Voltage clamp analysis of ethanol effects on pacemaker currents ofAplysia neurons

The effect of 4% ethanol on pacemaker currents of Aplysia neurons was studied under voltage clamp. In normal seawater the n-shape in the I-V disappeared and outward current increased. Ion substitution and drug blocking experiments determined that leakage current and the slow inward calcium current were decreased and that the outward currents, IA and IK, were increased. This knowledge can be used to explain ethanol effects on spontaneous firing patterns.     

Action of tetrodotoxin on pacemaker conductances in Aplysia neurons

Fresh tetrodotoxin (TTX, 30–100 μM) reversibly blocks bursting pacemaker activity and the negative slope region of the voltage clamp current-voltage curve in cell R15 of Aplysia californica. Leakage conductances and the calcium spike are unaffected by TTX.     

Differential effects of pentylenetetrazole on ion currents of Aplysia neurones

The effect of the convulsant drug pentylenetetrazole (PTZ) on separated membrane current components has been studied in identified voltage-clamped Aplysia neurones. External PTZ blocks the voltage-dependent Na+, Ca2+ currents and the delayed rectifier current (INa, ICa and IK,V, respectively). The amplitude of the Ca2+-activated K+ current (IK,Ca) is increased. The amplitude of the fast inactivating K+ current (IA) is transiently increased at low concentrations of PTZ but is depressed at higher concentrations or after long-lasting application of the drug. The effect of PTZ on leakage current (IL) seems to depend on the cell type. In some cells (R-15, L-7, LP-1) IL is decreased while it is increased in other cells (L-11, BL-1, BR-1). PTZ accelerates the inactivation of IK,V and IA and shifts the current-voltage relation of ICa to negative voltages by 5-8 mV. Pressure injection of PTZ into the neurone did not affect IK,V or IK,Ca. Thus PTZ seems to act on the outside of the plasma membrane. The effect of external PTZ on INa, ICa, IK,V and IL is also observed if the internal Ca2+ activity is buffered with EGTA suggesting that an increase in the internal Ca2+ activity is not involved. At -40 mV PTZ induces a tetrodotoxin-insensitive inward current carried by Na+ ions. PTZ transforms the beating pacemaker cell L-11 into a bursting pacemaker and the bursting pacemaker cell R-15 exhibits 'square-wave'-like oscillations of the membrane potential.     

Seawater osmolarity influences bursting pacemaker activity in intactAplysia california

Activity in cell R15 was monitored in a preparation ofAplysia californica in which all sensory inputs and the hemolymph were left intact. R15 exhibited bursting pacemaker activity in situ. Substitution of 93% sea water for normal sea water produced a hyperpolarization of R15 which either silenced the cell or significantly increased the interburst interval in 9 out of 12 of the experiments. Excitatory activity from head ganglia acted to antagonize this inhibitory response. The results provide support for a role of R15 in osmoregulation, and provide a model system in which the interaction of an identified neurosecretory cell, peptide release, and physiological response may be studied.     

Effects of sea water temperature on bursting pacemaker activity in cell R15 in the intactAplysia

Thermosensitivity of bursting pacemaker activity (BPA) in neuron R15 ofAplysia was compared in an intact preparation and after excision of the ganglion containing R15. We noted no differences indicative of an in situ antagonism of cooling-induced suppression of BPA. Interruption of circulation hastened hemolymph cooling during exposure of the animal to cold sea water, suggesting mechanisms to protect R15 and other cells from transient cooling of the animal.     

Mercury (Hg) decreases voltage-gated calcium channel currents in rat DRG and Aplysia neurons

Inorganic mercury (Hg²⁺) reduced voltage-gated calcium channel currents irreversibility in two different preparations. In cultured rat dorsal root ganglion (DRG) neurons, studied with the whole cell patch clamp technique, a rapid concentration-dependent decrease in the L/N-type currents to a steady state was observed with an IC₅₀ of 1.1 μM and a Hill coefficient of 1.3 T-currents were blocked with Hg²⁺ in the same concentration range (0.5–2 μM). With increasing Hg²⁺ concentrations a slow membrane current was additionally activated most obviously at concentrations over 2 μM Hg²⁺. This current was irreversible and might be due to the opening of other (non-specific) ion channels by Hg²⁺. The current-voltage (I–V) relation of DRG neurons shifted to more positive values, suggesting a binding of Hg²⁺ to the channel protein and/or modifying its gating properties. In neurons of the abdominal ganglion of Aplysia californica, studied with the two electrode voltage clamp technique, a continous decrease of calcium channel currents was seen even with the lowest used concentration of Hg²⁺ (5 μM). A steady state was not reached and the effect was irreversible without any change on resting membrane currents, even with high concentrations (up to 50 μM). No shift of the I–V relation of the calcium channel currents was observed. Effects on voltage-activated calcium channel currents with Hg²⁺ concentrations such low have not been reported before. We conclude that neurotoxic effects of inorganic mercury could be partially due to the irreversible blockade of voltage-activated calcium channels.     

Role of pedal ganglia motor neurons in pedal wave generation in Aplysia

Presumptive pedal ganglia motor neurons involved in pedal wave generation in the foot of Aplysia were examined. Pedal motor neurons fired tonicly in the absence of pedal waves, but exhibited bursting with constant phase angles during pedal wave generation. Motor neurons which burst both in and 180° out of phase with the pedal wave were recorded. The relative latency of neurons firing at large phase angles was more variable than that of neurons firing at small phase angles. Pedal neurons did not make monosynaptic connections among themselves. Their firing both during and in the absence of pedal waves was due to synaptic input, presumably from intemeurons which generate the oscillations underlying the pedal wave. Motor neurons firing in phase had common synaptic input of the same sign, while some motor neurons firing out of phase had common synaptic input of opposite sign. There also appeared to be polysynaptic connections among pedal motor neurons. Evidence suggesting polysynaptic feedback from the motor neurons to the oscillator interneurons was found. The results are consistent with a multineuronal oscillator in which different elements drive different groups of motor neurons. The pedal wave is the result of cyclic activation of the motor neurons by the oscillator network. Pedal motor neurons evoked several different types of tension changes in the foot when intracellularly stimulated. Fast and slow tension increases were observed. Some caused decreased tension in the foot when stimulated, suggesting that they might be inhibitory motor neurons. Other neurons appeared to be involved in the central control of foot tonus. Pedal motor neurons had conduction velocities ranging from 36 to 136 cm·sec⁻¹ and were found to have axons in more than one foot nerve branch. This property may contribute to the longitudinal spread of the pedal wave.     

A calcium-activated, nonselective cationic conductance in Aplysia silent neurons

We have previously reported the activation of a triphasic current response by calcium injection in voltage-clamped, nonbursting neurons of Aplysia californica. Present evidence indicates that the second phase, a delayed inward current that peaks 10–20 seconds after the end of the injection, is a calcium-activated, nonselective cationic conductance. It can be carried by both sodium and calcium, is not sensitive to chloride concentration changes, but is voltage sensitive, decreasing in amplitude with hyperpolarization.     

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 (<3 s), medium (3–6 s) and slow (>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 μM. 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 °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 < carbachol < arecoline. The carbachol response was best fitted by two exponential functions, while arecoline was best fitted by a single exponential plus a constant.     

Voltage-dependent current evoked by dopamine and octopamine in Aplysia

Octopamine and dopamine evoke a slow voltage-dependent inward current in neurons of Aplysia. The responses are present only at depolarized potentials (−40 mV). They are insensitive to changes in potassium, persist after removal of sodium, are not reduced by low chloride solution, and are blocked by cadmium. Because of the similarities in voltage-dependence, time course and ionic sensitivities, the ionic mechanism of the voltage-dependent responses to octopamine and dopamine are concluded to be the same as that of the voltage-dependent response to serotonin reported previously ¹³.     

Localized illumination of the Aplysia and Bulla eye reveals new relationships between retinal layers

The cellular organization of the opisthobranch retina is of interest since the eyes of several of these molluscs express circadian rhythms in optic nerve impulse frequency. In a model for retinal organization proposed by Audesirk, photoreceptors make electrical contacts with higher order cells which generate the compound action potential (CAP) recorded in the optic nerve. However, using micro-illumination on selected retinal regions, we now find that cells near the base of the retina are responsible for light transduction leading to CAPs. Illumination of the distal segments of photoreceptors surrounding the lens generates low-amplitude unitary activity in the optic nerve without CAPs. Furthermore, illumination of this region leads to inhibition of spontaneous CAPs or those generated by illumination of the retinal base. This inhibition is blocked by high magnesium-low calcium solutions and thus we conclude that inhibition and a secretory step comprise at least part of the pathway between the photoreceptor layer and neurons giving rise to the CAP.     

A neural pathway for learning that food is inedible in Aplysia

Aplysia can be taught to stop responding to inedible food, by pairing lip stimuli with stimuli arising from food struck in the buccal cavity⁸. When the esophageal nerves innervating the gut are cut, Aplysia cease responding to inedible food in a mean of 2.09 times longer than when these nerves are intact. Patterning of feeding movements is also changed. Cessation of responses in lesioned animals may be due to adaptation caused by lip stimulation. The data suggest that the esophageal nerves carry information about whether food is edible or inedible.     

Inhibitors of calcium-dependent enzymes prevent the onset of afterdischarge in the peptidergic bag cell neurons of Aplysia

Experiments with isolated bag cell neurons of Aplysia have produced evidence that changes in neuronal excitability may be brought about by the activation of calcium dependent enzymes such as calcium-dependent protein kinases. We have now examined the effects of agents which have been shown to inhibit several calcium-dependent enzymes on the properties of bag cell neurons in situ. In response to brief electrical stimulation the bag cell neurons of Aplysia generate an afterdischarge during which they release neuroactive peptides. We have found that the ability of stimulation to trigger an afterdischarge in the bag cell neurons is inhibited by trifluoperazine (TFP, 50-100 microM), N-(6-aminohexyl)-5-chloro-1-napthalenesulphonamide (W7) (25-50 microM) and calmidazolium (40-100 microM), each of which has previously been shown to inhibit calmodulin-dependent enzymes and the calcium-phospholipid-dependent protein kinase in the bag cell neurons. Further analysis of the effects of TFP showed that this inhibition occurs at concentrations which do not inhibit synaptic transmission or the endogenous bursting of another neurosecretory neuron, R15. Secretion of neuroactive peptides from the bag cell neurons was measured both electrophysiologically and biochemically. No attenuation of secretion could be observed at concentrations of TFP below those which inhibited the initiation of afterdischarge. Our results indicate that these agents inhibit secretion from these neurons primarily by inhibiting the onset of the afterdischarge and are consistent with the hypothesis that a calcium-dependent enzyme plays a role in triggering the stimulus-induced transformation in the electrical properties of these neurons.     

Effects of n-alkanols on the calcium current of intracellularly perfused neurons of Helix aspersa

The effects of n-alkanols on the calcium current (ICa) were studied in molluscan neurons perfused intracellularly and voltage clamped using a suction pipette technique. All n-alkanols employed in this experiment (methanol, ethanol and butanol) decreased the peak amplitude of ICa and caused acceleration of the decay of ICa in a dose-dependent manner at all membrane potentials. The concentrations of n-alkanols required for these actions decreased as the hydrocarbon chain increased in length. The results suggest that these effects on the ICa of molluscan neurons may be related to the lipophilic properties of n-alkanols.     

Synthesis and fast axonal transport of proteins in the isolated Aplysia nervous system

Fast axonal transport and neuronal protein synthesis was studied in the isolated nervous system of Aplysia californica. The abdominal ganglion with attached pleural-abdominal connectives (PAC) was removed and the ganglion pulse-labelled with [35S]methionine for 30 min in vitro. The axon containing connectives were ligated 24-28 mm from the ganglia and the system was perfused with chase media for 6-72 h to allow labelled rapidly transported proteins to accumulate at the ligature. One-dimensional polyacrylamide gel electrophoresis (PAGE) and fluorography was used to analyze the distribution of rapidly transported proteins along the right PAC. By 12 h, a significant accumulation of labelled proteins at the ligature was present but the build up was not complete until 48 h when almost no trailing of rapidly transported proteins was observed. Quantitation of the transport profiles of several rapidly transported proteins suggested a discontinuous release of proteins from the cell body. Analysis using two-dimensional PAGE revealed 10 major groups of rapidly transported proteins. These proteins were all identified among the total complement of newly synthesized proteins in cell R2. Not all rapidly transported proteins are cleared from the cell body at the same rate. Several of the major groups were no longer present in the neuron cell body 24 h after labelling, indicating that these species are selectively exported; others were still present after 3 days, suggesting that these proteins with a longer residence time have functions in both somatic and axonal regions of the neuron.     

Activation of a non-specific cation conductance by intracellular injection of inositol 1,3,4,5-tetrakisphosphate into identified neurons of Aplysia

The ionic mechanism of the effect of intracellulary injected inositol 1,3,4,5-tetrakisphosphate (IP₄) on the membrane of identified neurons (R9–R12) of Aplysia kurodai was investigated with conventional voltage-clamp, pressure injection, and ion-substitution techniques. Intracellular injection of IP₄ into a neuron voltage-clamped at −45 mV reproducibly induced a slow inward current (20–60 s in duration, 3–5 nA in amplitude) associated with a conductance increase. The current was decreased by depolarization and increased by hyperpolarization. The extrapolated reversal potential was −21 mV. The IP₄-induced inward current was sensitive to changes in the external Na⁺, Ca²⁺ and K⁺ concentration but not to changes in Cl⁻ concentration, and was resistant to tetrodotoxin (50 μM). When the cell was perfused with tetraethylammonium (5 mM) but not with 4-aminopyridine (5 mM), the IP₄-induced inward current recorded at −45 mV slightly increased. The IP₄-induced inward current was partially reduced by calcium channel blockers (Co²⁺ and Mn²⁺). These results suggest that intracellularly injected IP₄ can activate a non-specific cation conductance.     

Motor program for pedal waves during Aplysia locomotion is generated in the pedal ganglia

The activity in the nerves innervating the foot of Aplysia was examined during pedal wave generation. Cyclic patterned discharges (i.e. bursting) in the pedal nerves was associated with the pedal wave. Individual units exhibited bursting during pedal wave generation, but fired tonicly when the pedal wave was absent. Bursting in the nerves persisted after the foot was deafferented, confirming previous behavioral results that pedal waves were the result of a centrally generated motor program. Deafferentation caused increased burst durations and decreased spike frequencies within each burst. This suggests that sensory input from the foot excites the oscillator which underlies pedal wave generation and serves to increase the amplitude of the oscillations. Bursting in the nerves persisted after surgically isolating the pedal ganglia, showing that the neural circuitry necessary for pedal wave generation resides in the pedal ganglia. After isolating the pedal ganglia, bursts in the nerves were longer, less vigorous and less frequent. This suggests that input from the other central ganglia affects both the amplitude and the period of the oscillator. The firing of units in homologous pedal nerve branches, showed similar, but not identical patterns with the pedal commissure intact. Individual homologous units differed in both absolute timing and frequency of firing. After cutting the commissure, synchronization of activity in the homologous nerves was lost. This indicates that bilateral coordination of the pedal wave motor programs generated in each pedal ganglion is maintained via the pedal commissure. The possible mechanisms involved are considered.     

Further studies of bulk and orosensory decrement in producing satiation of feeding in Aplysia

Prior evidence has suggested that meal satiation in the marine mollusk Aplysia is associated with stretch of the crop. The current data, however, suggest that under some conditions, bulk in the crop can be dissociated from the propensity to feed. The crop was hyper-distended 6 h after a satiating meal of rehydrated seaweed; that is, the crop took in water and therefore contained a greater volume than it had contained immediately after satiation. Animals presented with food 6 h after an initial satiating meal consumed a new meal despite the fact that their crop was distended beyond the level at which they had previously terminated feeding. This unexpected result led to additional experiments designed to study possible orosensory decrement during presentation of food. Orosensory input was assessed by recording from the metacerebral cell (MCC) in free-moving animals. The MCC receives excitatory input in response to chemosensory stimulation of the lips, and exhibited a slow decrement during the course of a meal or during repeated lip stimulation without ingestion. Lesions of the cerebro-buccal connectives abolished the long-term MCC response decrement to lip stimulation. This result suggests that the MCC long-term response decrement to lip stimulation is a product of buccal-ganglion feedback and may not reflect sensory decrement of chemosensory pathways. Therefore, satiation may not produce a change in lip sensitivity to chemosensory input. Our data suggest that one important factor that determines satiation is a stretch stimulus of the posterior esophagus/anterior crop. This stretch stimulus may subside over several hours as the crop contents are redistributed or as receptors slowly adapt.