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The pedal ganglion is a peripheral ganglion which gives rise to the innervation for both the somatic and visceral organs of the Mytilus foot. In the present study, different histofluorescence methods for the demonstration of monamines (formaldehyde-glutaraldehyde followed by polyethylene glycol embedding; aluminium-formaldehyde; Falck) and acetylcholinesterase histochemistry were applied in order to characterize the neuronal population of the ganglion. The fluorescence methods employed showed that the cortical region of the pedal ganglion is composed of roundish cells; these mainly contained an orange autofluorescent pigment. Yellow-fluorescing cells were scattered in the anterior region of the cortex, but they were more numerous and arranged in clusters in the posterior region. Greenfluorescing cells were mainly located at the border between the cortex and neuropile and in the neuropile itself, where a rich plexus of beaded green-fluorescing fibres was also present. Of the three methods, that using formaldehydeglutaraldehyde followed by embedding in polyethylene glycol gave the best preservation of morphological details. Acetylcholinesterase histochemistry showed the presence of positive cells and fibres mainly in the anterior region of the ganglion. [ABSTRACT FROM AUTHOR]

Serotonergic cell bodies and fibers were identified in the pedal ganglia of Mytilus gattoprovincialis with a serum raised against serotonin and the unlabelled peroxidase-antiperoxidase pre- and post-embedding methods. Examination of Vibratome and serial semithin sections showed that most reactive perikarya are located in the ganglionic cortex, being mainly concentrated at the medial aspect of the postero-dorsal portion of the ganglia. Immunoreactive fibers form a dense network in the neuropil, extend throughout the commissure and run parallel in the nerves and connective tracts. The morphology of serotonin-positive cells compared with that of Golgi-impregnated neurons allows the identification of a main population of unipolar, probably projecting neurons and of smaller multipolar cells likely representing local circuit elements. The ultrastructure of labelled neurons is comparable to that of serotonergic cells described in both vertebrate and invertebrate nervous systems. [ABSTRACT FROM AUTHOR]

The purpose of this study was to characterize enzyme, receptor, and signaling involved in the synthesis and the activity of cysteinyl leukotrienes (cys-LTs) in human umbilical vein endothelial cells (HUVECs). We used primary cultures of HUVECs and evaluated the formation of cys-LTs by RP-HPLC. Suicide inactivation and subcellular localization of the enzyme responsible for the conversion of leukotriene (LT) A(4) into LTC(4) were studied by repeated incubations with LTA(4) and immunogold electron microscopy. The CysLT(2) receptor in HUVECs was characterized by equilibrium binding studies, Western blot analysis, and immunohistochemistry. Concentration-response curves in HUVECs and in transfected COS-7 cells were used to characterize a novel specific CysLT(2) receptor antagonist (pA(2) of 8.33 and 6.79 against CysLT(2) and CysLT(1) receptors, respectively). The results obtained provide evidence that the mGST-II synthesizing LTC(4) in HUVECs is pharmacologically distinguishable from the LTC(4)-synthase (IC(50) of MK886 <5 μM for LTC(4)-synthase and >30 μM for mGST-II), is not suicide-inactivated and is strategically located on endothelial transport vesicles. The CysLT(2) receptor is responsible for the increase in intracellular Ca(2+) following exposure of HUVECs to cys-LTs and is coupled to a pertussis toxin-insensitive G(q) protein. The synthesis of cys-LTs from LTA(4) by endothelial cells is directly associated with the activation of the CysLT(2) receptor (EC(50) 0.64 μM) in a typical autocrine fashion.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by motoneuron loss. Some familial cases (fALS) are linked to mutations of superoxide dismutase type-1 (SOD1), an antioxidant enzyme whose activity is preserved in most mutant forms. Owing to the similarities in sporadic and fALS forms, mutant SOD1 animal and cellular models are a useful tool to study the disease. In transgenic mice expressing either wild-type (wt) human SOD1 or mutant G93A-SOD1, we found that wtSOD1 was present in cytoplasm and in nuclei of motoneurons, whereas mutant SOD1 was mainly cytoplasmic. Similar results were obtained in immortalized motoneurons (NSC34 cells) expressing either wtSOD1 or G93A-SOD1. Analyzing the proteasome activity, responsible for misfolded protein clearance, in the two subcellular compartments, we found proteasome impairment only in the cytoplasm. The effect of G93A-SOD1 exclusion from nuclei was then analyzed after oxidative stress. Cells expressing G93A-SOD1 showed a higher DNA damage compared with those expressing wtSOD1, possibly because of a loss of nuclear protection. The toxicity of mutant SOD1 might, therefore, arise from an initial misfolding (gain of function) reducing nuclear protection from the active enzyme (loss of function in the nuclei), a process that may be involved in ALS pathogenesis.

We have examined the regional distribution of several chondroitin sulfate proteoglycans (neurocan, brevican, versican, aggrecan, phosphacan), of their glycosaminoglycan moieties, and of tenascin-R in the spinal cord of adult rat. The relationships of these molecules with glial and neuronal populations, identified with appropriate markers, were investigated by using multiple fluorescence labeling combined with confocal microscopy. The results showed that the distribution of the examined molecules was similar at all spinal cord levels but displayed area-specific differences along the dorso-ventral axis, delimiting functionally and developmentally distinct areas. In the gray matter, laminae I and II lacked perineuronal nets (PNNs) of extracellular matrix and contained low levels of chondroitin sulfate glycosaminoglycans (CS-GAGs), brevican, and tenascin-R, possibly favoring the maintenance of local neuroplastic properties. Conversely, CS-GAGs, brevican, and phosphacan were abundant, with numerous thick PNNs, in laminae III-VIII and X. Motor neurons (lamina IX) were surrounded by PNNs that contained all molecules investigated but displayed various amounts of CS-GAGs. Double-labeling experiments showed that the presence of PNNs could not be unequivocally related to specific classes of neurons, such as motor neurons or interneurons identified by their expression of calcium-binding proteins (parvalbumin, calbindin, calretinin). However, a good correlation was found between PNNs rich in CS-GAGs and the neuronal expression of the Kv3.1b subunit of the potassium channel, a marker of fast-firing neurons. This observation confirms the correlation between the electrophysiological properties of these neurons and the specific composition of their microenvironment.

Water balance between cells and extracellular compartments is essential for proper functioning of the central nervous system, as demonstrated by its perturbations in pathological conditions. Aquaporin 4 (AQP4) is the predominant water channel in brain and spinal cord, where it is present mainly on astrocytic endfeet contacting vessels. A role in water homeostasis control has been proposed also for the extracellular matrix, that in brain consists mainly of chondroitin sulfate proteoglycans (CSPGs). Using cytochemical and immunocytochemical techniques, we investigated their distribution in rodent spinal cord, to better understand the role of these two classes of molecules. The results show that in spinal gray matter AQP4 labeling is intense in all perivascular profiles and (1) displays a marked dorsoventral gradient in the neuropil; and (2) coexists extensively with glial glutamate transporter-1 (GLT-1) but scarcely with glial fibrillary acidic protein (GFAP). In white matter the overlap between AQP4, GLT-1, and GFAP is almost complete. Ultrastructural examination shows that AQP4-labeled astrocytic processes surround blood vessels, neuronal perikarya and processes, and both asymmetric and symmetric synapses, indicating that the protein may be involved in the regulation of water fluxes around both inhibitory and excitatory synapses. CSPGs, visualized by labeling with Wisteria floribunda agglutinin, show a distribution complementary to that of AQP4, being absent or weekly expressed in AQP4-enriched areas. These findings suggest that different mechanisms may contribute to the regulation of water homeostasis in different spinal cord regions.

The binding of Lycopersicon esculentum lectin (LEA) to the vascular endothelium was studied in the central nervous system of rat, mouse and guinea pig at different developmental ages, and in a gliosarcoma model. Our observations showed that LEA consistently stained the entire vascular tree in the spinal cord and in the brain of all animal species at all developmental ages investigated. In the tumor model, the staining of the vascular network was very reproducible, enabled an easy identification of vascular profiles and displayed a higher efficiency when compared to two other commonly used vascular marker (EHS laminin and PECAM-1). Moreover, our results showed that LEA staining was comparable in both vibratome and paraffin sections and could be easily combined with other markers in double labeling experiments. These observations indicate that LEA staining may represent an effective and versatile endothelial marker for the study of the vasculature of the central nervous system in different animal species and experimental conditions.

Our previous electron microscope study showed that chondrocytes from cephalopod cartilage possess a highly developed cytoskeleton and numerous cytoplasmic processes that ramify extensively through the tissue. We have now carried out a light microscope immunocytochemical study of chondrocytes from the orbital cartilage of Sepia officinalis to obtain indications as to the nature of the cytoskeletal components. We found clear positivity to antibodies against mammalian tubulin, vimentin, GFAP, and actin, but not keratin. The simultaneous presence of several cytoskeletal components is consistent with the hypothesis that cephalopod chondrocytes have the characteristics of both chondrocytes and osteocytes of vertebrates, which endow the tissue as a whole with some of the properties of vertebrate bone. We confirm, therefore, the presence in molluscs of the ubiquitous cytoskeletal proteins of metazoan cells that have remained highly conserved throughout phylogenetic evolution.

Isolated organ preparations represent valuable models for biomedical research, provided that the functional and morphological integrity of vascular and parenchymal compartments is preserved. In this investigation, we have studied the molecular organization of the cerebral microvessels in the isolated guinea-pig brain maintained in vitro by arterial perfusion, a preparation previously proposed as a model of blood-brain barrier (BBB). Using lectin cytochemistry and immunohistochemistry, we examined the microvasculature of the cerebral cortex after 5 h in vitro to assess: (a) the structure of the endothelial glycocalyx at microscopical and ultrastructural level; (b) the distribution of the junctional molecules occludin, ZO-1, PECAM-1 and vinculin; (c) the distribution of basal lamina molecules, such as collagen type IV, laminin and heparan sulfate proteoglycan. All these components of microvessel wall have been previously shown to be vulnerable to ischemic conditions and their organization could be altered in consequence of the transient hypoxia associated with the brain isolation procedure. Our observations demonstrate that the distribution pattern of the molecules considered (i) is comparable to that shown in the cerebral microvasculature of other mammals and (ii) is similar in brains maintained in vitro and in control brains perfused in situ with fixative. The complex of our observation indicates that the molecular organization of the cerebral microvessels is preserved in isolated guinea-pig brain, thus indicating that these preparations can be used to study the cerebrovascular structure and blood-brain barrier function in a variety of experimental conditions.

Sections of integument from gastropod, bivalve and cephalopod species were studied immunohistochemically to determine reactivity to antibody against the type I-like collagen from Sepia cartilage and antibodies against components of the extracellular matrix (ECM) of vertebrate connective tissue: type I, III, IV, V, and VI collagens, laminin, nidogen and heparan sulphate. All samples exhibited similar reactivities to the antibodies, although differences in the intensity and localization of the immunostaining were found that were clearly correlated with between-species differences in integumental ultrastructure. These findings indicate that the composition of the integumental ECM is similar in the three classes of molluscs examined and that several types of collagen are present. However molluscan subepidermal connective tissue differs from the ECM of vertebrate dermis: molluscan integumental ECM contains collagens similar to type I, V and VI collagens but has no type III-similar collagen. Furthermore molecules similar to the type IV collagen, laminin, nidogen and heparan sulphate of vertebrates were present ubiquitously in molluscan basement membrane, confirming the statement that the structure and composition of basement membrane have remained constant throughout the evolution of all animal phyla.

Theories and data do not always fit and sometimes are sources of conflicts among scientists. This is the case of a morphological structure, the perineuronal net, which was denied on the basis of an ideological conflict between two giants of neurosciences: Camillo Golgi and Santiago Ramon y Cajal. The perineuronal net is a reticular structure enveloping many neurons. Orginally reported by Golgi in 1893 and 1898 and confirmed by several authors before the turn of the century, the perineuronal net was used by Golgi to support the reticular theory of the organization of the nervous system. Ramon y Cajal, the paladin of the neuronal theory who had also observed this anatomical structure, denied its existence suggesting that it was a fixation artifact. After Cajal's statements, only a few Italian scientists continued to work in this field, and after the 1930s the perineuronal net was forgotten. Only the recent advances in histochemical and immunocytochemical technology confirmed the existence of this structure opening new fields in functional neuroanatomy and neuropathology.