Left Pleural Ganglion Research Articles

Specific Expression of Aplysia Phosphodiesterase 4 in Bag Cells Revealed by in situ Hybridization Analysis.

Phosphodiesterases (PDEs) play a key role in the regulation of cyclic adenosine monophosphate (cAMP), which in turn mediates various cellular functions including learning and memory. We previously cloned and characterized three PDE4 isoforms (ApPDE4) from Aplysia kurodai. Using reverse transcription polymerase chain reaction (RT-PCR), we found that ApPDE4 isoforms are primarily expressed in the central nervous system. However, the detailed distribution of ApPDE4 mRNA in Aplysia individual ganglions was not evident. In this study, to determine the distribution of ApPDE4 mRNAs in Aplysia ganglions, we performed in situ hybridization (ISH) using a probe targeting ApPDE4, including the PDE catalytic domain. Interestingly, we found the strongest ISH-positive signals in the symmetrical bag cell clusters of the abdominal ganglion. The R2, R14, L7, L2 and L11 neurons in the abdominal ganglion, LP1 neuron in pleural ganglion, and metacerebral (MCC) neurons were ISH-positive. Mechanosensory neurons of the sensory cluster were also stained on the ventral aspect of the right and left pleural ganglia. Taken together, we found the detailed distribution of ApPDE4 mRNA in Aplysia ganglion and support their roles in serotonin (5-HT)-induced synaptic facilitation of Aplysia mechanosensory neurons.

Effects of organophosphates on cholinesterase activity and neurite regeneration in Aplysia

In Aplysia, a marine mollusc, acetylcholinesterase (AChE) is present in cholinergic and non-cholinergic neurons and in hemolymph. Aplysia hemolymph has a very high level of AChE which promotes neurite growth in primary cultures of dopaminergic neurons via a non-catalytic mechanism. In contrast, AChE is known to facilitate neurite growth in cholinoceptive neurons by hydrolyzing ACh which inhibits neurite growth. In order to test whether AChE’s site-specific neurotrophic action varies with the neuronal phenotype, we investigated the effects of active-site inhibited hemolymph AChE on neurite growth of cholinergic neurons of Aplysia in primary culture. Organophosphates being long-acting active site inhibitors of AChE were chosen for this study. The effects of active site inhibited hemolymph AChE was tested on large cholinergic neurons, R2 (abdominal ganglion) and LPL1 (left pleural ganglion) as well as small cholinergic neurons (buccal ganglion) of Aplysia, maintained in culture. Partially purified hemolymph AChE was inhibited by either 10 μM of echothiophate or 5 μM of paraoxon. Neurons were maintained in (1) L15 (defined medium) alone; (2) L15+echothiophate; (3) L-15+paraoxon; (4) L-15+hemolymph AChE; (5) L15+hemolymph AChE+echothiophate; and (6) L-15+hemolymph AChE+paraoxon. Addition of uninhibited hemolymph AChE significantly increased neurite growth of cultured neurons compared to L15 alone. In the presence of echothiophate-inhibited or praoxon-inhibited AChE, neurite growth was significantly reduced when compared to L15+uninhibited AChE. While the presence of echothiophate by itself did not reduce survival or neurite growth when compared to L-15 alone, the presence of paraoxon by itself markedly reduced survival and neurite growth of cultured neurons. The results show that AChE’s catalytic action contributes to enhance neurite growth in cholinergic neurons and the effects of paraoxon appears to differ from that of echothiophate on cholinergic neurons of Aplysia.

Identified FMRFamide-immunoreactive neuron LPL16 in the left pleural ganglion of Aplysia produces presynaptic inhibition of siphon sensory neurons

The gill- and siphon-withdrawal reflex of Aplysia undergoes transient inhibition following noxious stimuli such as tail shock. This behavioral inhibition appears to be due in part to transient presynaptic inhibition of the siphon sensory cells, which can be mimicked by application of the peptide FMRFamide. Although FMRFamide is widespread in the Aplysia nervous system, an FMRFamide-containing inhibitory neuron has not previously been identified. We have searched for such a neuron by combining FMRFamide immunofluorescence with fluorescent dye backfilling from the abdominal ganglion, the location of the siphon sensory cells. These methods localized a neuron in the left pleural ganglion, which we have named LPL16. LPL16 is FMRFamide immunoreactive; it is excited by tail shock; and stimulation of LPL16 produces inhibition of siphon sensory cell-to-motor cell postsynaptic potentials and narrowing of action potentials in the sensory cells in tetraethylammonium solution. These results indicate that LPL16 participates in the inhibitory effects of tail shock, and support the idea that FMRFamide plays a physiological role in the inhibition.

Functional and morphological evidence for the existence of neurites from abdominal ganglion bag cell neurons in the head-ring ganglia of Aplysia

Three lines of evidence are presented indicating that axons of the Aplysia neuroendocrine bag cells extend into the head-ring ganglia of the CNS. When the abdominal ganglion was bisected longitudinally, separating the two bag cell clusters, an afterdischarge induced in one cluster generated an afterdischarge in the other via activity through the head-ring ganglia to which each half abdominal ganglion was attached by connective nerves. This suggests that some axons of bag cells in each cluster communicate through the head-ring ganglia. Retrograde labelling of bag cells occurred when rhodamine-conjugated latex microspheres were injected into the cerebral or either pleural ganglion, a direct demonstration that bag cell axons extend into these ganglia. Finally, cell LP1 in the left pleural ganglion was inhibited during a bag cell afterdischarge, an action mimicked by application of alpha-bag cell peptide (alpha BCP). Since alpha BCP can act only close to its site of release due to susceptibility to peptidase activity, it is likely that LP1 inhibition is dependent on the local release of alpha BCP from bag cell neurites in the pleural ganglion. These results open new possibilities for how bag cell afterdischarges may be initiated and broaden the distribution of their effects.

Neuromotor Bases of the Escape Behaviour of Nassa Mutabilis

ABSTRACT The complex sequence of movements in the escape behaviour of the snail Nassa mutabilis (L.) was described in detail and the neuromotor activity underlying the behaviour was investigated by extra- and intracellular recording. The escape reaction is triggered by a chemical stimulus to the animal ‘s foot, in these experiments either application of KC1 solution or contact with a starfish. It consists of a preliminary phase in which the shell tilts to its side, the actual locomotor phase, and a final righting movement. The snail performs leaps, in which the foot and the shell are repeatedly rotated with respect to one another. EMGs recorded from the columellar muscle during the escape reaction showed that bursts of potentials are coupled to the shell rotations. In the intact animal this burst activity ordinarily began 0 ·6 ± 0 ·3 s after stimulation with KC1. In an animal dissected for recording from the columellar nerve (which supplies the columellar muscle), KC1 stimulation of the dorsum of the foot induced burstlike neuronal activity with a latency of 0 ·5 ± 0 ·3 s. The dorsal foot region, the site at which the escape reaction can be triggered, was found to be supplied by the posterior pedal nerves; electrical stimulation of these nerves elicited bursts in the columellar nerve. The left pleural ganglion, which is known to contain neurones that project into the columellar nerve, was also found to contain neurones responsive to KC1 stimulation of the foot. These findings suggest that the left pleural ganglion contains a motor centre which is involved in control of activity of the columellar nerve, and is also active during the escape reaction.

Tail shock produces inhibition as well as sensitization of the siphon-withdrawal reflex of Aplysia: possible behavioral role for presynaptic inhibition mediated by the peptide Phe-Met-Arg-Phe-NH2.

Recent studies have shown that, in addition to being modulated by presynaptic facilitation, the sensory neurons of the gill- and siphon-withdrawal reflex of Aplysia are also capable of being modulated by transient presynaptic inhibition produced by the peptide Phe-Met-Arg-Phe-NH2. These two modulatory effects involve different second-messenger systems: the facilitation is mediated through cAMP-dependent protein phosphorylation, and the inhibition is mediated through the lipoxygenase pathway of arachidonic acid. To explore the behavioral function of this inhibition, we have carried out a parametric analysis of the effect of tail shock on the siphon-withdrawal reflex. In addition to producing sensitization of the withdrawal reflex, tail shock also transiently inhibits the reflex. The inhibition is produced by relatively weak shock, whereas sensitization is more prominent and may mask the inhibition with stronger shock. Furthermore, inhibition is not observed after habituation training. Cellular studies suggest that the behavioral inhibition is mediated, at least in part, by presynaptic inhibition of transmitter release from the siphon sensory neurons. Moreover, we have identified an interneuron within the left pleural ganglion (LPL16) that shows Phe-Met-Arg-Phe-NH2 immunoreactivity, is activated by tail shock, and simulates the presynaptic inhibitory actions produced by tail shock. Therefore, our results suggest that presynaptic inhibition mediated by Phe-Met-Arg-Phe-NH2 and its lipoxygenase second messenger contributes to behavioral inhibition of the siphon-withdrawal reflex.

Synaptic block of a transmitter‐induced potassium conductance in Aplysia neurones.

A voltage-clamp study was made of a slow excitatory post-synaptic potential (slow e.p.s.p.) that can be elicited in the medial cells of the left pleural ganglion of Aplysia californica by the firing of at least three different presynaptic neurones (labelled I, II and III). Each of these three neurones elicits other permeability changes in addition to the slow e.p.s.p., and all elements of these synaptic responses were shown to be mediated monosynaptically. The slow e.p.s.p., associated with an increase in membrane resistance, was shown to be due to a decrease in K permeability. When the slow e.p.s.p. was present spontaneously, it could be blocked by three compounds (tetraethylammonium (TEA), phenyltrimethylammonium (PTMA), or methylxylocholine (beta-TM 10], all previously shown to block the cholinergic receptor that mediates an increase in K conductance in the medial cells (see Kehoe, 1972b). Furthermore, in ganglia in which no slow e.p.s.p. was seen in response to firing of the neurones I, II, and III, such a response became manifest when agonists capable of activating the cholinergic receptor were applied (e.g. acetylcholine (ACh), carbachol, arecoline, or F2268). The slow e.p.s.p. thus appears to result from the reduction, induced by any one of three 'blocking neurones', of a cholinergically controlled K conductance. Finally, when presynaptic neurone I (the only neurone tested) was fired shortly before or during the activation of presynaptic neurone IV, previously shown to be cholinergic (Kehoe, 1972b), the K component of the cholinergic post-synaptic inhibitory potential was markedly reduced. The concentration at which a given agonist caused the manifestation of the synaptic diminution in K conductance (i.e. the slow e.p.s.p.) was found to be the same as that at which it caused a reduction in the synaptically activated, cholinergic, K-dependent conductance elicited by presynaptic neurone IV. Intracellularly injected adenosine 3',5'-cyclic monophosphate (cyclic AMP) imitated the effect of the 'blocking neurones' on the K conductance activated by bath-applied cholinomimetics. This effect was superimposed on a cyclic-AMP-induced, voltage-dependent inward current that disappeared when the cell was bathed in Na-free sea water, or when the extracellular Ca concentration was increased to 60 mM. The effect of cyclic AMP on the cholinergic K conductance remained even after this cyclic-AMP-activated inward current was eliminated.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterosynaptic facilitation in the giant cell of Aplysia.

1. Heterosynaptic facilitation, defined as an increase of the efficacy of synaptic transmission between a test interneurone and a post-synaptic neurone, produced by the stimulation of a separate pathway, was studied in the left pleural ganglion. The experimental procedure consisted of detecting the effects of a brief tetanus, applied to tentacular and tegumentary nerves, on the amplitude of monosynaptic and unitary post-synaptic potentials (p.s.p.s) recorded in the left giant cell and generated by stimulating the test interneurone every 10 sec. The membrane potential of the test interneurone was simultaneously recorded. 2. Following heterosynaptic stimulation, the amplitude of the test p.s.p. increased, after a delay of about 30 sec, up to 250% of its original size; this increase subsided after 2-3 min or more. 3. Only the interneurones producing in the giant cell the e.i.p.s.p. (excitatory-inhibitory post-synaptic potential) were affected by hetero-synaptic facilitation. Other interneuronal types showed no changes in their synaptic transmission on the giant cell after heterosynaptic stimulation. 4. Heterosynaptic stimulation did not produce either orthodromic or antidromic spikes in the test interneurones clearly indicating that facilitation of test p.s.p. did not result from increased spike activity in the test interneurone. 5. Often heterosynaptic facilitation of the test p.s.p. was observed due to spontaneous activity in the heterosynaptic pathway, demonstrating the normal occurrence of the phenomenon. 6. Iontophoretic injection of 5-HT at critical, presumably synaptic, sites in the neuropil, evoked a facilitation of the test p.s.p. similar to heterosynaptic facilitation. Only the e.i.p.s.p.s. were so affected by 5-HT. On the contrary, other p.s.p. types were depressed by 5-HT as a result of conductance changes in the left giant cells. 7. Both heterosynaptic facilitation and 5-HT facilitation were suppressed by the presence in the bath of 5-HT (10(-5) M) and of LSD-25 (3 X 10(-4) M). The action of injected 5-HT on the membrane conductance of the left giant cell was also depressed in the pressence of 5-HT in the bath, but was unaffected by LSD-25 (3 X 10(-4) M). 8. From the parallelism of properties of heterosynaptic and 5-HT facilitation, it is suggested that 5-HT is the probable transmitter mediating heterosynaptic facilitation. It seems likely that 5HT is released from the heterosynaptic pathway at the level of the synaptic ending of the test interneurone on to the giant cell and that it increases the efficacy of this synapse, probably acting on the quantity of synaptic transmitter liberated.

Facilitation of excitatory post-synaptic potentials in the giant cell in the left pleural ganglion of Aplysia californica

1. 1. Heterosynaptic facilitation (HSF) was found to begin to develop after a latency of between 1900 and 2000 msec after which the amplitude of HSF increased as a function of the prime test interval. 2. 2. Changes in the amplitude of the HSF could last as long as 42 hr. 3. 3. The HSF in the pleural giant cell was of greater amplitude than that in the abdominal giant cell.

A direct synaptic connexion between the left and right giant cells in Aplysia.

1. In Aplysia fasciata, intrasomatic stimulation of the giant cell (RGC) of the right upper quadrant of the abdominal ganglia is followed after a constant delay by the appearance of a synaptic potential recorded in the giant cell (LGC) of the left pleural ganglion.2. The synaptic potential recorded in the LGC soma has a biphasic form (hence biphasic post-synaptic potential or BPSP) consisting of a fast depolarizing phase of about 200-800 muV amplitude and 0.15-0.25 sec duration, followed by a slow hyperpolarizing phase of about 200-800 muV amplitude and 1-3 sec duration.3. During repetitive stimulation summation results in an over-all hyperpolarization at low frequencies (less than 5/sec) and an over-all depolarization for higher frequencies. Very high frequencies (25/sec) of stimulation of the RGC may elicit a spike in the LGC.4. Stimulation with two shocks showed increasing effects with shorter intervals on both the depolarizing and hyperpolarizing phase. These effects were progressive and there was no falling out of one of the two phases as might be expected if the BPSP was a composite of an inhibitory post-synaptic potential (IPSP) and an excitatory post-synaptic potential (EPSP).5. The effects of artificially imposed polarization of the LGC through a second micro-electrode suggest that the BPSP results from a chemical transmission mechanism for both its depolarizing and hyperpolarizing phases but electrical transmission cannot be excluded.6. Curare has no effect on the BPSP and thus excludes a cholinergic transmission mechanism. Chloride ions injected into the LGC soma do not appear to modify the BPSP and hence it is concluded that the hyperpolarizing phase is different from IPSPs of the same cell.7. No synaptic potential is recorded in the RGC following stimulation of the LGC, except in a single preparation in which the RGC soma was situated in the right pleural ganglion. In this case the synaptic potential recorded in both giant cells following stimulation of the other, was biphasic in form.8. It is concluded that the BPSP is a unitary monosynaptic potential which is a characteristic feature of the organization of these two giant cells.

Untersuchungen zur Spezifitätsfrage der heterosynaptischen Facilitation bei Aplysia californica

1. Heterosynaptic facilitation (H.S.F.) could be obtained in 10–15% of all tested nerve cells in the abdominal and left pleural ganglion of Aplysia californica.

Composite nature of the monosynaptic excitatory postsynaptic potential.

Composite nature of the monosynaptic excitatory postsynaptic potential.

Further Studies on the Electrophysiological Anatomy of the Left and Right Giant Cells in Aplysia

ABSTRACT Electrophysiological studies on cells of Aplysia central ganglia have become of general interest in recent years and have developed from work on the nature of integrative mechanisms (Arvanitaki & Chalazonitis, 1959; Tauc, 1955; Tauc & Hughes, 1963) to investigations of plastic change at a cellular level (Bruner & Tauc, 1966; Kandel & Tauc, 1965). Earlier work showed the range of phenomena found in a variety of cells, particularly in the abdominal ganglia, but research has now become more concentrated on certain cells which are readily identifiable in all preparations (Strumwasser, 1965 ; Hughes & Tauc, 1961 ; Kandel & Tauc, 1965). Of these cells, the large cell in the right upper quadrant, which is often called the giant cell, or right giant cell (RGC), has been used a great deal in studies on the basic phenomena of spike initiation in molluscan cells (Tauc, 1962). The anatomy of this cell was shown to be quite complex as a result of electrophysiological studies (Hughes & Tauc, 1963) which also revealed the presence of a comparable cell (LGC) on the left side of the C.N.S. Because of similarities in the general pattern of branching of these cells, they were considered homologous.