Melanin concentrating hormone depresses synaptic activity of glutamate and GABA neurons from rat lateral hypothalamus - PubMed (original) (raw)
Melanin concentrating hormone depresses synaptic activity of glutamate and GABA neurons from rat lateral hypothalamus
X B Gao et al. J Physiol. 2001.
Abstract
The neuropeptide melanin concentrating hormone (MCH) is synthesised only by neurons of the lateral hypothalamic (LH) area in the CNS. MCH cells project widely throughout the brain. Despite the growing interest in this peptide, in part related to its role in feeding, little has been done to characterise its physiological effects in neurons. Using whole-cell recording with current and voltage clamp, we examined the cellular actions in neurons from the LH. MCH induced a consistent decrease in the frequency of action potentials and reduced synaptic activity. Most fast synaptic activity in the hypothalamus is mediated by GABA or glutamate. MCH inhibited the synaptic activity of both glutamatergic and GABAergic LH neurons, each tested independently. MCH reduced the amplitude of glutamate-evoked currents and reduced the amplitude of miniature excitatory currents, indicating an inhibitory modulation of postsynaptic glutamate receptors. In the presence of tetrodotoxin to block action potentials, MCH caused a depression in the frequency of miniature glutamate-mediated postsynaptic currents, suggesting a presynaptic site of receptor expression. In voltage clamp experiments, MCH depressed the amplitude of calcium currents, suggesting that a mechanism of inhibition may involve a reduced calcium-dependent release of amino acid transmitter. Previous reports have suggested that MCH activated potassium channels in non-neuronal cells transfected with the MCH receptor gene. We found no effect of MCH on voltage-dependent potassium channels in LH neurons. Baclofen, a GABAB receptor agonist, activated G-protein gated inwardly rectifying potassium (GIRK)-type channels; in the same neurons, MCH had no effect on GIRK channels. MCH showed no modulation of sodium currents. Blockade of the Gi/Go protein with pertussis toxin eliminated the actions of MCH. The inhibitory actions of MCH on both excitatory and inhibitory synaptic events, coupled with opposing excitatory actions of hypocretin, another LH peptide that projects to many of the same loci, suggest a substantial level of complexity in neuropeptide modulation of LH actions.
Figures
Figure 1. MCH inhibits action potentials in LH neurons
The trace shown here was recorded under current clamp. Resting membrane potential is -60.4 mV. MCH (1 μ
m
) induced a dramatic depression of spontaneous action potentials, which was reversible after MCH washout. The mean membrane potential was slightly hyperpolarised in the presence of MCH.
Figure 2. MCH does not modulate membrane potential and conductance directly
A, the trace presented here was recorded under current clamp in the presence of AP5 (100 μ
m
), CNQX (10 μ
m
) BIC (30 μ
m
) and TTX (1 μ
m
). Hyperpolarising current (20 pA, 100 ms duration, 1 Hz) was injected at regular intervals to test membrane conductance (inset). MCH exerted no effect on membrane potential or conductance. Resting membrane potential was -63.5 mV. B and C, bar graphs showing resting membrane potential and conductance before, during and after MCH application in all 15 neurons. MCH had no effect. D, the trace was from one of seven typical neurons, whose resting membrane potential and conductance was not changed by MCH. The experiments were also performed in the presence of of AP5 (100 μ
m
), CNQX (10 μ
m
), BIC (30 μ
m
) and TTX (1 μ
m
). Baclofen application (4 μ
m
) exerted a substantial decrease in membrane potential, and a modest increase in conductance, shown by the smaller amplitude hyperpolarisation upon current injection. Initial resting membrane potential was -63.4 mV. E and F, bar graphs show baclofen evokes a hyperpolarisation of the resting membrane potential and a slight increase in conductance during application (n = 7).
Figure 3. MCH and hypocretin: opposing effects on the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs)
A and B, raw traces recorded in the presence of glutamate receptor antagonists CNQX (10 μ
m
) and AP5 (50 μ
m
) under voltage clamp. A, MCH (1 μ
m
) reduced the frequency of spontaneous IPSCs. B, hypocretin (1 μ
m
) increased frequency of IPSCs. C, normalised data from five neurons were plotted to show the time course of MCH application. The frequency of sIPSCs was normalised to the mean of control sIPSC frequency. During application of MCH (1 μ
m
), a depression of GABAergic synaptic transmission developed. D, bar graph showing the relative effect of MCH and hypocretin on the frequency of sIPSCs.
Figure 4. MCH and hypocretin: opposing effects on the frequency of spontaneous excitatory postsynaptic currents (sEPSCs)
A and B, raw traces recorded in the presence of GABAA receptor antagonist bicuculline (30 μ
m
) under voltage clamp. A, MCH (1 μ
m
) reduced the frequency of spontaneous EPSCs. B, hypocretin (1 μ
m
) increased the frequency of sEPSCs. C, normalised data from two neurons were plotted to indicate the time course of MCH treatment. D, comparison of the effect of MCH and hypocretin on the frequency of sEPSCs demonstrates the opposing actions of the two LH peptides.
Figure 5. MCH and miniature IPSCs
A, raw traces recorded in the presence of TTX (1 μ
m
), CNQX (10 μ
m
) and AP5 (50 μ
m
) under voltage clamp (-60 mV). B, normalised frequency of mIPSCs from all tested neurons was plotted. ANOVA test suggested insignificant change. C, mean normalised medians of detected synaptic events from all tested neurons were plotted. _D, c_umulative distribution of detected events before, during and after MCH application was plotted. Although a small MCH-induced depression in mIPSC frequency was noted, a Kolmogorov-Smirnov test revealed no statistically significant change during the application of MCH.
Figure 6. MCH-mediated depression of frequency and amplitude of mEPSCs
A, raw traces presented here were recorded in the presence of TTX (1 μ
m
) and BIC (30 μ
m
) under voltage clamp (-60 mV). B, normalised frequency of mEPSCs from two neurons was plotted to show the time course of MCH actions. C, normalised frequency of mEPSCs from all eight neurons was plotted in this bar graph. D, a representative cumulative distribution from five of eight neurons showing that the cumulative probability of detected events was changed during the application of MCH. E, a representative cumulative distribution from three of eight neurons showing that the cumulative probability of detected events was not changed during the application of MCH, although the frequency was reduced by 15 %. F, mean normalised median of mEPSC amplitudes before, during and after MCH treatment was plotted.
Figure 7. MCH-mediated depression of glutamate-evoked response
Raw data recorded under voltage clamp (-60 mV) showing an MCH-mediated depression of glutamate-evoked current.
l
-Glutamate (100 μ
m
) was applied via a flow pipe and pressure (8 p.s.i., 20 ms duration) to the recorded neurons before, during and after the application of MCH.
Figure 8. MCH-mediated depression of voltage-dependent calcium channels
Left traces in A, whole-cell current of calcium channel in BaCl2 bath solution. Traces show the pre-MCH control (1), inhibition induced by MCH (2), a partial return to control levels after 4 min of MCH application (3) and recovery after MCH washout (4). Right panel in A, mean peak _I_Ba before (□), during () and after (▪) MCH application from all nine neurons is shown. B, whole-cell current of voltage-dependent sodium channels. Experiments were done in the presence of 40 m
m
TEA and 200 μ
m
CdCl2 in all extracellular solutions and CsCl in the pipette solution. Left traces represent sodium currents prior to (1), during (2) and after (3) MCH application. Right, mean peak _I_Na before (□), during () and after (▪) MCH application from all seven neurons is shown. C, whole-cell current recordings of voltage dependent potassium channels. Experiments were done in the presence of TTX (1 μ
m
) and CdCl2 (200 μ
m
) in all extracellular solutions. Left traces represent potassium currents prior to (1), during (2) and after (3) MCH application. Right, mean peak _I_K before (□), during () and after (▪) MCH application from all seven neurons is shown.
Figure 9. MCH actions are dependent on pertussis toxin-sensitive Gi/Go pathway
LH neurons were treated with 300 ng ml−1 pertussis toxin (PTX) for 48 h before MCH was tested. The inhibitory effects of MCH on sEPSCs and sIPSCs were abolished by pertussis toxin.
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