A confocal Ca2+ imaging technique has been used to detect ATP release from individual sympathetic varicosities on the same nerve terminal branch. Varicose nerve terminals and smooth muscle cells in mouse vas deferens were loaded with the Ca2+ indicator Oregon Green 488 BAPTA‐1. Field (nerve) stimulation evoked discrete, focal increases in [Ca2+] in smooth muscle cells adjacent to identified varicosities. These focal increases in [Ca2+] have been termed ‘neuroeffector Ca2+ transients’ (NCTs). NCTs were abolished by α,β‐methylene ATP (1 μM), but not by nifedipine (1 μM) or prazosin (100 nm), suggesting that NCTs are generated by Ca2+ influx through P2X receptors without a detectable contribution from L‐type Ca2+ channels or α1‐adrenoceptor‐mediated pathways. Action potential‐evoked ATP release was highly intermittent (mean probability 0.019 ± 0.002; range 0.001‐0.10) at 1 Hz stimulation, even though there was no failure of action potential propagation in the nerve terminals. Twenty‐eight per cent of varicosities failed to release transmitter following more than 500 stimuli. Spontaneous ATP release was very infrequent (0.0014 Hz). No Ca2+ transient attributable to noradrenaline release was detected even in response to 5 Hz stimulation. There was evidence of local noradrenaline release as the α2‐adrenoceptor antagonist yohimbine increased the probability of occurrence of NCTs by 55 ± 21 % during trains of stimuli at 1 Hz. Frequency‐dependent facilitation preferentially occurred at low probability release sites. The monitoring of NCTs now allows transmitter release to be detected simultaneously from each functional varicosity on an identified nerve terminal branch on an impulse‐to‐impulse basis.

Release of endogenous dopamine elicited in slices of rat neostriatum or nucleus accumbens by a single electric pulse or by trains of 4 or 10 pulses was examined using fast cyclic voltammetry. Single electric pulses gave rise to a marked and transient increase in the extracellular concentration of dopamine in the neostriatum (by 0.43 mumol/l) and nucleus accumbens (by 0.39 mumol/l). The overflow elicited by subsequent pulses delivered at a frequency of 0.2 Hz caused separate but much smaller peaks of dopamine concentration, whereas the overflow elicited by subsequent pulses delivered at 1 Hz caused only a shoulder in the descending limb of the peak due to pulse 1. Four pulses at 5 Hz produced a monophasic response that was higher than the single pulse-evoked peak. Nomifensine 1 mumol/l greatly increased and prolonged the evoked overflow of dopamine. In the absence of nomifensine, metoclopramide 0.3 mumol/l did not change the response to a single pulse or 4 pulses delivered at 0.2 Hz but increased the response to 4 or 10 pulses at 1 Hz and to 4 pulses at 5 Hz. In the presence of nomifensine, metoclopramide increased the response to a single pulse as well as, to a greater extent, the response to 4 pulses at 0.2 Hz and 4 pulses at 1 Hz. Sulpiride 1 mumol/l produced effects similar to those of metoclopramide in the neostriatum in the presence of nomifensine.(ABSTRACT TRUNCATED AT 250 WORDS)

1 Dopamine efflux following single pulse or train of pulse stimulations was measured in slices of rat caudate putamen, nucleus accumbens and tuberculum olfactorium, using fast cyclic voltammetry at a carbon fibre microelectrode; 1, 5, 10, 20 The tuberculum olfactorium releases the least dopamine of the three regions following a single pulse stimulation (approximately 40 nM dopamine), but the ratio of peak dopamine release following trains of 20 pulses (50 Hz) when compared to single pulse can result in a value approaching 20. 6 When 20 pulses are applied to the caudate putamen at frequencies ranging from 10 to 500 Hz, the peak efflux is essentially the same (frequency/release profile is flat), with the maximum increase only 140% that of a single pulse. 7 By contrast, in the nucleus accumbens and the tuberculum olfactorium, well defined frequencyrelease relationships are seen following 20 pulses applied at 10, 20 and 50 Hz; at higher frequencies the dopamine release decreases as frequency is increased. 8 Experiments with sulpiride or metoclopramide (dopamine D2 receptor antagonists) indicate that dopamine release in the caudate putamen is not regulated by dopamine autoreceptor activation by endogenous dopamine under our experimental conditions (in the absence of reuptake inhibtion). By contrast, in the nucleus accumbens and in the tuberculum olfactorium, we present evidence to show that at frequencies of stimulation up to 20 Hz, endogenous dopamine acts at dopamine autoreceptors to inhibit further release of dopamine but a minimum exposure time of between 500 and 1000 ms is needed for the response to D2 autoreceptor activation by endogenously released dopamine to be seen.

Fast cyclic voltammetry using carbon fibre microelectrodes in rat brain slices, was used to investigate regional differences in electrically-evoked dopamine (DA) efflux at 10 different sites in the anterior caudate putamen (aCPu) and 10 sites in the posterior caudate putamen (pCPu). For each site DA overflow was evoked by both single pulse (1P) stimulation and by trains of 25 pulses applied at a frequency of 50 Hz (25P/50 Hz). Peak DA efflux evoked by 1P was about 58% greater in the aCPu (0.19 mumol/l DA) than in the pCPu (0.12 mumol/l DA), but showed no mediolateral variation in either region. Peak DA efflux evoked by 25P/50 Hz relative to 1P efflux also varied between the two regions; the aCPu contained predominantly low ratio (25P/50 Hz: 1P) sites ranging from 1.47 to 3.71, whereas in the pCPu these ratios were higher, ranging from 2.73 to 9.40, and were particularly high in the dorsomedial region of the pCPu. Efflux detected in low ratio sites of the aCPu showed little dependence on the frequency (10 to 500 Hz), or the number of pulses (5 to 20) in a train. By contrast DA efflux evoked in high ratio sites of the pCPu responded in a pulse and frequency dependent manner, the maximum ratio (approximately 8 times 1P) being at 20P/20 Hz. Interestingly the frequency response relationship obtained in the pCPu resembled the profile observed in the nucleus accumbens (NAc). Voltammetric evidence and experiments with selective reuptake blockers indicated that only DA was measured in our studies and 5-HT did not significantly contribute to the frequency dependent pattern of efflux detected in high ratio sites of the pCPu, where striatal 5-HT concentrations are highest. Experiments with the selective D2 receptor antagonists metoclopramide or (-)sulpiride revealed that under our experimental conditions, DA efflux in the aCPu was not modulated by DA autoreceptor activation. By contrast, autoreceptor modulation did occur in high ratio sites of the pCPu at stimulations lasting longer than approximately 1000 ms. These observations support the concept that the caudate putamen is heterogeneously organised with respect to the frequency characteristics of evoked DA release. The factors controlling frequency dependent release under these conditions may be a function of A10 innervation, since high ratio release sites occur in areas where the density of such innervation is greatest, for example, the dorsomedial pCPu. This is supported by the observation that high ratio release sites are also found in the NAc, which receives dopaminergic fibres predominantly from an A10 region.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Action potential‐evoked Ca2+ transients in postganglionic sympathetic axon bundles in mouse vas deferens have been characterized using confocal microscopy and Ca2+ imaging. Axonal Ca2+ transients were tetrodotoxin sensitive. The amplitude depended on both the frequency of stimulation and the number of stimuli in a train. Removal of extracellular Ca2+ abolished the Ca2+ transient. Cd2+ (100 μm) inhibited the Ca2+ transient by 78 ± 10 %. The N‐type Ca2+ channel blocker ω‐conotoxin GVIA (0.1 μm) reduced the amplitude by −35 ± 4 %, whereas nifedipine (10 μm; L‐type) and ω‐conotoxin MVIIC (0.1 μm; P/Q type) were ineffective. Caffeine (10 mm), ryanodine (10 μm), cyclopiazonic acid (30 μm) or CCCP (10 μm) had no detectable effects. Blockade of large and small conductance Ca2+‐dependent K+ channels with iberiotoxin (0.1 μm) and apamin (1 μm), respectively, or Ca2+‐dependent Cl− channels by niflumic acid (100 μm) did not alter Ca2+ transients. In contrast, the non‐specific K+ channel blockers tetraethylammonium (10 mm) and 4‐aminopyridine (10 mm) markedly increased the amplitude of the Ca2+ transient. Blockade of delayed rectifiers and A‐like K+ channels, by tityustoxin‐K (α) (0.1 μm) and pandinustoxin‐K (α) (10 nm), respectively, also increased the Ca2+ transient amplitude. Thus, Ca2+ transients are evoked by Na+‐dependent action potentials in axons. These transients originate mainly from Ca2+ entry through voltage‐dependent Ca2+ channels (80 % Cd2+ sensitive of which 40 % was attributable to N‐type). Twenty per cent of the Ca2+ transient was not due to Ca2+ entry through voltage‐gated Ca2+ channels. Intracellular stores and mitochondria were not involved in the generation of the transient. Ca2+ transients are modulated by A‐like K+ channels and delayed rectifiers (possibly KV1.2) but not by Ca2+‐activated ion channels.