L. Hiripi

A comparison of four techniques for mapping the distribution of serotonin and serotonin-containing neurons in fixed and living ganglia of the snail,Lymnaea

SummaryThe distribution of serotonin and serotonin-containing neurons was studied in the ganglia of the CNS of the snailLymnaea stagnalis. Results of the application of three different labelling techniques on wholemount preparations were compared with each other and with the serotonin content of the ganglia, measured by high-performance liquid chromatography. Serotonin immunocytochemistry resulted in the highest number of labelled neurons, but the more recently developedin vivo method of 5,6- or 5,7-dihydroxytryptamine-induced pigmentation also proved to be a reliable technique for the visualization of serotonin-containing cell bodies. In comparison with these two techniques, the glyoxylic acid fluorescence method appeared to be less sensitive. The distribution and number of serotonin-containing neurons and biochemically measured serotonin in specific ganglia showed a close correlation. By combining the results of the three labelling techniques, a detailed map of serotonin-containing neurons was constructed, and this was compared with maps of identified neurons prepared from earlier electrophysiological studies. Previously described serotonergic neurons were consistently found, as well as several new serotonin-containirig cell types in the cerebral, visceral and parietal ganglia. A network of serotonin-containing inter- and intraganglionic axon tracts, and thin serotonergic fibres in the perineurium were also demonstrated. Thisin vivo andin vitro identification of serotonin-containing neurons will facilitate further neurophysiological analysis of serotonergic neural mechanisms inLymnaea.

Development of catecholaminergic neurons in the pond snail, Lymnaea stagnalis: I. Embryonic development of dopamine-containing neurons and dopamine-dependent behaviors

The embryonic development of the catecholaminergic system of the pond snail, Lymnaea stagnalis, was investigated by using chromatographic and histochemical methods. High performance liquid chromatography suggested that dopamine was the only catecholamine present in significant concentrations throughout the embryonic development of Lymnaea. Dopamine first became detectable at about embryonic stage (E) 15 (15% of embryonic development) and then increased in amount during early development to reach about 120–140 fmol per animal by around E40. Dopamine content remained stable during mid‐embryogenesis (E40–65), increased slowing for the next couple of days, and then increased rapidly to culminate at about 400 fmol per animal by hatching. The detection of aldehyde‐ and glyoxylate‐induced fluorescence and of tyrosine hydroxylaselike immunoreactivity indicated that the first catecholaminergic cells appeared in the late trochophore or early veliger stage of embryonic development (E32–35). The paired perikarya of these transient apical catecholaminergic (TAC) neurons were located beneath the apical plate, remained outside of the central ganglia during embryogenesis, and no longer contained detectable catecholamines close to hatching. TAC neurons bore cilia on the ends of short processes that penetrated the overlying epithelium; their long processes branched repeatedly under the ciliated apical plate. Several smaller catecholaminergic cells first appeared in the anterior margin of the foot at a stage when the embryos began to metamorphose from the veliger form (E55). Similar bipolar cells later appeared in the tentacle and lips. The axons of all of these small peripheral cells projected centrally and terminated within the neuropil of different central ganglia. Central catecholaminergic neurons, including RPeD1, differentiated only after metamorphosis was complete (E75). Development of locomotor, respiratory, and feeding behaviors correlated with maturation of catecholaminergic neurons, as indicated by histology and chromatography. J. Comp. Neurol. 404:285–296, 1999.

Fluorimetric determination of 5-hydroxytryptamine and catecholamines in the central nervous system and heart of Locusta migratoria migratorioides

Abstract 5-Hydroxytryptamine (5HT) was measured in the nervous and heart tissues of Locusta migratoria migratorioides in concentrations of 2·34 and 2·69 μg/g wet weight respectively. Among catecholamines, dopamine was found in the nervous tissue (1·31 μg/g wet weight) as well as in the heart (2·42 μg/g wet weight). Noradrenaline was present only in the brain, but in smaller amounts (0·24 μg/g wet weight) than dopamine. Adrenaline was found neither in the ganglia nor in the heart. On the basis of their occurrence in the CNS, 5HT, dopamine, and noradrenaline, and in the heart 5HT and dopamine can be regarded as transmitter or modulator substances.

Development of catecholaminergic neurons in the pond snail, Lymnaea stagnalis: II. Postembryonic development of central and peripheral cells

Catecholamines have long been thought to play important roles in different mollusc neural functions. The present study used glyoxylate‐ and aldehyde‐induced histofluorescence to identify central and peripheral catecholaminergic neurons in the snail Lymnaea stagnalis. The majority of these cells were also found to react to antibodies raised against tyrosine hydroxylase. A minority of the catecholaminergic neurons, however, exhibited no such immunoreactivity. The number of central catecholaminergic neurons nearly doubled (from about 45 to about 80 cells) during the first 2–3 days of postembryonic development. Thereafter, catecholaminergic neurons again doubled in number and generally grew by about 100–200% in soma diameter as the snails grew by 1,000% in overall linear measurements. In contrast to the relatively meager addition of central catecholaminergic neurons, several thousand catecholaminergic somata were added to different peripheral tissues during postembryonic development. These small, centrally projecting neurons were particularly concentrated in the lips, esophagus, anterior margin of the foot, and different regions of the male and female reproductive tracts. Chromatographic analyses indicated that dopamine was the major catecholamine present in the central ganglia, foot, and esophagus, although detectable levels of norepinephrine (approximately 20% of dopamine levels) were also found in the ganglia. The total content but not the concentration of dopamine increased within the tissue samples during postembryonic development. The companion study (Voronezhskaya et al. [1999] J. Comp. Neurol. 404:285–296) and the present study furnish a complete description of central and peripheral catecholaminergic neurons from their first appearance in early embryonic development to adulthood. J. Comp. Neurol. 404:297–309, 1999.

Membrane effects of toxins isolated from a cyanobacterium, Cylindrospermopsis raciborskii, on identified molluscan neurones.

The effect of anatoxin (ANTX), the crude extract (AlgTX) and purified fraction (F1) isolated from cyanobacterium C. raciborskii was studied on the neurones of two snail species. ANTX and AlgTX exerted excitatory, inhibitory and biphasic effects on the spontaneous activity of identified neurones. Both ANTX and AlgTX elicited an inward current, which could be decreased by curare or amiloride. On the contrary, F1 had no direct effect on the spontaneous activity; it was not able to induce conductance changes of the neuronal membrane, but it did antagonise the acetylcholine (ACh)-induced inward current. We concluded that ANTX affects the neuronal membrane of neurones acting on ACh receptors. The AlgTX had similar effects, and therefore the extract of C. raciborskii may contain an ANTX-like component. The purified fraction prolonged and decreased the ACh-elicited response, but had no direct membrane effect. We suggest, therefore, that both AlgTX and the purified fraction F1 interact with the ACh receptor, but they have different binding sites on the neuronal ACh receptor-ion channel complex. The possible neurotoxic effects of the C. raciborskii extract and F1 are demonstrated for the first time; the molecular mechanism of their action, however, remains to be elucidated.

Seasonal and activity-dependent changes of the serotonin level in the C.N.S. and heart of the snail (Helix Pomatia L.)

Abstract 1. There is seasonal variation in the 5 HT level of the central nervous system and heart of the snail, Helic pomatia L.: in active animals the 5 HT content is higher in the cerebral ganglia, auricle, and ventricle in the autumn than at spring, while this pattern is reversed in the suboesophageal ganglion. 2. Snails kept in dry conditions at room temperature fall into a state of rest. Under such conditions an increase of the 5 HT level was observed in the C.N.S. and heart both in the summer as well as in the winter; the question remains as to what results from the decrease of the 5 HT utilization during the resting state of the animal. 3. There is a different seasonal variation of the 5 HT level in snails kept in a refrigerator: during the first winter months the 5 HT level of the ganglia increases; this is followed by a decrease, while in the summer an initial decrease with subsequent increase is observed. The 5 HT content of the heart showed an initial decrease both during the winter and the summer. 4. Arousal of the snails from hibernation in winter causes a decrease of the serotonin level of the ganglia. On cooling these animals again, the 5 HT level increases only to a moderate degree. 5. The variations of the 5 HT level in snails can be considered partly as the consequence of the change of the activity, but on the other hand the serotonin level can play a role in the determination of the activity of the animal.

Dopamine and serotonin receptors mediating contractions of the snail, helix pomatia, salivary duct

The combination of high performance liquid chromatography, bioassay and immunocytochemistry was applied to study the regulation of the salivary duct muscle of the snail, Helix pomatia. The major function of the duct appears to be to propel the saliva toward the buccal cavity during feeding. It has been established that serotonin and dopamine applied exogenously mimic the effect on the duct exerted by the stimulation of the salivary nerve. Immunohistochemistry revealed the presence of serotonin, but not dopaminergic nerve elements in the nerve and along the duct surface. However, both serotonin (14.9-15.5 pmol/mg) and dopamine (0.38-0.58 pmol/mg), as well as the synthesizing enzymes (tyrozine hydroxylase 0.28 pmol/mg tissue/h and DOPA 0.32 nmol synthesized DA/mg tissue/h) could regularly be assayed in the salivary duct by high performance liquid chromatography. When released following the stimulation of the salivary nerve, both monoamines were shown to interact with distinct membrane receptors. Dopamine elicited a sustained increase of the muscle tone in concentration-dependent manner (K(d)=1.5 microM). Mammalian D(1) receptor antagonist flupenthixol and fluphenazine attenuated, whereas the D(1) receptor agonist SKF-38393 mimicked the effect elicited by exogenous dopamine. Serotonin had a double effect on the salivary duct: a relaxing and a contracting one with different K(d) values 76 nM and 2.4 microM, respectively. 5-HT(2) receptor antagonist ritanserin and ketanserin attenuated the serotonin-induced relaxation. In contrast 5-HT(3) antagonist metoclopramide and MDL2222 decreased and 5-HT(3) receptor agonist 1-(m-chlorophenyl)-biguanide mimicked the serotonin-induced contraction, suggesting that serotonin exerted its action on two different receptor subtypes. The release of radiolabeled serotonin and dopamine upon nerve stimulation was found to be Ca-dependent. Furthermore, the increase in serotonin concentration induced a decrease of the potency of dopamine to elicit sustained contraction. These results provide evidence for the transmitter role of serotonin and dopamine in salivary duct. It is concluded that receptors reveal a pharmacological profile related to vertebrate D(1), 5-HT(2) and 5-HT(3) receptor subtypes. Moreover, it was found that the process of conveying the saliva is modulated by an interaction of dopamine and serotonin.

Actions of methionine enkephalin and morphine on single neuronal activity in Helix pomatia L.

1. Methionine enkephalin and morphine accelerated the firing frequency of the cells in the right parietal (RPa2) and visceral (V) ganglia of Helix pomatia L. The effect of both agents was blocked by naloxone. 2. Naloxone blocked the inhibitory effect caused by dopamine application to the RPa2 cell. 3. Dopamine had a stimulatory effect on the neurosecretory cell (NS) which was not altered by naloxone pretreatments. 4. Naloxone specificity may be extended to a type of dopamine receptor. 5. In Helix pomatia L. an opiate receptor with similar pharmacological properties to that reported in mammals exists.

The organization of serotonin-, dopamine-, and FMRFamide- containing neuronal elements and their possible role in the regulation of spontaneous contraction of the gastrointestinal tract in the snail Helix pomatia

SummaryThe distribution of serotonin-, tyrosine hydroxylase-, and FMRFamide-immunoreactive neuronal elements, as well as the concentrations of serotonin and dopamine in the different parts of the gastrointestinal tract, were studied in the snail Helix pomatia. The sensitivity of the spontaneous contractions of the alimentary tract to serotonin, dopamine, and FMRFamide was also tested. Serotonin-, tyrosine hydroxylase-, and FMRFamide-immunoreactive elements could be demonstrated in each part of the gastrointestinal tract, but they showed different innervation patterns. Serotonin- and tyrosine hydroxylase-immunoreactive elements were dominant in the submucosal layer, whereas FMRFamide-immunoreactive elements were dominant in both the mucosal and submucosal layers. Tyrosine hydroxylase-immunoreactive elements were confined to the longitudinal muscle trabeculae of submucosa, whereas serotonin-immunoreactive elements were distributed throughout the submucosal layer. No serotonin-immunoreactive cell bodies, but only fibers, could be detected in the gastrointestinal tract, and therefore they represent extrinsic elements. Tyrosine hydroxylase- and FMRFamide-immunoreactive cell bodies represent intrinsic elements of the tract. The occurrence and density of the serotonin- and tyrosine hydroxylase-immunoreactive elements showed significant differences in the different parts of the alimentary tract, in accordance with HPLC assays, which revealed a significant frontocaudal decrease in both the serotonin (from 2.11 to 1.21 pM/mg) and dopamine (from 3.28 to 0.52 pM/mg) contents of the different parts of the alimentary tract. Dopamine at 10-5 M concentration proved to be effective only on the longitudinal muscles by increasing the tone and frequency of contractions, but was ineffective on the circular muscles. Serotonin affected both the longitudinal and circular muscles. Serotonin at 10-5 M concentration decreased the tone and increased the frequency of low-amplitude contractions of the longitudinal muscles of the esophagus and the gizzard but increased both the tone and frequency of the crop. Serotonin at 10-9 M concentration slightly decreased the tone and blocked the contractions of the circular muscles in the crop but at 10-5 M concentration induced contractions of the circular muscles in the gizzard. FMRFamide at 10-6 M concentration decreased the tone and was shown to block the contractions of both the longitudinal and circular muscles.

PACAP has anti-apoptotic effect in the salivary gland of an invertebrate species, Helix pomatia.

Pituitary adenylate cyclase activating polypeptide (PACAP) shows a remarkable sequence similarity among species and several studies provide evidence that the functions of PACAP have also been conserved among vertebrate species. Relatively little is known about its presence and functions in invertebrates. The aim of the present study was to investigate whether the well-known anti-apoptotic effect of PACAP can also be demonstrated in invertebrates. This effect was studied in the salivary gland of a molluscan species, Helix pomatia. In this work, we first showed the presence of PACAP-like immunoreactivity in the Helix salivary gland by means of immunohistochemistry. Radioimmunoassay measurements showed that PACAP38-like immunoreactivity dominated in the salivary gland of both active and inactive snails and its concentration was higher in active than in inactive animals in contrast to PACAP27-like immunoreactivity, which did not show activity-dependent changes. PACAP induced a significant elevation of cAMP level in salivary gland extracts. Application of apoptosis-inducing agents, dopamine and colchicine, led to a marked increase in the number of terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells in the salivary gland, which was significantly attenuated by PACAP treatment. In a similar manner, the number of caspase-positive cells was reduced after co-application of dopamine and PACAP. Taken together, the data indicate that PACAP activates cAMP in a molluscan species and we show, for the first time, that PACAP is anti-apoptotic in the invertebrate Helix pomatia.