Calcium currents of rhythmic neurons recorded in the isolated respiratory network of neonatal mice

Abstract

To obtain a quantitative characterization of voltage-activated calcium currents in respiratory neurons, we performed voltage-clamp recordings in the transverse brainstem slice of mice from neurons located within the ventral respiratory group. It is assumed that this medullary region contains the neuronal network responsible for generating the respiratory rhythm. This study represents one of the first attempts to analyze quantitatively the currents in respiratory neurons. The inward calcium currents of VRG neurons consisted of two components: a high voltage-activated (HVA) and a low voltage-activated (LVA) calcium current. The activation threshold of the HVA current was at -40 mV. It was fully activated (peak voltage) between -10 and 0 mV. The half-maximal activation (V50) was at -27. 29 mV +/- 1.15 (n = 24). The HVA current was inactivated completely at a holding potential of -35 mV and fully deinactivated at a holding potential of -65 mV (V50, -52.26 mV +/- 0.27; n = 18). The threshold for the activation of the LVA current was at -65 mV. This current had its peak voltage between -50 and -40 mV (mean, V50 = -59. 15 mV +/- 0.21; n = 15). The LVA current was inactivated completely at a holding potential of -65 mV and deinactivated fully at a holding potential of -95 mV (mean, V50 = -82.40 mV +/- 0.32; n = 38). These properties are consistent with other studies suggesting that the LVA current is a T-type current. The properties of these inward currents are discussed with respect to their role in generating Ca2+ potentials that may contribute to the generation of the mammalian respiratory rhythm.

Fig. 1.

Preparation and definition of rhythmic versus nonrhythmic neurons of the ventral respiratory group (VRG).A, Schematic representation of the transverse slice containing the pre-Bötzinger complex (pBC;gray area), a region in the VRG that is presumably the site for respiratory rhythm generation. The right panelshows a video image obtained from the VRG. The image contains neurons that were visualized with infrared Nomarski optics. The electrode tip is visible and points to the voltage-clamp recording shown inC. B, Extracellular recording obtained from the rhythmically active hypoglossal rootlet (XII,bottom trace) and its integrated signal (top trace). C, Burst activity in the integrated XII recording (top trace) corresponds to rhythmic inward currents in the whole-cell patch recording (bottom trace). A neuron with this property is defined as rhythmic (R). D, Burst activity in the integrated XII recording (top trace) is not correlated with synaptic activity in the intracellularly recorded VRG neuron (bottom trace). A neuron with this characteristic is defined as nonrhythmic (nR). IO, Inferior olive; NA, nucleus ambiguus; NTS, nucleus tractus solitarius; Sp5, spinal trigeminal nucleus;XII, hypoglossus motor nucleus; XII nerve, XII rootlet.

Fig. 2.

A, Voltage-clamp current response traces from a holding potential of −60 mV to different test potentials (from −80 to 40 mV; see inset) obtained from a VRG neuron under control conditions [intracellular CsCl (110 mm) + TEA (30 mm); top panel] and after bath application of 30 mm tetraethylammonium (TEA) chloride (bottom panel). Possible remaining unblocked K⁺ currents were most obvious at the steady-state level. Therefore, current amplitudes were measured at the steady-state values (75 msec after onset of test pulse; seediamonds at top and bottom panels). B, Average current–voltage response curve from 25 VRG neurons. Filled diamonds represent current response under control conditions, and open diamonds correspond to current response after TEA application.

Fig. 3.

Voltage-clamp recordings from a VRG neuron after bath application of 0.5 μm TTX [intracellular solution contains CsCl (110 mm) and TEA (30 mm) to block potassium currents]. A, Current response traces from a holding potential (V_h) of −60 mV to different test potentials (see inset). B, Current–voltage response curve obtained from the current traces inA. Note that, in contrast to Figure 2, the current amplitude was measured at the peak response, as indicated inA (filled triangles).

Fig. 4.

Normalized current–voltage curve (for calculations, see Results) from current response traces obtained by depolarizing steps from a holding potential (V_h) of −60 mV to different test potentials (see inset). The filled triangles represent the percentage peak current amplitude under control conditions (only voltage-activated sodium and potassium currents are blocked), and the open triangles indicate the percentage of peak current amplitude after bath application of 200 μm cadmium chloride.

Fig. 5.

Calcium current response traces of a VRG neuron from different holding potentials. A, Current response traces obtained by steps from a holding potential of −90 mV to test potentials between −80 and 40 mV. B, Current response traces from a holding potential of −60 mV to the same test potentials as indicated for A. C, Current–voltage relation curve of the peak current amplitude from A(filled squares) and B(filled triangles). Filled triangles represent HVA calcium current, and filled squares indicate HVA plus LVA calcium currents.

Fig. 6.

A, Example for LVA calcium current response traces (for protocol description, see Results).B, Comparison of the LVA peak current amplitude (current–voltage relationship curves) between rhythmic (R, filled circles; n = 12) and nonrhythmic (nR, open circles; n = 9) VRG neurons. Inset, Example (I–V curves) for a nickel-induced (200 μm) reduction of the LVA current amplitude (see Results).

Fig. 7.

Comparison of voltage dependencies (activation and inactivation curves) of LVA calcium currents between rhythmic (R, filled circles) and nonrhythmic (nR, open circles) VRG neurons. All data points are normalized (see Results). A sigmoidal Boltzmann curve is fit through each data set (see Results). A, Voltage-dependent activation (data points on the right curve; n= 9) and inactivation (data points on theleft curve; n = 11) properties for the LVA calcium current in rhythmic neurons. B, Voltage-dependent activation (data points on theright curve; n = 6) and inactivation (data points on the left curve;n = 27) properties for the LVA calcium current in nonrhythmic neurons.

Fig. 8.

A, Comparison of the HVA calcium current amplitude (current–voltage relationship curves) between rhythmic (R, filled circles; n = 10) and nonrhythmic (nR, open circles; n= 21) VRG neurons. B, Two examples for a qualitative characterization of different subtypes of the HVA calcium current in VRG neurons (left figure, rhythmic;right figure, nonrhythmic). Current amplitude (for detailed protocol description, see Results) is plotted against time. Specific calcium channel blockers ω-conotoxin MVIIA (to block N-type calcium channels), ω-conotoxin MVIIC (to block Q-type calcium channels), ω-agatoxin IVA (to block P-type calcium channels), and nifedipine (to block L-type calcium channels) have been applied as indicated by the dotted lines. MVIIA, ω-Conotoxin MVIIA; MVIIC, ω-conotoxin MVIIC;AgaIVA, ω-agatoxin IVA; Nif, nifedipine; CdCl, cadmium chloride.

Fig. 9.

Comparison of voltage dependencies (activation and inactivation curves) of HVA calcium currents between rhythmic (R, filled circles) and nonrhythmic (nR, open circles) VRG neurons. All data points are normalized (see Results). A sigmoidal Boltzmann curve is fit through each data set (see Results). A, Voltage-dependent activation (data points on the right curve; n= 9) and inactivation (data points on theleft curve; n = 5) properties for the HVA calcium current in rhythmic neurons. B, Voltage-dependent activation (data points on theright curve; n = 15) and inactivation (data points on the leftcurve; n = 13) properties for the HVA calcium current in nonrhythmic neurons.

Fig. 10.

HVA voltage-dependent activation curves under control conditions (Ca²⁺ as charge carrier;filled diamonds) and after bath application of 5 mm barium chloride (open diamonds). The fit is a sigmoidal Boltzmann curve (control, solid line; barium, dotted line).