354 G. THE BERNOCCHI JOURNAL OF ET EXPERIMENTAL AL. ZOOLOGY 280:354–367 (1998) Bioactive Peptides and Serotonin Immunocytochemistry in the Cerebral Ganglia of Hibernating Helix aspersa G. BERNOCCHI,* C. VIGNOLA, E. SCHERINI, D. NECCHI, AND M.B. PISU Dipartimento di Biologia Animale, Centro di Studio per lIstochimica del C.N.R., Universita´ degli Studi di Pavia, I-27100 Pavia, Italy ABSTRACT The role of some neuromodulators and neurotransmitters in the functioning of molluskan cerebral neurons and in their metabolic changes during hibernation has been considered. The cerebral ganglion of mollusks is a center for the integration of different inputs from the sensory areas of the head and for the generation of motor command impulses. During hibernation, animals are deprived of many external sensory stimuli and do not have locomotion and feeding. Immunocytochemistry for bioactive peptides (BAPs), such as SP (Substance P), CCK8 (Cholecystokinin 8/ Gastrin), CGRP (Calcitonin-Gene-Related Peptide) and ET (Endothelin), and serotonin was performed on cerebral ganglia of active and hibernating Helix aspersa. The distribution of the immunopositivity was analyzed in different cell-containing areas (procerebrum, mesocerebrum, metacerebrum) and in the neuropiles. With all the antibodies raised against peptides, we observed that only a few neurons, mainly of small and medium size, had immunopositivity during the period of activity, the patterns of distribution being quite similar to those previously described in Helix or other gastropods. Fibers and varicosities with BAP immunopositivity were found in the procerebral and central neuropiles and sometimes around neurons. Serotonin-immunopositive neurons, including the giant neuron, were observed in the metacerebrum; numerous fibers and varicosities immunopositive for serotonin were present in the neuropile areas. In hibernating snails, the number of fibers with BAP and serotonin immunopositivity decreased in several areas of the neuropiles. Moreover, an increased number of neurons of the metacerebrum (two- to four-fold) and mesocerebrum (8- to 28-fold) had BAP-like immunopositivity, and the intensity of the immunoreaction for serotonin of the metacerebral neurons was also higher than in the active snails. These results are discussed, taking into account two hypotheses. The first hypothesis assumes that the increased immunocytochemical staining was really linked to accumulation of BAPs and serotonin. The second hypothesis considers that the antibodies for BAPs recognized a preprotein, the synthesis of BAPs being completed during the active period only. Both the hypotheses account for the co-occurrence and colocalization of two or ore peptides and serotonin and stress that the hibernation condition is of interest for studies on the actual function of single neurons in the cerebral ganglia. Finally, the data are consistent with the changes recently found in other markers of the morphological and functional activity of neurons, demonstrating that the neuromodulation and the neurotransmission are slowed during hibernation. J. Exp. Zool. 280:354367, 1998. © 1998 Wiley-Liss, Inc. A wide spectrum of biological problems can be investigated using hibernation as a model. In fact, studies of hibernating animals are of particular interest in learning about the physiology and metabolism of organisms (Hunter, ’64; Machin, ’75; Heller, ’79; Lyman et al., ’82), because they contribute to the understanding of some important aspects of the structure and function of cells (Kolaeva et al., ’80; Bernocchi et al., ’86; Barni et al., ’87; Giacometti et al., ’89; Anderson et al., ’90; Barni and Bernocchi, ’91; Fenoglio et al., ’92; Malatesta et al., ’94, ’95; March and Reisman, ’95). Our research was focused on studying the © 1998 WILEY-LISS, INC. mechanism by which neurons change their metabolism when there is suppression or reduction of some physiological activities of the organism (Hunter, ’64; Machin, ’75; Heller, ’79; Lyman et al., ’82). This may be considered a primitive form of cellular adaptability, similar to that used in a variety of situations associated with neuronal plasticity. *Correspondence to: Prof. Graziella Bernocchi, Dipartimento di Biologia Animale, Piazza Botta 10, I-27100 Pavia, Italy. E-mail: BERN@IPV36.UNIPV.IT Received 20 February 1997; Accepted 24 October 1997 HIBERNATION AND NEURONAL ACTIVITY A large number of biactive peptides (BAPs) has been demonstrated in specific cell populations of both vertebrates and invertebrates. In particular, in the nervous system, the amino acid sequence of specific peptides is often similar in different phyla, suggesting that this class of molecules is widely distributed and highly conserved (for a review, see Fasolo and Clairambault, ’91). BAPs have a pervasive role in governing essentially all physiological activities, and there are probably only a few biological processes in which they are not involved. In particular, in invertebrates, the role of BAPs in the regulation of a variety of activities has been extensively investigated in mollusks. They are involved in feeding (CCK8/ Gastrin, FMRF-amide, see Engelhardt et al., ’82; Vigna et al., ’84; Elekes and Naessel, ’90; Sonetti et al., ’90; Cottrel, ’92; Cottrel et al., ’92), pain perception, blood flow regulation, and vasodilation (CGRP, ET, SP, see Osborne et al., ’82; Osborne ’84; Breimer et al., ’88; Giaid et al., ’91). In addition, recent studies (Elekes et al., ’93, ’94) on the distribution of enkephalins and leucokinin in the central nervous system of Helix point to an involvement of opioid substances in several behavioral phenomena (Gutierrez and Asai, ’91; Marchang et al., ’91; Elekes et al., ’93, ’94). Peptides of the VIP (vasoactive intestinal peptide) family are present in the nervous system of Helix, where they are involved in several functions such as vasodilation, water and electrolyte secretion, regulation of metabolism, modulation of circadian rhythm, promotion of glycogenolysis, etc. (Kaufmann et al., ’95). Recent reports have demonstrated that the number of VIP-immunoreactive cells is lower in active snails collected in summer in comparison with hibernating animals (Kaufmann et al. ’95). In addition to BAPs, the biogenic amine serotonin, which occurs in relatively high concentrations in the central nervous system (CNS) of mollusks (Osborne et al., ’82; Osborne, ’84; Hernadi et al., ’89; Elekes, ’91; Vehovszky et al., ’93), can affect and modulate a variety of behavioral and physiological processes, such as feeding, withdrawal and escape reaction, blood circulation, and some forms of learning. Some data indicate that, in Helix, the number of serotonin-immunoreactive neurons is lower during hibernation (Hernadi et al., ’89). In order to have a histological and histochemical picture of CNS physiology during hibernation, we have carried out a comparative analysis of changes in immunopositivity for BAPs, whose distribution is at present unknown in hibernating Helix aspersa. The BAPs we have analyzed are 355 involved in many functions accomplished by the cerebral ganglion of H. aspersa, which is a center of integration of several stimuli (Kandel, ’76; Balaban, ’93) that are at least partially suppressed during hibernation. The BAP-immunoreactivity changes were compared with the immunocytochemical pattern of serotonin, one of the first neurotransmitters studied by fluorescence histochemistry (Cardot, ’71; Kerkut and Walker, ’75). A preliminary report of some of these findings has already appeared in abstract form (Bernocchi et al., ’93). MATERIALS AND METHODS Adult terrestral snails (Helix aspersa), collected in the garden of the Section of Ecology (Department of Genetics and Microbiology, University of Pavia) were used. The active animals were collected in September and remained active in the laboratory for 10 days before killing. The hibernating snails were collected in November with the shell closed by the epyphragm and kept in this condition at 4°C in a cage until February. A total of 54 animals of similar size was used. Fixation and sectioning The cerebral ganglia were fixed in Bouin solution (12 for both active and hibernating animals) for 12 h at room temperature or in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2 (12 for both active and hibernating animals), for 12 h at 4°C. After being washed in 70% ethanol for 3 days (Bouin) or in phosphate buffer for 12 h (paraformaldehyde), the ganglia were dehydrated and embedded in paraffin. Eight-µm thick horizontal or transverse sections were collected on silane-coated slides. Three other ganglia fixed in Bouin for each experimental group were cut serially (series of 4 slides) for counting immunoreactive neurons (see below). Immunocytochemistry Immunocytochemical reactions were carried out simultaneously for active and hibernating animals. For every antibody and fixative solution, two slides from at least three animals for both the active and hibernation periods were used. On Bouin- and paraformaldehyde-fixed specimens, the following rabbit polyclonal antibodies were used: 1:500 anti-Substance P (SP, Sera Lab, Sussex, U.K.), 1:500 anti-Cholecystokinin/Gastrin (CCK8/Gastrin, Incstar, MN), 1:500 anti-CalcitoninGene-related Peptide (CGRP, Peninsula Lab, CA), 1:500 anti-Endothelin (ET, Peninsula Lab). Seroto- 356 G. BERNOCCHI ET AL. nin immunocytochemistry (1:500 antiserotonin, Incstar) was performed on paraformaldehyde-fixed specimens only. For all antibodies, the incubation time was 18 h. The peroxidase-antiperoxidase (PAP) method of Sternberger et al. (’70) was used to reveal the sites of antigen-antibody reaction. Briefly, the sections were incubated in 3% H2O2 in 10% methanol for 7 min; 5% normal goat serum for 45 min; primary antibodies; 1:50 goat-anti-rabbit IgG for 90 min; 1:200 rabbit PAP for 60 min; and 0.05% 3,3´diaminobenzidine tetrahydrochloride in 0.05 M Tris/HCl buffer, pH 7.6, with 0.01% H2O2 for 20 min. All washings and incubations were in PBS and at room temperature (20°C). For negative controls, some sections were incubated in normal rabbit serum instead of the primary antibodies at the same dilution; for positive staining, control tissues and cells that contain the antigen were used. Counting neurons The number of immunoreactive for BAPs and immunonegative neurons of the metacerebrum and mesocerebrum was estimated on serial sections of Bouin-fixed ganglia. To avoid multiple counting of a neuron, its cell body was traced out on consecutive sections. The number of immunoreactive neurons was also expressed as percentage of the total neuronal population. The differences Fig. 1. Immunoreaction for CGRP in paraformaldehydefixed ganglia. (a) Active snails: the neurons (n) are immunonegative; in the central neuropile there are varicosities and strongly labeled fibers. (b) Hibernating snails: several small- between the active period and hibernation were significant by a Mann-Whitney test (P < 0.001). RESULTS All the antibodies recognized epitopes in the neuronal perikarya and/or in the fibers of neuropile and interneuronal area (trophospongium) of the land snail CNS. The staining pattern was consistent in the different animals of the same period (activity or hibernation). No immunohistochemical labeling was observed in the cerebral areas and neuropiles in negative control slides. In the following paragraphs we shall describe analytically the patterns of the metacerebrum and mesocerebrum (Figs. 1–7) and the central neuropile. In the procerebrum, with all the antibodies, no positivity was detectable in the cytoplasm of the small neurons during both the active period and the period of hibernation. During the active period, around the neurons and in the neuropile, there were fibers and varicosities immunopositive for some of the antibodies, e.g., serotonin (Fig. 5a), SP, CGRP, ET. This BAP and serotonin immunoreactivity was less detectable during the hibernation (Fig. 5b). Influence of fixatives Paraformaldehyde permitted a better detection of the immunopositivity to some BAPs of the fibers in the neuropiles and around neurons. For in- and medium-sized neurons of the metacerebrum are strongly immunopositive, but some areas (stars) of the central neuropile do not show immunopositive fibers or varicosities. a: ×90; b: ×100. HIBERNATION AND NEURONAL ACTIVITY 357 Fig. 2. Immunoreaction for CGRP in Bouin-fixed ganglia. (a) Active snails: immunopositivity is detectable in some small and large neurons and the giant neuron (GN, arrow) of the metacerebrum. (b) Hibernating snails: most neurons of the metacerebrum, including the GN (arrow) are strongly immunostained. a, b: ×100. Fig. 3. Immunoreaction for CCK8/gastrin in Bouin-fixed ganglia. (a, b) Active snails: the fibers around neurons are immunopositive (arrows). On the contrary, the neurons are negative. (c, d) Hibernating snails: in the meso (Ms) and metacerebrum (Mt), part of the cytoplasm of several neurons is immunopositive; the fibers around neurons maintain immunopositivity (arrows). a, c: ×90; b, d: ×360. 358 G. BERNOCCHI ET AL. Fig. 4. Immunoreaction for SP in Bouin-fixed sections. (a) Active snails: immunopositive fibers (arrows) are present around neurons that appear negative. (b) Hibernating snails: a part of the cytoplasm of some mesocerebral neurons is immunoreactive (stars). a, b: ×360. stance, after immunoreaction for CGRP (Fig. 1a), CCK8 (not shown) and SP (not shown) strong positivity was present in the varicosities and fibers of the central neuropile of active snails. This positivity was not observed in several areas of the neuropile during hibernation (Fig. 1b) and was decreased after immunoreaction for SP around neurons. In general, as analytically described below, Bouin solution allowed a better visualization of the BAP immunocytochemical reaction in the neuronal cytoplasms (see Figs. 2–6). of the metacerebrum and mesocerebrum were negative in the active period (Fig. 4a) but very reactive in the period of hibernation (Fig. 4b). In some of the large neurons, the positivity was located in a discrete area of the cytoplasm. The GN was weakly positive (not shown). Immunocytochemistry for CGRP In comparison with the active period (Fig. 2a), a greater number of neurons were immunopositive in both metacerebrum and mesocerebrum during hibernation (Fig. 2b). The metacerebral giant neuron (GN) was entirely stained. Immunocytochemistry for CCK8/gastrin A few weakly immunopositive neurons and immunopositive fibers around neurons were observed in the active snails (Fig. 3a,b). During hibernation, the fibers maintained some immunopositivity (Fig. 3c,d). Several large neurons of the metacerebrum and mesocerebrum (Fig. 3c,d), including GN (Fig. 6d), were reactive and had the immunopositivity often confined to a discrete area of the cytoplasm. Immunocytochemistry for SP Only a few of the small neurons were strongly positive in the active animals. The large neurons Immunocytochemistry for ET In active animals (Fig. 5a,b), the immunopositivity was in the fibers around the neurons and in the cytoplasm of some neurons (Fig. 5b). Hibernation determined a decrease in the immunoreactive fibers and an increase in the number of immunopositive neuronal cytoplasms (including the GN, Fig. 5c,d; 6c) in both the metacerebrum and mesocerebrum. The immunopositivity in neurons was mainly located in a distinct area of the cytoplasm (Fig. 5c,d). Coexistence/colocalization of ET and CGRP in the mesocerebrum Adjacent sections of hibernating snail ganglia showed that in both the left and right mesocerebra, ET-immunopositive neurons (Fig. 6a) were present. The positivity was stronger in the peripheral area of the left mesocerebrum. Almost all the neurons of the Fig. 5. Immunoreaction for ET in Bouin-fixed cerebral ganglia. (a, b) Active snails: immunopositive fibers or varicosities are found around neurons, of which only a few are labeled (arrows). (c, d) Hibernating snails: most of the neurons, including the GN (arrow), are immunoreactive. Fibers around neurons are less immunopositive. a, c: ×140; b, d: ×360. HIBERNATION AND NEURONAL ACTIVITY Figure 5. 359 360 G. BERNOCCHI ET AL. Fig. 6. Immunoreaction for ET, CGRP and CCK8/gastrin in Bouin-fixed ganglia of hibernating snails. (a) Immunoreaction for ET in the mesocerebrum: in the right (R) mesocerebrum, all the neurons are immunostained; in the left (L) mesocerebrum, the peripheral neurons are more strongly labeled than those of the central area. (b) Immunoreaction for CGRP in the mesocerebrum: in the right mesocerebrum, a group of central-lateral neurons are immunostained, while in the left, almost all the neurons are strongly positive. (c) Immunoreaction for ET in the GN: the immunoreaction (arrow) is confined to a segregated cytoplasmic area. (d) Immunoreaction for CCK8/gastrin in the GN: the same cytoplasmic area (arrow) as in c is weakly positive. a, b: ×90; c, d: ×580. HIBERNATION AND NEURONAL ACTIVITY Fig. 7. Immunoreaction for serotonin in paraformaldehyde-fixed cerebral ganglia. (a, b, c) Active snails: in the neuropile of the procerebrum (Pnp), there are immunopositive varicosities with granular appearance and fine fibers (a); the GN is immunopositive (b), and a large number of labeled fibers and varicosities (arrows) are present in the central neu- 361 ropile (c). (d, e, f) Hibernating snails: in the procerebrum, lesser granular varicosities and fine fibers are evident (d); increased immunoreactivity is shown in the GN (e). In the neuropile surrounding the GN (e) and in the central neuropile (f), a reduction in the positive varicosities can be observed. a, b, c, d, e, f: ×360. 362 G. BERNOCCHI ET AL. left mesocerebrum were also positive for CGRP (Fig. 6b). In the right mesocerebrum, on the contrary, only the central area was immunoreactive (Fig. 6b). The GN was immunopositive for ET (Fig. 6c) and CCK8 (Fig. 6d). For both the immunoreactions, the distribution of immunoprecipitates was confined to the same distinct cytoplasmic area. Immunocytochemistry for serotonin In the active snails, the immunopositivity was observed in the GN (Fig. 7b) and in some neurons of the metacerebrum; numerous positive varicosities and fibers were also present in the central neuropile (Fig. 7c). A marked increase in the intensity of the reaction was found in the same neurons, including the GN (Fig. 7e), of the hibernating animals, whereas immunopositive fibers of the neuropile adjacent the GN were decreased in number (Fig. 7e,f). Number and percentages of BAPimmunoreactive neurons For all BAPs, the number (Table 1) and the percentages (Fig. 8) of immunoreactive neurons during the active period were very low, mainly in the mesocerebrum (from 4 ± 0.9 to a maximum of 12 ± 2.5 that corresponded to about 2.5% and 7% of the neuronal population). In hibernating snails, the percentages were 2- to 4-fold in the metacerebrum and 28-fold in the mesocerebrum. The highest percentages during hibernation were in the mesocerebrum (64–79%). DISCUSSION Critical aspects Though we used antibodies that are raised against vertebrate antigens, they recognized epiTABLE 1. Changes in the number of BAP-like immunoreactive neurons of the metacerebrum and mesocerebrum during active period and hibernation* Active period CCK8 SP CGRP ET metacerebrum mesocerebrum metacerebrum mesocerebrum metacerebrum mesocerebrum metacerebrum mesocerebrum 26 10 46 12 40 4 39 4 ± ± ± ± ± ± ± ± 5 2 6 3 7 1 3 1 Hibernation 103 85 100 114 136 112 150 92 ± ± ± ± ± ± ± ± 5 8 11 6 10 7 14 3 *Values are given as mean ± S.D.; n = 3 per each period; MannWhitney test significant level P < 0.001. CCK8, cholecystokinin; SP, substance P; CGRP, calcitonin-gene-related peptide; ET, endothelium. Fig. 8. Percentages of BAP-like immunoreactive neurons in the metacerebrum (MT) and mesocerebrum (MS) during active period (Ac) and hibernation (Hi). CCK8: Cholecystokinin; SP: Substance P; CGRP: Calcitonin-gene-related peptide; ET: Endothelin. topes in the Helix aspersa CNS. Indeed, the data obtained with vertebrate antibodies to reveal invertebrate antigens must be interpreted with some caution. In particular, the results may be affected by cross-reaction with unrelated molecules, or immunocytochemical reaction may have detected parts of a molecule that can be either identical to or shorter or longer than the original antigen because we used polyclonal antibodies that contain several immunoglobulins against different epitopes of the same immunogenic antigen. As a consequence, we describe an “antigen-like immunoreactivity.” Nonetheless, as reported below, most of the BAPs considered here and serotonin have been chemically identified in Helix (for a review, see Elekes et al., ’94) or other gastropods (Vigna et al., ’84; Sonetti et al., ’90; Giaid et al., ’91). It is known that the type of fixation can influence immunoreactivity, in particular of peptidergic cells (Schot et al., ’84; Andriès et al., ’91). We emphasize that with the two fixatives we used immunolabeling for BAPs was often detected in different places. For instance, after immunoreaction to CGRP, CCK8 and SP, fibers and varicosities were better visualized in HIBERNATION AND NEURONAL ACTIVITY formaldehyde-fixed specimens, whereas neurons showed immunopositivity mainly after fixation with Bouin solution. Only for immunoreactions to CCK8 and ET were fibers and neurons labeled from samples fixed in formaldehyde and those fixed in Bouin solution. Occurrence and function of BAPs and serotonin in gastropods Several BAPs have been mapped in the nervous system of gastropods and in particular in Helix in order to increase the knowledge about putative peptidergic pathways. Some BAPs, such as CCK8/gastrin (Osborne, ’84; Vigna et al., ’84; Gesser and Larsson, ’85; Sonetti et al., ’90), FMRF-amide (Cottrel et al., ’92), myomodulin (Santama et al., ’94), and buccalin (Santama et al., ’94) are implicated in control of feeding behavior. SP, which has been described in some molluskan neurons (Osborne, ’84; Sonetti et al., ’90), is involved in sensory pathways concerned with pain and touch. In mammals, calcitonin and CGRP are involved in fundamental physiological functions such as pain perception, appetite and blood flow regulation, and vasodilation (Breimer et al., ’88), and in mollusks, CGRP has been found in slugs (Sasayama et al., ’91). First recognized as a potent vasoconstrictor, ET has been immunocytochemically demonstrated in neurons controlling the vascular system and water retention in Aplysia (Giaid et al., ’91). Opioid peptides are involved in a wide spectrum of physiological functions, such as thermoregulation, locomotion, pain resistance, and immunomodulation (Kavaliers and Hirst, 1984). In particular, neurons containing enkephalins have been recently mapped in ganglia of Helix (Marchand et al., ’91; Elekes et al., ’93). Finally, the distribution of peptides of the VIP-family, which are involved in many functions, has been described in the CNS of Helix (Kaufmann et al., ’95). With all the antibodies against the peptides we have considered, individual neurons and fibers were positive in the cerebral ganglia of H. aspersa during the active period; the patterns of distribution were quite similar to those previously described. In gastropods and in land snails of the genus Helix (Osborne et al., ’82; Osborne, ’84; Cottrel et al., ’92; Elekes and Naessel, ’90; Gutierrez and Asai, ’91; Marchand et al., ’91; Elekes et al., ’93, ’94; Kaufmann et al., ’95); several peptides have been localized in the circumesophageal ganglia, and we have found also fibers immunopositive for antibodies against SP, CCK8, CGRP, and ET 363 in the neuropile or around the neurons of the meta and mesocerebrum. Some neuron somata were also stained in these areas. It must be recalled that there are small, medium, and large neurons in the metacerebrum, whereas the mesocerebrum comprises only large neurons (for a review, see Kerkut and Walker, ’75). In the metacerebrum, the neurons that showed SP-, CCK8-, CGRP- and ET-like immunoreactivity were mainly small in size, and in the mesocerebrum, the immunopositive neurons occurred sporadically. In invertebrates, including mollusks, serotonin acts as neurotransmitter, modulator, and neurohormone (Hernadi et al., ’89; Hernadi and Elekes, ’93). In gastropods in particular, serotonin can affect and modulate a variety of behavioral and physiological processes, such as feeding (Weiss et al., ’82; Kupfermann and Weiss, ’82; Kemenes and Srozsa, ’87), withdrawal and escape reaction (Kupfermann and Weiss, ’82), blood circulation (Liebeswar et al., ’75), and some forms of learning (Kandel, ’76; Balaban, ’93). In the Helix central nervous system, it has been found by immunocytochemistry that both the neuropile and the cell body layer are richly innervated by serotonergic fibers (Hernadi et al., ’89; Elekes, ’91). Accordingly, during the active period, we have found neurons with serotonin immunoreaction in the Helix metacerebrum, which sends numerous projections to the central neuropile, the body wall, and different internal organs. The metacerebral giant neuron, which innervates buccal ganglia, also had serotonin immunoreactivity, as previously reported (Osborne et al., ’82; Osborne, ’84). BAP immunocytochemistry in hibernating pulmonate snails The most striking result of the present research was the observation that, in the hibernating Helix aspersa, a high number of neurons had immunopositive cytoplasm for all BAPs. The immunoreactivity of fibers decreased for some BAPs in several areas of the neuropile and for SP and ET around neurons. In the mesocerebrum, a high number of neurons (64% to 78%) were immunopositive for CGRP, ET, CCK8, SP, and in the metacerebrum (30% to 57%) several small- and medium-sized neurons were also immunopositive. In the metacerebrum, the GN was strongly immunopositive for CGRP and SP, and a part of the cytoplasm was also intensely immunoreactive for CCK8 and ET. The high number of immunopositive neurons and the increase in the intensity of staining in the immunoreactive cells in hibernating terrestrial snails 364 G. BERNOCCHI ET AL. suggest an accumulation of reactive epitopes and molecules in the cytoplasm. This probably gives rise to the co-occurrence of two or more peptides in the same cells, which were negative during activity, and to the co-localization of differently immunopositive neurons. Two hypotheses may be put forward as explanations for these points. The first one assumes that the immunopositivity is really linked to BAP presence. Actually, the segregation of immunoprecipitates—for instance, for CCK8 and ET—in discrete areas of the cytoplasm of neurons that are entirely positive for CGRP and SP (e.g., the GN) is consistent with a storage of BAPs (two or more) in hibernating snail neurons. Therefore, hibernation may be a tool for studies of the functional role of different cerebral neurons. In this context, the small nervous systems not only give lessons for the study of functional organization of cotransmission systems (Marder et al., ’95), but the seasonal cycle of some invertebrates also offers an unique opportunity to unravel the true function of brain areas. Though this was not the aim of our study, we cannot exclude the possibility of the co-occurrence of two or more peptides, which suggests that a neuron can be involved in different functions. Co-occurrence of neurotransmitters and neuropeptides has been widely documented (Osborne, ’84; Fasolo and Clairambault, ’91; Marder et al., ’95). Moreover, the co-localization of neurotransmitters and neuropeptides in the cerebral ganglia of Helix is evident in the paper by Hernadi and Elekes (’93). However, co-reactivity does not necessarily mean co-occurrence of two or more peptides. In fact, during the biosynthesis of neuropeptides, a preprotein is incorporated into dense-core vesicles and subsequently processed to give several BAPs and then transported to the cell terminals. Thus, the preprotein is a precursor for several bioactive peptides with distinct biological properties. In this way, all the peptidergic neurons can putatively manufacture different mixtures of peptides, each with a markedly different spectrum of biological activity. The recent findings of Kaufmann et al. (’95) show that hibernating snails contained the highest number of VIP and mainly of prepro-VIP immunoreactive neurons. In this light, the positivity of the neurons for two or more antibodies against bioactive peptides also may be ascribed to the presence of a preprotein molecule, synthesized in the cell cytoplasm and blocked in this localization. In this case, all the neurons may have the capability and possess the preteolytic enzymes to manufacture several peptides. The hypothesis will be tested by further research. According to this interpretation, during hibernation the neurons are ready to elaborate and release more than one bioactive peptide. The eventual diversification of neurons occurs only during the period of activity. This places under discussion the multipotential role of neurons and the compartmentalization of ganglia in invertebrates, as tentatively analyzed by Hernadi and Elekes (’93). During hibernation the large neurons of the mesocerebrum, which has a role in mating and avoidance behavior (for a review, see Chase and Li, ’94; Li and Chase, ’95), showed immunoreactivity for ET, CGRP, SP, and CCK8. This indicates that all these BAPs are involved in these activities or that the mesocerebral neurons have more numerous roles than expected. The same consideration may be made for GN, which controls the feeding behavior (Chase and Tidd, ’91; Chase and Tolloczko, ’92) and is immunopositive not only to CCK8, but also to CGRP and ET. Finally, it is particularly interesting that there are also differences in the immunoreactivity of the paired mesocerebral lobes during hibernation; this supports a different involvement of the right and left mesocerebrum in the hormonal and/or neuronal activities (for a review, see Chase and Li, ’94). Serotonin immunocytochemistry in hibernating pulmonate snails Another interesting result concerns the changes, not in only number but also in intensity in immunoreactivity for serotoninergic neurons of the metacerebrum. In particular, the metacerebral GN was described as a typical serotonin-containing neuron (Osborne et al., ’82; Osborne, ’84; Hernadi et al., ’89). This neuron increased its immunoreactivity during hibernation, suggesting an accumulation of serotonin in the neuronal cytoplasm, as observed for peptides. It has been shown, however, that 3H-serotonin injected into serotoninergic somata accumulates in both synaptic vesicles and large lysosomes (Cottrel and Osborne, ’70; Schkolnik and Schwartz, ’80). Consequently, it has been suggested that serotoninergic synaptic vesicles that are not released are ultimately transported back to the soma, where they are degraded by lysosomes. Electron immunocytochemistry will clarify whether the increase in the immunopositivity of the soma of the GN is due to the storage of the neurotransmitter before the release or before being degraded by lysosomes. It must be stressed that the immunoreactivity of serotonin was decreased in the nearby neuropile. HIBERNATION AND NEURONAL ACTIVITY Functional changes in the CNS during hibernation The physiology and metabolism of the organism change during hibernation in both vertebrates and invertebrates (Hunter, ’64; Machin, ’75; Heller, ’79; Lyman et al., ’82), to different extents, according to the modality of hibernation and, probably, to the systematic position. Studies carried out mainly on organs, tissues, and cells from vertebrate species (Kolaeva et al., ’80; Barni et al., ’87; Bernocchi et al., ’86; Giacometti et al., ’89; Anderson et al., ;90; Barni and Bernocchi, ’91; Fenoglio et al., ’92; Malatesta et al., ’94, ’95; March and Reisman, ’95) have explained some control mechanisms of adaptation of the animals to different environmental conditions and have provided interesting contributions to the knowledge of the structure and function of quiescent cells. In the vertebrate CNS, information has been acquired concerning some structural and functional changes of neurons during natural hibernation, mainly regarding the condensation state of chromatin (Bernocchi et al., ’86) and nucleolar and nuclear organization (Giacometti et al., ’89; Malatesta et al., ’94, ’95). These studies showed a lower activity of the nucleus both in amphibians and in mammals. There are few data about seasonal variation in serotonin, dopamine and enkephalin contents in gastropod ganglia (Cardot, ’71; Hiripi and Salanki, ’73; Hernadi et al., ’89, ’93; Gutierrez and Asai, ’91). The biochemical analysis demonstrated that levels of serotonin and dopamine were higher in the spring than in winter (Hernadi et al., ’89; Juhos et al., ’93). Furthermore, there are few studies about the histochemistry of circumesophageal ganglia during the seasonal cycle (Gesser and Larsson, ’85; Hernadi et al., ’89; Kaufmann et al., ’95). In particular, Hernadi et al. (’93) observed a notable amount of dopamine in the CNS of Helix, accompanied by a low number of dopamine-immunopositive neurons in summer, but the highest number of dopamineimmunopositive neurons was present in winter, when the dopamine content was lower. The different distribution and intensity of immunopositivity we have found in the neuropile and in neuronal somata could explain this discrepancy. The immunocytochemistry of some markers of systems that regulate neuronal morphology and activity showed clear changes in the cerebral ganglion of H. aspersa during hibernation in comparison with the active period (Vignola et al., ’95). In hibernating snails, there was an increased immunoreactivity for calmodulin, in agreement with 365 an increase of the intracellular Ca2+ concentration. Calmodulin is the major intracellular Ca2+ recipient in most eukaryotic cells and is thus an important mediator for many basic cellular processes, including the synthesis and release of neurotransmitters. This suggests that, during hibernation, neurons decrease or suppress the Ca2+dependent mechanisms. The lowered Ca2+-ATPase activity (Vignola et al., ’95), which also plays a role in regulating the intracellular ionized calcium concentration, supports this interpretation. Finally, the immunocytochemical expression of some cytoskeletal components also changed during hibernation (Vignola et al., ’95). 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