1. Electrical stimuli applied in the locus coeruleus/subcoeruleus (LC/SC) and raphe nuclei produce a profound depression of transmission in reflex pathways from group II muscle afferents. The present experiments were performed to determine whether presynaptic inhibitory mechanisms contribute to these effects. 2. Changes in the excitability of afferent terminals to electrical stimuli have been used as an indication of primary afferent depolarization (PAD) produced by conditioning stimuli applied within the LC/SC and raphe nuclei and, for comparison, in the nucleus ruber. Group II afferents originating from ankle flexor muscles and terminating in the midlumbar segments were used for testing. 3. Clear changes in excitability were observed in fourteen of nineteen group II fibres in which the effects of conditioning stimuli applied in the LC/SC were tested and in twelve of seventeen fibres in which the effects of stimuli applied within the raphe nuclei were tested. By comparison, only one of the twelve fibres tested with conditioning stimuli applied to the nucleus ruber was found to be influenced. These effects matched those of the same conditioning stimuli on field potentials evoked by group II afferents at the location at which the terminals of group II fibres were stimulated. 4. Stimuli applied in the LC/SC and in the raphe nuclei both produced a mean decrease in threshold stimulus current of 19 %. These effects are comparable to those produced by the most effective volleys in peripheral afferents which, in the same fibres, produced a mean decrease in threshold stimulus current of 24 %. 5. In all cases (twelve) in which the effects of stimuli applied in the LC/SC and raphe nuclei were tested on the same group II fibre, either both or neither were found to be effective. This strengthens previous indications that some populations of neurones might be activated by stimuli applied in each of these regions of the brain. 6. In contrast to group II afferents, group Ia afferents investigated in the same experiments were only exceptionally affected. Of seven fibres tested with stimuli applied in the LC/SC, six with stimuli applied in the raphe nuclei and seven with stimuli applied in the nucleus ruber, only one fibre showed any clear change in threshold and this was a single fibre which was similarly affected by stimuli in all three sites. 7. It is concluded that presynaptic inhibitory mechanisms contribute to the depression of transmission in spinal reflex pathways from group II muscle afferents produced by stimulation in the LC/SC and raphe nuclei. 38-735590 1. The whole-cell patch clamp and intracellular perfusion techniques were used for studying the effects of atropine and other muscarinic acetylcholine receptor (mAChR) antagonists on the L-type calcium currents (I(Ca)) in frog and rat ventricular myocytes, and on the mAChR-activated K+ current (I(K(ACh))) in frog atrial myocytes. 2. In frog ventricular myocytes, atropine (0.1 nm to 1 mum) reversed the inhibitory effect of acetylcholine (ACh, 1 nm) on I(Ca) previously stimulated by isoprenaline (Iso, 2 mum), a beta-adrenergic agonist. However, in the concomitant presence of Iso, ACh and atropine, I(Ca) was > 50 % larger than in Iso alone. 3 The effects of atropine were then examined in the absence of mAChR agonists. After a preliminary stimulation of I(Ca) with Iso (0. 1 or 2 mum), atropine induced a dose-dependent stimulation of I(Ca). EC50 (i.e. the concentration of atropine at which the response was 50 % of the maximum) and E(max) (i.e. maximal stimulation of I(Ca) expressed as percentage increase in I(Ca) with respect to the level in Iso alone) were respectively 0.6 nm and 35 %. The stimulatory effect of atropine on I(Ca) was not voltage dependent. 4. Atropine (1 muM) had no effect on frog I(Ca) (i) under basal conditions, (ii) upon stimulation of I(Ca) by the dihydropyridine agonist (-)-Bay K 8644 (1 mum), or (iii) when I(Ca) had been previously stimulated by intracellular perfusion with cyclic AMP (3 mum). However, atropine increased I(Ca) after a stimulation by forskolin (0.3 mum). Therefore, an increased adenylyl cyclase activity was required for atropine to produce its stimulatory effect on I(Ca). 5. The order of potency of mAChR antagonists to reverse the inhibitory effect of ACh on Iso elevated I(Ca) in frog ventricle was atropine > AF-DX 116 much greater than pirenzepine. In the absence of ACh, mAChR antagonists produced their stimulatory effect on Iso elevated I(Ca) with the same order of potency. 6. Intracellular substitution of Gpp(NH)p (5'-guanylylimidiphosphate) for GTP (420 muM) induced a strong inhibition of frog I(Ca) in the presence of Iso (2 mum). This effect was attributed earlier to the spontaneous and irreversible activation of the GTP-binding regulatory protein (G protein), G(i), responsible for adenylyl cyclase inhibition. Atropine (1 muM) slowed down by a factor of 2 the rate of I(Ca) inhibition induced by Gpp(NH)p. 7. In frog atrial myocytes, intracellular perfusion with 1 mm Gpp(NH)p induces spontaneous activation of I(K(ACh)). This effect was attributed earlier to the spontaneous and irreversible activation of the G protein, G(K). Atropine (1 muM) or propylbenzyl choline mustard (1 mum) slowed down by a factor of 2-3 the rate of spontaneous activation of I(K(ACh)). 8. In rat ventricular cells, atropine (I mum) exerted also a stimulatory effect on I(Ca). However, unlike in frog myocytes, atropine enhanced both basal and Iso-stimulated I(Ca) in rat. 9. lt is concluded that, in the absence of mAChR agonist, atropine and other 'M2-selective' mAChR antagonists exert a stimulatory effect on I(Ca) and an inhibitory effect on I(K(ACh)) after binding to the mAChR. Both effects are due to a reduction in the spontaneous activation of the G proteins, G(i) and G(K), respectively. Therefore, in addition to displacing the agonist from its binding site, mAChR antagonists may induce a conformational change of the receptor which impairs spontaneous G protein activation by the receptor.
