Localization of connexins in neurons and glia cells of the Helix aspersa suboesophageal brain ganglia by immunocytochemistry

The aim of the present study was to examine the distribution of cells expressing connexin 26 (Cx26) in the suboesophageal visceral, left and right parietal and left and right pleural ganglia of the snail Helix aspersa by immunocytochemistry. Altogether we have found approximately 452 immunoreactive neurons which represent the 4.7% of the total neurons counted. The stained large neurons (measured diameter 55-140 microm) occurred mostly on the peripheral surface of the ganglia while the small immunostained cells (5-25 microm diameter) were observed in groups near the neuropil. The number of large neurons giving positive Cx26-like immunostaining was small in comparison with that for medium (30-50 microm diameter) and small sized cells. The expression of Cx26 was also observed in the processes of glia cells localized among neurons somata and in the neuropil showing that the antiserum recognized epitopes in both protoplasmic and fibrous glia cells of Helix aspersa. The neuropils of all ganglia showed fibers densely immunostained. While we have observed a good specificity for Cx26-antiserum in neurons, a lack of reaction for Cx43 antiserum was observed in neurons and glia cells. The reaction for enolase antiserum in neurons was light and non-specific and a lack of reaction in glia cells and processes for GFAP antiserum was observed. Although the percentage of positive neurons for Cx26 antiserum was low is suggested that in normal physiological conditions or under stimulation the expression of connexin could be increased. The observed results can be considered of interest in the interpretation of Helix aspersa elemental two neuron networks synchronizing activity, observed under applied extremely low frequency magnetic fields.     

Actions of the molluscan neuropeptide FMRF-amide on neurones in the suboesophageal ganglia of the snail Helix aspersa

The effects of the molluscan neuropeptide FMRF-amide were tested on several neurones in the suboesophageal ganglia of the snail Helix aspersa. Almost all neurones tested responded to the peptide, some being hyperpolarized (H response) and others depolarized (D response). The H response is due primarily to an inward potassium current and may be blocked in 20 microM 4-aminopyridine. The hyperpolarizing actions of FMRF-amide and dopamine may be separated by ergometrine which blocks the response to dopamine but not to FMRF-amide. The D response is due mainly to an inward sodium current but this is not blocked by d-tubocurarine, morphine or TTX. It appears to be mediated by a distinct receptor/ionophore as excitation by ACh and 5-HT are both antagonized by d-tubocurarine. The Leu2-substituted analogue FLRF-amide was found to produce similar H responses to FMRF-amide, but was much less potent at producing D responses. It did, however, produce cross-desensitization of the D response to FMRF-amide, suggesting that it does bind to the FMRF-amide receptor.     

Morphological and electrophysiological mapping of giant neurons in the suboesophageal ganglia of Helix pomatia

1. To reveal the morphology of the suboesophageal ganglia of Helix pomatia, the connective tissue was completely removed and the preparations stained whole-mount with methyl green-pyronin G to display the relative locations of the neurons. 2. Fifteen large cells which could be recognized as individuals in at least 75% of the preparations investigated, were morphologically identified by size, position and color. 3. The cells were electrophysiologically characterized with respect to spontaneous activity, synaptic input from peripheral nerves, and response to application of drugs (e.g. ACh, DA and 5-HT). 4. The peripheral axonal projections of eight of the major identified cells were investigated by intracellular CoCl2 injection and by cobalt backfilling of the peripheral nerves.     

Specific insulin binding sites in snail (Helix aspersa) ganglia

1. Insulin binding sites were characterized and quantified in snail (Helix aspersa) ganglia by incubation of tissue sections with 125I-porcine insulin, autoradiography with [3H]Ultrofilm, image analysis coupled to computer-assisted microdensitometry, and comparison with 125I-standards. Cellular localization was performed in the same sections by emulsion autoradiography. 2. Specific insulin binding sites were demonstrated in discretely localized groups of neurons of the cerebral, pleural, parietal, visceral, and pedal ganglia and in nerves. Scatchard analysis performed with consecutive sections from single animals revealed a single class of high-affinity insulin binding sites (Kd, 0.13 +/- 0.01 nM; Bmax, 157 +/- 10 fmol/mg protein). 3. Our results suggest that insulin may play a role as a neurotransmitter or neuromodulator in snail ganglia.     

IR-Met and IR-Leu-enkephalin content in the perioesophageal ganglia of Helix aspersa seasonal variations

1. In order to determine the presence of opioid peptides and their possible variation throughout the year in the different ganglia of the perioesophageal ring of the terrestrial snail Helix aspersa, radioimmunoassays with highly specific antisera risen for Met-enkephalin (ME) and Leu-enkephalin (LE) were carried out monthly.2. Clear monthly variations in both IR-enkephalins' concentrations were found, following a seasonal trend. IR-LE concentrations were higher than IR-ME throughout the year in all the regions, however, no clear regional differences were found for each.3. IR-LE/IR-ME ratios presented denned seasonal peaks for each region.     

The ionic mechanism associated with the action of putative transmitters on identified neurons of the snail, Helix aspersa

Intracellular recordings were made from identified neurons in the suboesophageal ganglionic mass of the snail, Helix aspersa. The ionic mechanisms associated with acetylcholine excitation and inhibition, dopamine excitation and inhibition, gamma-aminobutyric acid (GABA) excitation and inhibition and serotonin excitation were investigated. Acetylcholine excitation was found to involve an initial increase in sodium conductance while acetylcholine inhibition was a pure chloride event which reversed at membrane potentials more negative than the chloride equilibrium potential. Dopamine excitation appeared to involve only an increase in sodium conductance while serotonin excitation involved an increase in conductance to both sodium and calcium ions. Dopamine inhibition was associated with an increase in potassium conductance but failed to reverse at membrane potentials more negative than the potassium equilibrium potential. GABA excitation involved conductance increases to both sodium and chloride ions while GABA inhibition was a pure chloride event. An attempt was made to estimate the degree of co-operativity of the putative transmitters with their receptors using log-log and Hill plots. The slopes of the line for the log-log plots for acetylcholine excitation and inhibition were 0.88 and 1.1, respectively, suggesting the interaction of one molecule of acetylcholine with the receptor. The slope of the log-log plot for dopamine inhibition was 0.46 while that for serotonin excitation was 0.75. The Hill plots for GABA excitation and inhibition were 1.64 and 1.42, respectively, suggesting that two molecules of GABA are required for receptor activation.     

IR-Met and IR-leu-enkephalin content in the perioesophageal ganglia of Helix aspersa seasonal variations.

1. In order to determine the presence of opioid peptides and their possible variation throughout the year in the different ganglia of the perioesophageal ring of the terrestrial snail Helix aspersa, radioimmunoassays with highly specific antisera risen for Met-enkephalin (ME) and Leu-enkephalin (LE) were carried out monthly. 2. Clear monthly variation in both IR-enkephalins' concentrations were found, following a seasonal trend. IR-LE concentrations were higher than IR-ME throughout the year in all the regions, however, no clear regional differences were found for each. 3. IR-LE/IR-ME ratios presented defined seasonal peaks for each region.     

FMRFamide receptors in Helix aspersa

An in vitro receptor binding assay and an isolated heart bioassay were developed and used to characterize the structure-activity relations (SAR) of FMRFamide receptors in a land snail, Helix aspersa. In the radioreceptor assay, binding of ¹²⁵I-desaminoTyr-Phe-norLeu-Arg-Phe-amide (¹²⁵I-daYFnLRFamide) at 0°C to Helix brain membranes was reversible, saturable, and specific, with a K_D of 14 nM and a B_max of 85 fmol/mg brain. A lower affinity site was also observed (K_D=245 nM; B_max=575 fmol/mg brain). In the heart bioassay, daYFnLRFamide and other FMRFamide analogs increased myocardial contraction force. The SAR of cardiostimulation correlated with the specificity of high affinity ¹²⁵I-daYFnLRFamide binding to brain and heart receptors. The SAR was also similar to that described for other molluscan FMRFamide bioassays, except for a marked preference for N-blocked analogs. Peptides with N-terminal extensions of desaminoTyr, Tyr, Tyr-Gly-Gly, and acetyl, exhibited the highest potency in both radioligand displacement and cardiostimulation. The endogenous Helix heptapeptide analogs of FLRFamide (pQDP-, NDP-, and SDP-FLRFamide) were stimulatory on the heart at low doses, but were inhibitory at moderate to high doses. These peptides were 20 times weaker than FMRFamide in both the brain and heart receptor binding assays, with IC₅₀s about 10 μM. The results suggest that the effects of FMRFamide in Helix are receptor-mediated, and that the heptapeptides do not interact at FMRFamide receptors.     

Demonstration of the presence and localization of the synthesis of a somatostatin-like substance by immunocytochemistry and hybridization in situ in the brain of Helix aspersa]

The immunocytological method has revealed the presence of somatostatin-like substance (SSI) in the brain of the snail Helix aspersa Müller. The Cerebral Green Cells (CeGC) in the mesocerebron and some neurons in parietal and visceral ganglia react positively with an antibody raised against Vertebrate somatostatin-14. The hybridization in situ with an oligonucleotide probe labelled with 35S-dATP complementary to the 3'-coding region of rat preprosomatostatin mRNA seems to show a colocalization between synthesis and stocking sites of SSI in the nervous ganglia. These results suggest for the first time that the codage of a SSI seems to be realized in the same way in Helix aspersa and Mammals.     

Intracellular pH regulation in neurons from chemosensitive and nonchemosensitive regions of Helix aspersa

We used 2',7'-bis(carboxyethyl)-5(6)-carboxyflourescein (BCECF), a pH-sensitive fluorescent dye, to study intracellular pH (pH(i)) regulation in neurons in CO(2) chemoreceptor and nonchemoreceptor regions in the pulmonate, terrestrial snail, Helix aspersa. We studied pH(i) during hypercapnic acidosis, after ammonia prepulse, and during isohydric hypercapnia. In all treatment conditions, pH(i) fell to similar levels in chemoreceptor and nonchemoreceptor regions. However, pH(i) recovery was consistently slower in chemoreceptor regions compared with nonchemoreceptor regions, and pH(i) recovery was slower in all regions when extracellular pH (pH(e)) was also reduced. We also studied the effect of amiloride and DIDS on pH(i) regulation during isohydric hypercapnia. An amiloride-sensitive mechanism was the dominant pH(i) regulatory process during acidosis. We conclude that pH(e) modulates and slows pH(i) regulation in chemoreceptor regions to a greater extent than in nonchemoreceptor regions by inhibiting an amiloride-sensitive Na(+)/H(+) exchanger. Although the phylogenetic distance between vertebrates and invertebrates is large, similar results have been reported in CO(2)-sensitive regions within the rat brain stem.     

Histochemical localisation of FMRFamide-gated Na+ channels in Helisoma trivolvis and Helix aspersa neurones

FMRFamide-gated Na⁺ channels of molluscan neurones belong to the ENa/Deg family of channels which have diverse functions. FMRFamide (Phe-Met-Arg-Phe-NH₂) Na⁺ channels were detected electrophysiologically in specified neurones of Helix (Helix aspersa) and Helisoma (Helisoma trivolvis), and clones (FaNaCs) subsequently identified. We have now made a study to determine the distribution of mRNA for the clones HaFaNaC (Helix) and HtFaNaC (Helisoma) in the nervous systems of these species using standard in situ hybridization techniques. Immunohistochemical experiments were also made using an HtFaNaC antibody to detect the channel protein in Helisoma neurones. Many neurones in the central ganglia, including those which exhibit the FMRFamide Na⁺ current, stained for FaNaC-mRNA, suggesting a much wider distribution of the channel than was indicated by the earlier work. An immunoreactive response to the channel antibody was also observed in some Helisoma neurones, such as the giant dopamine neurone of the left pedal ganglion, also shown to possess HtFaNaC-mRNA and to exhibit the FMRFamide Na⁺ current. Taken together, these experiments suggest that the clones HaFaNaC and HtFaNaC are major, if not the only, subunits of the FMRFamide-gated Na⁺ channel detected electrophysiologically in the identified neurones of these species. However, fewer neurones in Helisoma reacted with the HtFaNaC-antibody than those which exhibited message for the channel. This discrepancy may be due to a difference in sensitivity of the two techniques, or because not all of the channel mRNA is normally expressed as a membrane protein.     

Nucleotidase activities in membrane preparations of nervous ganglia and digestive gland of the snail Helix aspersa

Nucleotide-metabolizing enzymes play an important role in the regulation of nucleotide levels. In the present report, we demonstrated an enzyme activity with different kinetic properties in membrane preparations of the nervous ganglia and digestive gland from Helix aspersa. ATPase and ADPase activities were dependent on Ca2+ and Mg2+ with pH optima approximately 7.2 and between 6.0 and 8.0 in digestive gland and nervous ganglia, respectively. The enzyme activities present in membrane preparations of these tissues preferentially hydrolyzed triphosphate nucleotides. In nervous ganglia, the enzyme was insensitive to the classical ATPases inhibitors. In contrast, in digestive gland, N-ethylmaleimide (NEM) produced 45% inhibition of Ca(2+)-ATP hydrolysis. Sodium azide, at 100 microM and 20 mM, inhibited Mg(2+)-ATP hydrolysis by 36% and 55% in digestive gland, respectively. The presence of nucleotide-metabolizing enzymes in these tissues may be important for the modulation of nucleotide and nucleoside levels, controlling their actions on specific purinoceptors in these species.     

On the regeneration of the eye in Helix aspersa and Cryptomphallus aspersa

Summary The distal half of the posterior tentacle of adult Helix aspersa and Cryptomphallus aspersa was removed and the proximal half was studied with light and electron microscopy after different intervals. The tentacle itself does not regenerate, but the receptor organs at the distal end of the normal tentacle differentiate at the level of the section. The newly formed eye is smaller than the control; however, its components and subcellular characteristics resemble those of the normal eye.Work supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina, and from National Institutes of Health (2 RO1 NS 06953-07 NEUA) USA.We are greatly indebted to Miss Margarita López for her skilful technical assistance and to Mr. Alberto Saénz for the electron micrographs.     

Mechanisms of up-regulation of single calcium channels by serotonin in Helix pomatia neurons

Action of serotonin (5-HT) on single Ca(2+) channel activity was studied in identified neurons of snail Helix pomatia. Only one type of Ca(2+) channels of 5 pS unitary conductance was determined under patch-clamp cell-attached mode. Kinetic analysis have shown a monotonically declining distribution of channel open times (OT) with mean time constant of 0.2 ms. The distribution of channel closed times (CT) could be fitted by double-exponential curve with time constants 1 and 12 ms. We established that 5-HT acts on Ca(2+) channel activity indirectly via cytoplasm. 5-HT prolonged the OT (up to 0.3 ms) and shortened the CT proportionally for both constants to 0.4 and 6 ms correspondingly. A conclusion is made that enhancement of Ca(2+) macro-current by 5-HT is determined by kinetic changes, increase of the number of active channels, and increase of the probability of OT. At the same time the transmitter did not affect the unitary channel conductance.