DOPAMINERGIC TRANSMISSION BETWEEN IDENTIFIED NEURONS FROM THE MOLLUSK, LYMNAEA-STAGNALIS

1. Dopaminergic transmission was investigated in the central nervous s ystem (CNS) of the freshwater snail, Lymnaea stagnalis. 2. The giant p edal neuron, designated as right pedal dorsal one (RPeD1), makes chemi cal, monosynaptic connections with a number of identifed follower cell s in the CNS. Previous work has shown that RPeD1 is an interneuron and a important component of the Lymnaea respiratory central pattern gene rator. In this study, the hypothesis that RPeD1 uses dopamine as its n eurotransmitter was tested by chromatographic, pharmacological, and el ectrophysiological methods. Characterization of RPeD1's transmitter ph armacology is essential to clearly understand its role in Lymnaea. 3. Earlier studies demonstrated that the soma of RPeD1 contains dopamine. This was quantitated in the present study by high-performance liquid chromatography (with electrochemical detection) of isolated RPeD1 soma ta and growth cones, which yielded 0.8 +/- 0.3 and 0.10 +/- 0.08 pmol of dopamine per soma and growth cone, respectively. 4. Bath or pressur e application of dopamine to follower cells of RPeD1, in situ, mimicke d the effects of RPeD1 stimulation. Dose-response curves were construc ted for the excitatory effect of dopamine on follower cells, visceral dorsal two and three (VD2/3) (ED(50) = 39 mu M; Hill coefficient = 1.0 3), and the inhibitory effect of dopamine on follower cell, visceral d orsal four (ED(50) = 33 mu M; Hill coefficient = 0.92). 5. The followi ng dopamine agonists (100 mu M) were tested by bath application: -amin o-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN), apopmorphine, 2- bromo-alpha-ergocryptine, deoxyepinephrine (DE), mesulergine, (-)quinp irole, SKF 38393, and tyramine. Only the general dopamine agonists, AD TN and DE, mimicked RPeD1's effects on its follower cells. 6. When VD2 /3 was isolated and plated in vitro, it maintained a depolarizing resp onse to dopamine. This response was reduced by intracellular injection of the G-protein blocker, GDP-beta-S (2 mM in electrode). Similarly, incubation of VD2/3, in vitro for similar to 18 h, with pertussis toxi n (PTX; 5 mu g/ml), the G-protein inactivating exotoxin, also reduced the dopamine response. Injecting GDP or incubating in heat-inactivated PTX did not effect the response. 7. Several dopamine antagonists were used in an attempt to block RPeD1's synapses: chlorpromazine, ergonov ine, fluphenazine, haloperidol, 6-hydroxydopamine, SCH 23390, (+/-) su lpiride, and tubocurarine. Only the D-2 dopamine receptor antagonist, (+/-) sulpiride, reversibly blocked synaptic transmission from RPeD1 t o its follower cells. Both the (+) and the (-) enantiomer of sulpiride also antagonized synaptic transmission. A dose-inhibition curve for ( +/-) sulpiride was constructed (IC50 = 47 mu M). (+/-) Sulpiride also blocked the effects of bath applied dopamine. 8. Collectively, these d ata suggest that RPeD1 uses dopamine as a neurotransmitter and that do pamine may elicit its effects via a G-protein coupled, D-2-like dopami ne receptor.