We introduced radioactive precursors directly into identified neurons of Aplysia californica. [3HJ-Choline and L-[3H]tryptophan were injected with pressure into nerve cell bodies to study synthesis of acetylcholine and serotonin. We confirmed the cholinergic nature of R2, LIO, and Lii, identified neurons of the abdominal ganglion. Cells in the LD cluster (which contains motor neurons to the heart and gill) also converted most of the injected choline into acetylcholine. Neurons in the RB cluster (which contains an excitatory motor neuron to the heart) and the two metacerebral cells of the cerebral ganglion converted injected tryptophan to serotonin.No cell studied could convert both choline to acetylcholine and tryptophan to serotonin. Pressure permits rapid injection of precursors, from small amounts to amounts large enough to saturate intracellular synthetic pathways. In contrast to the results with injection, we found far less synthesis of acetylcholine and serotonin in identified nerve cell bodies when ganglia were incubated in the presence of the radioactive precursors.

Sensitization of the gill withdrawal reflex in Aplysia californica is an elementary form of learning, in part resulting from presynaptic facilitation of the LE mechanoreceptor neurons of the abdominal ganglion. It has previously been established that either application of serotonin or direct stimulation of a group of facilitatory neurons, the L29 cells of the abdominal ganglion, can simulate the effect of physiological stimulation in producing presynaptic facilitation. Because the evidence that serotonin serves as a facilitatory transmitter was indirect, we examined the distribution of serotonin-immunoreactive fibers and cell bodies in the abdominal ganglion in order to answer two questions: (1) do the sensory neurons receive serotonergic innervation and (2) are the L29 cells serotonergic? We observed two distinctive patterns of serotonergic innervation within the ganglion, sparse and dense. The sparse pattern is correlated with a serotonin-stimulated increase in cAMP in identified target cells, while the dense innervation is not. We found a sparse distribution of serotonin-immunoreactive fibers with varicosities close to both cell bodies and processes of identified LE sensory cells. It therefore is likely that the sensory neurons do receive serotonergic innervation. We also mapped the population of serotonergic neuronal cell bodies in the ganglion, and found five clusters of neurons. Cells in one of these clusters, the identified RB neurons, had previously been shown to synthesize serotonin from tryptophan and to contain the neurotransmitter in high concentration. Identified L29 facilitator cells marked by injection with Lucifer Yellow do not contain serotonin immunoreactivity and therefore evidently are not a source of serotonergic input onto sensory cells.

The R20 neurons of Aplysia exhibit frequency-dependent spike broadening. Previously, we had used two-electrode voltage clamp to examine the mechanisms of this spike broadening (Ma and Koester, 1995). We identified three K ϩ currents that mediate action-potential repolarization: a transient A-type K ϩ current (I Adepol ), a delayed rectifier current (I K-V ), and a Ca 2ϩ -sensitive K ϩ current (I K-Ca ). A major constraint in that study was the lack of completely selective blockers for I Adepol and I K-V , resulting in an inability to assess directly the effects of their activation and inactivation on spike broadening. In the present study, the dynamic-clamp technique, which employs computer simulation to inject biologically realistic currents into a cell under current-clamp conditions (Sharp et al., 1993a,b), was used either to block I Adepol or I K-V or to modify their inactivation properties.The data in this paper, together with earlier results, lead to the following hypothesis for the mechanism of spike broadening in the R20 cells. As the spike train progresses, the primary responsibility for spike repolarization gradually shifts from I Adepol to I K-V to I K-Ca . This sequence can be explained on the basis of the relative rates of activation and inactivation of each current with respect to the constantly changing spike durations, the cumulative inactivation of I Adepol and I K-V , and the progressive potentiation of I K-Ca . Positive feedback interactions between spike broadening and inactivation contribute to the cumulative inactivation of both I Adepol and I K-V . The data also illustrate that when two or more currents have similar driving forces and partially overlapping activation characteristics, selectively blocking one current under current-clamp conditions can lead to a significant underestimate of its normal physiological importance.Key words: spike broadening; dynamic clamp; K ϩ current; I Adepol ; inactivation; Aplysia ; R20 Frequency-dependent spike broadening, an endogenously generated increase in spike duration that increases as a function of firing rate, has been shown in neurons from a variety of species to be correlated with enhanced transmitter release (Gillette et al., 1980;Coates and Bulloch, 1985) and to result primarily from inactivation of K ϩ currents (Aldrich et al., 1979a,b;Jackson et al., 1991;Bielefeldt et al., 1992;Crest and Gola, 1993;Quattrocki et al., 1994). Previously we had described the facilitatory synaptic effects and the mechanisms of frequency-dependent spike broadening in the two electrically coupled peptidergic R20 neurons of Aplysia (Ma and Koester, 1995). In addition, to analyze the mechanisms underlying spike broadening, several voltageactivated currents were isolated from the R20 cells by conventional voltage-clamp methods. These included a Na ϩ current (I Na ), a multi-component Ca 2ϩ current (I Ca ), and three K ϩ currents-a high-threshold transient A-type current (I Adepol ), a delayed rectifier current (I K-V ), and a two component Ca 2ϩ -sensitive K ϩ current...

1. The release of ink from the ink gland of Aplysia californica in response to noxious stimuli is mediated by three electrically coupled motor neurons, L14A, L14B, L14C, whose cell bodies are located in the abdominal ganglion. The initial synaptic input to the ink motor neurons is relatively ineffective in firing the cells. As a result, a pause of 1--3 s often occurs before the cells attain their maximum firing frequency and cause the release of ink. Using current and voltage-clamp techniques we have analyzed the mechanisms underlying the firing pattern of these cells. 2. The presence of a fast transient K+ current appears to play an important role in mediating the firing pattern of the ink motor neurons. Their high resting potential (-75 mV) ensures that the steady-state level of inactivation of the conductance channels for the fast K+ current will normally be low. Thus a train of EPSPs or a depolarizing current pulse can activate this current maximally, thereby reducing the initial effectiveness of the excitatory input. 3. In addition to the fast transient K+ current, four other currents were identified: 1) a fast transient tetrodotoxin-sensitive inward current, presumed to be carried by Na+; 2) a slower tetrodotoxin-insensitive inward current, presumed to be carried by Ca2+; 3) a slow transient outward tetraethylammonium- (TEA) sensitive current; and 4) a very slow TEA-insensitive outward current. 4. A decreased conductance EPSP, which turns on over a several-second period, contributes to a late acceleration of spike discharge in the L14 cells. 5. The results suggest that a unique combination of biophysical properties of the L14 cells and the features of the synaptic input cause them to act as a low-pass filter in the reflex pathway for inking. Their high resting potential, which ensures minimal inactivation of the fast transient K+ current channel, makes these cells preferentially responsive to strong and long-lasting stimuli. The delayed recruitment of a decreased conductance EPSP augments the tendency of the L14 cells to fire in an accelerating burst pattern.

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