Monoamine receptors and immature cerebellum cytoarchitecture after cisplatin injury.

The experimental model of cisplatin treatment provides the opportunity to identify the precise function of the neurotransmitters in some crucial events of brain development, and their interactions or modulatory roles. The serotonin and noradrenaline monoamines influence the formation of the cerebellar cortex circuitry. In this study we found changes in the expression of the serotonin and noradrenaline receptors after a single injection of cisplatin in 10-day-old rats. The growth of Pc dendrites was early altered in lobules VI-VIII of cerebellum vermis. In these lobules, at postnatal day (PD) 17, the cisplatin-induced increase of the serotoninergic receptor 5-HT2AR, a factor that inhibits Pc dendrite growth by acting post-synaptically, occurred in all cerebellar layers, suggesting also alteration of granule cell proliferation and migration. The decreased labelling of beta l adrenergic receptor (beta1AR) in the soma of some Pc at PD11 can be correlated with the altered expression of glutamate receptors and GAD65 (glutamic acid decarboxylase) of and on Pc we have previously described [Pisu, M.B., Guioli, S., Conforti, E., Bernocchi, G., 2003. Signal molecules and receptors in the differential development of cerebellum lobules. Acute effects of cisplatin on nitric oxide and glutamate system in Purkinje cell population. Dev. Brain Res. 145, 229-240; Pisu, M.B., Roda, E., Avella, D., Bernocchi, G., 2004. Developmental plasticity of rat cerebellar cortex after cisplatin injury: inhibitory synapses and differentiating Purkinje neurons. Neuroscience 129, 655-664]. Moreover, beta1AR seems to be the key factor in the cerebellar reorganization between PD17 and PD30. The expression of this receptor was maintained in the molecular layer (ML), in particular in the inhibitory interneurons, despite their different distributions. The labelling of 5-HT1AR in the ML areas lacking Pc dendrite branches could contribute to the recovery phase of the cerebellar cytoarchitecture in cisplatin-treated rats. In general these findings should be taken into consideration in therapeutic interventions for developmental CNS disorders with a morphological basis.

Tissue damage after acute intoxication by polychlorinated biphenyls in cockroaches.

It is common knowledge that polychlorinated biphenyls (PCBs) represent a serious threat to the health of both vertebrates and invertebrates. As far as the former are concerned, especially as regards human beings, a broad literature describes the direct and indirect effects induced by the PCBs on their systems and organs. Among invertebrates, the information available is mostly related to arthropods and is, however, very scarce. The aim of this work was to evaluate the effects of polychlorinated biphenyls (PCBs) on tissues and organs of individuals belonging to a species of Blattaria (Blattella germanica) treated with various doses of this toxic material. The pathologies found became more serious as the dosage increased and were present throughout the entire digestive system, in the fat body and in the male gonads: in these areas cell and tissue breakdown and severely damaged spermiogenesis were observed. In particular, the testes, Malpighian tubules and fat body accumulated an amorphous basophilic PAS-positive substance. Furthermore, the NOS-dependent NADPH diaphorase activity pattern in the retina and optic lobes was more evident in the treated than in the control insects.

Nitric oxide-containing neurons in the nervous ganglia of Helix aspersa during rest and activity: immunocytochemical and enzyme histochemical detection.

Nitric oxide synthase (NOS) immunoreactivity and staining for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase) activity are two cytochemical markers for nitric oxide (NO)-containing neurons. The authors examined the changes in the distribution of NOS immunolabeling and NADPH-diaphorase reactivity in the cerebral and buccal ganglia of the terrestrial snail Helix aspersa during resting and active phases. During inactivity and after 1 day of activity, in the mesocerebrum and metacerebrum of the snails, there were several reactive neurons for both markers; after 7 days of activity, the number of reactive neurons was lower. Opposite results were obtained in the buccal ganglia, in which increased staining and numbers of reactive neurons were present in the active snails (after 1 day and 7 days of activity). Although the staining patterns for the two reactions were similar, colocalization was not always observed. The comparison between inactive and active animals provided a more precise survey of NOS-containing neurons in the snail cerebral ganglia than previously described. Moreover, it suggested that not only is NO involved in distinct nervous circuits, but, as a ubiquitous molecule, it also plays a role in neuroprotection and neuropeptide release.

Developmental patterns in the rat cerebellum after cis-dichlorodiammineplatinum treatment.

A cytochemical study was made of some metabolic enzymes in the cerebellar neurons during postnatal ontogenesis after injection of cis-dichlorodiammineplatinum into 10-day-old rats. The profiles during development of neuron-specific enolase immunoreactivity (involved in the glycolytic pathway), dihydrofolate reductase activity (involved in the metabolism of nucleic acids and folate) and dipeptidylaminopeptidase II activity were determined in lobules V-VII of cerebellar vermis. At different developmental stages, treated rats had folia in which the morphology and cytochemical responses of Purkinje neurons were greatly affected. On postinjection day 1 (PD 11), only neuron-specific enolase immunoreactivity was changed, reactions being more intense at the basal pole, which was abnormally enlarged in several neurons. Seven days after treatment (PD 17), the dihydrofolate reductase reaction showed weakly positive cells with small grains of formazan in the perinuclear regions and dipeptidylaminopeptidase II activity, which had appeared at this time in some cells of the controls, was not observed. On PD 25 and PD 35, Purkinje cells, probably undergoing clear degeneration, were negative or very weakly positive in all the reactions. Some tracts of folia had no Purkinje cells. These results suggest that cis-dichlorodiammineplatinum affects the differentiation of Purkinje neurons and interferes first with the glycolytic enzyme and then with some enzymes of the synthetic and catabolic machinery, leading to cellular dysfunction and degeneration.

The annual cycle of Erinaceus europaeus L. as a model for a further study of cytochemical heterogeneity in Purkinje neuron nuclei.

The cytochemical heterogeneity of nuclei in Purkinje cells, which reflects differences in metabolic states within the population, has been studied by comparing cerebellar hemispheres of the hedgehog during periods of activity and hibernation. Cerebellar granule cells and hepatocytes served as controls for diploid cells. Three different cytochemical stains (Feulgen reaction, Propidium iodide and Hoechst 33342 fluorochromes) were used in order to evaluate by microdensitometry and microfluorometry how much the heterogeneity is dependent on the variations in DNA content and degree of chromatin compaction. In the active period, Feulgen-DNA values of Purkinje cells were more widely dispersed than in control cells. Some values (18-40% of the population) exceeded (hyperdiploid) the diploid value. With Propidium iodide and Hoechst 33342 (under conditions for quantitative evaluation of DNA), a few values also exceeded the maximum value for control cells. We conclude that there is nuclear heterogeneity (diploid-hyperdiploid) in the Purkinje neuron population during normal activity. The percentage of hyperdiploid nuclei changes according to the technique used; in particular, the comparison of the different stains indicates that a different chromatin compaction is mainly responsible for hyperdiploid Feulgen-DNA values, but in a few hyperdiploid cells there is an actual surplus of DNA. In view of the modalities of detection of DNA by the different cytochemical approaches utilized, this may be mainly single-stranded DNA. During hibernation nuclear heterogeneity is still present in Purkinje neurons, but with a shift of the distribution towards lower values; hyperdiploid nuclei were not detected with all the procedures used. Moreover, when Propidium iodide is used at the concentration suitable for qualitative studies, the dye intercalation is lower than during activity. Data suggest that both decreased DNA and increased chromatin compaction occur in Purkinje cells, thus pointing to a lower metabolic activity of these neurons in hibernating animals. The changes in diploid granule cells were always less than those in Purkinje neurons and could be ascribed to a further increase in chromatin compaction only, not requiring changes in DNA content.

Cis-dichlorodiammineplatinum alters GABAergic structures in the immature rat cerebellum.

It has been reported that injection of the antitumoral drug cis-dichlorodiammineplatinum at 10 days of life affects cerebellar development in rats. After a single dose of 5 micrograms/g of body weight, the formation of granule cells is decreased and the maturation of postmitotic neurons is slowed down. A substantial time after treatment, reduced cell packing density of the internal granule layer and atrophy of the molecular layer can be observed. In addition, there is degeneration of some Purkinje cells and Golgi neurons. In spite of all these alterations, the regular architecture of the cerebellar folia is retained in many places. In the present study, we used immunocytochemistry with an immune serum raised against glutamic acid decarboxylase to further characterize the cis-dichlorodiammineplatinum-induced alterations of GABAergic neurons. The aim was to examine cerebellar development and to test for factors controlling the settling of GABAergic circuits. At all post-treatment intervals, most of the Purkinje and Golgi neurons and molecular layer interneurons showed stronger anti-glutamic acid decarboxylase immunoreactivity than in controls; this may have been due to altered fixation because of cis-dichlorodiammineplatinum-induced damages to the blood vessels; but could also reflect cellular retention of the enzyme, maybe due to cis-dichlorodiammineplatinum-induced damage of the microtubular apparatus. After seven days, large roundish immunoreactive varicosities were present in the molecular layer adjacent to the Purkinje cell dendritic poles. These varicosities, which were not observed in control animals, may be terminals of Purkinje cell axon recurrent collaterals contributing to the supraganglionic plexus, whose abnormal development would compensate for the reduced inhibitory inputs from inhibitory interneurons and/or Purkinje cells, which degenerated at early post-treatment intervals. At later post-treatment intervals (15 and 21 days), there were also alterations in the pericellular basket at the Purkinje cell axon hillock, which was poorly developed in or absent from the majority of cells. The finding was confirmed by morphological observation of basket cells in Golgi-Cox preparation and immunocytochemistry with an antibody raised against 200,000 mol. wt phosphorylated neurofilaments. It is concluded that early changes in anti-glutamic acid decarboxylase immunoreactivity of neurons may be due to a direct interference of the drug with the cellular metabolic pathways. The late anomalies in the anti-glutamic acid decarboxylase immunoreactivity appear to be secondary to changes in the tissue cytoarchitecture rather than being primary cis-dichlorodiammineplatinum-induced lesions of the cells.

Developmental plasticity of rat cerebellar cortex after cisplatin injury: inhibitory synapses and differentiating Purkinje neurons.

A single injection of cisplatin, a cytostatic agent, (5 microg/g body weight) in 10-day old rats leads later to the reorganization of the cerebellar cortex in lobules VI-VIII of the vermis. Double immunofluorescence reaction for glutamate receptor (GluR)2/3, a ionotropic glutamate receptor that labels postsynaptically Purkinje neurons, and glutamic acid decarboxylase (GAD)65, an isoform of the GABA synthesis enzyme that labels presynaptically inhibitory terminals in the molecular layer, were employed. Less-differentiated Purkinje cells were present in rats treated on postnatal day (PD)11 at the top of lobule VI and in lobules VII-VIII, in comparison with the deep zones of the same lobules and lobule III. The changes were interpreted as due to loss of trophic factors of Purkinje cell growth, e.g. signaling molecules and granule cells. However, we have shown that a remodelling of Purkinje cell dendrites occurred on PD30 (20 days after cisplatin). In fact, despite of the GluR2/3 labeling of the entire Purkinje cell dendrites, the GAD65 immunofluorescent terminals were adjacent to the proximal parts of the dendrite, while they were scarce in the distal dendritic branchlets. The findings were discussed in relation to the changed cytoarchitecture of the cerebellar cortex, which from PD17 to PD30 includes regeneration of the external germinal layer, reorientation of the main dendritic branches and of the Purkinje cell branchlets, and the presence of ectopic cells.

Immunocytochemical changes of cytoskeleton components and calmodulin in the frog cerebellum and optic tectum during hibernation.

During hibernation, variation in the metabolism of nerve cells occurs. Since the cytoskeleton plays an important role in nerve cell function, we have analyzed the immunocytochemical expression of two cytoskeleton components, i.e. phosphorylated 200 kDa neurofilament protein, and microtubule-associated protein 2 in the cerebellum and optic tectum of hibernating frogs (Rana esculenta) in comparison with active animals. In addition, we have considered the immunocytochemical expression of calmodulin, which is known to be involved in neurofilament phosphorylation. In hibernating animals, there was a decrease in the immunoreactivity for phosphorylated 200 kDa neurofilament protein and microtubule-associated protein 2 of fibers in both the cerebellum and in the optic tectum. In contrast, in the large neurons of the cerebellum, i.e. Purkinje neurons, there was an increase in the immunoreactivity for microtubule-associated protein 2. The changes in the cytoskeleton components were accompanied by a decrease in calmodulin immunoreactivity in the cytoplasm of nerve cells of the cerebellum. All the changes observed are consistent with a low neuronal activity during hibernation, as also indicated by previous microdensitometric and microfluorometric data. This shows a higher degree of chromatin condensation in hibernating animals and suggests that hibernation represents a simple form of neuronal plasticity.

Distribution of calretinin-like immunoreactivity in the brain of Rana esculenta.

The distribution of calretinin-like immunoreactivity has been analyzed in the brain of Rana esculenta. Several neurons of nuclei belonging to sensory pathways, subhabenular area and left habenula were immunopositive. Immunoreactivity was present in fibers of motor and sensory pathways, thalamus, tegmentum and isthmus. The immunolabeling pattern partially overlapped that previously described in the rat. However, in comparison with the rat, fewer cells and fibers were immunoreactive and there were less positive brain nuclei. especially in the pallium, septum and striatum, that were totally negative. Taking into consideration that these regions are rather simple in the frog, the presence of calretinin seems to be consistent with the degree of complexity of brain areas and segregation of different nuclei.

Inhibitory cerebellar circuits: GAD immunoreactivity in the frog cerebellum.

The presence of GABAergic structures in the frog cerebellum with simple nervous circuits was investigated with an immune serum raised against glutamic acid decarboxylase (GAD). GAD immunoreactivity is detectable in all the cerebellar layers. The most obvious difference in comparison with mammals is the scarcity of GAD-positive terminal boutons on Purkinje cells and this is consistent with neurophysiological data indicating that the inhibition on frog Purkinje cells is weaker than in mammals. However, there are numerous GAD synapses on cerebellar and vestibular nuclei. A large number of GAD immunoprecipitates are present in the molecular and granular layers, though their morphological organization is different from that in mammals. In the upper part of the molecular layer GAD immunoreactive cells and fibres form a plexus-like structure and in the internal granule layer immunoreactive varicosities are not arranged in the ring-like assemblies of mammals.

Electron-cytochemical localization of succinic semialdehyde dehydrogenase activity in Purkinje neurons and hepatocytes of the rat.

A method is presented for the ultrastructural demonstration of succinate semialdehyde dehydrogenase (SSADH) activity in cerebellar Purkinje neurons and liver hepatocytes; SSADH is an enzyme involved in the degradation of gamma-aminobutyric acid (GABA). Incubation media originally used for light microscopy were considered. Reaction products were mainly detected when fresh tissue was used. In Purkinje cells, grains ascribable to SSADH activity were localized on the mitochondria (especially on the outer membrane); some extramitochondrial formazan deposits were also found. After brief fixation by immersion or perfusion, only a few formazan granules were detected in the cytoplasm. A similar distribution pattern was observed in hepatocytes, in which extramitochondrial grains and grains on the nuclear membrane were frequent. The actual existence and the possible meaning of extramitochondrial SSADH activity is critically discussed on the basis of the data in the literature.

A re-evaluation of the ultrastructural localization of 5'-nucleotidase activity in the developing rat cerebellum, with a cerium-based method.

The membrane ectoenzyme 5'-nucleotidase converts 5'-AMP into adenosine which, in the nervous tissue, plays an important role as intercellular messenger. Moreover, during histogenesis, 5'-nucleotidase seems to be related to cell proliferation and migration. Conflicting data are reported in the literature about the localization (neuronal or glial) of 5'-nucleotides in the rat cerebellum. In the present report we have analyzed the distribution of 5'-nucleotidase activity with electron microscopy, using a cerium-based method, at different postnatal histogenetic stages (postnatal days (PND) 11, 17, 28). On PND 11 and 17, rims of reaction product outlined the plasma membranes of some neuroblasts in the external granular layer and of parallel fibers and some migrating cells in the developing molecular layer. Positivity was frequently observed on membranes of adjacent neuronal cells and glial processes. Moderate activity was also present on the membranes of granule cells and of mossy fiber rosettes and granule cell dendrites constituting the cerebellar glomeruli within the internal granule cell layer. At PND 28, the reaction product was slightly reduced in some localizations. Cytochemical patterns prove that the cerium-based method is suitable for demonstration of 5'-nucleotidase-specific activity. In fact, a continuous and fine reaction product appears strictly linked to the cell membranes, and no unevenly scattered precipitates can be observed. Data suggest that, during cerebellar histogenesis, 5'-nucleotidase may be involved in the mechanisms of cell migration and proliferation. However, in adulthood, prominent localization of the reaction product on neuronal elements suggests a major role in neuromodulation processes for the enzyme.

Seasonal changes in the nucleoli of Purkinje cells of the hedgehog cerebellum.

A relationship between size and shape of nucleolus and cellular metabolic demands can be seen from measurements of Purkinje cell nucleoli in cerebellar hemispheres during the annual cycle of the hedgehog. During hibernation, nucleoli are smaller than during activity. The extent of the associated heterochromatin increases from activity to the beginning of hibernation. Moreover, during activity it is mostly distributed in small masses all around the nucleolus, while during hibernation it is clumped in a single mass. Data indicating a lesser protein synthesis by neurons during hibernation agree with electrophysiological indications that during hibernation the cerebral cortex, linked to the cerebellar hemispheres via afferent systems, is silent.

Mature and developing visual system of Ceratitis capitata (Diptera, Tephritidae): histochemical evidence of nitric oxide synthase in the wild type and the white eye mutant strains.

Nitric oxide (NO) is acknowledged as a messenger molecule in the nervous system. It has a role in the modulation of the chemosensory information and seems implicated also in visual processes and visually guided behaviour of some insects. In the present study, we used two different strains of the medfly Ceratitis capitata (Diptera, Tephritidae), a wild type eye colour and a white eye mutant line, as models to clarify the involvement of NO in the mature and developing visual system. The comparison between the pattern of enzyme histochemical localization of NO synthase (NOS), through NADPH diaphorase (NADPHd) staining, in the optic lobes of the two strains revealed for adults a stronger intensity of reaction in all the neuropiles and the sub-retinic monopolar cell layer of the wild type flies, with respect to the white eye mutant correspondent areas. Anti-NOS immunocytochemistry correlated with these results, underlying reactivity both in fine fibres and varicosities and in cell bodies and supporting the idea of presence of NOS also in the retina of the medfly optic lobes. NADPHd reactivity was present in the first developmental stages of the white eye mutant also, but at lower intensity than wild type, and it decreased in some areas during the transition to adult fly stage both in the wild type and in the white eye mutant. All these observations together indicate that changes in the NO system of C. capitata could be related to the visual information processing, when the visual response or discrimination are altered. Furthermore, NO may be involved in the establishment of the retinal projection pattern and in the control of optic lobes morphogenesis.

Alteration of complex IV and acetylcholine-related enzymes in experimental spinal cord injury.

Acute, severe injury of the rabbit spinal cord, induced by the weight-drop method, causes alterations of the enzyme activities related to cholinergic and energy metabolism. Morphological examinations at the trauma site show degenerative processes in neurons 0.5 hr posttrauma and a marked decrease in the number of living cells 24 hrs later. Both biochemical and cytochemical findings show that the tissue metabolic and morphologic derangement, caused by severe spinal cord injury, is mostly confined to the gray matter at an early stage (0.5 hr), whereas 24 hrs later the white matter is also involved. The decrease in choline acetyl-transferase and acetylcholinesterase activities in the gray matter parallels the impairment of complex IV (cytochrome c oxidase) of the respiratory chain and the presence of morphological alteration in neurons. The dramatic drop in the enzyme activities, observed 24 hrs after the induction of the severe trauma is clearly associated with the loss of cells.

Morphohistochemical changes in hepatocytes during the life cycle of the European eel.

The comparative analysis of morphological, histochemical and cytochemical patterns of eel (Anguilla anguilla L.) hepatocytes reveals clear differences between two stages of its life cycle, i.e. the trophic stage (yellow eel) and reproductive stage (silver eel). The storage of glycogen prevails in the yellow eel, whilst lipids appear to be remarkably increased in the silver eel, in which some hepatocytes also show glycogen-rich areas. Generally, in the silver eel dehydrogenase and acid phosphatase activities seem greater and different distribution of the reaction products is present; on the contrary, a lower G6PDH activity is observed. The electron microscopy characteristics and distribution of both cellular organelles and reserve materials reflect the modifications found at light microscopy. The ultrastructural patterns provide further evidence for the heterogeneity of liver parenchyma in silver eel. In particular, the coexistence of nuclei showing a different degree of chromatin compactness is also accounted for by the quantitative cytochemical data on the nuclear DNA after Feulgen reaction and intercalation with propidium iodide at low and high concentrations. With regard to the DNA content, the hepatocytes in the silver eel as well as in the yellow eel are mainly 2c. However, some 4c values are also found, which according to the literature can be ascribed to cells in G2 phase. The present data may express the onset of different functional requirements during the reproductive stage in comparison with the trophic one. Moreover, our results are consistent with modifications found by other authors as a consequence of interruption of nourishment and during gonad maturation, i.e. two phenomena characterizing the transition from yellow to silver eel.

Frog hepatocyte modifications induced by seasonal variations: a morphological and cytochemical study.

A correlated morphological and cytochemical approach was employed to study frog hepatocytes in different periods of their annual cycle, including the natural hibernating period. There were considerable changes in the distribution and organization of hepatic glycogen in different phases of the annual cycle, and distribution of organelles as well. The most striking findings were glycogen storage during the prehibernation and hibernation phases, followed by drastic glycogen depletion. Cytochemical staining of a number of enzymes (succinate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, paranitrophenyl phosphatase, acid phosphatase, and glucose-6-phosphatase) involved in a variety of metabolic pathways, showed various cytoplasmic localizations and differences in intensity of the reaction products as a function of seasonality. Morphological and cytochemical data were interpreted as evidencing different functional requirements during seasonal changes in the frog.

Nuclear changes and morphology of the epidermis in the hibernating frog.

Cytochemical changes of chromatin and DNA in frog epidermal cells were correlated with some morphological features to investigate the skin physiology during hibernation in comparison with the active period. The epidermal cells of hibernating frogs showed less condensed chromatin in all the layers; a greater loss of DNA was found during the transition from the middle to the superficial layer. In the germinative layer, a lesser frequency of hyperdiploid cells and a remarkably low amount of mitoses were detected; this is accompanied by the increase of epidermal thickness and the presence of two layers of cornified cells. The slowing of tissue differentiation and cell renewal kinetics during hibernation can be related to lowered activity of the frog skin. Further, the smaller intercellular spaces as well as the scarcity of puffed ER and vacuoles may be indicative of a lower ion transport in epidermal cells during hibernation.

The cerebral neurons of Helix aspersa during hibernation. Changes in the cytochemical detection of calmodulin, cytoskeletal components and phosphatases.

Some markers of the intracellular systems that regulate neuronal activity and morphology were analyzed in the cerebral ganglion of hibernating snails (Helix aspersa), in comparison with active animals. The immunocytochemical expression of a calcium-binding protein, i.e. calmodulin, and some cytoskeletal components, i.e. 200 kDa phosphorylated neurofilament protein (pNFH), microtubule associated protein 2 (MAP2) and alpha-tubulin were analyzed by the use of a panel of antibodies raised against mammal antigens. Moreover, by enzymatic reactions the Ca(2+)-ATPase and alkaline phosphatase (AIPase) activities were demonstrated. In comparison with the active phase, the hibernation induced an increase in the immunopositivity for calmodulin in all the neurons. The increase may be linked to unmasking of immunoreactive epitopes due to conformational changes of the protein, which in turn may be a consequence of a reduction or absence of binding with calcium ions or of a real increase in the amount of calmodulin in the somata of neurons. In any event, both the hypotheses indicate that neurons have decreased or suppressed the Ca(2+)-dependent mechanisms as also shown by the lower Ca(2+)-ATPase activity. Nevertheless, the AIPase activity, which was localized in the epineural sheat, was not significantly changed during hibernation and this supports that some metabolic activities are preserved in the hibernated animals. Changes in the immunopositivity for cytoskeletal components were found. There was an increase in the epitopes recognized by the mammalian pNF antibody, that concerned both the positivity of the entire cytoplasm of some clusters of metacerebral neurons and the intensity of the reaction. This would be aimed to improve the stability of the somata and primary neurites. Moreover, the decrease of alpha-tubulin and MAP2 immunopositivity, suggests that a disassembly of microtubules have occurred. The findings indicate that the transport of vesicles in the axons is slowed down during hibernation. In fact, research in progress show that the patterns of neurotransmission and neuromodulation are also deeply modified.

Perineuronal glial system in the cerebral ganglion of active and hibernating Helix aspersa.

The electron microscopical changes in the glial lacunar network that surrounds the large neurons of meso- and metacerebrum of land snail cerebral ganglia were considered, in order to get an insight into the functional role of this peculiar structure in invertebrates. Compared with snails during the active period, in the hibernating animals the extension of glial processes was reduced and the glial processes appeared more regular and stacked around neurons. Moreover, they did not form deep, long interdigitations with neuronal infoldings as during the active period. In particular, data on the ultracytochemical detection of alkaline phosphatase and Ca(2+)/Mg(2+)-ATPase enzyme activities, point to a correlation between the extension of the glial system and its function in the regulation of the extracellular environment. In fact, in hibernating snails, lower reactivity was found on the glial membranes, including those of the trophospongium.