Currents generated by depolarizing voltage pulses were recorded in neurons from the pyramidal cell layer of the CA1 region of rat or guinea pig hippocampus with single electrode voltage-clamp or tight-seal whole-cell voltageclamp techniques. In neurons in situ in slices, and in dissociated neurons, subtraction of currents generated by identical depolarizing voltage pulses before and after exposure to tetrodotoxin revealed a small, persistent current after the transient current. These currents could also be recorded directly in dissociated neurons in which other ionic currents were effectively suppressed. It was concluded that the persistent current was carded by sodium ions because it was blocked by TIX, decreased in amplitude when extraceUular sodium concentration was reduced, and was not blocked by cadmium. The amplitude of the persistent sodium current varied with clamp potential, being detectable at potentials as negative as -70 mV and reaching a maximum at ~-40 mV. The maximum amplitude at -40 mV in 21 cells in slices was -0.34 • 0.05 nA (mean • 1 SEM) and -0.21 • 0.05 nA in 10 dissociated neurons. Persistent sodium conductance increased sigmoidally with a potential between -70 and -30 mV and could be fitted with the Boltzmann equation, g = g~,/{1 + exp[(V' -V)/k)]}. The average g~, was 7.8 • 1.1 nS in the 21 neurons in slices and 4.4 • t.6 nS in the 10 dissociated cells that had lost their processes indicating that the channels responsible are probably most densely aggregated on or close to the soma. The half-maximum conductance occurred close to -50 mV, both in neurons in slices and in dissociated neurons, and the slope factor (k) was 5-9 mV. The persistent sodium current was much more resistant to inactivation by depolarization than the transient current and could be recorded at >50% of its normal amplitude when the transient current was completely inactivated.

1. Intracellular stimulation of single neurons in the Lymnaea CNS was carried out to identify heart motoneurons. 2. Two of the identified motoneurons, the E heart excitor (Ehe) cells, were shown to contain Phe-Met-Arg-Phe-NH2 (FMRFamide)-like peptides by immunocytochemical staining of dye-marked neurons and by radioimmunoassay (RIA) applied to extracts of single dissected cells. 3. Bursts of spikes in the Ehe cells increased heart rate, beat amplitude and muscle tonus. This response was mimicked by perfusion of exogenous FMRFamide at low concentration (10(-6) to 5 x 10(-8) M) through the interior of the intact heart. 4. Application of selective antagonists to 5-hydroxytryptamine (5-HT) and dopamine failed to block Ehe cardiac effects. 5. Detailed evidence that the Ehe cells were heart motoneurons was obtained. 1) Anatomic mapping using the dye Lucifer yellow showed Ehe cells had peripheral projections restricted mainly to the intestinal nerve, the only nerve known to innervate the heart. 2) Perfusion of the CNS with a saline containing Co2+ blocked central chemical synapses but did not affect activity of Ehe cells on the heart. 3) Simultaneous intracellular recordings from Ehe cells and auricle muscle fibers showed unitary excitatory junction potentials following with constant latency from spikes in Ehe cells. 6. The present study elucidates the role of FMRFamide in cardioregulation and provides the first evidence that it acts as an excitatory neurotransmitter on the snail heart.

1. The present paper extends the model of neuronal control of the Lymnaea heart by the use of intracellular recording techniques to identify further types of cardioactive neurons in the CNS that, like the previously described E heart excitor (Ehe) cells, influence the myogenic heartbeat. 2. Four new types of neuron that act on the heart are described. These are excitatory Hhe and She cells (H and S heart excitors) and the inhibitory Khi cell (K heart inhibitor). The fourth class, tonus pericardium excitor (Tpe), modulates the heart by action on pericardial tissue. 3. Pharmacologic, electrophysiological, and anatomic evidence is presented that shows that these cells are motoneurons, innervating heart muscle fibers directly: blocking central chemical synapses failed to prevent the actions of the neurons on the heart; simultaneous intracellular recordings showed unitary EJPs in heart muscle after 1:1 and with constant delay from evoked neuronal action potentials; intracellular injection of the dye Lucifer yellow showed all cells had axonal branches entering the intestinal nerve (which innervates the heart). 4. The use of selective antagonists to 5-hydroxytryptamine (5-HT) (cinanserin), dopamine (ergonovine), and acetylcholine (alpha-bungarotoxin) provided evidence that the actions of She and Hhe cells are mediated by 5-HT, whereas those of the Khi cell are mediated by acetylcholine. 5. A cyclically active network of three interneuronal inputs acting on the heart motoneurons is described. 6. One of these, input 3, is responsible for periodic excitation of the heart via its effects on the Hhe cells.

1. We have used pharmacologic, immunologic, and biochemical techniques to examine the role of neurochemicals in modulating the myogenic heart of the snail, Lymnaea. 2. 5-HT [high-pressure liquid chromatography (HPLC) and immunocytochemistry], dopamine (HPLC), FMRFamide-related peptides (radioimmunoassay and immunocytochemistry) and substance P-related peptides (immunocytochemistry) were shown to be localized within heart tissue. 3. The pharmacologic actions of these substances on the auricle from an isolated heart preparation were examined together with other putative modulators, acetylcholine (ACh), small cardioactive peptides A and B (SCPA and SCPB), [Arg]8vasotocin (AVT), and Lymnaea native FMRFamide-related peptides [Phe-Met-Arg-Phe-NH2 (FMRFamide), Ser-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (SDPFLRFamide) and Gly-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (GDPFLRFamide)]. 4. The response to each substance could be distinguished by different effect on beat rate, amplitude, and diastolic tonus, as well as by the duration of responses to standard 1-min applications. ACh was inhibitory at low concentrations (threshold less than 10(-10) M) but excitatory at high concentrations (10(-6) M). AVT was alone in producing no dose-dependent response. At high concentrations (10(-4) M), AVT caused a massive tonic contraction and cessation of auricle beat. All other substances examined were excitatory. 5. Antagonists to 5-HT (cinanserin), dopamine (ergonovine), and ACh (alpha-bungarotoxin) were identified. 6. ACh, 5-HT, dopamine, and FMRFamide-related peptides all acted on the auricle at low concentrations, and the rapid onset and short duration of their excitatory effects (ACh inhibitory at low concentrations) suggested that they may have roles as neurotransmitters. SCPA and SCPB were also potent (threshold less than 10(-10) M) but produced long-duration responses suggesting a modulatory or hormonal role.

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