 21 currents and not simply a leak current. Thus, U-92032-sensitive currents may contribute to at least one component of the resistant current found at 120 mV in IC neurons. These results also suggest that in addition to LVA, and MVA currents, as well as N-, L-, and P/Q-type currents, additional as of yet, uncharacterized currents may contribute to whole-cell Ba 21 currents in dissociated neonatal IC neurons. DISCUSSION

Previous studies have indicated the presence of voltageactivated Ca 21 currents in neonatal IC neurons. 26 The present study demonstrates that acutely dissociated neonatal rat IC neurons express LVA, MVA, and HVA Ca 21 currents on the basis of their biophysical characteristics and pharmacological sensitivities. LVA and MVA currents were observed in subsets of IC neurons whereas HVA currents were recorded in all IC neurons tested. HVA currents in IC neurons could be pharmacologically dissected into four

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Fig. 8. High threshold voltage-activated barium currents were insensitive to a combination of calcium channel blockers. (A) An example of current vs time illustrating the reduction in the peak HVA current in presence of v-Aga IVA (300 nM) alone, and in the presence of a combination of 300 nM v-Aga IVA, 1 mM v-CTx GVIA and 1 mM NIM. (B) Representative traces (upper traces) of control (a), in the presence of v-Aga IVA (b) and in the presence of a combination of v-Aga IVA, v-CTx GVIA and NIM (c). Note that the current resistant to v-Aga IVA, v-CTx GVIA and NIM exhibited inactivation (lower trace). (C) Examples of I–V relationships in control, in the presence of v-Aga IVA alone, and in the presence of a combination of v-Aga IVA, v-CTx GIVA and NIM. Five millimolar Ba 21 was used as the charge carrier.

components including NIM-, v-CTx GVIA- and v-Aga IVAsensitive currents. A current that was insensitive to a combination of NIM and toxin blockers was also present. A component of this current was sensitive to U-92032. The diversity of Ca 21 channel types that appear to contribute to whole-cell Ba 21 currents in neonatal IC neurons is supported by binding and in situ hybridization studies, and a1 subunit mRNA expression data which indicate that adult IC neurons express various types of HVA Ca 21 channels including L-, N-, and P/Q-types. 12,20,42,63–65

An MVA current was also present in IC neurons defined by a threshold of activation at 250 mV, no slow tail current, inactivating current kinetics at a test potential of 220 mV, and pharmacological sensitivities to Ni and U-92032. It has been reported that MVA currents can be observed when the a1E class of mRNA is expressed in either Xenopus oocytes or COS-7 cells. 59,60 These currents are similar to MVA currents in IC neurons in that they activated near 250 mV, peaked around 210 mV, and were Ni-sensitive, partially sensitive to v-Aga IVA, but insensitive to v-CTx GVIA. 59,60

Low threshold voltage-activated and high threshold voltageactivated currents

High threshold voltage-activated currents

Only 33% of IC neurons had currents that were activated at low threshold and exhibited a slow rate of deactivation at 270 mV, two key characteristics that suggest the presence of LVA currents. The relatively low incidence of LVA currents in neonatal IC neurons is consistent with current-clamp studies which reported that the majority of adult IC neurons did not spontaneously discharge in a bursting pattern at the resting membrane potential, as is characteristic of neurons possessing significant densities of LVA Ca 21 channels. 13,27,33,34,57 As in other CNS neurons, LVA currents in neonatal IC neurons were sensitive to Ni, a blocker of LVA current. However, U-92032, the reported LVA T-type current blocker in hippocampal CA1 neurons, 5 had only a minimal effect on IC neurons raising the possibility of different pharmacological sensitivities of LVA currents between these CNS sites.

In the present study, HVA currents were comprised of four current types including L-, N- and P/Q-type currents as well as a current resistant to a combination of NIM, v-CTx GVIA and v-Aga IVA. NIM partially reduced the amplitude of the control peak currents measured at 1 20 mV in most IC neurons tested. In a number of IC neurons, the tail current recorded at 240 mV exhibited both a fast and a long-lasting component. NIM partially reduced the amplitude of the longlasting component of the tail current, suggesting that NIMsensitive L-type channel activity contributes to these currents. Consistent with this hypothesis is the finding that FPL increased the amplitude of the long-lasting component of the tail current. The mechanisms underlying the FPL-induced increase in the amplitude of the long-lasting component of the tail current has not been completely elucidated. Our preliminary studies on single L-type channel activity in

Calcium currents in neonatal inferior colliculus neurons

neonatal IC neurons demonstrated that FPL elicited long openings (unpublished data). Thus, the long-lasting component of the tail current could be due to maintained openings at 240 mV and/or re-openings of L-type channels at or below normal threshold. Similar openings and/or reopenings have been observed previously in studies of unitary L-type Ca 21 currents with or without a DHP agonist in some CNS neurons. 8,21,23,30,31,48,56,66 Taken together these findings indicate that IC neurons express L-type channels. In addition to L-type currents, N- and P/Q-type currents also contribute to whole-cell HVA currents in neonatal IC neurons. v-CTx GVIA partially reduced the amplitude of peak currents in the present study. v-Aga IVA at a concentration reported to inhibit P-type and Q-type currents, partially reduced the amplitude of the peak current. Because 300 nM v-Aga IVA was used in this study, it was difficult to determine whether both P-type and Q-type currents are present in IC neurons. However, the v-Aga IVA sensitive current showed little inactivation, suggesting that the majority of this current is of the P-type. 44 Pharmacologically resistant currents Approximately 40% of the total Ba 21 current measured at 120 mV was resistant to a combination of NIM, v-CTx GVIA and v-Aga IVA, indicating further heterogeneity of Ca 21 currents in IC neurons. Resistant currents exhibited inactivation, similar to the DHP- and toxin blocker-resistant HVA currents first described in cerebellar granule neurons. 53,71 Antisense studies indicate that some of the resistant current in cerebellar granule neurons can be attributed to Ca 21 channels produced from a1E message. 52 However, the contribution of a1E protein to the resistant current is still controversial because a1E currents in recombinant expression systems exhibit different permeation and inactivation sensitivities as compared to the resistant currents in neurons. 15,71 In addition, currents unaffected by

763

NIM, v-CTx GVIA and v-Aga IVA with inactivation kinetics distinct from those reported in cerebellar granule neurons have been found in other CNS neurons. 22,50,62,68 Thus, resistant currents could have different molecular identities in different CNS neurons. In this study, U-92032, a putative LVA T-type channel antagonist that inhibited MVA current and a component of current at 1 20 mV in neonatal IC neurons, partially reduced the amplitude of resistant currents, suggesting that U-92032-sensitive channels may contribute to at least a component of the resistant current. Whether the persistent residual current represents an additional uncharacterized type(s) of Ba 21 current or a combination of residual L-, Nand P/Q-type currents as well as LVA and MVA currents is unknown. To our knowledge this study is the first in describing the composition of voltage-activated Ca 21 channels in neonatal IC neurons. The identification of LVA and MVA as well as HVA currents including a NIM-sensitive L-type, v-CTx GVIA-sensitive N-type and v-Aga IVA-sensitive P/Q-type current, and a current resistant to NIM, v-CTx GVIA and v-Aga IVA in acutely dissociated neonatal IC neurons is an important step towards a better understanding of the ionic currents present in neonatal IC. Biophysical, pharmacological and molecular characterization of Ca 21 currents in different nuclei of the adult IC are currently underway and should yield additional detail about the heterogeneous expression of Ca 21 currents in the IC.

Acknowledgements—This publication was made possible by Grants R29-NS34195 (A. R. R.) and 1F32AA05497 (P. N’G.) from the National Institutes of Health and its contents are the responsibility of the authors and do not necessarily represent the official views of the NINDS and NIAAA. A. R. R. is the recipient of an Established Investigator Award from the American Heart Association. We are grateful to Professor Gregory J. Brewer for his critical input in the cell dissociation, and to Dr Liwang Liu for his critique of the manuscript.

REFERENCES

1. Aitkin L. (1986) The Auditory Midbrain: Structure and Function in the Central Auditory Pathway, pp. 75–100, Humana, Clifton, NJ. 2. Altman J. and Bayer S. A. (1981) Time of origin of neurons of the rat inferior colliculus and the relations between cytogenesis and tonotopic order in the auditory pathway. Expl Brain Res. 42, 411–423. 3. Amico C., Marchetti C., Nobile M. and Usai C. (1995) Pharmacological types of calcium channels and their modulation by baclofen in cerebellar granules. J. Neurosci. 15, 2839–2848. 4. Armstrong C. M. and Matteson D. R. (1985) Two distinct populations of calcium channels in a clonal line of pituitary cells. Science 227, 65–67. 5. Avery R. B. and Johnston D. (1997) Ca 21 channel antagonist U-92032 inhibits both T-type and Ca 21 channels and Na 1 channels in hippocampal CA1 pyramidal neurons. J. Neurophysiol. 33, 1023–1028. 6. Avery R. B. and Johnston D. (1996) Multiple channel types contribute to the low-voltage-activated calcium current in hippocampal CA3 pyramidal neurons. J. Neurosci. 16, 5567–5582. 7. Bech-Hansen N. T., Naylor M. J., Maybaum T. A., Pearce W. G., Koop B., Fishman G. A., Mets M., Musarella M. A. and Boycott K. M. (1998) Loss-of-function mutations in a calcium-channel a1-subunit gene in Xp11.23 cause incomplete X-linked congenital stationary night blindness. Nature Genet. 19, 264–267. 8. Bossu J. L., DeWaard M., Fagni L., Tanzi F. and Feltz A. (1994) Characteristics of calcium channels responsible for voltage-activated calcium entry in rat cerebellar granule cells. Eur. J. Neurosci. 6, 335–344. 9. Brewer G. J., Torricelli J. R., Evege E. K. and Price P. J. (1993) Optimized survival of hippocampal neurons in B27-supplemented neurobasale, a new serum-free medium combination. J. Neurosci. Res. 35, 567–576. 10. Brown A. M., Schwindt P. C. and Crill W. E. (1993) Voltage dependence and activation kinetics of pharmacologically defined components of the highthreshold calcium current in rat neocortical neurons. J. Neurophysiol. 70, 1530–1543. 11. Brown A. M., Sayer R. J., Schwindt P. C. and Crill W. E. (1994) P-type calcium channels in rat neocortical neurones. J. Physiol. 475, 197–205. 12. Corte´s R., Supavilai P., Karobath M. and Palacios J. M. (1984) Calcium antagonist binding sites in the rat brain: quantitative autoradiographic mapping using the 1,4-DHP [ 3H]PN 200-110 and [ 3H]PY 108-068. J. neural Transm. 60, 169–197. 13. Coulter D. A., Huguenard J. R. and Prince D. A. (1989) Calcium currents in rat thalamocortical relay neurones: kinetic properties of the transient lowthreshold current. J. Physiol. 414, 587–604. 14. Cribbs L. L., Lee J-H., Yang J., Satin J., Zhang Y., Daud A., Barclay J., Williamson M. P., Fox M., Rees M. and Perez-Reyes E. (1998) Cloning and characterization of a1H from human heart, a member of the T-type Ca 21 channel gene family. Circulation Res. 83, 103–109. 15. De Leon M., Wang Y., Jones L., Perez-Reyes E., Wei X., Soong T. W., Snutch T. P. and Yue D. T. (1995) Essential Ca 21-binding motif for Ca 21-sensitive inactivation of L-type Ca 21 channels. Science 270, 1502–1506. 16. Dunlap K., Luebke J. I. and Turner T. J. (1995) Exocytotic Ca 21 channels in mammalian central neurons. Trends Neurosci. 18, 89–98.

764

P. N’Gouemo and A. R. Rittenhouse

17. Eliot L. S. and Johnston D. (1994) Multiple components of calcium current in acutely dissociated dentate gyrus granule neurons. J. Neurophysiol. 72, 762–777. 18. Ertel S. I. and Ertel E. A. (1997) Low voltage-activated T-type Ca 21 channel. Trends pharmac. Sci. 18, 37–42. 19. Faye-Lund H. and Osen K. K. (1985) Anatomy of the inferior colliculus in rat. Anat. Embryol. 171, 1–20. 20. Filloux F., Karras J., Imperial J. S., Gray W. R. and Olivera B. M. (1994) The distribution of v-conotoxin MVIICnle-binding sites in rat brain measured by autoradiography. Neurosci. Lett. 178, 263–266. 21. Fisher R. E., Gray R. and Johnston D. (1990) Properties and distribution of single voltage-gated calcium channels in adult hippocampal neurons. J. Neurophysiol. 64, 91–104. 22. Foehring R. C. and Armstrong W. E. (1996) Pharmacological dissection of high-voltage activated Ca 21 current types in acutely dissociated rat supraoptic magnocellular neurons. J. Neurophysiol. 76, 977–983. 23. Forti L. and Pietrobon D. (1993) Functional diversity of L-type calcium channels in rat cerebellar neurons. Neuron 10, 437–450. 24. Fujita Y., Mynlieff M., Dirksen R. T., Kim M.-S., Niidome T., Nakai J., Friedrich T., Iwabe N., Miyata T., Furuichi T., Furutama D., Mikoshiba K., Mori Y. and Beam K. G. (1993) Primary structure and functional expression of the v-conotoxin-sensitive N-type calcium channel from rabbit brain. Neuron 10, 585–598. 25. Hamill O. P., Marty A., Neher E., Sakmann B. and Sigworth F. J. (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflu¨gers Arch. 391, 85–100. 26. Hosomi H., Mori M., Amatsu M. and Okada Y. (1997) GABA-activated conductances in cultured rat inferior colliculus neurons. J. Neurophysiol. 77, 994–1002. 27. Huguenard J. R. (1996) Low-threshold calcium currents in central nervous system neurons. A. Rev. Physiol. 58, 329–348. 28. Jones S. W. and Elmslie K. S. (1997) Transmitter modulation of neuronal calcium channels. J. Membrane Biol. 155, 1–10. 29. Jones S. W. and Jacobs L. S. (1990) Dihydropyridine actions on calcium currents of frog sympathetic neurons. J. Neurosci. 10, 2261–2267. 30. Kavalali E. T. and Plummer M. R. (1994) Selective potentiation of a novel calcium channel in rat hippocampal neurones. J. Physiol. 480, 475–484. 31. Kavalali E. T. and Plummer M. R. (1996) Multiple voltage-dependent mechanisms potentiate calcium channel activity in hippocampus neurons. J. Neurosci. 16, 1072–1082. 32. Lee J-H., Daud A. N., Cribbs L. L., Lacerda A. E., Pereverzev A., Klo¨ckner U., Schneider T. and Perez-Reyes E. (1999) Cloning and expression of a novel member of the low voltage-activated T-type calcium channel family. J. Neurosci. 19, 1912–1921. 33. Li Y., Evans M. S. and Faingold C. L. (1998) In vitro electrophysiology of neurons in subnuclei of rat inferior colliculus. Hearing Res. 121, 1–10. 34. Liu S. J. and Kaczmarek L. K. (1998) Depolarization selectively increases the expression of the Kv3.1 potassium channel in developing inferior colliculus neurons. J. Neurosci. 18, 8758–8769. 35. Llina´s R., Sugimori M., Lin J.-W. and Cherksey B. (1989) Blocking and isolation of a calcium channel from neurons in mammals and cephalopods utilizing a toxin fraction (FTX) from funnel-web spider poison. Proc. natn. Acad. Sci. U.S.A. 86, 1689–1693. 36. Llina´s R., Sugimori M., Hillman D. E. and Cherksey B. (1992) Distribution and functional significance of P-type voltage-dependent Ca 21 channels in the mammalian central nervous system. Trends Neurosci. 15, 351–355. 37. Lorenzon N. M. and Foehring R. C. (1995) Characterization of pharmacologically identified voltage-gated calcium channel currents in acutely isolated rat neocortical neurons I. Adult neurons. J. Neurophysiol. 73, 1430–1442. 38. Lorenzon N. M. and Foehring R. C. (1995) Characterization of pharmacologically identified voltage-gated calcium channel currents in acutely isolated rat neocortical neurons II. Postnatal development. J. Neurophysiol. 73, 1443–1451. 39. Marchetti C., Carignani C. and Robello M. (1991) Voltage-dependent calcium currents in dissociated granule cells from rat cerebellum. Neuroscience 43, 121–133. 40. McCown T. J. and Breese G. R. (1991) The role of inferior collicular cortex in the neonatal rat: sensorimotor modulation. Devl Brain Res. 59, 1–5. 41. McCown T. J. and Breese G. R. (1992) The developmental profile of seizure genesis in the inferior collicular cortex of the rat: relevance to human neonatal seizures. Epilepsia 33, 2–10. 42. McIntosh J. M., Adams M. E., Olivera B. M. and Filloux F. (1992) Autoradiographic localization of the binding of calcium channel antagonist [ 125I]vagatotoxin IIIA in rat brain. Brain Res. 594, 109–114. 43. Mintz I. M., Venema V. J., Swiderek K. M., Lee T. D., Bean B. P. and Adams M. E. (1992) P-type calcium channels blocked by the spider toxin v-Aga IVA. Nature 355, 827–829. 44. Mintz I., Adams M. E. and Bean B. P. (1992) P-type calcium channels in rat central and peripheral neurons. Neuron 9, 85–95. 45. Morest D. K. and Oliver D. L. (1984) The neuronal architecture of the inferior colliculus in the cat: defining the functional anatomy of the auditory midbrain. J. comp. Neurol. 222, 209–236. 46. N’Gouemo P. and Rittenhouse A. R. (1997) Pharmacological characterization of voltage-sensitive calcium currents in inferior colliculus neurons. Soc. Neurosci. Abstr. 23, 1187. 47. Nooney J. M., Lambert R. C. and Feltz A. (1997) Identifying neuronal non-L Ca21 channels-more than stamp collecting. Trends pharmac. Sci. 18, 363–371. 48. O’Dell T. J. and Alger B. E. (1991) Single calcium channels in rat and guinea-pig hippocampal neurons. J. Physiol. 436, 739–767. 49. Olivera B. M., Miljanich G. P., Ramachandran J. and Adams M. E. (1994) Calcium channel diversity and neurotransmitter release: the v-conotoxins and v-agatoxins. A. Rev. Biochem. 63, 823–867. 50. Penington N. J. and Fox A. P. (1995) Toxin-insensitive Ca 21 current in dorsal raphe neurons. J. Neurosci. 15, 5719–5726. 51. Perez-Reyes E., Cribbs L. L., Daud A., Lacerda A. E., Barclay J., Williamson M. P., Fox M., Rees M. and Lee J.-H. (1998) Molecular characterization of neuronal low-voltage-activated T-type calcium channel. Nature 391, 896–900. 52. Piedras-Renterı´a E. S. and Tsien R. W. (1998) Antisense oligonucleotides against a1E reduce R-type calcium currents in cerebellar granule cells. Proc. natn. Acad. Sci. U.S.A. 95, 7760–7765. 53. Randall A. and Tsien R. W. (1995) Pharmacological dissection of multiple types of Ca 21 currents in rat cerebellar granule neurons. J. Neurosci. 15, 2995–3012. 54. Roberts R. C. and Ribak C. E. (1987) An electron microscopic study of GABAergic neurons and terminals in the central nucleus of the inferior colliculus of the rat. J. Neurocytol. 16, 333–345. 55. Sather W. A., Tanabe T., Zhang J.-F., Mori Y., Adams M. E. and Tsien R. W. (1993) Distinctive biophysical and pharmacological properties of class A (BI) calcium channel a1 subunits. Neuron 11, 291–303. 56. Slesinger P. A. and Lansman J. B. (1991) Reopening of Ca 21 channels in mouse cerebellar neurons at resting membrane potentials during recovery from inactivation. Neuron 7, 755–762. 57. Smith P. H. (1992) Anatomy and physiology of multipolar cells in the rat inferior collicular cortex using the in vitro brain slice technique. Neuroscience 12, 3700–3715. 58. Snutch T. P., Leonard J. P., Gilbert M. M., Lester H. A. and Davidson N. (1990) Rat brain expresses a heterogeneous family of calcium channels. Proc. natn. Acad. Sci. U.S.A. 87, 3391–3395. 59. Soong T. W., Stea A., Hodson C. D., Dubel S. J., Vincent S. R. and Snutch T. P. (1992) Structure and functional expression of member of the low voltageactivated calcium channel family. Science 260, 1133–1136. 60. Stephens G. J., Page K. M., Burley J. R., Berrow N. S. and Dolphin A. C. (1997) Functional expression of rat brain cloned a1E calcium channels in COS7 cells. Pflu¨gers Arch. 433, 523–532. 61. Strom T. M., Nyakatura G., Apfelstedt-Sylla E., Hellebrand H., Lorenz B., Weber B. H. F., Wutz K., Gutwillinger N., Ru¨ther K., Drescher B., Sauer C.,

Calcium currents in neonatal inferior colliculus neurons

62. 63. 64. 65. 66. 67. 68. 69. 70. 71.

765

Zrenner E., Meitinger T., Rosenthal A. and Meindl A. (1998) An L-type calcium-channel gene mutated in incomplete X-linked congenital stationary night blindness. Nature Genet. 19, 260–263. Sundgren-Anderson A. K. and Johansson S. (1998) Calcium spikes and calcium currents in neurons from the medial preoptic nucleus of rat. Brain Res. 783, 194–209. Takemura M., Kiyama H., Fukui H., Tohyama M. and Wada H. (1989) Distribution of the v-conotoxin receptor in rat brain. An autoradiographic mapping. Neuroscience 32, 405–416. Talley E. M., Cribbs L. L., Lee J-H., Daud A., Perez-Reyes E. and Bayliss D. A. (1999) Differential distribution of the three members of a gene family encoding low voltage-activated (T-type) calcium channels. J. Neurosci. 19, 1895–1911. Tanaka O., Sakagami H. and Kondo H. (1995) Localization of mRNAs of voltage-dependent Ca 21-channels: four subtypes of a1- and b-subunits in developing and mature rat brain. Molec. Brain Res. 30, 1–16. Thibault O., Porter N. M. and Landfield P. W. (1993) Low Ba 21 and Ca 21 induce a sustained high probability of repolarization openings of L-type Ca 21 channels in hippocampal neurons: physiological implications. Proc. natn. Acad. Sci. U.S.A. 90, 11792–11796. Usowicz M. M., Sugimori M., Cherksey B. and Llina´s R. (1992) P-type calcium channels in the somata and dendrites of adult cerebellar Purkinje cells. Neuron 9, 1185–1199. Wang X., McKenzie J. S. and Kemm R. E. (1996) Whole-cell calcium currents in acutely isolated olfactory bulb output neurons of the rat. J. Neurophysiol. 75, 1138–1151. Williams M. E., Washburn M. S., Hans M., Urrutia A., Brust P. F., Prodanovich P., Harpold M. M. and Stauderman K. A. (1999) Structure and functional characterization of a novel human low-voltage activated calcium channel. J. Neurochem. 72, 791–799. Yu B. and Shinnick-Gallagher P. (1997) Dihydropyridine-and neurotoxin-sensitive and insensitive calcium currents in acutely dissociated neurons of the rat central amygdala. J. Neurophysiol. 77, 690–701. Zhang J.-F., Randall A. D., Ellinor P. T., Horne W. A., Sather W. A., Tanabe T., Schwarz T. L. and Tsien R. W. (1993) Distinctive pharmacology and kinetics of cloned neuronal Ca 21 channels and their possible counterparts in mammalian CNS neurons. Neuropharmacology 32, 1075–1088. (Accepted 7 January 2000)