The effects of carbachol on rat neostriatal neurons were examined in the slice and the freshly dissociated neuron preparations using intracellular and whole-cell voltage-clamp recording methods. Superfusion of carbachol (30 μM) produced a depolarization concomitant with an increase in the rate of spontaneous action potentials. This depolarization was associated with an increase in the input resistance. The carbachol-induced membrane depolarization was blocked by pirenzepine (1 μM), a selective M1 muscarinic receptor antagonist. In other experiments, we observed that carbachol induced a transient inward current on the freshly dissociated neostriatal neuron at a holding potential of -60 mV in a concentration-dependent manner underlying the whole-cell voltage-clamp mode. The inward current caused by carbachol was not reduced by tetrodotoxin (1 μM), calcium-free recording solution or Cd2+ (100 μM). However, it was blocked by Ba2+ (100 μM). In addition, the carbachol-induced inward current reversed polarity at about the potassium equilibrium potential. The whole-cell membrane inward current in response to voltage-clamp step from -90 to -140 mV was reduced by 30 μM carbachol. With stronger hyperpolarization beyond the potassium equilibrium potential, carbachol produced a progressively greater reduction in membrane current. This inhibitory effect was also abolished by Ba2+ (100 μM). A concentration of 30 μM carbachol-induced inward current could be reversibly antagonized by the M1 muscarinic receptor antagonist pirenzepine (0.1-1 μM), with an estimated IC50 of 0.3 μM. However, other muscarinic receptor subtype (M2 or M3) antagonists could also block the carbachol-induced inward current. The rank order of antagonist potency was: pirenzepine (M1 antagonist)> 4-diphenylacetoxy-N,N-methyl-piperidine methiodide (M3/M1 antagonist) > gallamine (M2 antagonist). Based on these pharmacological data, we concluded that carbachol can act at M1-like muscarinic receptors to reduce the membrane K+ conductances and excite the neostriatal neurons.
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