pituitrin and iberiotoxin

Release of arginine vasopressin (AVP) from magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) is controlled by the electrical activities of the MNCs. Ca(2+)-activated K(+) channels, such as the BK and SK channels, are K(+)-selective ion channels that are activated in response to increased intracellular calcium concentrations. Intrinsic affinities for Ca(2+) permit these channels to exert a negative feedback effect on cellular excitability. In the present study, we used the whole-cell patch-clamp technique to examine the effects of BK or SK channel modulators on neuronal activity in single isolated rat SON MNCs that express an AVP-enhanced green fluorescent protein (eGFP) transgene. Application of BK or SK channel activators abolished the action potentials and induced hyperpolarization. In contrast, the number of action potentials was significantly increased after application of BK or SK channel blockers. Our results suggest that BK and SK channels in AVP neurons may play a role in the regulatory mechanisms of neural activity.

Topics: Action Potentials; Animals; Apamin; Charybdotoxin; Large-Conductance Calcium-Activated Potassium Channels; Male; Membrane Potentials; Neurons; Patch-Clamp Techniques; Peptides; Rats; Rats, Transgenic; Small-Conductance Calcium-Activated Potassium Channels; Supraoptic Nucleus; Vasopressins

The contribution of Ca(2+)-activated K(+) channels to hyperpolarizing after-potentials (HAP) of action potentials, to spike-frequency adaptation and thus to the shaping of discharge pattern, was examined in rat supraoptic magnocellular neurosecretory cells. In addition, the expression of BK channels and SK3 subunits of SK channels was studied using double immunofluorescence detection. The presence of BK channels and SK3 subunits was detected in many supraoptic neurones containing either vasopressin or oxytocin. Current-clamp recordings of current-induced spike trains revealed that HAPs comprise a fast and a slow HAP (fHAP and sHAP). Correlation analyses revealed that the increase of the fHAP in amplitude and spike broadening were correlated to a moderate gradual increase of the interspike interval and thus to weak spike-frequency adaptation. By contrast, marked prolongation of the interspike interval and strong spike-frequency adaptation depended on the appearance and on the amplitude of the sHAP. The sHAP and spike-frequency adaptation were blocked by cadmium, as well as by the SK channel antagonist apamin. The fHAP was attenuated by the BK channel antagonist iberiotoxin (IbTX), by the BK/IK channel antagonist charybdotoxin (ChTX) and by apamin. ChTX attenuated fHAPs throughout the entire spike train. By contrast, the IbTX-induced attenuation of the fHAP was restricted to the initial part of the spike train, while the apamin-induced attenuation slowly increased with the progression of the spike train. These results suggest that strong spike-frequency adaptation in supraoptic neurones essentially depends on the generation of the sHAP by activation of SK channels. Comparison of effects of IbTX, ChTX and apamin suggests a complementary contribution of SK-, BK- and IK-channels to fHAPs.

Topics: Action Potentials; Adaptation, Physiological; Animals; Apamin; Charybdotoxin; Fluorescent Antibody Technique; Male; Neurons; Neurosecretory Systems; Neurotoxins; Oxytocin; Peptides; Potassium Channels, Calcium-Activated; Protein Subunits; Rats; Rats, Sprague-Dawley; Supraoptic Nucleus; Vasopressins

A slow posttrain afterhyperpolarization (sAHP) was studied in rat magnocellular neurosecretory cells (MNCs) in vitro. The sAHP was isolated from other afterpotentials by blocking the depolarizing afterpotential (DAP) with Cs(+) and the medium afterhyperpolarization (mAHP) with apamin. The sAHP amplitude increased logarithmically with activity ( approximately 3 mV per e-fold increase in number of impulses) and, when firing stopped, decayed exponentially with a time constant of 2 sec. The sAHP was associated with increased membrane conductance, and its amplitude varied linearly with voltage, reversing at the K(+) equilibrium potential. The sAHP was blocked by Cd(2+) but not by charybdotoxin or iberiotoxin, blockers of intermediate- and big-conductance-type Ca(2+)-dependent K(+) (K(Ca)) channels. The sAHP was reversibly inhibited by muscarine, an effect antagonized by atropine, indicating involvement of muscarinic cholinergic receptors. Muscarine did not affect Ca(2+)-dependent features of action potentials, DAPs, or the mAHP in MNCs, indicating selective modulation of K(Ca) channels causing the sAHP. Muscarinic inhibition of the sAHP enhanced plateau potentials and increased the mean firing rate and duration of afterdischarges that followed spike trains evoked from voltages near threshold. Similarly, the frequency and duration of the spontaneous phasic bursts that characterize physiologically activated vasopressin-releasing MNCs were enhanced by muscarine. MNCs thus express apamin- and voltage-insensitive K(Ca) channels that mediate an sAHP. The activity dependence and kinetics of the sAHP cause it to mask DAPs in a manner that attenuates the amplitude of plateau potentials. Muscarinic inhibition of the sAHP provides an effective mechanism for promoting phasic firing in MNCs.

Topics: Action Potentials; Animals; Apamin; Atropine; Cadmium; Calcium; Cells, Cultured; Charybdotoxin; Ion Channels; Ion Transport; Male; Membrane Potentials; Muscarine; Neurons; Organ Culture Techniques; Peptides; Potassium; Rats; Rats, Long-Evans; Receptors, Muscarinic; Supraoptic Nucleus; Tetrodotoxin; Vasopressins