Calretinin in the peripheral nervous system of the adult zebrafish

Calretinin is a calcium-binding protein found widely distributed in the central nervous system and chemosensory cells of the teleosts, but its presence in the peripheral nervous system of fishes is unknown. In this study we used Western blot analysis and immunohistochemistry to investigate the occurrence and distribution of calretinin in the cranial nerve ganglia, dorsal root ganglia, sympathetic ganglia, and enteric nervous system of the adult zebrafish. By Western blotting a unique and specific protein band with an estimated molecular weight of around 30 kDa was detected, and it was identified as calretinin. Immunohistochemistry revealed that calretinin is selectively present in the cytoplasm of the neurons and never in the satellite glial cells. In both sensory and sympathetic ganglia the density of neurons that were immunolabelled, their size and morphology, as well as the intensity of immunostaining developed within the cytoplasm, were heterogeneous. In the enteric nervous system calretinin immunoreactivity was detected in a subset of enteric neurons as well as in a nerve fibre plexus localized inside the muscular layers. The present results demonstrate that in addition to the central nervous system, calretinin is also present in the peripheral nervous system of zebrafish, and contribute to completing the map of the distribution of this protein in the nervous system of teleosts.     

Six cadm/SynCAM genes are expressed in the nervous system of developing zebrafish.

The Cadm (cell adhesion molecule) family of cell adhesion molecules (also known as IGSF4, SynCAM, Necl and TSLC) has been implicated in a multitude of physiological and pathological processes, such as spermatogenesis, synapse formation and lung cancer. The precise mechanisms by which these adhesion molecules mediate these diverse functions remain unknown. To investigate mechanisms of action of these molecules during development, we have identified zebrafish orthologs of Cadm family members and have examined their expression patterns during development and in the adult. Zebrafish possess six cadm genes. Sequence comparisons and phylogenetic analysis suggest that four of the zebrafish cadm genes represent duplicates of two tetrapod Cadm genes, whereas the other two cadm genes are single orthologs of tetrapod Cadm genes. All six zebrafish cadms are expressed throughout the nervous system both during development and in the adult. The spatial and temporal patterns of expression suggest multiple roles for Cadms during nervous system development.     

Maturation of shoaling behavior is accompanied by changes in the dopaminergic and serotoninergic systems in zebrafish

The zebrafish has been one of the preferred vertebrate model organisms of developmental biology, and is becoming an important research tool for behavioral neuroscience and behavior genetics. A prominent feature of zebrafish is their strong shoaling tendency. Most recently, the first study investigating the development of shoaling in zebrafish demonstrated that a few days after hatching zebrafish do not shoal, but that shoaling tendency gradually increases during development. The current study investigates this phenomenon using the nearest neighbor distance, a measure most frequently employed for the quantification of shoal cohesion in fish. We demonstrate that shoal cohesion increases with age, while thigmotaxis, "wall hugging," does not show a consistent age-dependent change. The mechanisms underlying the maturation of shoaling are unknown. HPLC analysis of whole brain extracts finds the concentration of dopamine, DOPAC, serotonin, and 5-HIAA normalized to total brain protein weight to increase with age. Although the behavioral and neurochemical results are only correlative at this point, they may open a new avenue into the investigation of the mechanisms and development of social behavior in zebrafish.     

Permanent effector phenotype of neuroantigen-specific T cells acquired in the central nervous system during experimental allergic encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is regarded as an animal model of the human autoimmune disease multiple sclerosis (MS). Autoreactive T cells are present in the peripheral T cell repertoire of healthy mice and mediate clinical autoimmune disease only after activation by immunization or pathogens and migrate into the central nervous system (CNS). Because it is not known whether autoreactive T cells are regulated differentially once entering the CNS we investigated cytokine regulation in T cells from peripheral lymphatic organs and from the inflamed CNS ex vivo obtained from SJL mice after inducing relapsing-remitting EAE with PLP peptide 139–151. We show here that during acute EAE, an interleukin-2 (IL-2) biased T cell response exists in the spleen, while an interferon-γ (IFN-γ) biased T cell response prevails in the CNS of mice with acute EAE. The IFN-γ biased phenotype was stable with optimized costimulation and even after in vitro stimulation with IL-2. After adoptive transfer into naïve syngeneic mice these T cells were only partially reversed to an IL-2 biased phenotype. These findings of our work suggest that a permanent effector phenotype of neuroantigen-specific T cells is finally acquired in the CNS in EAE.     

The role of the central serotonergic system in pilocarpine-induced seizures: receptor mechanisms

Modification of central serotonergic transmission resulted in alterations of pilocarpine convulsive activity in male Wistar rats. Seizure activity was increased after pizotifen injection and the latency period to onset of convulsions was shortened in animals pretreated with mianserine and quipazine. Stimulation of 5-HT1A receptors with 8-hydroxy-di-N,N-propylaminotetralin (8-OH-DPAT) and blockade of 5-HT1B receptors with cyanopindolol resulted in seizure protection. Intracerebroventricular injections of 5,6-dihydroxytryptamine (5,6-DHT) did not change the protective effect of cyanopindolol. Other agents specifically affecting serotonergic receptors, the agonists 1-(3-chlorophenyl)piperazine (mCPP) and 5-methoxytryptamine (5-MT) and the antagonists spiperone, metergoline, methysergide, cyproheptadine and metoclopramide, did not influence pilocarpine-induced seizures. In conclusion, the present study suggests that the inhibition of pilocarpine-induced seizures may be mediated by stimulation of 5-HT1A and by blockade of 5-HT1B receptors, located probably on the cholinergic terminals.     

Associations among central nervous system serotonergic function and neuroticism are moderated by gender

Serotonergic dysregulation is associated with negative affect. Plasma prolactin responses to a tryptophan enhancement challenge are used as a measure of central nervous system serotonergic activity. We examined prolactin responses to a tryptophan challenge as they relate to the personality domains of neuroticism, extraversion, openness, agreeableness, and conscientiousness. Participants were 67 volunteers. Regression models assessed peak prolactin response to intravenous tryptophan infusion as a predictor of neuroticism, extraversion, openness, agreeableness, and conscientiousness. Prolactin × gender product terms were included to examine moderation by gender. Models were adjusted for baseline levels of prolactin, age, and race. Gender moderated the association between N and prolactin level (p < .03). Higher levels of N were associated with decreased levels of prolactin responses in females, whereas the opposite was true for males. Remaining personality domains were not related to prolactin levels. Findings add to literature suggesting the serotonin system functions differently, in important ways, in males and females.     

The expression and localization of Prune2 mRNA in the central nervous system

A family of Bcl-2/adenovirus E1B 19 kDa-interacting proteins (BNIPs) plays critical roles in several cellular processes such as cellular transformation, apoptosis, neuronal differentiation, and synaptic function, which are mediated by the BNIP2 and Cdc42GAP homology (BCH) domain. Prune homolog 2 (Drosophila) (PRUNE2) and its isoforms - C9orf65, BCH motif-containing molecule at the carboxyl terminal region 1 (BMCC1), and BNIP2 Extra Long (BNIPXL) - have been shown to be a susceptibility gene for Alzheimer's disease, a biomarker for leiomyosarcomas, a proapoptotic protein in neuronal cells, and an antagonist of cellular transformation, respectively. However, precise localization of PRUNE2 in the brain remains unclear. Here, we identified the distribution of Prune2 mRNA in the adult mouse brain. Prune2 mRNA is predominantly expressed in the neurons of the cranial nerve motor nuclei and the motor neurons of the spinal cord. The expression in the dorsal root ganglia (DRG) is consistent with the previously described reports. In addition, we observed the expression in another sensory neuron in the mesencephalic trigeminal nucleus. These results suggest that Prune2 may be functional in these restricted brain regions.     

Characterization and comparative expression of zebrafish calpain system genes during early development

The classic calpain system has been implicated in regulating a variety of cellular processes including cell adhesion, migration, and intracellular signaling; however, little is known regarding the function of this system in vivo. Two heterodimeric Ca(2+)-dependent cysteine proteases, mu-calpain (CAPN1) and m-calpain (CAPN2), and the endogenous inhibitor calpastatin (CAST) comprise the classic/ubiquitous calpain system in mammals. Recently, knockout of two murine classic calpain genes, Capn2 and Capn4/Capns1, revealed that components of the classic system are indispensable for preimplantation development. We identified four classic calpain catalytic subunit genes (capn1a, 1b, 2a, 2b), two regulatory subunit genes (capns1a, 1b), and calpastatin (cast) from the zebrafish. Our data suggest that the components of the classic mammalian system are both conserved and expanded in the teleost lineage. In contrast to the classic/ubiquitous mammalian system, zebrafish calpain system genes acquire unique, tissue-specific patterns of expression within the first 2 days of development.     

The effects on the central nervous system of nitroglycerin--putative mechanisms and mediators

Nitroglycerin is an organic nitrate that has been used as a vasodilator in the treatment of cardiac diseases for over a century. Only recently it has been demonstrated that the vasodilator effect of this drug depends upon the formation of nitric oxide in the blood vessel wall. However, clinical and research data gathered during the last decades have suggested that nitroglycerin possesses, besides its peripheral vasodilator effect, additional, puzzling biological activities. This organic nitrate compound provokes reflex cardiovascular activities via its interaction with the central sympathetic system. Its cerebrovascular effect, on the other hand, is probably mediated by the local release of neuropeptides. The direct application of nitroglycerin onto brain nuclei causes a prompt increase in the neuronal discharge rate. From a neurological point of view, nitroglycerin consistently induces a specific headache attack in patients suffering from migraine. Because of its temporal pattern and clinical characteristics, nitroglycerin-induced headache cannot be solely ascribed to the a drug-induced vasorelaxation. The demonstration that systemic nitroglycerin administration activates a widespread set of vegetative, nociceptive and neuroendocrine structures in the central nervous system seems to further support the occurrence of central mechanisms in the biological activity of nitroglycerin. Double labeling immunocytochemical and neuropharmacological studies have provided information on the putative neurotransmitters and neurochemical mechanisms involved in nitroglycerin-induced neuronal activation.     

Podoplanin is expressed in subsets of tumors of the central nervous system

Immunohistochemical analyses with the monoclonal antibody D2-40 were performed to ascertain the expression of podoplanin (a.k.a. T1-alpha, gp36, or aggrus) in tumors of the central nervous system (CNS) and to determine the diagnostic utility of the antibody. The analyses were performed on 325 tumors of various histologic types. The chief finding was almost constant immunoreactivity in ependymal tumors (37/40, 92.5%), choroid plexus papillomas (8/8, 100%), and meningiomas (100/100, 100%). The reactivity was considered “tissue-specific,” as the corresponding normal tissue of each tumor was also found to express podoplanin. In addition, expression, not committed to the lineages, was found in many other tumor types, including astrocytic tumors, medulloblastomas, and hemangioblastomas, with variable frequency and intensity. The way of expression was not fully understood, but the expression in astrocytic tumors seemed to be associated with pronounced fibrous properties or malignant phenotype, as was shown by high-frequent expression in pilocytic astrocytomas (12/12, 100%) and glioblastomas (29/35, 82.9%). The present study has shown that podoplanin is expressed in several types of CNS tumors with variable frequency and intensity. Given the widespread expression of podoplanin, the antibody D2-40 is of little use in diagnostic practice for CNS tumors.     

The ultrastructure of prosternal sensory hair afferents within the locust central nervous system

The sensory neurones innervating long prosternai hairs of Locusta migratorioides were backfilled with horseradish peroxidase through their dendrites. The neurones' central projections in and around the medial ventral tract were examined with electron microscopy. Most synapses occur on axon collaterals which ramify through the neuropile around the tract where both input and output synapses were observed. Serial sectioning methods were used to determine the relative distribution of inputs and outputs which often lie in close proximity to one another on the axon terminals. The prosternai hair terminals contain agranular synaptic vesicles approximately 37 nm in diameter. Surrounding unidentified neuropilar profiles contain vesicles which are either statistically indistinguishable in size, or are larger, 45 nm diameter agranular vesicles. Neurones which are pre- or postsynaptic to labelled terminals generally contain vesicles of the second type.Input synapses onto the central terminals of primary afferent neurones can be recognised as a widespread phenomenon in the nervous systems of both invertebrates and vertebrates which will allow a fine degree of control of sensory inflow into the central nervous system.     

The chemokine receptor CCR5 in the central nervous system

The expression and the role of the chemokine receptor CCR5 have been mainly studied in the context of HIV infection. However, this protein is also expressed in the brain, where it can be crucial in determining the outcome in response to different insults. CCR5 expression can be deleterious or protective in controlling the progression of certain infections in the CNS, but it is also emerging that it could play a role in non-infectious diseases. In particular, it appears that, in addition to modulating immune responses, CCR5 can influence neuronal survival. Here, we summarize the present knowledge about the expression of CCR5 in the brain and highlight recent findings suggesting its possible involvement in neuroprotective mechanisms.     

Non-coplanar polychlorinated biphenyl (PCB)-induced immunotoxicity is coincident with alterations in the serotonergic system

Attention to non-coplanar polychlorinated biphenyl (PCB) congeners in immunotoxicological research is increasing. However, the exact mechanism by which these congeners may induce immune dysfunction is still undefined. Because the serotonergic nervous system has been shown to be involved in the regulation of some immune responses, and also serves as a sensitive target for PCBs, the relationship (if any) between non-coplanar PCB exposure, immune responsiveness and the neurotransmitter serotonin (5-HT) was examined. Using bluegill sunfish (Lepomis macrochirus) as a model, changes in brain 5-HT levels, 5-HT synthesis and metabolism, and innate and cell-mediated immune parameters were evaluated following a single intraperitoneal injection of PCB 153 (5.0 or 50?μg/g body weight). Results revealed that 3?d following administration, PCB exposure decreased brain 5-HT levels (in the absence of effects on some enzymes involved in 5-HT synthesis and metabolism), increased oxyradical production by kidney phagocytes, and reduced splenic T- and B-lymphocyte proliferation. In vivo treatment of PCB-exposed fish with 5-hydroxy-L-tryptophan (the immediate precursor to 5-HT) ameliorated the observed PCB-induced immunotoxicity; in vitro treatment of immune cells from PCB-exposed fish with 5-HT failed to reverse the effects. Taken together, results from this study could suggest a link between PCB-induced alterations of brain 5-HT levels and subsequent immune dysfunction. These studies highlight the importance of indirect mechanisms of immunotoxicity, and, specifically, suggest a role for the neuroimmune axis in non-coplanar PCB-induced immune alterations.     

Inflammatory pseudotumors of the central nervous system

Inflammatory pseudotumor is a disease with unsettled pathogenesis. The brain is a rare site of occurrence. The aim of this study is to investigate ALK-1 protein expression and IgG4-positive plasma cells detection in 4 intracranial inflammatory pseudotumors. Three dural-based and 1 intraventricular inflammatory pseudotumors were retrieved from the hospitals' archive. The data on clinical presentation, radiological findings, procedure undertaken, and patients' progress were collected. Sections from the excised lesions were examined under hematoxylin and eosin, histochemical, and immunohistochemical staining including ALK-1 and IgG4. All 4 cases displayed typical histological features of inflammatory pseudotumor with dense lymphoplasmacytic infiltrate admixed with small number of benign-looking spindle cells in a collagenous stroma. Three cases exhibited high density of IgG4-positive plasma cells per high-power field. ALK-1 was negative. ALK expression was not found in any of our cases. On the contrary, the detection of significant number of IgG4-positive plasma cells in 3 inflammatory pseudotumors suggests that a considerable proportion of intracranial inflammatory pseudotumor may belong to the IgG4-related subgroup. Hence, a trial of corticosteroid after histological confirmation may be valid to avoid unnecessary risk-taking neurosurgical procedures or in cases with incomplete tumor removal.     

Heat shock proteins: Cellular and molecular mechanisms in the central nervous system

Emerging evidence indicates that heat shock proteins (HSPs) are critical regulators in normal neural physiological function as well as in cell stress responses. The functions of HSPs represent an enormous and diverse range of cellular activities, far beyond the originally identified roles in protein folding and chaperoning. HSPs are now understood to be involved in processes such as synaptic transmission, autophagy, ER stress response, protein kinase and cell death signaling. In addition, manipulation of HSPs has robust effects on the fate of cells in neurological injury and disease states. The ongoing exploration of multiple HSP superfamilies has underscored the pluripotent nature of HSPs in the cellular context, and has demanded the recent revamping of the nomenclature referring to these families to reflect a re-organization based on structure and function. In keeping with this re-organization, we first discuss the HSP superfamilies in terms of protein structure, regulation, expression and distribution in the brain. We then explore major cellular functions of HSPs that are relevant to neural physiological states, and from there we discuss known and proposed HSP impacts on major neurological disease states. This review article presents a three-part discussion on the array of HSP families relevant to neuronal tissue, their cellular functions, and the exploration of therapeutic targets of these proteins in the context of neurological diseases.     

Germline transgenesis of zebrafish using the medaka Tol1 transposon system

Tol1 is a DNA-based transposable element first identified from an albino mutant medaka fish. It has been demonstrated to function as an efficient gene transfer vector in mammalian cells. We now demonstrate Tol1 germline transgenesis in zebrafish. A construct containing the green fluorescence protein (GFP) reporter gene inserted between the Tol1 arms was microinjected together with Tol1 transposase mRNA into fertilized eggs. Sustained GFP expression was observed in 88% of 1-month-old fish, suggesting efficient transposon integration into somatic cells. Eleven of 24 adult GFP-positive fish yielded GFP-positive progeny. Sequencing analysis of Tol1 insertion sites in GFP-positive progeny confirmed Tol1 transposition-mediated integrations into zebrafish chromosomes. We also observed functional independence of the Tol1 transposase-substrate system from that of Tol2, another medaka-derived transposon. Coupled with its previously demonstrated maximal cargo capacity of >20 kb, Tol1 could serve as a useful addition to the zebrafish genetic engineering toolbox.