Morphological traits - desiccation resistance - habitat characteristics: a possible key for distribution in woodlice (Isopoda, Oniscidea).

Terrestrial isopods, as successful colonizers of land habitats, show a great variety in species distribution patterns on a global, continental, or regional scale. On a local, within-habitat level these patterns reflect the species' tolerance limits and the presence of suitable hiding places (shelter sites, refugia). Humidity preference reflects a species' capability for water retention which, in turn, depends on the integumental barrier. Desiccation resistance is a key feature in isopod survival under different environmental conditions. The present study shows a correlation between cuticle thickness and desiccation resistance under three relative humidity (RH) ranges (about 30, 75 and 100% RH) in nine species, relating these to the species' differences in meso- and microhabitat choices. Habitat preferences are also associated with differences in cuticle surface morphology. The results support our hypothesis that species distribution and desiccation resistance are associated with particular cuticular morphological traits. Phylogenetic relations seem to be less important in desiccation resistance than cuticle thickness and external morphology.

Fine structure and synaptology of the nitrergic neurons in medial septum of the rat brain.

The nitrergic neuron population and certain aspects of their connectivity (peptidergic inputs, co-localization with GABA, synaptic target distribution) were studied in the medial septum of the rat brain. The histochemical localization of NADPH diaphorase and immunohistochemical identification of nNOS at light and electron microscopic level was applied. Double-labeling experiments with galanin and leucine enkephalin, moreover the postembedding GABA immunogold staining was also carried out. NADPH diaphorase- and nNOS-immunopositive neurons could be identified inside the borders of medial septum. Out of their peptidergic inputs galanin- and leucine enkephaline-immunopositive varicose fibers were found in close apposition with nNOS-immunopositive neurons. Based on fine structural characteristics (large indented nucleus, thin cytoplasmic rim, lack of axosomatic synapses) the nitrergic neurons are suggested to be identical with the septal cholinergic nerve cells. Their boutons established asymmetrical synapses mainly on dendritic shafts and spines, some of which were also nNOS-immunopositive. A lower amount of nNOS-immunopositive boutons of presumably extrinsic origin were found to be GABAergic.

Histological studies on the marsupium of two terrestrial isopods (Crustacea, Isopoda, Oniscidea).

The marsupium, a brood pouch in peracarid crustaceans (Crustacea, Malacostraca) has evolved in terrestrial environment for providing nutrition and optimal conditions for embryogenesis. In the present study we give details on the histology and ultrastructure of its constituting elements such as oostegites and cotyledons. Marsupia of two different eco-morphological types of woodlice, namely the non-conglobating species Trachelipusrathkii Brandt, 1833 and the conglobating species Cylisticusconvexus De Geer, 1778 were investigated. Light microscopic (LM) studies showed some differences in the main structure of the two species' brood pouch: in Trachelipusrathkii, a 'clinger' type woodlice, the oostegites bend outwards during brood incubation as growing offspring require more space, while in Cylisticusconvexus, a 'roller' type isopod, the sternites arch into the body cavity to ensure space for developing offspring and still allowing conglobation of the gravid females. The quantitative analysis of the oostegites' cuticle proved that the outer part is about 2.5 - 3 times thicker compared to the inner part in both species. Electron microscopic (TEM) examinations show only small histological differences in the oostegites and cotyledon structure of the two species. Cellular elements and moderately electron dense fleecy precipitate are found in the hemolymph space between the two cuticles of oostegites. The cells contain PAS positive polysaccharide areas. TEM studies revealed some differences in the cotyledon ultrastructure of the two species. Cotyledons of Trachelipusrathkii consist of cells with cristate mitochondria and granular endoplasmic reticulum with cisterns. Cotyledons of Cylisticusconvexus consist of cells with densely cristate mitochondria and ribosomes attached to vesicular membrane structures. In both species cells with electron dense bodies were observed. We conclude that - besides the differences in marsupial shapes - the fine structure of the oostegites and cotyledons is hardly affected by the eco-morphological type, specifically the conglobating or non-conglobating character of the studied species.

The effect of partial food deprivation on the astroglia in the dorsal subnucleus of the lateral septum of the rat brain.

The effect of 40% partial food deprivation was studied on the immunohistochemically detectable amount of glial fibrillary acidic protein (GFAP) - the specific marker of astroglia - in the dorsal subnucleus of lateral septum (LS) of male, intact and ovariectomized (OVX) female rats. Animals were either fed ad libitum (control) or 40% food deprived for one week, then perfusion-fixed, their brains removed, and serial vibratome sections were processed for the immunocytochemical localization of GFAP. Computeraided densitometry was carried out on digital photographs.The results showed that ovariectomy alone did not exert any effect on the density of GFAPimmunoreactivity (GFAP-IR) as compared to the values detected in intact females. Food deprivation increased the density of GFAP in each experimental group. The difference was most pronounced in males, significant in females and much less in ovariectomized females. Parietal cortex chosen as reference area did not show any increase in the local GFAP-IR.It was previously shown that the dorsal subnucleus of the lateral septum reacts with plastic neurochemical changes to food deprivation. Our results prove that these changes affect not only neuronal but also glial elements.

Eco-morphological studies on pleopodal lungs and cuticle in Armadillidium species (Crustacea, Isopoda, Oniscidea).

Terrestrial isopods (Crustacea, Isopoda, Oniscidea) have adapted to land life by diverse morphological, physiological and behavioral changes. Woodlice species exhibit a large variety in this respect, their preferences ranging from moist to dry habitats. These moisture preference values are related to various morphological adaptations, rendering terrestrial isopods amenable to studying morphological adaptations to terrestrial life. We performed a comparison of four Armadillidium species (Armadillidium zenckeri, Armadillidium nasatum, Armadillidium versicolor, Armadillidium vulgare), by quantifying two morphological traits: the extent of the interfacial endothelium between the respiratory space and the hemolymph within pleopodal lungs and the thickness of tergite cuticle, which are 'key factors' in determining protection from desiccation. These values were measured from light micrographs of cross-sectioned lungs. The cosmopolitan A. vulgare, as a habitat generalist, seems to be the most resistant against desiccation and other environmental conditions, while A. zenckeri is the most sensitive one. Light microscopic studies revealed that the four species can be ordered similarly, if we compare them by the extension of the endothelial interface and cuticle thickness, suggesting that these morphological traits are important determinants of their distribution on habitat, microhabitat scales and through the existence of suitable habitats - together with many other factors - the geographical pattern of species occurence.

Cocaine- and amphetamine-regulated transcript (CART) peptide-immunopositive neuronal elements in the lateral septum: rostrocaudal distribution in the male rat.

The morphological features and distribution of cocaine- and amphetamine-regulated transcript peptide immunoreactivity (CART-IR) were studied in the lateral septum (LS) of male rats using light and electron microscopic immunocytochemistry and computer-aided densitometry. CART-IR was detected along the rostrocaudal axis of the LS in varicose axonal fibers only, although immunoreactive cell bodies and dendrites were not detected. Pericellular basket-like arrangements around immunonegative cell bodies were present. From among the targets of such pericellular baskets, glutamic acid decarboxylase (GAD)-immunopositive and NPY-immunoreactive somata were identified. Thin varicose axons were present in each section, whereas thick varicose axons were restricted to the sections of rostral position only. CART-IR was observed in varicose fibers forming a dense subependymal plexus, from which solitary varicose fibers entered the ependymal layer. The fine structure of varicosities was similar to that of other neuropeptide-containing fibers. Small varicosities established asymmetrical synaptic contacts mainly with dendrites and dendritic spines, and larger varicosities established symmetrical synapses with somata and dendritic shafts. CART-to-CART connections were not revealed. The density curve of the CART-IR along the rostrocaudal axis of LS was found to be paraboloid. CART is known as one of the most anorexigenic peptides. These results serve as basis for further physiological studies concerning the biological significance of lateral septal CART peptide in the regulation of food intake.

Synthetic calpain activator boosts neuronal excitability without extra Ca2+.

Earlier we have shown that an equimolar mixture of calpastatin subdomains A and C (19 amino acids each) strongly activates m-calpain in vitro. In the present work we developed a membrane-permeable activator system, by conjugating an oligo-arginine tail to both peptides. We tested calpain activation as well as synaptic excitability on rat brain slices ex vivo. In hippocampal slices both basic excitability and long-term synaptic efficacy were significantly increased upon treatment with the activator. We propose that the activator peptide conjugates can be used with any mammalian cell, to specifically challenge the calpain system apparently without raising cytoplasmic Ca2+. Such an effector may be a useful tool in dissecting intracellular mechanisms involving the calpain system.

Synaptic alpha-dystrobrevin: localization of a short alpha-dystrobrevin isoform in melanin-concentrating hormone neurons of the hypothalamus.

The expression of the two members of the dystrobrevin (DB) family in the adult brain was thought to be highly specific for the two main cell types: alpha-dystrobrevin (alpha-DB) and beta-dystrobrevin (beta-DB) has been identified as glial and neuronal proteins, respectively. In the present work we show that a subset of neurons in the hypothalamus contains alpha-DB. Comparative immunohistochemical studies with two alpha-DB antibodies of different specificity indicate that the neurons contain short alpha-DB isoform(s) alpha-DB-2 and/or alpha-DB-4. Immunoreactive multipolar or spindle-shaped neurons form clusters with bilateral symmetry, localized predominantly in the lateral hypothalamic area, with extensions into the zona incerta and the dorso-medial and ventro-medial hypothalamic region. alpha-DB immunoreactivity was localized in cell processes and at postsynaptic densities, furthermore in the endoplasmic reticulum within the perikarya. alpha-DB-positive neurons are beta-dystrobrevin immunoreactive, but alpha- and beta-DB do not co-localize with their usual molecular anchors like dystrophins or high molecular weight forms of utrophin. Colocalization with nNOS was also not observed. The pattern of alpha-DB immunoreactive neurons gave a perfect colocalization with melanin-concentrating hormone (MCH) neurons throughout the whole region studied. We propose that alpha-DB plays a role in a structure or regulation mechanism unique to MCH-expressing neurons.

Chronic fasting-induced changes of neuropeptide Y immunoreactivity in the lateral septum of intact and ovariectomized female rats.

The effect of 40% food deprivation for 1 week on the immunohistochemically detectable amount of neuropeptide Y (NPY) was studied in the lateral septum (LS) of intact and ovariectomized (OVX) female rats. Animals were either fed ad libitum or 40% food-deprived. Densitometric analysis of immunostained material showed a significant decrease in NPY-immunoreactivity (NPY-IR) in OVX rats compared to the control group. Food deprivation increased the density of punctate NPY-IR profiles in both intact and OVX animals, however, the density in food-deprived OVX animals was increased compared to baseline but remained reduced compared to intact rats. Our study indicates that the lack of gonadal hormones - most likely estrogen - results in a decrease in the density of NPY-IR axonal fibers within the LS, while food deprivation induced considerable elevation in NPY density. Food restriction-induced changes in the density of NPY-containing neural elements suggest that the LS may play a crucial role in the regulation of food intake and energy balance, in concert with the relevant hypothalamic areas.

Dystrophin splice variants are distinctly localized in the hippocampus.

It has previously been demonstrated that Dp71, the most abundant dystrophin protein in the brain, is mainly localized in the postsynaptic densities. Here we show the localization of Dp71f, one of the splice variants of this protein, within the CA3 region of the hippocampus. Immunopositivity occurs in the postsynaptic density of small asymmetrical axospinous and axodendritic synapses, while it is absent in the postsynaptic densities of the axospinous synapses of the large mossy fiber terminals. Dp71f immunoreactivity was found to be attached to the membranes of the mossy fibers in the stratum lucidum of the CA3 area. In a certain population of thin myelinated axons the protein seems to be present within the axon proper. These data support the notion of a physiological role of Dp71f distinct from other dystrophin isoforms present in the central nervous system.

Fasting-induced changes of neuropeptide immunoreactivity in the lateral septum of male rats.

The objective of the present study was to determine the rostrocaudal distribution and the effect of reduced food intake (60% of the average daily food intake for 1-4 weeks) on the amount of leucine-enkephalin (Leu-enk), neuropeptide Y (NPY) and galanin (Gal) in the lateral septum of male rat brain. Using pre-embedding immunocytochemistry combined with densitometry on 60 microm serial vibratome sections we found that in control animals Leu-enk-immunoreactive elements showed an increasing density from rostral towards the medial part of the septum, then a gradual decrease towards the caudal direction. The distribution of NPY proved to be rather even along the examined sequence of sections with two smaller peaks roughly at the 1/3 and 2/3 of the rostrocaudal axis. Gal showed similar distribution but the peaks were shifted to more caudal direction. We also found that Leu-enk forms the most dense plexus followed by a moderate amount of NPY-positive axonal meshwork. Gal was present in the lowest amount along the lateral septal nuclei. The effect of reduced food intake was marked and differential in the case of the three examined peptides. During the first 2 weeks of reduced food intake NPY-immunoreactivity was upregulated as compared to the control, then it was reduced close to the control value by the 4th week. The changes in Gal immunoreactivity showed similar pattern. The average relative density of Leu-enk-immunoreactive elements immediately decreased as a result of reduced food intake for 1 week and it gradually decreased by the end of the 4th week. These results indicate that reduced food intake affects the expression of NPY, Gal and Leu-enk not only in the relevant hypothalamic neuroendocrine centres, but also in the lateral septal area.

Somato-dendritic nicotinic receptor responses recorded in vitro from the medial septal diagonal band complex of the rodent.

The medial septal diagonal band area (MS/DB), made up of GABAergic and cholinergic neurones, plays an essential role in the generation and modulation of the hippocampal theta rhythm. To understand the part that the cholinergic neurones might play in this activity, we sought to determine whether postsynaptic nicotinic receptor responses can be detected in slices of the rodent MS/DB by puffing on acetylcholine (ACh). Neurones were characterized electrophysiologically into GABAergic and cholinergic neurones according to previous criteria. Responses of the MS/SB neurones to ACh were various combinations of fast depolarizations (1.5-2.5 s), fast hyperpolarizations (3-4 s) and slow depolarizations (20-30 s), the latter two being blocked by atropine. The fast depolarizations were partially or not blocked with cadmium and low calcium, tetrodotoxin, and antagonists of other ionotropic receptors, and were antagonized with 25 microm mecamylamine. Pharmacological investigation of the responses showed that the alpha 7* nicotinic receptor type is associated with cholinergic neurones and 10% of the GABAergic neurones, and that non alpha 7* nicotinic receptor subtypes are associated with 50% of the GABAergic neurones. Pharmacological dissection of evoked and spontaneous postsynaptic responses, however, did not provide evidence for synaptic nicotinic receptor transmission in the MS/DB. It was concluded that nicotinic receptors, although prevalent on the somatic and/or dendritic membrane compartments of neurones in the MS/DB, are on extrasynaptic sites where they presumably play a neuromodulatory role. The presence of alpha 7* nicotinic receptors on cholinergic neurones may also render these cells specifically vulnerable to degeneration in Alzheimer's disease.

Distribution and origin of vesicular glutamate transporter 2-immunoreactive fibers in the rat hippocampus.

This study examined the distribution of vesicular glutamate transporter 2 (VGLUT2)-immunoreactive neuronal structures in the ipsilateral and contralateral hippocampi of unilateral fimbria/fornix transected, unilateral entorhinal cortex ablated, and intact female and male rats. In the hippocampi of intact animals, the highest density of VGLUT2-positive boutons was observed in the supragranular layer of the dentate gyrus, followed by the CA2 pyramidal and oriens layers, and the stratum lacunosum-moleculare of the CA1 field. This staining pattern was identical both in males and in females. Electron microscopic examination revealed that the immunolabeling was confined to axon terminals forming exclusively asymmetric synaptic contacts. The quantitative analysis of the synaptic targets of VGLUT2-positive terminals showed that in the dentate gyrus, 59% of the synaptic targets were dendritic spines, followed by dendritic shafts (22%) and granule cell somata (19%). In the pyramidal layer of the CA2 field, VGLUT2-immunoreactive boutons contacted mostly dendritic shafts (85%), only some of which (15%) synapsed with spines. The synaptic targets of VGLUT2-positive varicosities were dendritic spines (71%) and shafts (29%) in the stratum lacunosum-moleculare of the CA1 field. The fimbria/fornix transection caused a significant reduction in the density of VGLUT2-positive boutons only in the CA2 field, while entorhinal cortex ablation elicited no change in fiber density in any of the areas analyzed. Furthermore, our latest experiments on colchicine-treated animals revealed a large population of VGLUT2-positive neurons in the hippocampus that may be a possible intrinsic source of hippocampal VGLUT2 boutons. Our results suggest that the most likely sources of VGLUT2-positive boutons in the dentate supragranular layer, the CA2 area, as well as in the stratum lacunosum-moleculare of the CA1 field, might be the mossy cells, the supramammillary area, and the nucleus reuniens thalami, respectively.

Differential distribution of calpain small subunit 1 and 2 in rat brain.

Calpains, the Ca(2+)-dependent thiol proteases, are abundant in the nervous tissue. The ubiquitous enzyme forms in mammals are heterodimers consisting of a specific, micro or m, large (catalytic) subunit and, apparently, a common small (regulatory) subunit (CSS1). Recently, however, we described a second form of small subunit (CSS2), which is of restricted occurrence [Schád, E., Farkas, A., Jékely, G., Tompa, P. & Friedrich, P. (2002) Biochem. J., 362, 383-388]. Here we analysed the distribution of immunoreactivity in various parts of rat brain against two anti-CSS1 and two anti-CSS2 antibodies by correlated light and electron microscopy. Remarkably, the antibodies showed differential distribution in various parts of rat cortex: anti-CSS1 reacted mainly with perikarya and dendrites, whereas anti-CSS2 was more prominent in axons. In serial sections CSS2 and synaptophysin gave very similar patterns, i.e. these epitopes seem to colocalize. Electron microscopy confirmed that CSS1 was mainly localized postsynaptically in dendrites and somata, whereas CSS2 was found presynaptically. The hypothesis is advanced that these distinct distributions of calpain subunits may be related to the transport of these enzymes in nerve cells.

Kappa-opioid receptor in the rodent hippocampus: a comparative immunocytochemical study in the rat, guinea pig, hamster and gerbil.

Pre-embedding light microscopic immunocytochemistry, using a monoclonal antibody (mAb-KA8) raised against a frog brain kappa receptor preparation, recognising selectively the kappa-opioid receptor, was used for studying the occurrence, distribution, and species-specificity of the kappa-opioid receptor in the hippocampal formation of four rodent species (rat, guinea pig, hamster and gerbil). MAb-KA8 immunoreactivity was detectable in the rat, hamster and gerbil hippocampus, however the distribution of the labelled structures was heterogeneous. In the rat and hamster the hilus of dentate gyrus and the stratum oriens of the CA1 area contained immunoreactive cell bodies and proximal dendrites. In the gerbil mAb-KA8 immunopositive cell bodies were recognisable in the stratum radiatum of the CA1 and CA3 areas and in the subiculum. In the hamster varicose axon-like elements were also detected in the CA3 pyramidal layer. With the mAb-KA8 antibody there was no detectable kappa opioid receptor labelling in the hippocampus of the guinea pig. The results confirm the high degree of species-specific heterogeneity characterising the distribution of opioid peptides and their receptors in the hippocampal formation. The receptor was found in most cases postsynaptically, however in the hamster the immunopositive axons may refer to a presynaptic localisation.

Kappa opioid receptor is expressed by somatostatin- and neuropeptide Y-containing interneurons in the rat hippocampus.

In our previous studies (J. Chem. Neuroanat. 2000;19:233-241), kappa opioid receptors were immunocytochemically identified in inhibitory interneurons of the dentate hilus and CA1 area of the rat hippocampus. From among the known interneuron subtypes, somatostatin- (SOM) and neuropeptide Y- (NPY) immunoreactive (IR) hippocampal interneurons show morphology and distribution similar to the kappa opioid receptor (KOR) immunopositive cells. In the present study, with the help of double immunocytochemical labelling, we provide direct evidence that the majority of the interneurons immunoreactive for SOM and/or NPY also express the kappa opioid receptor. The receptor was localized on the perikaryal and proximal dendritic region of the SOM- and NPY-immunopositive neurons in the dentate hilus and the CA1 region. From among the SOM-immunoreactive cells, 77% in the dentate hilus and 51% in the CA1 stratum oriens was double labelled. In the case of NPY-immunoreactive neurons this proportion was 56 and 65%, respectively. The co-expression of KOR and SOM/NPY suggests that hippocampal interneurons can selectively be activated by the different opioids under different physiological circumstances.