CHARACTERIZATION OF A MUSCARINIC CURRENT THAT REGULATES EXCITABILITY OF AN IDENTIFIED INSECT MOTONEURON

1. Application of the muscarinic agonist oxotremorine-M (oxo-M) to iso lated abdominal ganglia of larval Manduca sexta excited an identified proleg retractor motoneuron called PPR. This excitation consisted of a persistent depolarization and an increased tendency to generate actio n potentials. Previous work has established that the action of oxo-M i s probably mediated by muscarinic acetylcholine receptors (mAChRs) on PPR and that oxo-M mimics an afferent-induced long-lasting depolarizat ion called the slow excitatory postsynaptic potential (sEPSP). 2. Acti on potentials in the ganglion could be blocked by applying tetrodotoxi n (TTX) in the bath saline. Under these conditions all excitatory post synaptic potentials in PPR were also blocked, but the depolarizing act ion of oxo-M was unaffected. In the absence of background activity PPR could be voltage clamped using a single-electrode switching clamp to study the currents underlying the response to oxo-M. 3. At a membrane potential of -50 mV, application of oxo-M to the ganglion in the bath saline (3-6 x 10(-7) M) or by brief (20-40 ms) pulses from a micropipe tte into the neuropil(1 X 10(-5) h(-1)) evoked an apparently inward cu rrent called I-ox. The mean peak current change in response to pulses was -0.80 +/- 0.04 nA (n = 48 preparations). 4. The voltage dependence of I-ox was determined by subtracting the current-voltage relationshi p for PPR in control saline from that during a response to oxo-M. I-ox was maximal near the resting potential of PPR (-45 to -40 mV), decrea sing slightly with hyperpolarization and strongly with depolarization. 5. Peak I-ox was directly dependent on the bath Na+ concentration. Co mplete replacement of Na+ with N-methyl-D-glucamine in the saline bloc ked I-ox. Changes in the bath K+ concentration (extracellular K+ conce ntration, [K+](o)) had only a small effect on I-ox. Reducing [Cl-](o) from 140 to 74.5 mM had no significant effect on I-ox during a 15-min exposure. Intracellular injections of Cl- from a KCl-containing electr ode also had no measurable effect on I-ox. 6. Changes in the bath Ca2 concentration above or below 2 mM inhibited I-ox. Furthermore, the di valent cations Ni2+, Co2+ Mg2+, and Ba2+ at millimolar concentrations and the Ca2+ channel blocking agents nifedipine and Cd2+ at micromolar concentrations inhibited I-ox. 7. These results suggest that mAChRs o n PPR activate an inward current that is persistent, TTX insensitive, voltage dependent and carried predominantly by Na+. However, the resul ts cannot eliminate the possibility that changes in K+ or Cl- conducta nces might also be involved. The effects of Ca2+ and Ca2+ channel bloc king agents suggest that Ca2+ is also required for activating, maintai ning, or controlling the effects of muscarinic stimulation. This novel insect current may underlie the previously described sEPSP and may se rve to regulate the excitability of PPR in response to afferent stimul ation.