Histamine and Gamma-aminobutyric acid in the nervous system of Pygospio elegans (Annelida: Spionidae). Structure and recovery during reparative regeneration

doi:10.21203/rs.3.rs-2071620/v1

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Histamine and Gamma-aminobutyric acid in the nervous system of Pygospio elegans (Annelida: Spionidae). Structure and recovery during reparative regeneration

https://doi.org/10.21203/rs.3.rs-2071620/v1

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Background

In recent two decades, studies of the annelid nervous systems were revolutionized by modern cell labeling techniques and state-of-the-art microscopy techniques. However, there are still huge white fields in our knowledge on nervous system organization and functioning. Though most recent studies were focused on the distribution of now routine serotonin and FMRFamide, data about many basic neurotransmitters such as histamine (HA) and gamma-aminobutyric acid (GABA) are scarce.

Results

Using immunohistochemistry and confocal microscopy we studied distribution of histamine and gamma-aminobutyric acid in nervous system of a spionid annelid Pygospio elegans and followed their redevelopment during reparative regeneration. Both neurotransmitters show specific patterns in central and peripheral nervous systems. HA-positive cells are concentrated mostly in the brain, while GABA-positive cell somata contribute equally to brain and segmental ganglia. Some immunoreactive elements were found in peripheral nerves. Both substances revealed high number of bipolar sensory cells in the palps. The first signs of regenerating HA- and GABA-ergic systems were detected only by 3 days after the amputation. Further redevelopment of GABA-ergic system proceeds faster than HA-ergic.

Conclusions

Comparisons with other studied annelids and mollusks reveal both similar and unique features in HA- and GABA-ergic systems organizations. Overall, difference between the full redevelopment of various neurotransmitters correlates with neuronal development during embryogenesis. All these results state the high importance of investigating different neurotransmitter distribution in comparative morphological and developmental studies.

Histamine

gamma aminobutyric acid

Annelida

Pygospio elegans

confocal microscopy

nervous system

regeneration

The studies of annelid neuroanatomy have reached a new level and broadened in two past decades. Many studies revealed in great detail the neuromorphology of different annelid groups that, together with new phylogenomic data, have led to a reconsideration of some former theories of nervous system evolution in the whole phylum (1–3). However, even now there is a great incompleteness in our knowledge about some aspects of annelid neuroanatomy. First, is the insufficient taxon sampling with modern microscpic techniques, hindering the comprehensive comparative analysis. Second, is the incompleteness of the existing data on already studied groups. Since confocal microscopy came into zoological research, most studies use only a restricted number of antibodies against neuroactive substances and structural proteins. The majority of modern studies concerned with the annelid nervous system were made using antibodies against acetylated α-tubulin, serotonin, and FMRFamide (4–13). Acetylated α-tubulin is a cytoskeletal protein that is characteristic of neuronal microtubules as well as ciliary structures (14,15). It is widely used to highlight the overall scaffold of the nervous system (16). Serotonin is a monoamine that acts as an important signal molecule in a wide range of processes like regulation of muscle contraction and participates in sensory and regeneration processes of the CNS and neurogenesis (17–19). FMRFamide is a neuropeptide described first as a cardio excitatory peptide from bivalve mollusk Macrocallista nimbosa (20). Later on, a number of FMRFamide-like peptides were described (21, 22). There is evidence that anti-FMRFamide antibody could bind with structurally related RFamides (23).

Apart from serotonin and FMRFamide little is known about the distribution of other basic neuroactive substances in annelids. Most studies are concentrated on different neuropeptides. The antibodies developed against conserved neuropeptide motifs DLamide, FVamide, FLamide, GWamide, and RYamide have shown cross-species reactivity in a wide range of marine invertebrates (24). The immunostained cells had projections to larval ciliary bands indicating the conserved role of neuropeptides in the regulation of ciliary locomotion. In Platynereis a full neuropeptide complement was characterized and for some of them, specific antibodies were raised to visualize their distribution in development (25, 26). The same antibodies were successfully used in studying the brain anatomy of Dinophilidae (27). Surprisingly, a high variation in the expression patterns of neuropeptides across species was revealed suggesting a high plasticity in neuropeptide expression.

Besides neuropeptides, a few data are available on other neurotransmitters such as histamine (HA) and γ-aminobutyric acid (GABA) in annelids. Nevertheless, these neuroactive substances have a widespread distribution in the animal kingdom, playing crucial roles both in physiology and behavior. Histamine is a biogenic monoamine known as a neurotransmitter as well as an inflammatory modulator (28). HA-containing cells are widespread in the CNS of different invertebrates, and HA-immunoreactive elements play an important role in the visual, mechanosensory, and vestibular sensory systems, which were studied in mollusks (29–31), annelids (32), and arthropods (33). Cellular inflammatory responses in annelids are comparable to the inflammatory process in vertebrates (34). Histamine, as one of the humoral factors in inflammation, has also been found in extracts of earthworms Lumbricus terrestris and Eisenia fetida (34).

Gamma-aminobutyric acid (GABA) as a neurotransmitter is widely represented in the nervous system of mammals as well as various groups of invertebrates (35). In Hydra vulgaris, GABA decreases the activity of impulse-generating systems (36), and in the flatworm Gastrothylax crumenifer, GABA has a general inhibitory effect (37). The presence of GABA in the nervous tissues in Nereis (38), and Lumbricus (39) was demonstrated for the first time by autoradiographic techniques and paper chromatographic methods. 5-hydroxytryptamine (5-HT) and GABA regulate the body wall muscle activity in annelids and nematodes (40). Research on cerebral brain extirpation (41) and embryogenesis (42) of Eisenia fetida showed that GABA-IR elements have been found primarily in the development and regeneration of the nervous system.

Pygospio elegans is a tube-dwelling worm belonging to one of the largest annelid families Spionidae, widely distributed in intertidal sand flats in bays and estuaries of the Arctic Ocean and northern regions of the Pacific and Atlantic oceans as well (43, 44). Its small size and outstanding regenerative capacities make P. elegans a perspective annelid model to study reparative processes (44). The general morphology of anterior and posterior regeneration after traumatic amputation or asexual reproduction of P. elegans is well described (45, 46). There are also some data on the structure and general course of nervous system regeneration visualized with antibody staining against acetylated α-tubulin, serotonin, and FMRFamide (47), as well as with histochemical staining for catecholamines (44).

In order to broaden the repertoire of used antibodies to provide new data for comparative analysis and to widen the knowledge about nervous system morphology and stages of regeneration, we used the commercially available antibodies against HA and GABA to study the nervous system of Pygospio elegans. We also followed the nervous system reorganization during reparative regeneration to provide morphological background for further comparative morphological and molecular studies.

Histamine-like immunoreactivity

The HA-like immunoreactive elements were found primarily in the central nervous system. In the brain, about 30 HA-like neurons were located (Fig. 1 a–c). Most of them contribute to a pair of cell clusters near the anterior eyespots. Two pairs of neurons were located closer to the rear pair of eyespots. The pigmented cups of the eyespots in most specimens have bright autofluorescence and may be thus easily located. The fluorescent signal here is stronger than in neurons and the separated granules or vesicles are clearly distinguishable. The projections of HA-like immunopositive cells were found in the central neuropil in both dorsal and ventral commissures, ventral roots of the cerebral commissure, circumoesophageal connectives, stomatogastric nerves, nuchal nerves, palp nerves, and frontal prostomial nerves (Fig. 1 b–f). A pair of cell somata was found at the anterior part of the prostomium (Fig. 1 c). Their projections contribute to the frontal prostomial nerves.

Inside the palps HA-immunopositive elements are part in the main palp nerve, and could be traced to the very tip of the palp (Fig. 1 d–f). Numerous regularly arranged HA-like immunopositive bipolar cells facing the food groove were located along the entire length of the palps. They possess a relatively thick dendrite, which is intensively labeled by anti-acetylated α-tubulin antibody.

In body segments, HA-like immunopositive elements were found mainly in the longitudinal nerves of the ventral nerve cord (Fig. 2; Fig. 3 a, h, j). In the abdominal and tail segments we did not find any other signal in CNS besides these trunks (Fig. 3 h, j). HA-like fibers were located mainly in the paramedian nerves. Numerous varicoses were seen in the region of the segmental ganglia neuropil. The HA-like immunopositive cell somata were not found in segmental ganglia.

Thoracic segments are unequal in distribution of HA-like elements. A pair of neurons was found at the posterior ventral side of 5^th and 6^th thoracic segments at the bases of the parapodia (Fig. 2; Fig. 3 a, b). The cells are unipolar, sending their projections to the segmental nerves and could be traced to the contralaterally located longitudinal nerves of the ventral nerve cord, thus making a chiasm inside two nerve trunks.

Starting from the 6^th thoracic segment, paired HA-like cell clusters were located at the ventral side of the worm along the posterior segmental border closer to the ventral nerve cord (Fig. 2; Fig. 3 c–f). The clusters contained 2-5 cells of irregular shape with numerous thin processes and a dense HA-like immunoreactive meshwork around them. There are also some HA-like immunoreactive cells of irregular shape in the body wall of abdominal and posterior thoracic segments, located at the lateral sides closer to the segmental margins (Fig. 3 i).

HA-like immunopositive elements were not found in the pygidial region (Fig. 3 j). At the border between the body segments and the pygidium in most specimens paired HA-positive fibers ended with large vesicles.

HA-like immunopositive signal was also detected in the longitudinal nerves supplying the intestine, though its intensity was relatively low (Fig. 3 g).

GABA-like immunoreactivity

The study of GABA-positive nerve elements was troubled by strong non-specific antibody binding. A weak background signal was registered in the body wall, making it difficult to isolate some thin immunoreactive elements in the nervous system. Nevertheless, careful slice-by-slice analysis allowed us to find GABA-like immunopositive cells and their projections in CNS and peripheral nerves.

GABA-ergic system of P. elegans is well developed and represented by numerous fibers and somata in both central and peripheral parts of the nervous system (Fig. 4; 5). In the brain the GABA-antibody gave immunostaining of approximately 20 cell somata and numerous fibers in central neuropil. The cell somata do not form distinct clusters in the brain and show bilateral symmetry in their distribution (Fig. 4 d, f). They project axons to the ventral and dorsal brain commissures which are extensively labeled by GABA antibody. The fibers in ventral commissure look like a nerve tract connecting the ventral roots of circumoesophageal connectives. It also gives rise to paired of frontal prostomial nerves (Fig. 4 g). The dorsal commissure is thicker than ventral and has a butterfly-like shape. From its posterior parts the fibers are projected that are connected with the posterior commissure of the prostomium which is situated between the posterior pair of eyespots and the nuchal organs (Fig. 4 d, e). The posterior commissure of the prostomium gives rise to longitudinal lateral peripheral nerves (Fig. 4 e, f). Many GABA-like immunopositive elements were found along the palps. In the palp basements a pair of intensively labeled cell somata was found. The cells have unipolar shape and lie closer to the main palp nerves. Numerous immunoreactive fibers contribute to the main palp nerves. In the additional palp nerves the immunoreactive signal was not detected (Fig. 4 a, b). Along the food groove two rows of bipolar GABA-like immunopositive cells were located (Fig. 4 a). Their projections face the body surface inside the food groove closer to the midline. The strong fluorescence of cilia in the food groove makes it impossible to determine whether these cells have their own cilia. In addition, a row of regularly spaced GABA-positive cells was found along the main palp nerve (Fig. 4 b). The cells are of closed type (do not reach the epithelial surface) and are located in the inner lateral sides of the palps.

The nerve plexus, constituting the stomatogastric system was also labeled by GABA antibody (Fig. 4 h–j). It is more conspicuous in regerenerating worms which possess thinner body wall facilitating the visualization of the inner structures. Most cells contributing the plexus are bipolar closed type cells with rounded somata. There are also pyriform cells with short processes facing the intestinal lumen.

The suboesophageal connectives possess strong GABA-like immunopositive fluorescence (Fig. 4 c). From the ventral side they give rise to the paired pharyngeal nerves that further branch and innervate the surface of the lower lip from the ventral side and then going to the pharynx. The suboesophageal ganglion has strong GABA-positive innervation. It has a number of cells and a thick commissure, which were not detected in the ganglia of following segments. In subsequent body segments GABA-like immunopositive elements were found mostly in the ventral nerve cord (Fig. 5 a–c). Up to 14 neuronal somata can be detected in each ganglion. Their projections form a well-developed neuropil. However, the GABA-positive commissures were relatively thin and contained only single fibers (Fig. 5 a). They were situated at the posterior part of the ganglion.

Three peripheral longitudinal nerves possess fibers of GABA-positive cells. A paired lateral longitudinal nerves originate from the posterior part of the prostomium and runs in the dorso-lateral sides of the body segments above the parapodia (Fig. 5 a, d, e). The unpaired dorsal nerve runs along the dorsal midline and can be traced from the second thoracic segment to the pygidium (Fig. 5 d–f).

Fibers of GABA-like immunopositive cells were detected in two segmental nerves. The first one is located in the anterior part of the segment and usually had a faint fluorescent signal. The second GABA-positive segmental nerve supplies the parapodium (Fig. 5 a). It goes along the posterior side of the parapodium, cross the lateral longitudinal nerve and proceeds to the dorsal side connecting the dorsal unpaired longitudinal nerve (Fig. 5 d–f).

At the dorsal side of the body closer to the posterior segment boundary the GABA antibody labeled a row of bipolar cells (fig. 5 g–i). They are spindle-shaped with a thick and short processes faced to the body surface. Bipolar GABA-like immunopositive cells were found in the body wall at the parapodia bases (Fig. 5 k–m). They project fibers to the body wall plexus.

GABA-like immunopositive fibers enter the pygidial longitudinal nerves as parts of the ventral nerve cords. However, we did not find any other elements in this body region (Fig. 6 b, f).

Regeneration of HA-ergic system

In the area of the anterior regenerate HA-like immunofluorescent elements appeared for the first time after 3 days post amputation (Fig. 6 a, d). These were fibers contributing the longitudinal nerves of the VNC, stretching from the first intact segmental ganglion. By that time HA-like fibers did not yet reach the prostomium. The fibers of the intestinal nerves also entered the regenerate at 3 dpa. Surprisingly, we did not find any accompanying anti-acetylated α-tubulin signal in the intestinal nerve either in the intact worms or during regeneration (Fig. 6 d). By the 7^th dpa HA-like immunopositive fibers outlined all the main nerves and tracts of the prostomium (Fig. 6 c). In the brain first one or two pairs of cells appear (Fig. 6 c, e). The first HA-positive elements in the palps also become visible after 7 dpa (Fig. 6 c).

In the posterior regenerate, HA-like immunopositive fibers started to grow in only after 3 or even 4 dpa (Fig. 6 b). On 7^th days after the operation, the HA-ergic system of the posterior end regenerate was fully reorganized, although the border between the intact and regenerated part was still clearly visible (Fig. 6 f).

Regeneration of GABA-ergic system

The first signs of reparation of GABA-like immunopositive elements in both anterior and posterior regenerates become visible starting from 3d dpa. In the anterior regenerate, the longitudinal fibers of the ventral nerve cord coming from the first intact segment appeared first (Fig 7 a). At 4^th dpa, GABA-positive fibers could be traced up to the head lobe (Fig. 7 c), and the first cells (2-4 neurons) appeared in the brain (Fig. 7 c’), though their fluorescence was faint and their projections were almost impossible to follow. After that, the active differentiation of neurons in the newly formed segmentary ganglia and in the brain began. The process was very fast and by 7^th dpa almost all neurons were differentiated, including neuronal elements of the palps (Fig. 7 e, f).

In the posterior regenerate, the difference between 3d and 4^th dpa was almost indiscernible. At the ventral side, GABA-positive fibers were traced as a part of the newly formed longitudinal nerves of the ventral nerve cord going up to the pygidium (Fig. 7 b, d). Besides the ventral nerve cord, GABA-like immunopositive fibers start to grow into the posterior regenerate from dorsal and lateral longitudinal nerves (Fig. 7 b). By the 7^th dpa, GABA-ergic system of the posterior regenerate did not differ from the intact segments (Fig. 7 g). However, the fibers in the commissure and segmentary nerves were not clearly distinguishable in newly formed segments.

In this study we have mapped the distribution of histamine and gamma-aminobutyric acid in the nervous system of P. elegans using the commercially available antibodies. The shematic representations of the studied neuroactive substances distribution is presented on Figures 8 and 9. We have also studied the involvement of this neuroactive substances in the course of nervous system reparative regeneration. Both studied neurotransmitters show unequal distribution in the central and peripheral parts of the nervous system. The results suggest the specific, apparently non-overlaping, localization of each type of labeled cells in central and peripheral parts of the nervous system, and the existence of a specific order in specification of neurons and nerve fibers in the regenerates of P. elegans.

Histamine

The most intriguing feature of HA distribution in CNS of P. elegans is that the neuronal somata were found exclusively in the brain (Fig. 8 a, c). The ventral nerve cord is devoid of cell somata and contains only fibers in longitudinal nerves (Fig. 9 a, f). No segmental nerves contained HA-like immunoreactive fibers. Thus, we conclude that HA-ergic cells in CNS are predominantly interneurons performing integrative functions and coordinating the neuronal activity between segments. Among annelids, the distribution of HA in CNS was described only in Nereis diversicolor (32), though only the brain was studied. The distribution of HA-positive elements in the brain of P. elegans is broadly similar to those of N. diversicolor indicating the general homology of HA-positive elements in both species.

HA-like immunoreactivity was revealed in palp contributing the main palp nerves. This is also in accordance with data from N. diversicolor (32). The antibody also labeled a number of bipolar cells with relatively thick and long processes facing the lateral sides of the food groove. By their morphology and localization these cells should be sensory. This observation is corresponds to findings of HA-positive cells in clitellates (48). In the body wall epithelium of L. terrestris and E. fetida bipolar HA-immunoreactive sensory cells as well as long processes coming from deeper regions of the body wall were demonstrated. Similar HA-immunoreactive sensory cells are characteristic to mollusks sense organs (30,49–51) thus corroborating the idea that HA plays similar sensory functions in both phyla.

Another interesting finding is the presence of paired unipolar HA-positive cells at the bases of parapodia in 5^th and 6^th thoracic segments (Fig. 9 d). To date we have no data about the origin and functions of these neurons. Probably they are related with reproduction, reception, or tube building. Further studies of these cells and related structures as well as the broad taxon sampling are highly needed.

Starting from 6^th body segment paired HA-positive cells appear at the ventral posterior parts of each segment. They possess numerous processes and may be recognized as fibroblasts or glial cells, but not the neurons. In vertebrates histamine-containing cells are necessary for inflammation response (52). Mast-like cells were also described for mollusks (53). According to our observations, there is no specific involvement (degranulation or migration) of these cells in wound healing and further reparative processes in P. elegans. Further studies can clarify the nature of these cells and their functions.

GABA

Among studied neuroactive substance GABA was the most widely distributed in P. elegans. The antibody labeling revealed a large number of cells in the brain and segmental ganglia as well as in palps and the body wall (Fig. 8 b, d; Fig. b, c). This finding is in great accordance with results from clitellates Lumbricus terrestris (54) and Eisenia fetida (48), although the number of labeled cells in P. elegans is significantly lower. In L. terrestris GABA-like cells form distinct clusters in the brain and ventral ganglia. In P. elegans the cell somata are more or less evenly distributed that can be partially explained by the fewer cell number. Another remarkable observation is the relatively low GABA content in segmental nerves (Fig. 9 e, g). According to our data, Pygospio possess at least 5 pairs of segmental nerves revealed by acetylated α-tubuline immunoreactivity (Fig 4. g), but only two of them demonstrate weak GABA-like immunoreactivity. At the same time GABA-like immunoreactive fibers in the neuropil are abundant. In L. terrestris many GABA-positive cells in VNC were recognized as interneurons sending their projections either to the neuropil of the same ganglion, or to the other ganglia, and only a few neurons are projected to the periphery (54). This may be also similar to P. elegans. In mollusks GABA-like immunoreactive cells are also located generally in the central nervous system and their peripheral projections are scarce (55–57) indicating primary central action of GABA-ergic neurons (58). Altogether this may indicate a conserved functions of GABA-positive cells in annelids, however this hypothesis needs further testing.

One of the most valuable result is the finding of GABA-like immunopositive cells along the palps. Moreover, there are apparently two different types of them (Fig. 8 b, d). The cells of the first type are bipolar, projected to the surface with short and thin processes, and lying in two rows inside the food groove and thus can be regarded as sensory. Similar GABA-positive sensory cells were found in the body wall of clitellates Eisenia fetida and Lumbricus terrestris (48). Clitellates have no palps, so the sensory cells involved in feeding behavior should be located directly in the body wall of the anterior segments. This corroborates with the experimental data: the number sensory cells in body wall gradually decreased in anterio-posterior direction, thus supporting the idea of the common origin and similar functions of GABA-positive sensory cells in spionids and clitellates (48). In gastropod mollusks sensory functions of GABA-positive cells were also described supporting the chemoreceptive functions (59,60).

The cells of the second type are less numerous and located at inner lateral sides of the palps. Unlike the cells of the firs type, they have no distinct projections to the surface and lie closer to the main palp nerve. We suggest these cells are neurons that modulate the palp activity. However, their sensory nature also may not be excluded since it was shown the presence of sensory cilia on abfrontal sides of the palps in P. elegans (61). In osphradium of a gastropod Limnaea stagnalis the colocalization of GABA with FMRFamide was demonstrated in ganglion neurons as well as in sensory cells penetrating the epithelium (59). In P. elegans FMRFamide-like immunoreactive cells were also found in palps (61), however their localization differs from GABA-like cells found in this study.

A number of GABA-like cells was also found in the stomatogastric nervous system. Similar cells were reported for E. fetida and L.terrestris (54,62). Based on localization and morphology sensory, but also endocrine function was suggested for these cells. We suggest similar functions of GABA-positive cells in stomatogastric system in P. elegans. However, all of these conclusions are based on indirect evidences, further studies are needed to discover the exact functions of GABA in stomatogastric system of annelids.

Investigations on the role of GABA in regulation the muscular activity in the body wall of annelids have led to contradictory results. In a clitellate Pheretima communissima, both inhibitory and excitatory effects have both been detected on spontaneous muscle contraction (63,64). In another clitellate, Eisenia fetida, GABA had no effect on muscle contraction in the body wall but was capable of inhibiting serotonin-mediated contractions (48). GABA-like immunopositive fibers in the wall of body segments of P. elegans are restricted to a few longitudinal and trasversal nerves running along the main muscular bundles suggesting the involvement of GABA in neuromuscular transmission. GABA-immunoreactive fibers were also observed in the body wall musculature of L. terrestris (54) indicating the possible common function of GABA in annelids.

Regeneration

The overall pattern of nervous system regeneration is similar to those described for other annelids (8,12,47,65). The redevelopment of the posterior end of the body is in general similar to the anterior one, though the speed of the process is faster. This difference is explained by different modes of anterior and posterior regeneration. The posterior regeneration is in fact restricted to reconstitution of the pygidium and the growth zone, while the anterior one is related with redevelopment of the full set of lost thoracic segments in addition to the prostomium (44). The difference in nervous system regeneration in different body parts reflects the overall difference in anterior and posterior body segments regeneration. Nevertheless, in both anterior and posterior regenerates the studied neuroactive substances become detectable starting from the third day after the operation (stage 3 according to (44)). At this stage the nerve fibers, are already infiltrated to the regenerate and the scaffold of regenerating CNS is well-recognizable. This is in contrast to the studies of serotoninergic system, whose nerve fibers were detected in regenerating parts since the first steps of nervous system redevelopment (8,65,66). In P. elegans the nervous system regenerations was previoustly studied by Lindsay et al. (47). The authors have shown that the after 3 days post-ablation FMRFamide-immunoreactive elements were already detected in the brain in P. elegans and Dipolydora quadrilobata. However, no data about earlier stages were presented and the experiments were performed at lower temperatures comparing to ours that complicates the direct comparison. Our preliminary data on serotonin- and FMRFamidergic systems regeneration, support the results by Lindsay et al., and demonstrate that these two neurotransmitters appear in the area of both anterior and posterior regenerates starting from the very first steps of nervous system redevelopment (data unpublished). Thus, in the course of regeneration the specification of neurons with different chemism proceeds non-simultaneously.

Further regenerational steps of P.elegans nervous system are characterized by the unequal rates of redevelopment of different neurotransmitters. HA-positive neuronal somata in the brain become visible only at 7 dpa, while for GABA this process is much faster. First neuronal somata are detectable since 4 dpa, and by the 7 dpa almost all GABAergic system is redeveloped. Previously we studied regeneration of catecholaminergic (CA) system of P. elegans using glyoxylic acid induced fluorescence method (44). The first CA-positive elements in regenerates were detected staring from 2 dpa. After the 4 dpa the regeneration speeds up, and by 7 dpa the reparation of both central and peripheral parts of nervous system are almost fully finished. Such dynamics resemble those of GABAergic system regeneration, though the reconstitution of the peripheral nervous system begins prior to the CNS. Thus, for different neuroactive substances the process of the full redevelopment takes different time. Perhaps this difference correlates with neuronal development during embryogenesis. In the most of investigation, where serotonin and FMRF-amide-positive nervous systems were studied, was shown that there is a clear sequence of NS development (67–69). In clitellates, for example, GABA-positive cells were reported prior to serotonin-positive (42,70) that corroborates the data of brain regeneration (41,71).

Data on neurotransmitter content and their distribution add new valuable features to a comparative analysis, and may act as a bridge between form and function. Our results provide new data on nervous system organization in spionid annelids and distribution of HA- and GABA-immunopositive cells. Comparison with clitellates and mollusks shows general similarity in HA and GABA distribution that may be a result of common basic functions of these neurotransmitters in Trochozoa. At the same time, we found numerous unique features in HA and GABA distribution in P. elegans. This indicates evolutionary plasticity of the nervous system. We also found a high representation of HA- and GABA-immunopositive receptor cells in palps of P. elegans, suggesting high complication of these sensory organs as well as high diversity of chemical specificity of sensory cells. The sequential process of nervous system regeneration provides new insights on correlation of reparative and developmental processes. Difference between the full redevelopment of various neurotransmitters correlates with neuronal development during embryogenesis.

Taken together our results illustrate the importance of using different neurotransmitter distribution in comparative morphological and developmental studies. Unfortunately, there is almost no data on the distribution, development and regeneration of HA and GABAergic cells in nervous system of other annelid families. Further comparative studies of various neuroactive substances in different annelid clades are urgently needed.

Animals collection and keeping

Pygodpio elegans Claparède, 1863 were collected at the Barents Sea region, at the marine biological station Dalnie Zelentsi (69°07′ N, 36°05′ E). The animals were collected at low tide from mud and sand flats. After collection, worms were placed in plastic containers with prepared sieved sand (≥0.5mm). The worms were transported to the laboratory, where they were kept at 18 °C in the same containers. We used artificial seawater (Red Sea Coral Pro salt, Israel) with a salinity of about 30-32‰. In such conditions, worms were successfully kept for several months, with regular water changing and feeding with powdered algae collected from the same intertidal zone.

Sampling, experiments, fixation

For regeneration experiments, worms were dissected with razor blades after the 20^th body segment. After that, both halves were kept in separate Petri dishes filled with artificial seawater at 18 °C. To describe the whole regeneration process, we used following fixation points: 0 hours post-amputation (hpa), 4 hpa, 10 hpa, 18 hpa, 24 hpa, 48 hpa, 3 days post-amputation (dpa), 4 dpa, 7 dpa.

All animals were relaxed in a 7.5% solution of MgCl₂• 6H₂O before fixation. Two different fixatives were used for immunochemistry: freshly prepared 3.7% paraformaldehyde + 0.3% glutaraldehyde on 0.1M phosphate-buffered saline (PBS) for staining with GABA antibodies and 4% 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide following with the postfixation with 4% paraformaldehyde for labeling with histamine antibody (32). Totally, about 10 worms were fixed for each type of antibody at each time point of regeneration. After fixation, samples were washed 3x20 min in PBS, and then kept in 1x PBS containing 0.03% sodium azide at 4 °C.

Immunochemistry

Before staining, worms were rinsed in PBS and preincubated overnight in PBS with 0.25% bovine serum albumin (BSA). As primary antibodies, we used the following primary antibodies: polyclonal rabbit anti-histamine (Immunostar, 22939) and polyclonal rabbit anti-GABA (Immunostar, 20094). The primary antibodies were diluted 1:100 in PBS with 0.25% BSA. To label the overall nerve scaffold the monoclonal mouse anti-acetylated α-tubulin antibody (Sigma, T-6793) was added in dilution 1:2000. Specimens were incubated in primary antibodies for 16-24h at 4 °C, then washed 3x20 min in PBS and incubated with secondary antibodies Alexa Fluor 488 Donkey Anti-Rabbit (Molecular probes, A-21206) and Alexa Fluor 647 Donkey Anti-Mouse (Molecular probes, A-31571) both diluted 1:500-1:1000 overnight at 4 °C. Then specimens again were washed several times in PBS, mounted in Mowiol 4-88 with DAPI (Carl Roth, 6335.1) between two coverslips and then stored at 4 °C for 1-7 days before microscopy.

Microscopy and image processing

Specimens were examined using a laser-scanning microscope Leica TCS SP5 (Leica Microsystems, Wetzlar, Germany). The specimens were scanned in 50-100 slices with a thin of 0.5 μm and then processed with Fiji (72) and Bitplane IMARIS. When necessary the brightness and contrast of the resulting images were adjusted with Krita. The schemes were drawn in Inkscape.

Ethics approval and consent to participate

All applicable international, national, and institutional guidelines for the care and use of animals were followed by the authors. We neither used endangered species nor were the investigated animals collected in protected areas.

Consent for publication

Not applicable

Availability of data and materials

The datasets analyzed during the current study are available from the corresponding author on reasonable request. All data needed are included in the paper.

Competing interests

The authors declare that they have no competing interests

Funding

The research was performed with financial support of the Russian Scientific Foundation grant No 21-14-00304.

Authors contribution

All authors participated in animal collection and handling. VVS and ELN designed the study and planned the experiments. ZIS and KVS performed experimental procedures, fixations, immunohistochemical staining, and microscopy. ZIS, KVS and VVS analyzed the results, prepared figures and drafted the manuscript. ZIS, KVS, ELN and VVS participated in manuscript discussion and editing. All authors have read and approved the manuscript.

Acknowledgements

The authors thank the administration of the Murmansk Marine Biological Institute of the Russian Academy of Sciences for providing opportunities for work at the biological research station in Dalnie Zelentsy and personally to Dr. M.V. Makarov. The research was performed at the center “CHROMAS” and center “Culture Collection of Microorganisms” of the Research park of Saint Petersburg State University (Saint Petersburg, Russia), and Core Facilities Centre “Taxon”, Zoological Institute of Russian Academy of Sciences (https://ckp-rf.ru/catalog/ckp/3038/). The research was carried out using the collection materials of the Zoological Institute RAS (Saint Petersburg) (http://www.ckp-rf.ru/usu/73561/).

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No competing interests reported.

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Histamine and Gamma-aminobutyric acid in the nervous system of Pygospio elegans (Annelida: Spionidae). Structure and recovery during reparative regeneration

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