Melanin-concentrating hormone depresses L-, N-, and P/Q-type voltage-dependent calcium channels in rat lateral hypothalamic neurons

Abstract

Melanin-concentrating hormone (MCH), a cyclic 19-amino-acid peptide, is synthesized exclusively by neurons in the lateral hypothalamic (LH) area. It is involved in a number of brain functions and recently has raised interest because of its role in energy homeostasis. MCH axons and receptors are found throughout the brain. Previous reports set the foundation for understanding the cellular actions of MCH by using non-neuronal cells transfected with the MCH receptor gene; these cells exhibited an increase in cytoplasmic calcium in response to MCH, suggesting an excitatory action for the peptide. In the study presented here, we have used whole-cell recording in 117 neurons from LH cultures and brain slices to examine the actions of MCH. MCH decreased the amplitude of voltage-dependent calcium currents in almost all tested neurons. The inhibition desensitized rapidly (18 s to half maximum at 100 nM concentration) and was dose-dependent (IC(50) = 7.8 nM) when activated with a pulse from -80 mV to 0 mV. A priori activation of G-proteins with GTPgammaS completely eliminated the MCH-induced effect at low MCH concentrations and reduced the MCH-induced effect at high MCH concentrations. Inhibition of G-proteins with pertussis toxin (PTX) blocked the MCH-induced inhibitory effect at high MCH concentrations. Pre-pulse depolarization resulted in an attenuation of the MCH-induced inhibition of calcium currents in most neurons. These data suggest that MCH exerts an inhibitory effect on calcium currents via PTX-sensitive G-protein pathways, probably the G(i)/G(o) pathway, in LH neurons. L-, N- and P/Q-type calcium channels were identified in LH neurons, with L- and N-type channels accounting for most of the voltage-activated current (about 40 % each); MCH attenuated each of the three types (mean 50 % depression), with the greatest inhibition found for N-type currents. In contrast to previous data on non-neuronal cells showing an MHC-evoked increase in calcium, our data suggest that the reverse occurs in LH neurons. The attenuation of calcium currents is consistent with an inhibitory action for the peptide in neurons.

Figure 1. Melanin-concentrating hormone (MCH) depresses voltage-dependent calcium currents in cultured neurons and brain slices from the lateral hypothalamus (LH)

A, time course of MCH-induced inhibition of calcium currents in a cultured LH neurone, typical of the 76 LH neurons that responded to MCH. a, b and c, raw recordings at corresponding time points during the experiment. The concentration of MCH was 100 nm in this example. B, time course of MCH-induced inhibition of calcium current in an LH slice (n = 4). MHC (1 μM) was bath-applied to the recorded slice.

Figure 2. Desensitization of MCH-induced inhibition of calcium currents

Time course of desensitization of the calcium current inhibition induced by 1 μM MCH (n = 6).

Figure 3. Dose-dependence of the MCH effect

A, raw data recorded from different neurons showing the effect of MCH at five different concentrations. B, dose-response relationship obtained from 23 neurons.

Figure 4. Voltage dependence of the MCH-induced inhibition of calcium currents

A, a, b and c represent raw data recorded before, during and after the application of MCH (1 μM), respectively. The test protocol was not designed to specifically test a T-type current. B, I-V relationship of calcium currents before (•), during (▪) and after (♦) the application of MCH (1 μM). The amplitude of current was measured at the peak of the calcium current. C, pooled data from eight neurons showing inhibition of calcium currents by MCH when recorded neurons were depolarized to each membrane potential. D, left, pre-pulse depression experiments before and during the application of MCH (1 μM); right, The attenuation of (relief from) MCH-mediated inhibition of calcium current for each of the 10 neurons tested is shown here by the 10 lines. The thick bar on the right is the mean pre-pulse attenuation for all 10 neurons.

Figure 5. Pre-activation of G-proteins reduces or eliminates MCH-induced inhibition

All experiments began 10 min after the formation of the whole-cell recording configuration to allow the complete diffusion of GTPγS (0.5 mm) from the pipettes. A, time course of a typical experiment with a low concentration (10 nm) of MCH after activation of G-proteins by GTPγS (n = 5). a, b and c are raw recordings before, during and after the application of 10 nm MCH, respectively. B, time course of a typical experiment with a high concentration of MCH (100 nm) after activation of G-proteins (n = 7). a, b and c represent the recordings before, during and after the application of MCH (100 nm), respectively. C, bar graph comparing MCH actions on calcium currents with and without GTPγS (n = 8 for 10 nm and n = 8 for 100 nm) in the recording pipette.

Figure 6. Pre-treatment with PTX blocks the MCH-evoked reduction in calcium currents

Bar graph showing the effect of MCH (1 μM) on calcium currents in non-PTX-treated (n = 14) and PTX-treated cultures (n = 5).

Figure 7. Relative contribution of L-, N- and P/Q-type channels to somatic calcium currents in LH neurons

A: a, b and c show raw traces. Trace 1 shows control calcium currents. Trace 2 shows calcium currents in the presence of a blocker (8 μM nimodipine in a, 4 μM ω-conotoxin GVIA in b, and 2 μM ω-conotoxin MVIIC + 50 nm ω-agatoxin IVA in c). B, bar graph showing the percentage of each of three subtypes of calcium channels in the whole-cell calcium currents (n = 8 for L-type, n = 7 for N-type and n = 6 for P/Q-type currents).

Figure 8. MCH depresses N-type calcium currents (an example)

A-D, calcium currents recorded in our experiments in control bath, MCH (100 nm), ω-conotoxin GVIA (4 μM) solution and ω-conotoxin GVIA (4 μM) plus MCH (100 nm) solution. E, N-type calcium current. This trace was obtained by subtracting trace C from trace A. F, N-type calcium current in the presence of MCH. This trace was obtained by subtracting trace D from trace B. Inset, time course of a typical experiment. Rundown of calcium currents was consistently < 10 %. After the whole set of experiments, 200 μM CdCl₂ was applied to the recorded neurons to block the recorded current to ensure the recording of calcium current as described in Methods. CdCl₂ completely blocked calcium currents.

Figure 9. Inhibition of L-, N- and P/Q-type calcium currents by MCH

A: a, b and c represent L-, N- and P/Q-type calcium currents, respectively, before (represented by dotted lines) and during (represented by continuous lines) application of 100 nm MCH. These traces were obtained using the procedure described in Fig. 8. B, bar graph comparing the percentage depression of L-type (n = 8), N-type (n = 6) and P/Q-type (n = 6) calcium currents to their control levels during the application of MCH.