Dopamine-immunoreactive neurones in the central nervous system of the pond snail Lymnaea stagnalis.

The distribution of dopamine and dopamine-immunoreactive neurones was studied in the central nervous system of the snail Lymnaea stagnalis. The results from immunocytochemical labelling were compared with those from the application of the glyoxylic acid fluorescence method and 6-hydroxydopamine-induced pigment labelling. Comparisons were also made between the number of dopamine immunoreactive neurones and the dopamine content of the ganglia, measured by high-performance liquid chromatography. Dopamine immunocytochemistry proved to be superior to the other two histochemical techniques in terms of specificity and sensitivity. The 6-hydroxydopamine-induced pigment labelling failed to prove a useful tool for the in vivo identification of all dopamine-containing neurones. The distribution and number of dopamine-immunoreactive neurones and levels of biochemically measured dopamine in specific ganglia showed a close correspondence. By using the results of the dopamine immunocytochemistry and glyoxylic acid technique, a detailed map of dopamine-containing neurones was constructed. Dopamine-containing inter- and intra-ganglionic axon tracts were also demonstrated. The mapping of dopamine-containing neurones will facilitate further neurophysiological analysis of dopaminergic neural mechanisms in Lymnaea.     

Evidence for an enkephalinergic system in the nervous system of the pond snail, Lymnaea stagnalis

Evidence for the presence of an enkephalinergic system in the ganglia of the pond snail, Lymnaea stagnalis, has been obtained with 3 experimental approaches. Scatchard analysis with [3H]etorphine reveals a monophasic high-affinity opiate binding site (Kd 2.3 nM) which is naloxone-sensitive. Immunocytochemical localization of Met- and Leu-enkephalin-like substances as well as alpha-MSH- and ACTH-like materials was demonstrated within specific populations of neurons. Substances with Met- and Leu-enkephalin and Met-enkephalin sulfoxide RIA reactivities were detected also in HPLC fractions corresponding to the retention times of authentic enkephalin standards. Together, the results provide strong evidence for the presence of enkephalinergic mechanisms in the nervous system of Lymnaea stagnalis. Additionally, the report provides indirect evidence for the existence of a macromolecular opioid precursor. This enkephalinergic system shows striking similarities to opioid mechanisms found in vertebrates and bespeaks a common evolutionary origin.     

Postembryonic development of serotoninlike immunoreactivity in the central nervous system of the snail, Lymnaea stagnalis

Posthatching growth in the pond snail Lymnaea stagnalis involves approximately a 20-fold increase in the linear dimensions of the ganglia composing the central nervous system. Developmental change within the population of neurons exhibiting serotoninlike immunoreactivity (SLIR) was examined in order to explain this growth in cellular terms. The study indicates that at least two factors contribute to the growth of the nervous system. First, SLIR cells approximately double in number from the 200-250 cells in hatchlings to the complement found in animals approaching sexual maturity. Much of this increase in cell number occurred within identifiable discrete clusters of neurons with different clusters adding cells at different rates and at different times. The number of SLIR cells also increased in more diffuse populations, particularly along the medial aspects of the paired pedal and the right parietal ganglion. No identified cells were added postembryonically. In addition to the increases in neuron numbers, posthatching development in Lymnaea also involves the growth of individual cells. All cells examined showed continuous somatic growth during posthatching development, but different identified cells and different cell clusters were characterized by different rates of relative growth. Together, the results highlight the complexity of postembryonic development in the snail by indicating the temporal and spatial specificity for both cell addition and cell growth within the nervous system.     

Non-ocular dermal photoreception in the pond snail Lymnaea stagnalis

Based on the results of previous behavioral experiments, researchers believe that sensitivity to light stimuli is not restricted to the eyes in the pond snail Lymnaea stagnalis. To determine the presence of a non-ocular dermal photoreception system and to examine the synaptic connections between this peripheral system and the central nervous system, we electrophysiologically examined the activities of the pedal nerves in L. stagnalis by light stimulation. The results demonstrated that light stimulation evokes non-ocular dermal photosensitive responses in the foot, that these responses exert inhibitory, afferent influences through the inferior pedal nerves to the pedal ganglia, and that these responses were independent of the ocular photoreception system in L. stagnalis.     

Distribution of the VIP-like immunoreactive neurons in the cat central nervous system

Immunohistochemical topographic localization of the vasoactive intestinal polypeptide (VIP)-like immunoreactive neurons in the cat brain was investigated using a peroxidase anti-peroxidase technique. VIP-like immunoreactive neurons were mainly localized in the cerebral cortex, limbic cortex, hypothalamic nuclei; suprachiasmatic nucleus, supraoptic nucleus, paraventricular nucleus, periventricular nucleus and arcuate nucleus, and in the midbrain; such as the central grey and the raphe nucleus. It was demonstrated that VIP-like immunoreactive neurons were widely distributed in the cat brain, particularly in the hypothalamus, compared with those of the rat and mouse; though whether these differences were species-related or due to differences in the physiological conditions remains to be determined. This is the first report of VIP neuronal perikarya in the arcuate nucleus of mammalian species, although these cells are present in the arcuate nucleus of birds.     

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. © 1999 Wiley-Liss, Inc.     

Development of serotoninlike immunoreactivity in the embryonic nervous system of the snail Lymnaea stagnalis.

In our initial effort to study the ontogeny of the gastropod nervous system, we used histological techniques to examine the post-embryonic development of cells which exhibit serotoninlike immunoreactivity in Lymnaea (Croll and Chiasson, J. Comp. Neurol. 230:122-142, '89). The present study complements that report by examining the embryonic development of these neurons. The first serotoninlike immunoreactive (SLIR) cells to be detected in the embryos are the paired C4 neurons of the cerebral ganglia. These cells are faintly visible at about 37-38% of embryonic development and have already produced axons which traverse the cerebral commissure. By about 2-3% later the axon tips reach the pedal ganglia and appose the next SLIR cells to appear, the EPe1 neurons. Over the next 30% of development four more pairs of cerebral neurons are added adjacent to the C4 neurons and over ten cells are added to each of the pedal ganglia. At about 70% of development SLIR fibers are first detected in the parietal and visceral ganglia forming the abdominal ring. Around this time the somata of the C1 neurons also first appear in the cerebral ganglia together with their prominent axons projecting to the buccal ganglia. The last 30% of development is marked by a massive addition of SLIR cells (up to 60) in each pedal ganglion. The early appearance of the first SLIR cells suggests that they may be among the first nerve cells to differentiate and that they may play central roles in the formation of the CNS. We hypothesize that most of the animal's neural circuitry is laid down during embryogenesis by a stereotypic ontogenetic program with post-embryonic neurogenesis subserving mostly compensatory and modulatory purposes.     

Distribution of catecholamines and of immunoreactivity to substances like vertebrate enzymes for the synthesis of catecholamines within the central nervous system of the snail, Lymnaea stagnalis

Catecholamines (CAs) were detected histochemically within over 185 cell bodies in the central nervous system (CNS) of juvenile and young adult Lymnaea. This distribution of CA-containing cells in all central ganglia except the pleural ganglia is more widespread than previously described but is consistent with other reports suggesting numerous roles for CAs within the nervous system. This study also describes the distribution of substances which are antigenically similar to four bovine enzymes for catecholamine synthesis, but the distribution patterns showed little or no overlap with each other or with CA. These results suggest the need for caution in the interpretation of such immunohistochemical studies.     

Photoinactivation of neurones in the pond snail, Lymnaea stagnalis: estimation of a safety factor

Neurones were irradiated with blue laser light (440 nm). The intensity of light for reliable cell killing (0.5 MW.m-2) was much greater than that used to kill arthropod neurones. In wild snails, there was no difference in the intensity to kill Lucifer yellow-filled neurones and unfilled neurones, probably because of the red pigments in the cell bodies. In laboratory-reared snails, which have much less pigmentation, only the filled cells were killed.     

Glutamate transporters in the central nervous system of a pond snail

Previous studies on glutamate (GLU) and its receptors in the pond snail Lymnaea stagnalis have suggested that GLU functions as a neurotransmitter in various behaviors, particularly for generation of feeding rhythm. The uptake mechanism of GLU is not yet known in Lymnaea. In the present study, we characterized the GLU transporters and examined their functions in the feeding circuits of the central nervous system (CNS) in Lymnaea. First, measurement of the accumulation of ³H‐labeled GLU revealed the presence of GLU transport systems in the Lymnaea CNS. The highest accumulation rate was observed in the buccal ganglia, supporting the involvement of GLU transport systems in feeding behavior. Second, we cloned two types of GLU transporters from the Lymnaea CNS, the excitatory amino acid transporter (LymEAAT) and the vesicular GLU transporter (LymVGLUT). When we compared their amino acid sequences with those of mammalian EAATs and VGLUTs, we found that the functional domains of both types are well conserved. Third, in situ hybridization revealed that the mRNAs of LymEAAT and LymVGLUT are localized in large populations of nerve cells, including the major feeding motoneurons in the buccal ganglia. Finally, we inhibited LymEAAT and found that changes in the firing patterns of the feeding motoneurons that have GLUergic input were similar to those obtained following stimulation with GLU. Our results confirmed the presence of GLU uptake systems in the Lymnaea CNS and showed that LymEAAT is required for proper rhythm generation, particularly for generation of the feeding rhythm. © 2009 Wiley‐Liss, Inc.     

Proctolin-like immunoreactive neurons in the blowfly central nervous system

The pentapeptide proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) is a well-studied bioactive substance in insects. With an antiserum against proctolin we have mapped proctolinlike-immunoreactive (PLI) neurons in the nervous system of the blowfly Calliphora erythrocephala. In the brain, including the suboesophageal ganglia, 80-90 neurons were found to be PLI. A further 200-250 PLI neurons innervate the lobula of the optic lobe. The thoracic ganglia contain 100-130, and the abdominal ca. 60 PLI neurons. In the brain and ventral ganglia the immunoreactive neurons are of different types: interneurons, efferents (possibly some motorneurons), and neurosecretory cells. Some of these neurons are individually identifiable; others can be identified collectively as clusters. Identifiable neurons innervate protocerebral neuropil associated with the pars intercerebralis and the beta-lobes of the mushroom bodies as well as tritocerebral neuropil. Some of the prominent clusters innervate the central body of the protocerebrum, tritocerebrum, and possibly leg motor neurons. One abdominal cluster is of special interest because it consist of efferent neurons with processes in the lateral abdominal nerves. Some of these processes are located in the neural sheath in neurohaemal regions, and electron microscopy demonstrates that their terminals are outside the blood-brain barrier. The PLI processes in the protocerebrum contain large granular vesicles and form chemical synapses with different kinds of nonimmunoreactive neural elements. Thus, in Calliphora the proctolinlike substance may be used as a central transmitter/modulator, a neuromuscular transmitter, and a neurohormone released into the circulation.     

Distribution and developmental expression of octopamine-immunoreactive neurons in the central nervous system of the leech

Octopamine, a biogenic amine analogous to norepinephrine, plays an important role in the orchestration and modulation of invertebrate behavior. In the leech, the behavioral actions of octopamine have been demonstrated; however, identification of octopaminergic neurons had not been determined by using immunohistochemical techniques. Thus, we used an antibody highly specific to octopamine to examine the distribution of octopamine-immunoreactive neurons in the segmental ganglia of American and European medicinal leeches (Macrobdella decora and Hirudo medicinalis). One pair of octopamine-immunoreactive neurons was located in the dorsolateral ganglionic region of anterior ganglia 1–6 and posterior ganglia 15–21. No corresponding octopamine-immunoreactive neurons were found in midbody ganglia 7–14. Using Neutral Red staining in combination with intracellular Neurobiotin injections and octopamine immunostaining, we determined the identity of the dorsolateral octopamineimmunoreactive cells. The dorsolateral octopamine-immunoreactive neuron (the DLO) was not cell 21, the only previously reported Neutral Red staining neuron in the dorsolateral position. We also determined that the Leydig neuron was not octopamine immunoreactive in either of the two medicinal leech species. Octopamine immunostaining in the sex ganglia revealed hundreds of immunoreactive neurons in sexually mature leeches. Such neurons were not observed in juvenile leeches. The developmental time course of octopamine immunoreactivity in the dorsolateral octopamine-immunoreactive neurons was also investigated by staining embryonic Hirudo medicinalis, Octopamine expression occurred relatively late as compared with the detectable onset of serotonin expression. Octopamine expression in the dorsolateral octopamine-immunoreactive cells was not detectable at early to mid-embryonic stages, and must commence during late embryonic to early juvenile stages. The identification of octopamineimmunoreactive cells now sets the stage for further investigations into the functional role of octopamine in leech behavior and the development of behavior. © 1995 Wiley-Liss, Inc.     

GTP-dependent dopamine reception in central nervous system tissues in the pond snail]

Guanosine-5'-triphosphate (GTP)-dependent binding of the agonist D1 dopamine (DA) receptor ligand [H3]-SKF 38393 is described. It is shown that binding of [H3]-SKF 38393 with two different populations takes place in the presence of guanylyl nucleotides, when they are absent--with one population. It is demonstrated using GDP-alpha-P33 binding analysis that the GDP in equilibrium with GTP exchange rate gets higher as a result of activation of DA receptors in the membrane from the mollusc nervous tissues. Influence of the catalytic subunit of the protein kinase A (cPKA) on the [H3]-SKF 38393 and GDP-alpha-P33 is investigated. Obvious influence of the cPKA on [H3]-SKF 38393 binding is not observed, while basal and DA-induced GDP in equilibrium with GTP exchange in membranes of the mollusc nervous tissues is considerably inhibited.     

异色瓢虫(Hanmonia axyridis)视叶中GABA阳性神经元的分布

目的:探讨异色瓢虫视叶中γ-氨基丁酸(GABA)能神经元的类型、形态特点、投射走向及分布规律.方法:树脂石蜡(colophony-paraffin,CP;专利号:ZL98125709.7)组织包埋切片技术结合免疫细胞化学链酶菌抗生物素蛋白-过氧化物酶(streptavidin-peroxidase,SP)双染法.结果:GABA阳性纤维起源于大量细胞体,在视叶髓区具有广泛的分布,染色程度互不相同,并有分层现象,几条神经通路染色较浅;主要观察到5种细胞类型,GABA能阳性细胞体染色显著,呈深褐色,根据其定位及神经突的走向可以区分为8个细胞群.在本模型昆虫中还观察到视网膜后交叉和内髓板结构.结论:GABA作为神经递质在传递视觉信息方面发挥重要的调节作用,且主要抑制局部神经活动,并在特定区域和5-羟色胺(5-HT)共同起作用.     

异色瓢虫（Hanmonia axyridis）视叶中GABA阳性神经元的分布

目的：探讨异色瓢虫视叶中γ-氨基丁酸(GABA)能神经元的类型、形态特点、投射走向及分布规律。方法：树脂石蜡(eolophony-paraffin，CP；专利号：ZL98125709．7)组织包埋切片技术结合免疫细胞化学链酶菌抗生物素蛋白-过氧化物酶(streptavidin-peroxidase，SP)双染法。结果：GABA阳性纤维起源于大量细胞体，在视叶髓区具有广泛的分布，染色程度互不相同，并有分层现象，几条神经通路染色较浅；主要观察到5种细胞类型，GABA能阳性细胞体染色显著，呈深褐色，根据其定位及神经突的走向可以区分为8个细胞群。在本模型昆虫中还观察到视网膜后交叉和内髓板结构。结论：GABA作为神经递质在传递视觉信息方面发挥重要的调节作用，且主要抑制局部神经活动，并在特定区域和5．羟色胺(5-HT)共同起作用。     

Polycyclic neuromodulation of the feeding rhythm of the pond snail Lymnaea stagnalis by the intrinsic octopaminergic interneuron, OC

We have examined the role of the octopamine-containing buccal OC interneuron in the fictive feeding rhythm generated by depolarizing a modulatory interneuron, SO, in the isolated central nervous system (CNS) of Lymnaea stagnalis. Before stimulating the SO, the initial fictive feeding rate was 2.0+/-0.37 bites/min (mean+/-S.E.). When the SO was stimulated, the fictive feeding rate more than doubled, increasing by 5.4+/-2.6 bites/min. Prestimulation of OC facilitates the ability of the modulatory neuron SO to drive fictive feeding 4 s later. Following OC stimulation, the increase in SO-driven feeding rate was 10.8+/-1.6 bites/min, significantly more than when only the SO was stimulated (P<0.02, paired t-test on five preparations). OC activity is not required during the SO stimulation for this enhancement. The maximum of the SO driven rhythm occurs between 6 and 12 s after the end of the OC stimulation at 20 bites/min. This is the maximum feeding rate of intact Lymnaea in sucrose. Facilitation is mimicked by bath applied octopamine at 5 microM. Facilitation is specific to OC interneurons, as the same prestimulation of the electrically coupled neuron N3P (central pattern generator) interneurons does not affect the feeding rhythm. The OC interneuron acts as a long term, polycyclic modulator, which peaks several feeding cycles after the OC activity.     

Complement receptor 3-like immunoreactivity in the light green cells and the canopy cells of the pond snail, Lymnaea stagnalis

We observed CR3-like immunoreactivity in the central nervous system (CNS) and its surrounding peripheral nerves of the pond snail, Lymnaea stagnalis. In the CNS of L. stagnalis, the immunoreactivity presenting meshwork-like structure was detected in some neurosecretory cells, which are the light green cells (LGCs) and the canopy cells (CCs), both controlling the body growth. The immunoreactivity was also observed along the edges of median lip nerves. The immunoreactive regions in the median lip nerves appeared to form the axonal plates, from which the LGCs and the CCs release molluscan insulin-related peptides (MIPs) into the blood. By contrast, no immunoreactivity was detected in other neurosecretory cells or their release sites, for example the caudodorsal cells and the cerebral commissure, which release ovulation hormones. The present findings, therefore, suggested that CR3 expresses only in the neurosecretory cells releasing MIPs in L. stagnalis.     

SKPYMRFamide, a novel FMRFamide-related peptide in the snail Lymnaea stagnalis.

In Lymnaea stagnalis, three members of the FMRFamide peptide family have been chemically identified in the central nervous system, and other members of the family are predicted by cDNA studies. The present study demonstrates the occurrence of even more FMRFamide-related peptides in this species by identifying a novel member of this family. The peptide was purified from brain extracts by three different HPLC steps. Its amino acid sequence has been determined as Ser-Lys-Pro-Tyr-Met-Arg-Phe-amide (SKPYMRFamide).