Low-threshold calcium currents contribute to locomotor-like activity in neonatal mice

Abstract

In this study, we examined the contribution of a low-threshold calcium current [I(Ca(T))] to locomotor-related activity in the neonatal mouse. Specifically, the role of I(Ca(T)) was studied during chemically induced, locomotor-like activity in the isolated whole cord and in a genetically distinct population of ventromedial spinal interneurons marked by the homeobox gene Hb9. In isolated whole spinal cords, cycle frequency was decreased in the presence of low-threshold calcium channel blockers, which suggests a role for I(Ca(T)) in the network that produces rhythmic, locomotor-like activity. Additionally, we used Hb9 interneurons as a model to study the cellular responses to application of low-threshold calcium channel blockers. In transverse slice preparations from transgenic Hb9::enhanced green fluorescent protein neonatal mice, N-methyl-d-aspartate-induced membrane potential oscillations in identified Hb9 interneurons also slowed in frequency with application of nickel when fast, spike-mediated, synaptic transmission was blocked with TTX. Voltage-clamp and immunolabeling experiments confirmed expression of I(Ca(T)) and channels, respectively, in Hb9 interneurons located in the ventromedial spinal cord. Taken together, these results provide support that T-type calcium currents play an important role in network-wide rhythm generation during chemically evoked, fictive locomotor activity.

Fig. 1.

Fictive locomotor-like activity in the isolated whole cord is eliminated by exogenous nickel (Ni⁺⁺) application. A: paired extracellular recordings from ipsilateral lumbar ventral roots 2 and 5 (iL2 and iL5) in an isolated whole-cord preparation with “cocktail” [3–10 μM N-methyl-d-aspartic acid (NMDA), 6–12 μM 5-hydroxytryptamine (5-HT), and 15–20 μM dopamine hydrochloride (DA)] applied to produce fictive locomotor-like activity. Top panel: cocktail-induced motor pattern; middle panel: exogenous application of NiCl₂ (+Ni⁺⁺) slows the rhythmic bursting of the motor pattern and reduces the burst strength; bottom panel: washout of NiCl₂ (−Ni⁺⁺) reverses the effects and coordinated pattern of motor activity returns. B: time course of cycle frequency with Ni⁺⁺ application. C: plot of normalized cycle frequency against treatment: Control, +Ni⁺⁺ (application), −Ni⁺⁺ (washout). D: plot of normalized burst strength against treatment, as in C. Data are normalized to “Control” condition prior to application of Ni⁺⁺. *Significant differences.

Fig. 2.

Concentration response of NiCl₂ on fictive locomotor-like activity in the isolated whole cord. A: extracellular recordings of cocktail (3–10 μM NMDA, 6–12 μM 5-HT, and 15–20 μM DA)-induced fictive locomotor-like activity from a single ventral root (L2) at various NiCl₂ concentrations. Cycle frequency decreases and is ultimately eliminated as the concentration of NiCl₂ increases. B: concentration-response curve showing the effects of NiCl₂ on cycle frequency, which is normalized to NMDA condition prior to application of NiCl₂. The curve was fit with the equation 1/(1 + {[D]/IC₅₀}ⁿ), where (D) is the NiCl₂ concentration, IC₅₀ is the dose for 1/2 inhibition, and n is the Hill coefficient; IC₅₀ = 110 μM. Data are normalized to control condition prior to application of Ni⁺⁺. C: in some preparations (2 of 6; 33%), high concentrations of NiCl₂ (∼200 μM) produced a transient, unorganized motor pattern. Note that the motor pattern was disrupted under these conditions: 1) the left and right ventral root recordings from L2 [iL2 and contralateral (cL2)] produced a synchronous activity pattern rather than the typical alternating pattern, and 2) the bursting occurred at a frequency higher than normally observed.

Fig. 3.

Application of the selective R-type calcium channel blocker SNX-482 does not alter fictive locomotor-like activity. A: paired extracellular recordings from iL2 and iL5 in an isolated whole-cord preparation with cocktail (3–10 μM NMDA, 6–12 μM 5-HT, and 15–20 μM DA) applied to produce fictive locomotor-like activity. Left: cocktail-induced motor pattern; right: exogenous application of SNX-482 (+SNX-482) does not alter the rhythmic motor pattern. B: time course of cycle frequency with SNX-482 application. C: plot of normalized cycle frequency against treatment: control and +SNX-482 (application). D: plot of normalized burst strength against treatment, as in C. Data are normalized to control condition prior to application of SNX-482.

Fig. 4.

Application of the selective T-type calcium channel blocker NNC 55-0396 eliminates fictive locomotor-like activity. A: single extracellular recording from ventral root L2 in an isolated whole-cord preparation with cocktail (3–10 μM NMDA, 6–12 μM 5-HT, and 15–20 μM DA) applied to produce fictive locomotor-like activity. Top trace: cocktail-induced motor pattern; middle trace: exogenous application of NNC 55-0396 (+NNC 55-0396) slows the rhythmic bursting of the motor pattern and reduces the burst strength; bottom trace: exogenous application of NNC 55-0396 ultimately (∼15 min) eliminates the motor pattern; this effect is irreversible over the time course monitored (data not shown). B: time course of cycle frequency with NNC 55-0396 application.

Fig. 5.

Chemically induced membrane potential oscillations in Hb9 interneurons are sensitive to Ni⁺⁺. A: whole-cell, current-clamp recordings of membrane potential oscillations in an Hb9 interneuron in the presence of a cocktail of chemicals (21 μM NMDA, 21 μM 5-HT, and 50 μM DA). Top trace: chemically induced membrane potential oscillations when fast, spike-mediated, synaptic events are blocked by TTX; middle trace: exogenous application of Ni⁺⁺ reduces cycle frequency and voltage amplitude; bottom trace: the effects of Ni⁺⁺ reverse following washout. B: time course of cycle frequency with Ni⁺⁺ application from a representative Hb9 interneuron. C: plots of normalized cycle frequency (raw mean at 0 min = 0.7 ± 0.2 Hz; range = 0.3–1.0 Hz; n = 10) against treatment condition. D: plot of normalized voltage amplitude (raw mean at 0 min = 17.8 ± 10.4 mV; range = 5.8–34.0 mV; n = 10) against treatment condition. Data are normalized to NMDA condition prior to application of Ni⁺⁺. *Significant differences.

Fig. 6.

Ni⁺⁺ reversibly blocks T-type calcium current in Hb9 interneurons. A: calcium currents elicited in response to voltage steps from −90 mV to −30 mV. Ni⁺⁺ (200 μM) reduced the calcium current. The reduction was reversed by a washout of Ni⁺⁺. Each step is the average of 4 identical voltage steps during the given application. Arrows represent peak initial inward current within the first 20 ms of the voltage step. B: average I-V plot of elicited currents. Points represent the amplitude of the peak initial inward current (mean ± SD) elicited by steps from −90 mV to potential on the x-axis. V_m, membrane potential. Black squares: control; black triangles: 200 μM Ni⁺⁺; gray squares: washout. Note the −30 and −20 mV steps with a reversible reduction of current. Points are offset on the x-axis to highlight error bars (SD).

Fig. 7.

Ca_V3 channels are expressed in Hb9 interneurons. Immunohistochemistry in Hb9::enhanced green fluorescent protein (eGFP);Hb9^nlz/+ mice demonstrates the distribution of Ca_V3.1 and Ca_V3.2 channels in the ventral spinal cord (A₁, B₁). At high magnification of the region of Hb9 interneurons (indicated by arrows), Ca_V channel expression (red; A₂, B₂) can be studied in Hb9 interneurons, which can be identified by expression of eGFP in the cytoplasm (green; A₃ and B₃) and lacZ (β-galactosidase) in the nucleus (blue; A₄ and B₄). Hb9 interneurons express Ca_V3.1 channels in the cytoplasm and possibly membrane (Overlay; A₅). On the other hand, Ca_V3.2 channels are seen in the nucleus of Hb9 interneurons (Overlay; B₅).