The voltage-dependent, slow inward current induced by the neuropeptide FMRFamide in Aplysia neuron R14

The effects of the peptide FMRFamide (Phe-Met-Arg-Phe-NH2) on the soma of neuron R14 in the abdominal ganglion of Aplysia californica and A. brasiliana were characterized. Pressure-ejected FMRFamide caused 3 types of responses, (1) a fast outward current (duration, less than 30 sec), (2) a fast inward current (duration, less than 20 sec), and (3) a slow inward current (peak at 0.5-1 min; duration, 2-3 min). The slow inward current, the chief object of this study, arises from a voltage-dependent conductance increase. The FMRFamide-elicited slow inward current is largest between -40 mV and -20 mV, the region of a negative slope resistance in the normal current-voltage relationship for R14. The slow FMRFamide-induced inward current is largely carried by Na+. This current is independent of external [K+] but depends inversely on external [Ca2+] and [Cl-]. The concentrations of the latter ions may influence the voltage dependence of the response. The slow inward current has many properties in common with inward currents induced in other molluscan neurons by applications of neuropeptides or intracellular injections of cyclic nucleotides.     

Desensitization kinetics of a K+ acetylcholine response in Aplysia

We have studied the process of acetylcholine receptor desensitization on Aplysia medial pleural neurons under voltage clamp conditions. Acetylcholine, applied by microperfusion, elicits a biphasic response on these neurons, a rapid component which reverses polarity at about -60 mV and is Cl-dependent, and a slower component which reverses at about -85 mV and is K-dependent. Both components show desensitization, and the present study focuses on the K-dependent component, which could be isolated by maintaining membrane potential at the Cl equilibrium potential or by blocking the Cl component pharmacologically. K-dependent acetylcholine responses on these neurons varied in regard to time to peak of response and rate of desensitization. While the rising phase of the response was always fitted by a single exponential process, times to peak were divided somewhat arbitrarily into three broad groups of fast (less than 3 s), medium (3-6 s) and slow (greater than 6 s). Desensitization of fast responses was best described by two exponential processes plus a constant, medium responses by a double exponential, and slow responses by single exponential plus a constant. The apparent dissociation constant of acetylcholine was 17.3 +/- 1.6 microM. The best fit of responses for a given cell remained constant over a range of acetylcholine doses, but the kinetics of both fast and slow components accelerated with dose and depolarization. The fast component of desensitization was very temperature dependent. In neurons where it was present it was abolished by cooling, while in neurons with no fast component at room temperature it would appear with warming. The time constant of the fast component varied inversely with temperature. The time constant of the slow component was maximal at 22-24 degrees C, and fell on either side of this temperature. These results suggest that receptor desensitization for acetylcholine K responses is, like Na-dependent responses, composed of two independent processes. When responses to the acetylcholine agonists, carbachol and arecoline, were compared to those of acetylcholine on fast-type neurons, the times to peak varied in the order acetylcholine less than carbachol less than arecoline. The carbachol response was best fitted by two exponential functions, while arecoline was best fitted by a single exponential plus a constant.     

Sensory function and gating of histaminergic neuron C2 in Aplysia

This paper explores the possible sensory function of the identified histaminergic neuron C2. Mechanical stimulation of a narrow region around the mouth of the animal (perioral zone) elicits brief depolarizing potentials in C2. Extracellular recordings from the peripheral axons of C2 indicate that the depolarizing potentials are due to action potentials that are conveyed from the periphery but do not invade the cell body, since they fail at a region with a low safety factor within the cerebral ganglion. These blocked axonal spikes (A-spikes) function as if they were excitatory synaptic inputs to C2, since the synaptic output of C2 does not occur unless the A-spikes succeed in evoking full action potentials in the soma (or an electrically close initial segment) of C2. Furthermore, like synaptic potentials, the A-spikes exhibit temporal and spatial summation, and facilitation. C2 receives both tonic and phasic inhibitory synaptic potentials, which can decrease the summation of A-spikes and thereby alter the frequency-filtering properties of C2 or block its synaptic output. Thus, C2 appears to be an unusual proprioceptive afferent that has a high degree of integrative function and may provide critical gating that is dependent on a variety of external and internal conditions.     

Dopamine reduces slow outward current and calcium influx in burst-firing neuron R15 of Aplysia

Dopamine's effect on calcium influx into the bursting neuron, R15, of Aplysia californica was tested by tail current measurements and by measurement of absorbance of intracellular Arsenazo III, a calcium-sensitive indicator. Slow outward tail currents were elicited by subthreshold depolarization in voltage clamp and were demonstrated to be dependent upon transient increases in intracellular calcium activity, (Ca)i), using calcium-free seawater, calcium blockers (Mn2+ and La3+), and intracellular injection of EGTA. Dopamine reduces these tail currents as it reduces the slow inward current. Next, the transient elevations of (Ca)i accompanying subthreshold depolarization were measured directly in Arsenazo III-loaded neurons. Dopamine did not reduce the rise in (Ca)i measured in the soma during depolarization. However, when absorbance of the axodendritic region was monitored, dopamine did reduce calcium influx. Voltage monitoring in the axon indicated that the reduced calcium influx could not simply be ascribed to altered space clamp. In keeping with the apparent axodendritic location of dopamine action, isolation of the soma by ligation of the axon markedly reduced the dopamine response. Dopamine seems to reduce calcium influx into R15, but this effect is topographically limited to nonsomatic membrane, an area of the neuron not usually monitored in optical studies of (Ca)i.     

Giant Aplysia neuron R2 reliably forms strong chemical connections in vitro

The giant cholinergic neuron R2 of Aplysia was cultured in combination with identified neurons L11 and R15 and members of a group of left upper quadrant (LUQ) cells L2 to L6 from the abdominal ganglion. All of these neurons receive cholinergic input from other cells in vivo, but not from R2. In vitro, R2 reliably formed unidirectional chemical connections with these cells. Single action potentials in R2 produced a dual fast and slow inhibitory response in LUQ cells (L2 to L6), a dual fast inhibitory-slow excitatory response in L11, and a slow inhibitory response in R15. The connections formed on LUQ cells were characteristic of their cholinergic input, but the R2-L11 and the R2-R15 connections also had noncholinergic properties. Thus, unlike L10 which forms connections only with its normal targets in vitro, R2 forms strong chemical connections with other neurons which are not found in vivo. The properties of the R2 connections also suggest that it may release another neurotransmitter besides acetylcholine.     

Reciprocal modulation of calcium current by serotonin and dopamine in the identified Aplysia neuron R15

Voltage-clamp methods were employed to study the effects of serotonin (5-HT) and dopamine on the pharmacologically isolated calcium current in the identified Aplysia neuron R15 grown in cell culture. Neurons were obtained from juvenile animals and had not yet developed the bursting pacemaker pattern of activity characteristic of R15 in mature animals. In R15 5-HT elicits a biphasic response consisting of excitatory depolarization followed by an inhibitory hyperpolarization and dopamine elicits an inhibitory hyperpolarization. 5-HT increased the Ca2+ current without affecting its voltage dependence. The 5-HT effect persisted when Ba2+ was employed to carry current through Ca2+ channels. 5-HT did not affect the rate of Ca2+-dependent Ca2+ current inactivation other than through its effect on the magnitude of the Ca2+ current. The adenylate cyclase activator forskolin, in the presence of a phosphodiesterase inhibitor, also increased the magnitude of the Ca2+ or Ba2+ current. This result suggested that the 5-HT-induced enhancement of Ca2+ current was mediated by cAMP. Dopamine inhibited Ca2+ current when either Ca2+ or Ba2+ was employed as the current carrier. Dopamine did not affect the rate of Ca2+-dependent inactivation of Ca2+ current other than through its effect on the magnitude of the Ca2+ current. Intracellular injection of the Ca2+ chelator EGTA inhibited serotonergic modulation of the Ca2+ current but not dopaminergic modulation. These results indicated that the putative neurotransmitters 5-HT and dopamine may regulate bursting activity in mature R15 neurons through modulation of Ca2+ current.     

Mechanism of calcium-dependent inactivation of a potassium current in Aplysia neuron R15: interaction between calcium and cyclic AMP

In the preceding paper (Kramer and Levitan, 1988), we presented evidence that an inwardly rectifying K+ current (IR) is inactivated by Ca2+ influx accompanying spontaneous bursting activity in the Aplysia neuron R15. In this paper we examine the mechanism that enables Ca2+ to inactivate IR. Since IR is enhanced by cyclic AMP in neuron R15 (Drummond et al., 1980; Benson and Levitan, 1983), we examined the Ca2+-dependent inactivation of IR after application of either serotonin (5-HT), the adenylate cyclase activator forskolin, or a membrane-permeable cAMP analog, all agents that increase cAMP and hence the magnitude of IR. Even though more active IR channels are available under these conditions, less Ca2+-dependent inactivation is observed. This is contrasted with the Ca2+-dependent inactivation of the voltage-gated Ca2+ current (ICa). Elevating cAMP enhances ICa in R15 and also increases its Ca2+-dependent inactivation. Hence the mechanisms whereby Ca2+ inactivates IR and ICa appear to differ from each other. Elevating internal Ca2+ by repeatedly depolarizing the neuron suppresses the response of IR to brief applications of 5-HT, and speeds the relaxation of the response, suggesting that Ca2+ can interfere with the cAMP-dependent activation of IR. One biochemical site where Ca2+ can reduce cellular cAMP is by activating the Ca2+/calmodulin-sensitive form of phosphodiesterase. We have detected such enzyme activity in homogenates of Aplysia abdominal ganglia and extracts of single R15 somata. Inhibitors of the phosphodiesterase activity suppress the Ca2+-dependent inactivation of IR. Finally, we have used a radioimmunoassay to measure cAMP in individual R15 somata, and have found that R15 neurons hyperpolarized for prolonged periods contain more cAMP than do R15 neurons allowed to burst, consistent with the hypothesis that Ca2+ influx reduces cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inward current responses to urinary substances in rat vomeronasal sensory neurons

No study has yet demonstrated an inward current in response to pheromonal substances in vomeronasal sensory neurons. Using female rat vomeronasal sensory neurons, we here successfully recorded inward currents in response to urine from various sources. Of the neurons that responded to urine, 77% responded to only one type of urine. Male Wistar urine induced responses preferentially in the apical layer of the sensory epithelium, whilst male Donryu and female Wistar urine induced responses mainly in the basal layer of the epithelium. The amplitude of inward currents induced by application of male Wistar urine was voltage-dependent with average amplitude of -47.1+/-6.2 pA at -74 mV. The average reversal potential for male Wistar urine was -9.3 +/-6.1 mV, which was not apparently different from the reversal potentials for urine from different species. It is likely that the urine-induced inward currents in response to different types of urine are mediated via a similar channel. The simultaneous removal of Na+ and Ca2+ from extracellular solution eliminated the response. The magnitude of the urine-induced inward current in Cl--free external solution was similar to that in normal solution, suggesting that the urine-induced current is cation selective. Removal of external Ca2+ enhanced the amplitude of the urine-induced current and prolonged the response. Application of the constant-field equation indicated a very high permeability coefficient for Ca2+. This study first demonstrated that substances contained in urine elicited inward currents, which induce an excitatory response in vomeronasal sensory neurons, through cation-selective channels.     

Nitric oxide donor sodium nitroprusside inhibits the acetylcholine-induced K+ current in identified Aplysia neurons

The effects of bath-applied sodium nitroprusside (SNP), a nitric oxide (NO) donor, on an acetylcholine (ACh)-induced K⁺ current recorded from identified neurons (R9 and R10) of Aplysia kurodai were investigated with conventional voltage-clamp and pressure ejection techniques. Bath-applied SNP (25–50 μM) reduced the ACh-induced K⁺ current in the neurons without affecting the resting membrane conductance and holding current. The suppressing effects of SNP on the current were completely reversible. Intracellular injection of 1 mM guanosine 3′,5′-cyclic monophosphate (cGMP) or bath-applied 50 μM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase (PDE) inhibitor, also inhibited the ACh-induced current, thus mimicking the effect of the NO donor on the ACh-induced current. In contrast, pretreatment with methylene blue (10 μM), an inhibitor of guanylate cyclase, and hemoglobin (50 μM), a nitric oxide scavenger, decreased the SNP-induced inhibition of the ACh-induced current. These results suggest that SNP, a NO donor, inhibits the ACh-induced K⁺ current, and that the mechanism of NO inhibition of the ACh-induced current recorded from identified Aplysia neurons involves cGMP-dependent protein kinase. © 1996 Wiley-Liss, Inc.     

Clustered distribution and variability in kinetics of transient K channels in molluscan neuron cell bodies

The spatial distribution of transient K current, IA, was studied using a combination of patch-clamp and whole-cell voltage-clamp techniques. The average IA current density in somatic patches is 0.64 times the current density in the entire axotomized cell body, a finding which suggests that the axon hillock or initial segment of the axon has a higher concentration of IA channels than much of soma. The highest density of active channels during the peak IA is 1/micron2 at a membrane voltage of -20 mV. There is no evidence for a gradient in the distribution of IA channels in the cell body, but the channels are not evenly distributed. The variability in the number of channels per patch for multiple patches on the same neuron is much higher than expected for a random distribution. Statistical analysis of the data yields a coefficient of dispersion of 8.1, a value indicating a high degree of clustering. The utility of this statistic for evaluating channel distributions is discussed. Several lines of evidence suggest that the upper limit for the area of IA channel clusters is approximately 250 micron2. Single-channel currents attributed to IA were recorded in the cell-attached configuration. The voltage dependence of channel opening and inactivation are the same as measured in whole-cell voltage-clamp experiments. The single-channel conductance is about 9 pS in normal saline. Patches 9-30 micron2 in areas that contain IA channels are often devoid of other K channel types, suggesting that IA channels can occur in isochannel clusters. IA inactivation follows an exponential time course in all of the neurons examined, but the time constant of inactivation ranges from 25 to 560 msec in different cells. The voltage dependence of activation and inactivation and the reversal potential of the current are approximately the same in all cells. When multiple patches on the same neuron are studied, it is found that IA inactivates exponentially with approximately the same time constant in each patch, regardless of patch area. The data suggest that each neuron expresses predominantly, and perhaps exclusively, a single type of IA channel with distinct kinetic properties. The wide range of IA inactivation time constants observed in different cell suggests that a large number of channel types are available for expression. Possible mechanisms for generating diversity in channel types are discussed.     

Interleukin-2 inhibits the GABA-induced Cl- current in identified Aplysia neurons.

The effects of extracellularly applied recombinant human interleukin-2 (rhIL-2) on the gamma-aminobutyric acid (GABA)-induced Cl- current recorded from identified neurons (R9 and R12) of Aplysia kurodai were investigated with conventional voltage-clamp and pressure ejection techniques. Bath-applied rhIL-2 (10-40 U/ml) reduced the GABA-induced current in the neurons without affecting resting membrane conductance and the holding current. The suppressing effect of rhIL-2 on the current was completely reversible. Heat-inactivated rhIL-2 was without effect. These results suggest that the immunomodulator IL-2 can modulate the GABA-induced response in the nervous system.     

Activity of an identified serotonergic neuron in free moving Aplysia correlates with behavioral arousal

Extracellular recordings of the metacerebral cell (MCC), a serotonergic neuron inAplysia, were obtained in free moving, undrugged animals. MCC activity was evoked by exposure to food. Arousal level was manipulated by satiating the animals or exposing them to a noxious stimulus. We found that the amount of evoked MCC activity correlated with the level of arousal of the animal.     

Development of the fast, transient outward K current in embryonic sympathetic neurones

Voltage-activated outward potassium (K+) currents in developing sympathetic neurones, dissociated from the rat superior cervical ganglion (SCG), were studied using the whole-cell patch clamp recording technique. In voltage-clamped neonatal SCG cells, two voltage-dependent K+ currents were measured: the fast, transient K+ current, IA; and, the slower activating, non-inactivating delayed rectifier, IK. Only IK, however, appeared to be present in SCG neurones isolated from early embryonic (E14.5-16.5) rat pups; IA was not observed in these cells. When these embryonic neurones were maintained in cell culture, IA developed over a time course (approximately 4-6 days) similar to that seen in vivo. IA, therefore, which appears to facilitate the fast repolarization phase of the action potential in rat SCG neurones, is the last voltage-activated current to develop in these cells.     

Biochemical and morphological correlates of transmitter type in C2, an identified histaminergic neuron in Aplysia

There is compelling evidence that histamine serves as a neurotransmitter in C2, a pair of symmetrical neurons in the cerebral ganglion of Aplysia californica. These cells had previously been shown to contain high concentrations both of histamine and of its biosynthetic enzyme, histidine decarboxylase; in addition, 3H-histamine injected intrasomatically was found to move along C2's axons by fast transport. Furthermore, several actions of C2 on identified follower cells were simulated by the application of histamine. We have now characterized this identified neuron further. C2 converts 3H-histidine to histamine: 16% of the labeled precursor was converted to histamine 1 hour after intrasomatic injection. Synthesis of 3H-histamine is specific, since no conversion occurred after injection of other identified Aplysia neurons that are known to use other neurotransmitter substances. We also examined the fine structure of C2's cell body, axons, and axon terminals within the cerebral ganglion and in the nerves that carry its three peripheral branches, identified after injection of Lucifer Yellow, 3H-histamine, or horseradish peroxidase. Characteristic dense-core vesicles are present in all regions of the neuron, and are labeled after intrasomatic injection of 3H-histamine. These 100-nm vesicles together with 60-nm electron-lucent vesicles fill the varicose extensions of C2's neurites that are widely distributed within the ganglion, but only the smaller vesicles cluster at the membrane specializations presumed to be active zones that make contact with many neurons. The widespread distribution of axon terminals and varicosities is consistent with the idea that C2 is modulatory in function; 3H-histamine is taken up selectively by the cell body and axons of C2 and of several other putative histaminergic neurons in a Na+ -dependent manner. Characterization of these biochemical and morphological features of C2 adds to the large amount of information already available to make this identified cell a standard for identifying other neurons that use histamine as a transmitter.     

Forskolin's effect on transient K current in nudibranch neurons is not reproduced by cAMP

Forskolin, a diterpene extracted from Coleus forskolii, stimulates the production of cAMP in a variety of cells and is potentially an important tool for studying the role of cAMP in the modulation of neuronal excitability. We studied the effects of forskolin on neurons of nudibranch molluscs and found that it caused characteristic, reversible changes in the amplitude and waveform of the transient K current, IA, and also activated an inward current similar to the cAMP-dependent inward current previously described in molluscan neurons. Forskolin altered the time course of IA activation and inactivation but did not affect the voltage dependence or the reversal potential of the current. IA normally inactivates exponentially, but in forskolin the time course of inactivation can be fit by the sum of 2 exponentials with an initial rate that is faster than the control and a final rate that is much slower. On depolarization in forskolin, IA begins to activate at the normal rate, but a slower component of activation is also seen. The changes in IA in the nudibranch cells were qualitatively different than the changes caused by forskolin in Aplysia bag cell neurons (Strong, 1984). Experiments were performed to determine whether these effects of forskolin require cAMP. Intracellular injection of cAMP, application of membrane-permeable analogs of cAMP, application of phosphodiesterase inhibitors, and intracellular injection of the active catalytic subunit of cAMP-dependent protein kinase did not affect the amplitude or waveform of IA. Also, the changes in IA that are caused by forskolin were not prevented or reversed by intracellular injection of an inhibitor of cAMP-dependent protein kinase. Cyclic AMP did, however, activate inward current at voltages near the resting potential. We conclude that the changes in IA and the activation of inward current represent separate affects of forskolin. The inward current appears to depend on an increase in intracellular cAMP, while the changes in IA do not. These experiments show that, in addition to activating adenylate cyclase, forskolin may have a separate direct affect on the transient K current.     

Biochemical and immunocytological localization of the neuropeptides FMRFamide, SCPA, SCPB, to neurons involved in the regulation of feeding in Aplysia

The localization of the neuropeptide FMRFamide in the buccal ganglia and buccal muscles of Aplysia was studied by immunocytology and high-pressure liquid chromatography (HPLC) combined with either a sensitive bioassay or 35S-methionine labeling. Immunocytology with an antiserum directed to FMRFamide stained a large number of fibers, varicosities, and neuronal somata. Two groups of stained neurons were of particular interest. One was the S cells, a group comprised of many small neurons, the majority of which were stained. HPLC of pooled labeled S cells confirmed that at least some of these neurons synthesize FMRFamide. The other group of stained neurons were in the ventral cluster, a group comprised of a small number of large neurons, many of which are motor neurons that innervate the buccal muscles involved in producing biting and swallowing movements. Several of the ventral neurons were previously shown to contain 2 other neuropeptides, the small cardioactive peptides SCPA and SCPB. These neurons are sufficiently large to permit HPLC analyses of the neuropeptides synthesized by individual neurons. This procedure confirmed that individual ventral neurons synthesized FMRFamide, or the SCPs, or all 3 peptides. The coexistence of FMRFamide and the SCPs in the same neuron was confirmed by simultaneous staining of sections from the buccal ganglia with a monoclonal antibody to the SCPs and an antiserum to FMRFamide. The coexistence of the 3 peptides in the same neuron was surprising in light of the observations that these peptides often have opposite biological activity. The ventral neurons are large and potentially identifiable as individuals. Thus, these neurons may be particularly useful for studying the physiological and behavioral roles of neuropeptides in generating complex behaviors.     

Giant Aplysia neuron R2 has distal dendrites: evidence for protein sorting and a second spike initiation site

Because of its anatomy, the neuron R2 of Aplysia has been used to study how proteins are distributed to their appropriate destinations within the cell. The R2 cell body resides in the abdominal ganglion, while its axons terminate on glands in the skin. Using intracellular injection of HRP and intraxonal recordings, we found that R2 has a dendritic (receptive) arborization in the pleural ganglion. The structure of these dendrites was examined after injecting the soma with 3H-L-fucose, thereby labeling glycoproteins that are transported to all regions of the cell. Light- and electron-microscope autoradiography show that the openings to the dendrites are not on the periphery, but are suspended inside the axon by glial cell infoldings. All of the organelles seen in the axon are found in the dendrites, including 2 types of vesicles. Neither the axon nor the dendrites contain ribosomes. Thus, R2 has 3 functionally distinct regions--cell body, dendrites, presynaptic terminals--that are separated from each other by at least 4 cm. This implies that pre- and postsynaptic proteins made in the cell body are transported along the axon to the pleural ganglion, where they are sorted. To investigate this idea, we exposed the abdominal ganglion to 35S-methionine to label R2's proteins. Analyses by SDS-PAGE of the rapidly transported labeled proteins from R2 consistently showed a 78 kDa band that accumulated in the pleural ganglion and did not move into the peripheral nerves. This then is a putative dendritic constituent.     

Human Dopamine D3 and D2L Receptors Couple to Inward Rectifier Potassium Channels in Mammalian Cell Lines

The molecular mechanisms coupling the D3 dopamine receptor to downstream effectors have neither been well defined nor well characterized. Here we examine the coupling of the human D3 receptor to G-protein coupled inward rectifier potassium channels (GIRKs) in mammalian cells. Human D3 receptors couple strongly to homomeric human GIRK2 channels coexpressed in Chinese hamster ovary (CHO) cells, with a coupling efficiency comparable to that of D2L receptors. The coupling between D3 receptors and native GIRK channels was examined in an AtT-20 mouse pituitary cell line stably expressing the human D3 receptor. AtT-20 cells endogenously express somatostatin and muscarinic receptors coupled to GIRK channels. RT-PCR and Western blot analyses revealed that AtT-20 cells natively express Kir3.1 and Kir3.2 channel isoforms, but not D2 or D3 dopamine receptors. In D3 receptor expressing AtT-20 cells, application of the D2/D3 receptor agonist, quinpirole, induces pertussis toxin-sensitive inward rectifying K⁺currents that are blocked by barium. Activation of D3 receptors leads to both homologous desensitization of this receptor and an unusual unidirectional heterologous desensitization of somatostatin receptors. AtT-20 cells may be a good model to examine the functional role of D3 dopamine receptors in regulating neurotransmitter secretion.     

Cyclic AMP-induced slow inward current: its synaptic manifestation in Aplysia neurons.

Three presynaptic neurons, monosynaptically connected to the medial cells of the pleural ganglion of Aplysia californica and previously shown to elicit cAMP-mediated diminutions in K conductance in those cells (Kehoe, 1985a, b), were shown to elicit still another slow synaptic current that resembles the cAMP-induced cationic current described in the preceding paper (Kehoe, 1990). The synaptic current elicited by these so-called "blocking" neurons was compared, in hyperpolarized medial cells, with the current induced by an intracellular injection of cAMP. It was found that (1) both currents show an outward rectification, (2) both currents are enhanced and prolonged by phosphodiesterase inhibitors (as well as by intracellular acidification of the postsynaptic neuron and by bath-applied caffeine), and (3) both currents react in the same way to changes in (Ca)0, showing a net enhancement when (Ca)0 is reduced and, conversely, a marked diminution when extracellular (Ca)0 is increased. The increase in amplitude of the slow synaptic current in low-Ca solutions and its decrease in high-Ca seawater are contrary to the changes that would be expected from the known effects of Ca on transmitter release at chemical synapses, revealing the overriding importance of the postsynaptic block by Ca. The data presented here strongly suggest that both the slow inward current and the diminutions in K conductance induced by the firing of the 3 blocking neurons are mediated by cAMP. Like the 2 cAMP-mediated diminutions in K conductance (Kehoe, 1985a, b), the cAMP-activated slow inward current, because of its atypical voltage dependence, both depolarizes the medial cell and causes an increase in its input resistance at resting potential. Consequently, the synaptically activated increase in cAMP prolongs the excitability of the medial cells for up to tens of seconds after the end of presynaptic firing.