Suboesophageal Ganglia Of Helix Aspersa Research Articles

Structure–activity and possible mode of action of S-Iamide neuropeptides on identified central neurons of Helix aspersa

Intracellular recordings were made from identified neurons from the suboesophageal ganglia of Helix aspersa. The inhibitory action of nine S-Iamide peptides was investigated. Structure–activity studies suggest that all act through a common receptor, which normally requires FVRIamide at the C terminal, with a preferred length of seven amino acids. Substitution at the N-terminal with alanine (A), threonine (T), proline (P) or leucine (L) results in little change in potency, suggesting the N-terminal requirements are relatively flexible. Ion substitution experiments suggest that potassium is the main ion involved in the inhibitory response to S-Iamide application. Studies using a range of compounds, which modify second messenger systems, would suggest that S-Iamide peptides may interact with adenylate cyclase. No evidence was found for an interaction with either guanylate cyclase or nitric oxide synthase.

Morphological and electrophysiological features of F76 and D1 neurones of the sub-oesophageal ganglia of Helix aspersa in vitro and in culture.

Identified neurones F76 and D1 of the suboesophageal ganglia of Helix aspersa were studied in the isolated ganglia in vitro and in culture. The neurones were examined electrophysiologically with current clamp and morphologically either with intracellular injections of Lucifer Yellow or biocytin. These nerve cells had very similar resting membrane potentials and responses to injected current. The projections of D1 and F76 have been characterised, with both neurones having two main axons. The F76 neurones project to the left pallial, right pallial, anal, and visceral nerves as well as to the left and right pleural ganglia. The D1 neurones have similar projections except that they do not project to the anal and visceral nerves. The bilateral symmetry to the pallial nerves and pleural ganglia is discussed. These cells were also studied electrophysiologically after mechanical isolation and culture. F76 and D1 neurones were separated by dissection (no enzymes) and cultured in three ways. In normal snail Ringer they remained viable for up to two weeks with no development. In Ringer preincubated with a ganglia or containing endothelial growth factor, neurite outgrowths were seen. Membrane potentials were significantly lower in cultured neurones than in vitro and the after hyperpolarization never went below resting in cultured cells but it did in vitro.

Cell volume changes upon sodium pump inhibition in Helix aspersa neurones.

1. Identified neurones of the suboesophageal ganglia of Helix aspersa were loaded with tetramethylammonium (TMA+). Experimentally induced changes in cell water volume and membrane potential were measured continuously by monitoring changes in intracellular [TMA+] using ion-sensitive double-barrelled microelectrodes. The technique allowed measurements of cell water volume changes of less than 5%. 2. Exposure to hyperosmotic (up to +24%) or hyposmotic (up to about -10%) solutions caused reversible decreases and increases in cell water volume respectively, which agreed with near-ideal osmometric behaviour. Upon exposure to hyposmotic solutions whose osmolality was decreased by 30-40%, the cell water volume increased to maximum values below those expected for ideal osmometric behaviour and exhibited partial regulatory volume decrease. 3. The sodium pump was inhibited in twenty identified neurones by sustained exposure to 1 mM ouabain. In every case ouabain caused cell membrane depolarization, as expected for inhibition of an electrogenic sodium pump. 4. Upon pump inhibition most cells (n = 14) shrank by up to 13% of their initial water volume. In five of these cells, shrinkage was preceded by one or more short-lived swelling phases. In two other neurones short-lived swelling was followed by cell volume recovery without appreciable shrinkage. In four out of the twenty cells, there were no measurable volume changes. 5. The lack of an initial swelling phase in the cells that shrank, as well as the absence of detectable volume changes in some of the neurones, was not due to loss of ion-selective electrode sensitivity since predictable changes in cell volume elicited by osmotic challenges were monitored in the same cells. 6. It is concluded that neurones can be endowed with ouabain-insensitive mechanisms of volume control, whose activation following Na+ pump inhibition prevents them from short-term swelling and lysis.

An initial screen of a series of neuroactive peptides for activity on identified central neurones of Helix aspersa

1. Intracellular recordings were made from identified neurones in the suboesophageal ganglia of Helix aspersa. Seven neuropeptides were tested for activity and their actions compared with acetylcholine and FMRFamide. 2. Three peptides isolated from nematodes, AF-1, AF-2 and PAN-1 had mainly inhibitory effects with thresholds of around 1 nM. This inhibition was due to an increase in potassium conductance. 3. The molluscan neuropeptides LSSFVRIamide, CARP and ACEP-1 were all active on certain neurones; the first two showed only inhibitory effects while ACEP-1 was mainly excitatory. The thresholds in each case were 0.1–10 μM. When norleucine replaced methionine in CARP, the potency was reduced by at least 100 times. 4. The echinoderm peptide, SALMF-1, only excited neurones but with a very low threshold, around 1.0 fM. 5. There was no obvious correlation between the action of these peptides and either acetylcholine or FMRFamide.

The actions of phorbol esters upon isolated calcium currents of Helix aspersa neurones

1. 1. Voltage-clamp recordings have been made from identified neurones in the suboesophageal ganglia of Helix aspersa. 2. 2. Calcium currents were isolated pharmacologically and studied under two-electrode voltage-clamp. 3. 3. PdBu but not PMA caused a transient enhancement followed by an irreversible inhibition of the calcium current. 4. 4. It is concluded that activation of protein kinase C within these neurones may show selectivity with respect to the exogenous activator used.

Localization of ARG-vasopressin-like material in central neurones and mechanism of action of ARG-vasotocin on identified neurones of the snail Helix aspersa

Immunocytochemical and electrophysiological studies were made on neurones in the suboesophageal ganglia of Helix aspersa. A group of neurones in the anterior dorsal surface of each parietal ganglion showed positive immunoreactivity to an antibody to arginine vasopressin (AVP), as did the neuropile and the nerves associated with the parietal and visceral ganglia. The only peripheral tissue to exhibit positive immunoreactivity to AVP was the optic tentacle, where positive immunoreactive fibres were present in the optic nerve and cell bodies below the retinal cell layer. Two central neurones, Fl and E13 were excited by arginine vasotocin (AVT), threshold 1–10 nM, and the ionic mechanism associated with this response was investigated. This excitation involved both sodium and calcium ions with an overall increase in resistance. In low sodium Ringer, the AVT response was greatly reduced, while in calcium-free 3 mM cobalt Ringer, the response was potentiated. Changing the external concentrations of potassium and chloride ions had no apparent effect on the response. The amplitude of the excitation induced by AVT was enhanced when the cells were hyperpolarized. Most of the other neurones tested failed to respond to AVT though the excitatory response of certain cells to acetylcholine was enhanced by 0.1 μM AVT. Excitation by 5-hydroxytryptamine was unaffected in the cells tested. This study provides further evidence for a role for an AVT-like peptide in the nervous system of Helix.

Comparison of the effects of FMRF-amide and pQDPFLRF-amide on identified Helix neurons

1. 1. Intracellular recordings were made from a selection of identified neurons in the sub-oesophageal ganglia of Helix aspersa, and their responses to the molluscan neuropeptides FMRF-amide and pQDPFLRF-amide compared. 2. 2. While FMRF-amide excited certain neurons and inhibited others, pQDPFLRF-amide did not produce excitation in any of the cells tested. A few cells were unresponsive to pQDPFLRF-amide, but most were inhibited with varying potency. 3. 3. FMRF-amide was generally 10–100 times more potent than pQDPFLRF-amide, but one cell, E13 in the visceral ganglion, showed equal sensitivity to the two peptides. 4. 4. On most cells there was no clear evidence for cross desensitization between the two peptides, although it is possible that in some cases where both peptides are inhibitory they may be acting on a single receptor.

Intracellular free magnesium in neurones of Helix aspersa measured with ion-selective micro-electrodes.

Cytoplasmic free Mg2+ concentration, [Mg2+]i, was measured in identified neuronal cell bodies of the suboesophageal ganglia of Helix aspersa, using Mg2+-selective micro-electrodes. In calibration solutions, the electrodes showed significant interference from K+, but not from Na+, or Ca2+, at concentrations found intracellularly. Therefore, in order to calibrate the electrodes properly, it was necessary first to obtain an accurate value for intracellular free K+ concentration [( K+]i). The mean value for [K+]i was 91 mM (S.E. of the mean +/- 2.2 mM, n = 8), measured with K+-sensitive 'liquid ion exchanger micro-electrodes'. In seven experiments, which met stringent criteria for satisfactory impalement and electrode calibration, the mean [Mg2+]i was 0.66 mM (S.E. of the mean +/- 0.05 mM). The mean [Mg2+]i in cells that had spontaneous spike activity was not significantly different from that in quiescent cells. If Mg2+ was in electrochemical equilibrium, the ratio [Mg2+]i/[Mg2+]o would be about 55. Mg2+ is therefore not passively distributed across the neuronal membrane and an outwardly directed extrusion mechanism must exist to keep [Mg2+]i low and constant, even in cells undergoing spike activity.

Free calcium ions in neurones of Helix aspersa measured with ion-selective micro-electrodes.

1. Intracellular free calcium concentration, [Ca2+]i, was measured in giant neurones of the sub-oesophageal ganglia of Helix aspersa, using Ca-selective micro-electrodes containing a PVC-gelled, neutral-ligand sensor. 2. In calibration solutions the electrodes had a virtually ideal, Nernstian, response down to 1 microM-Ca2+ in the presence of 0.125 M-K+, 18-24 mV from 1 to 0.1 microM-Ca2+ and 8-14 mV from 0.1 to 0.01 microM-Ca2+. Interference from H+ and Mg2+ was negligible. The small response to Na+ at sub-micromolar Ca2+ was taken into account, when necessary, in measurement of [Ca2+]i. 3. Measurements of basal [Ca2+]i were made in ganglia from animals kept only a few weeks in captivity, in a bathing solution equilibrated with air and containing 2 mM-Ca2+. In thirteen measurements from impalements which met stringent criteria for electrode performance and cell viability, the mean basal pCa (--log10[Ca2+]) was 6.77 +/- 0.07 (S.E.), corresponding to a mean free Ca2+ concentration of 0.17 microM. 4. The basal [Ca2+]i in neurones from a group of snails kept hibernating for several months was higher, mean pCa 6.15, for ganglia handled in 2 mM-Ca2+ solution. 5. Intracellular injections of Ca2+ or EGTA raised and lowered, respectively, the indicated basal [Ca2+]i, showing that the electrodes responded appropriately inside the cells and that unknown or untested components of cytoplasm were not significantly interfering with the Ca-sensor. 6. Altering the external Ca2+ concentration between 0.1 and 10 mM usually produced only small, +/- 0.1 pCa units, changes in basal [Ca2+]i of satisfactorily impaled, quiescent cells. 7. In cell 1F, which has repetitive spikes with a substantial Ca current, changes in Ca gradient or blockade of voltage-dependent Ca channels sometimes markedly altered [Ca2+]i, showing that Ca entry with the spikes was elevating [Ca2+]i. 8. Replacing external Na+ with Li+ or bis(2-hydroxyethyl)dimethylammonium had little effect on [Ca2+]i. 9. Elevating CO2 to 5% or 79% lowered [Ca2+]i by an average of 0.16 and 0.26 pCa units respectively.

The pA 2 values of cholinergic antagonists on identified neurons of the snail Helix aspersa

1. Seven identified neurons in the suboesophageal ganglia of Helix aspersa have been studied. 2. These neurons respond to bath and iontophoretic application of ACh. The cells were classified into D (depolarized-excited) and H (hyperpolarized-inhibited) by ACh. 3. Comparison of pD 2 values within each group of ACh-sensitive cells showed that the affinity of the silent cells to ACh is greater than that of the active cells showing action potentials (see Table 2). 4. The ACh H receptors (Cl′-mediated response) were similar to the nicotinic receptors found in the mammalian neuromuscular junction. 5. The antagonists studied had the following pA 2 values for H receptors. Decamethonium 5.2; d-tubocurarine 5.0; succinylcholine 4.4; pancuronium 4.2; atropine < 3; hexamethonium 0; pentolinium 0. 6. The ACh D receptors (Na +-mediated response) were similar to the nicotinic receptors of the mammalian autonomic ganglia. 7. The antagonists studied had the following pA 2 values for D receptors: pentolinium 5.3; hexamethonium 5.0; pancuronium 4.3; d-tubocurarine 4.0.

Structure-activity studies on glutamate receptor sites of three identifiable neurones in the sub-oesophageal ganglia of Helix aspersa

1. 1. The effect of glutamate analogues was investigated on three identifiable cells in the sub-oesophageal ganglia of the common garden snail, Helix aspersa. 2. 2. The three cells gave different responses to glutamate. Cell E4 gave a biphasic response—hyperpolarization (H response) followed by a depolarization (D response); cell F30 was depolarized whereas cell F1 gave only a hyperpolarization. 3. 3. The equipotent molar ratios (epmrs) of a range of glutamate analogues, as compared with glutamate, were calculated. 4. 4. D-Glutamate was found to be eight to ten times less potent than L-glutamate on all three cells whereas DL-glutamate was equipotent with L-glutamate. 5. 5. Substitution on the α or β carbon of glutamate reduced the activity more than did substitution on the γ carbon. 6. 6. In general, the receptor sites were able to accommodate an increase or reduction of one carbon in the glutamate carbon chain without too severely affecting the glutamate activity. 7. 7. The results support the hypothesis that the glutamate receptors require three ionically charged moieties. The evidence also suggests there are two different receptors: an excitatory receptor with the preferred glutamate conformation folded to resemble L-aspartic acid and an inhibitory receptor for which the preferred model for glutamate activity resembles the rigid structure of Ibotenic acid.

Mapping of nerve cells in the suboesophageal ganglia of Helix aspersa

Abstract o 1. More than 100 different neurones have been investigated in three suboesophageal ganglia (left parietal, visceral and right parietal ganglia) of the snail Helix aspersa . 2. Diagrams and photographs of the ganglia are presented so that the cells may be identified and localized. 3. The responses following stimulation of the main peripheral nerve trunks are analysed and described, in terms of EPSP, IPSP and antidromic potentials. 4. The cells' responses to iontophoretic application of acetylcholine, dopamine, 5-HT and glutamate are described. The majority of responses to the drugs are as follows: H to ACh; H to dopamine; H to 5-HT and D to glutamate. 5. The anatomical position, spontaneous electrical activity and responses to stimulation of the main peripheral nerves and drug application make it possible to identify specific nerve cells from preparation to preparation. 6. The axons from some cells have been traced following intracellular injections of CoCl 2 and precipitation of CoS. This supported the electrophysiological method of tracing axons by recording antidromic spikes in the cell body following stimulation of a peripheral nerve. 7. It is suggested that some of the nerve cells will be homologous with other nerve cells throughout the Gastropod Molluscs.

Action potential shape and frequency as criteria for neuron identification in the snail, Helix aspersa

1. 1. Intracellular recordings were made from 24 identifiable cells in the suboesophageal ganglia of Helix aspersa. 2. 2. The action potential shape for each cell was analysed; action potential duration ranged from 3 to 20 mseconds; positive after potential values ranged from 2 to 30 mV. 3. 3. The frequency and firing patterns of the cells' action potentials were also analysed. The spontaneous firing rate ranged from 3–4 per second to zero but was constant for a given cell. 4. 4. It is suggested that, in Helix, neuron action potential shape and firing pattern are useful criteria in cell identification.