MOBP-specific cellular immune responses are weaker than MOG-specific cellular immune responses in patients with multiple sclerosis and healthy subjects.

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). Myelin oligodendrocyte glycoprotein (MOG) and myelin oligodendrocyte basic protein (MOBP) were both shown to be highly encephalitogenic in animal models of MS. In contrast, the association of MOG- and MOBP-specific humoral or cellular immune responses and MS in humans is far less established. In this study, we sought to analyse MOG- and MOBP-specific T-cell responses in a large cohort of patients with various stages of the disease. Patients with other neurological diseases and healthy subjects were enrolled to serve as control study subjects. We determined the proliferation and the secretion of IFN-γ secretion in our cohort. We found that MOG-specific T-cell responses were higher and more frequent as compared to MOBP-specific ones. However, both MS patients and control study subjects had similar myelin-specific T-cell responses at the periphery, thus calling for more precise studies at CNS level.

Glucose-dependent insulinotropic polypeptide (GIP) and its receptor (GIPR): cellular localization, lesion-affected expression, and impaired regenerative axonal growth.

Glucose-dependent insulinotropic polypeptide (GIP) was initially described to be rapidly regulated by endocrine cells in response to nutrient ingestion, with stimulatory effects on insulin synthesis and release. Previously, we demonstrated a significant up-regulation of GIP mRNA in the rat subiculum after fornix injury. To gain more insight into the lesion-induced expression of GIP and its receptor (GIPR), expression profiles of the mRNAs were studied after rat sciatic nerve crush injury in 1) affected lumbar dorsal root ganglia (DRG), 2) spinal cord segments, and 3) proximal and distal nerve fragments by means of quantitative RT-PCR. Our results clearly identified lesion-induced as well as tissue type-specific mRNA regulation of GIP and its receptor. Furthermore, comprehensive immunohistochemical stainings not only confirmed and exceeded the previous observation of neuronal GIP expression but also revealed corresponding GIPR expression, implying putative modulatory functions of GIP/GIPR signaling in adult neurons. In complement, we also observed expression of GIP and its receptor in myelinating Schwann cells and oligodendrocytes. Polarized localization of GIPR in the abaxonal Schwann cell membranes, plasma membrane-associated GIPR expression of satellite cells, and ependymal GIPR expression strongly suggests complex cell type-specific functions of GIP and GIPR in the adult nervous system that are presumably mediated by autocrine and paracrine interactions, respectively. Notably, in vivo analyses with GIPR-deficient mice suggest a critical role of GIP/GIPR signal transduction in promoting spontaneous recovery after nerve crush, insofar as traumatic injury of GIPR-deficient mouse sciatic nerve revealed impaired axonal regeneration compared with wild-type mice.

A tumor-specific cellular environment at the brain invasion border of adamantinomatous craniopharyngiomas.

Craniopharyngiomas (CP) are benign epithelial tumors of the sellar region and can be clinicopathologically distinguished into adamantinomatous (adaCP) and papillary (papCP) variants. Both subtypes are classified according to the World Health Organization grade I, but their irregular digitate brain infiltration makes any complete surgical resection difficult to obtain. Herein, we characterized the cellular interface between the tumor and the surrounding brain tissue in 48 CP (41 adaCP and seven papCP) compared to non-neuroepithelial tumors, i.e., 12 cavernous hemangiomas, 10 meningiomas, and 14 metastases using antibodies directed against glial fibrillary acid protein (GFAP), vimentin, nestin, microtubule-associated protein 2 (MAP2) splice variants, and tenascin-C. We identified a specific cell population characterized by the coexpression of nestin, MAP2, and GFAP within the invasion niche of the adamantinomatous subtype. This was especially prominent along the finger-like protrusions. A similar population of presumably astroglial precursors was not visible in other lesions under study, which characterize them as distinct histopathological feature of adaCP. Furthermore, the outer tumor cell layer of adaCP showed a distinct expression of MAP2, a novel finding helpful in the differential diagnosis of epithelial tumors in the sellar region. Our data support the hypothesis that adaCP, unlike other non-neuroepithelial tumors of the central nervous system, create a tumor-specific cellular environment at the tumor-brain junction. Whether this facilitates the characteristic infiltrative growth pattern or is the consequence of an activated Wnt signaling pathway, detectable in 90% of these tumors, will need further consideration.

Cellular distribution of glucose and monocarboxylate transporters in human brain white matter and multiple sclerosis lesions.

To ensure efficient energy supply to the high demanding brain, nutrients are transported into brain cells via specific glucose (GLUT) and monocarboxylate transporters (MCT). Mitochondrial dysfunction and altered glucose metabolism are thought to play an important role in the progression of neurodegenerative diseases, including multiple sclerosis (MS). Here, we investigated the cellular localization of key GLUT and MCT proteins in human brain tissue of non-neurological controls and MS patients. We show that in control brain tissue GLUT and MCT proteins were abundantly expressed in a variety of central nervous system cells, particularly in microglia and endothelial cells. In active MS lesions, GLUTs and MCTs were highly expressed in infiltrating leukocytes and reactive astrocytes. Astrocytes manifest increased MCT1 staining and maintain GLUT expression in inactive lesions, whereas demyelinated axons exhibit significantly reduced GLUT3 and MCT2 immunoreactivity in inactive lesions. Finally, we demonstrated that the co-transcription factor peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α), an important protein involved in energy metabolism, is highly expressed in reactive astrocytes in active MS lesions. Overexpression of PGC-1α in astrocyte-like cells resulted in increased production of several GLUT and MCT proteins. In conclusion, we provide for the first time a comprehensive overview of key nutrient transporters in white matter brain samples. Moreover, our data demonstrate an altered expression of these nutrient transporters in MS brain tissue, including a marked reduction of axonal GLUT3 and MCT2 expression in chronic lesions, which may impede efficient nutrient supply to the hypoxic demyelinated axons thereby contributing to the ongoing neurodegeneration in MS. GLIA 2014;62:1125-1141.

Molecular and cellular analysis of genodermatoses

Summary Skin is the essential interface between our body and its environment; not only does it prevent water loss and protect us from external insults it also plays an essential role in the central nervous system acting as a major sense organ primarily for touch and pain. The main cell type present in skin, keratinocyte, undergoes a differentiation process leading to the formation of this protecting barrier. This work is intended to contribute to the understanding of how keratinocyte differentiates and skin functions. To do this, we studied two genetic skin diseases: Erythrokeratodermia variabilis and Mal de Meleda. Our approach was to examine the expression and localization of proteins implicated in these two pathologies in normal and diseased tissues and to determine the influence of mutant proteins at the molecular and cellular levels. Connexins are major components of gap junctions, channels allowing direct communication between cells. Our laboratory has identified mutations in both connexin 30.3 (Cx30.3) and 31 (Cx31) to be causally involved in erythrokeratodermia variabilis (EKV), an autosomal dominant disorder of keratinization. In the first chapter, we show a new mutation of Cx31, L209P-Cx31, in 3 EKV patients, extending the field of EKV-causing mutations although the mechanism by which connexin mutations lead to the disease is unclear. In the second chapter, we studied the effect of F137L-Cx30.3 on expression, trafficking and localization of cotransfected Cx31 and Cx30.3 in connexin-deficient HeLa cells. The F137 amino acid, highly conserved in connexin family, is oriented towards the channel pore and F137L mutation in either Cx30.3 or Cx31 lead to EKV. As two genes can lead to EKV when mutated, our hypothesis was that Cx31 and Cx30.3 might cooperate at a molecular level. We were able to demonstrate a physical interaction between Cx31 and Cx30.3. The presence of F137L-Cx30.3 disturbed the trafficking of both connexins, less connexins were integrated into gap junctions and thus, the coupling between cell was diminished. Connexins formed in the presence of F137L-Cx30.3 are degraded at their exit from the endoplasmic reticulum. In conclusion, our results indicate that the genetic heterogeneity of EKV is due to mutations in two interacting proteins. F137L-Cx30.3 has a dominant negative effect and affects Cx31, disturbing cellular communication in epidermal cells. Mal de Meleda is an autosomal recessive inflammatory and a keratotic palmoplantar skin disorder due to mutations in SLURP1 (secreted LY6/PLAUR-related protein 1). SLURP1 belongs to the LY6/PLAUR family of proteins and has the particularity of being secreted instead of being GPI-anchored. The high degree of structural similarity between SLURP1 and the three fingers motif of snake neurotoxins and LYNX 1-C suggests that this protein could interact with the neuronal acetylcholine receptors. In the third chapter, we show that SLURP1 potentiates responses of the a7 nicotinic acetylcholine receptor (nAchR) to acetylcholine. These results identify SLURP1 as a secreted epidermal neuromodulator that is likely to be essential for palmoplantar skin. In the fourth chapter, we show that SLURP1 is expressed in the granular layer of the epidermis but is absent from skin biopsies of Mal de Meleda patients. SLURP1 is also present in secretions such as sweat, tears or saliva. An in vitro analysis on two mutant of SLURP-I demonstrates that W15R-SLURP1 is absent in cells while G86R-SLURP1 is expressed and secreted, suggesting that SLURP1 can lead to the disease by either an absent or an abnormal protein. Finally, in the fifth chapter, we analyse the expression and biological properties of other LY6/PLAUR members, clustered around SLURP] on chromosome 8. Their GPI-anchored or secreted status were analysed in vitro. SLURP1, LYNX1-A and -B are secreted while LYPDC2 and LYNX 1-C are GPI anchored. Three of these proteins are expressed in the epidermis and in cultured keratinocytes. These results suggest that these LY6/PLAUR members may have an important role in skin homeostasis. Résumé Résumé La peau est la barrière essentielle entre notre corps et l'environnement, nous protégeant des agressions extérieures, de la déshydratation et assurant aussi un rôle dans le système nerveux central en tant qu'organe du toucher et de la douleur. Le principal type de cellules présent dans la peau est le kératinocyte qui suit un processus de différenciation aboutissant à la formation de cette barrière protectrice. Ce travail est destiné à comprendre la différenciation des kératinocytes et le fonctionnement de la peau. Pour cela, nous avons étudié deux maladies génodermatoses : l'Erthrokeratodermia Variabilis (EKV) et le Mal de Meleda. Nous avons examiné l'expression et la localisation des protéines impliquées dans ces deux pathologies dans des tissus normaux et malades puis déterminé l'influence des protéines mutantes aux niveaux moléculaires et cellulaires. Les connexines (Cx) sont les composants majeurs des jonctions communicantes, canaux permettant la communication directe entre les cellules. Notre laboratoire a identifié des mutations dans les Cx30.3 et Cx31 comme responsables de l'EKV, génodermatose de transmission autosomique dominante. Dans le ler chapitre, nous décrivons une nouvelle mutation de Cx31, L209-Cx31, et contribuons à l'établissement du catalogue des mutations de Cx31 entraînant cette maladie. Cependant, le mécanisme par lequel les mutations de Cx31 et C3x0.3 provoquent l'EKV est inconnu. Dans le 2ème chapitre, nous étudions les effets de la mutation F137L-Cx30.3 sur l'expression, le trafic et la localisation des Cx31 et Cx30.3 transfectées dans des cellules HeLa, déficientes en connexines. Comme deux gènes peuvent causer une EKV quand ils sont mutés, notre hypothèse était que Cx31 et Cx30.3 pourraient coopérer au niveau moléculaire. Nous avons montré l'existence d'une interaction physique entre ces deux connexines. La présence de la mutation F137L-Cx30.3 perturbe le trafic des deux connexines, moins de connexines sont intégrées dans les jonctions communicantes et donc le couplage entre les cellules est diminué. Les connexons formés en présence de cette mutation sont dégradés à leur sortie du réticulum endoplasmique. En conclusion, nos résultats indiquent que l'hétérogénéité génétique de EKV est due à des mutations dans deux protéines qui interagissent. F137L-Cx30.3 a un effet dominant négatif et affecte Cx31, perturbant la communication entre les cellules épidermiques. Le Mal de Meleda est une maladie récessive de la peau palmoplantaire due à des mutations dans SLURP1. SLURP1 appartient à la famille des protéines contenant un domaine LY6/PLAUR et a la particularité d'être sécrétée. La grande homologie de structure existant entre SLURP1, les neurotoxines de serpent et LYNX1-C suggère que la protéine pourrait interagir avec des récepteurs à acétylcholine (Ach). Dans le 3ème chapitre, nous montrons que SLURP1 module la réponse à l'Ach du récepteur nicotinique α7. Ces résultats identifient SLURP1 comme un neuromodulateur épidermique sécrété, probablement essentiel pour la peau palmoplantaire. Dans le 4ème chapitre, nous montrons que SLURP1 est exprimé dans la couche granuleuse de l'épiderme et qu'il est absent des biopsies des patients. SLURP1 a aussi été détecté dans des sécrétions telles que la sueur, les lamies et la salive. Une analyse in vitro de deux mutants de SLURP1 a montré que W15R-SLURP1 est absent des cellules tandis que G86R-SLURP1 est exprimé et sécrété, suggérant qu'une absence ou une anomalie de SLURP1 peuvent causer la maladie. Finalement, dans le 5ème chapitre, nous analysons l'expression et les propriétés biologiques d'autres membres de la famille LY6/PLAUR localisés autour de SLURP1 sur le chromosome 8. Leur statut de protéines sécrétées ou liées à la membrane par une ancre GPI est analysé in vitro. SLURP1, LYNXI-A et -B sont sécrétées alors que LYPDC2 et LYNX1-C sont liés à la membrane. Trois de ces protéines sont exprimées dans l'épiderme et dans des kératinocytes cultivés. Ces résultats suggèrent que la famille LY6/PLAUR pourrait avoir un rôle important dans l'homéostasie de la peau.

Cellular localization, expression and regulation of neuropeptide Y in kidneys of hypertensive rats.

Neuropeptide Y (NPY) is a key modulator of the autonomic nervous system playing pivotal roles in cardiovascular and neuronal functions. In this study, we assessed the cellular localization and gene expression of NPY in rat kidneys. We also examined the relationship between NPY gene expression and renin in two rat models of hypertension (two-kidney, one-clip renal hypertension (2K1C), and deoxycorticosterone-salt-induced hypertension (DOCA-salt)) characterized by a similar blood pressure elevation. In situ hybridization and immunohistochemistry, using anti-NPY or anti-C-flanking peptide of NPY (CPON) antibodies, showed that NPY transcript and protein were colocalized in the tubules of rat kidneys. During experimental hypertension, NPY mRNA was decreased in both kidneys of the 2K1C animals, but not in the kidney of DOCA-salt rats. In 2K1C rats, renal NPY content was also decreased. The difference in NPY gene expression between 2K1C rats (a high renin model of hypertension) and DOCA-salt rats (a low renin model of hypertension) suggests that circulating angiotensin II plays a role in local renal NPY gene expression and that the elevated blood pressure per se is not the primary factor responsible for the control of NPY gene expression in the kidney.

Cellular and viral factors implicated in persistent infection in canine distemper virus

Summary SLAM (signalling lymphocyte activation molecule, CD150) serves as a cellular receptor for different morbiliviruses, including measles virus and canine distemper virus. Laboratory cell lines that do not express dog SLAM are therefore quite refractory to infection by wildtype CDV. SLAM expression is not only required for CDV virion attachment, but also for the establishment of cytolytic infection characterized by syncytia formation. In order to determine if SLAM has a direct influence on CDV replication, we compared wild-type and mutated SLAM variants for their capacity to influence viral polymerase activity and syncytia formation. Deletion of immunoreceptor tyrosine-based signalling motif (ITSM) in the cytoplasmic tail of SLAM did not seem to influence viral replication, viral polymerase activity or cell-to cell fusion. Instead, it was the level of cell surface expression of SLAM, which was important. Additional experiments corroborated the importance of SLAM for efficient cell-to cell fusion: Both SLAM, as well as viral fusion (F) and attachment (H) glycoproteins, were found to be required for efficient cell-to-cell fusion, which, in turn, enhanced the activity of the viral polymerase and, viral replication. Wild-type A75/17 canine distemper virus (CDV) strain is known to induce a persistent infection in the central nervous system and in dog footpad keratinocytes in vivo. Recently, it has been shown that the A75/17 virus could also infect canine footpad keratinocytes (CFKs) in vitro. CFK infection with A75/17 was initially inefficient and produced very little virus progeny, however, after only three passages the adapted virus produced more progeny and induced limited syncytia formation. Sequence comparison between the A75/17 and the CFKadapted A75/17-K virus revealed three amino acid differences, one in the phosphoprotein (P), one in the matrix protein (M) and one in the H protein. In order to identify viral determinants of A75/17-K adaptation, recombinant viruses containing one, two or three nucleotides substitutions were analyzed. The amino acid substitution in the M protein was without effect on viral particle formation. In contrast, the amino acid substitution in the cytoplasmic tail of H protein was clearly important for syncytia formation. Concerning the mutation in the P protein, it led to an increase in viral replication. However, we cannot rule out that the observed effect is due to the amino acid substitutions in the overlapping accessory proteins C and V, also affected by the P mutation. The adaptation of wild-type CDV strains to cell culture almost always involves modifications of M protein. In order to understand the influence of these modifications, we tested recombinant A75/17 viruses bearing different M proteins. Preliminary results demonstrated that the M protein from the Vero-adapted strain reduced syncytia formation. Future studies will focus on the M mRNA and protein stability, its expression level, localisation and its effect on viral particles formation and on the phenotype of infection. Résumé La protéine SLAM (signalling lymphocyte activation molecule ou CD150) est utilisée comme récepteur cellulaire par les morbillivirus parmi lesquels on trouve le virus de la rougeole (VR) ainsi que le virus de la maladie de Carré (CDV). Les lignées cellulaires qui n'expriment pas la protéine SLAM du chien à leur surface sont réfractaires à l'infection par les souches sauvages de CDV. Le récepteur SLAM n'est pas seulement requis pour l'attachement du virion à la surface de la cellule, mais il participe également de façon active à l'établissement d'une infection cytolytique à travers la formation de syncytia. Afin de déterminer si la protéine SLAM exerce une influence directe sur la réplication virale du virus de la maladie de Carré, nous avons généré différentes protéines tronquées de SLAM et comparé leurs capacités à influencer l'activité de la polymérase ainsi que la formation de syncytia. Nos résultas ont montré que la réplication virale, l'activité de la polymérase ainsi que la fusion cellulaire ne semblent pas être influencées par les délétions dans les régions cytoplasmiques du récepteur SLAM. Cependant, ces délétions agissent sur l'expression de la protéine SLAM à la surface des cellules. Les expériences additionnelles ont permis de souligner l'importance de la protéine SLAM dans le phénomène de fusion entre cellules. En effet, la protéine SLAM ainsi que les deux glycoprotéines virales F et H sont requises pour la formation de syncytia, laquelle induit une augmentation de l'activité de la polymérase ainsi que de la réplication virale. La souche virulente A75/17 du virus, de la Maladie de Carré est connue pour induire une infection persistante au niveau du système nerveux central ainsi que dans les kératinocytes de pattes chez le chien. Des études récentes ont montré que des cultures primaires de kératinocytes de chien pouvaient aussi êtres infectées par la souche A75/17 de CDV. En effet, le virus induit une infection persistante en produisant très peu de progéniture. Cependant, trois passages du virus sauvage A75/17 dans ces cultures aboutissent à la sélection d'un virus produisant plus de progéniture et favorisant la formation limitée de syncytia. La comparaison des séquences génomique entre la souche A75/17 et la souche adaptée A75/17-K montre une différence de trois nucléotides. La première mutation, située dans le gène P, modifie la phosphoprotéine (P) ainsi que les protéines V et C. La deuxième se situe dans le gène de la protéine matricielle (M) et la dernière dans celui de la protéine d'attachement (H). Afin de déterminer les facteurs viraux impliqués lors de l'adaptation virale dans la culture primaire de kératinocytes, des virus recombinants contenant une, deux ou trois de ces mutations ont été analysés. La substitution d'un acide aminé dans la protéine M reste sans effet sur la production de particules virales. En revanche, la substitution d'un acide aminé dans la queue cytoplasmique de la protéine H s'avère clairement importante pour la formation de syncytia. Quant à la mutation dans le gène P, elle permet une augmentation de la réplication virale. Cependant, nous ne pouvons pas écarter l'hypothèse que l'augmentation de la réplication virale soit due aux substitutions d'un acide aminé dans les protéines accessoires V et C qui sont, elles aussi, affectées par la mutation dans le gène P. L'adaptation des souches sauvages de CDV aux cultures de cellules induit presque toujours des modifications de la protéine matricielle M. Afin de comprendre l'influence de ces modifications, nous avons testé 'des virus A75/17 recombinants contenant différentes protéines M. Les résultats préliminaires ont démontré que la protéine M de la souche adaptée aux cellules Vero réduisait la formation de syncytia. Les études futures seront axées sur la stabilité de l'ARN messager, celle de la protéine M, de son niveau d'expression, de sa localisation cellulaire et de son effet sur la formation de particules virale ainsi que sur le phénotype de l'infection.

Role of the cellular prion protein in oligodendrocyte precursor cell proliferation and differentiation in the developing and adult mouse CNS

There are numerous studies describing the signaling mechanisms that mediate oligodendrocyte precursor cell (OPC) proliferation and differentiation, although the contribution of the cellular prion protein (PrPc) to this process remains unclear. PrPc is a glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein involved in diverse cellular processes during the development and maturation of the mammalian central nervous system (CNS). Here we describe how PrPc influences oligodendrocyte proliferation in the developing and adult CNS. OPCs that lack PrPc proliferate more vigorously at the expense of a delay in differentiation, which correlates with changes in the expression of oligodendrocyte lineage markers. In addition, numerous NG2-positive cells were observed in cortical regions of adult PrPc knockout mice, although no significant changes in myelination can be seen, probably due to the death of surplus cells.

Cell-selective modulation of the neuromuscular system in Drosophila

The capacity for all living cells to sense and interact with their environment is a necessity for life. In highly evolved, eukaryotic species, like humans, signalling mechanisms are necessary to regulate the function and survival of all cells in the organism. Synchronizing systemic signalling systems at the cellular, organ and whole-organism level is a formidable task, and for most species requires a large number of signalling molecules and their receptors. One of the major types of signalling molecules used throughout the animal kingdom are modulatory substances (e.x. hormones and peptides). Modulators can act as chemical transmitters, facilitating communication at chemical synapses. There are hundreds of circulating modulators within the mammalian system, but the reason for so many remains a mystery. Recent work with the fruit fly, Drosophila melanogaster demonstrated the capacity for peptides to modulate synaptic transmission in a neuron-specific manner, suggesting that peptides are not simply redundant, but rather may have highly specific roles. Thus, the diversity of peptides may reflect cell-specific functions. The main objective of my doctoral thesis was to examine the extent to which neuromodulator substances and their receptors modulate synaptic transmission at a cell-specific level using D. melanogaster. Using three different modulatory substances, i) octopamine - a biogenic amine released from motor neuron terminals, ii) DPKQDFMRFa - a neuropeptide secreted into circulation, and iii) Proctolin - a pentapeptide released both from motor neuron terminals and into circulation, I was able to investigate not only the capacity of these various substances to work in a cell-selective manner, but also examine the different mechanisms of action and how modulatory substances work in concert to execute systemic functionality . The results support the idea that modulatory substances act in a circuit-selective manner in the central nervous system and in the periphery in order to coordinate and synchronize physiologically and behaviourally relevant outputs. The findings contribute as to why the nervous system encodes so many modulatory substances.

Dysregulation of REST-regulated coding and non-coding RNAs in a cellular model of Huntington's disease

Huntingtin (Htt) protein interacts with many transcriptional regulators, with widespread disruption to the transcriptome in Huntington's disease (HD) brought about by altered interactions with the mutant Htt (muHtt) protein. Repressor Element-1 Silencing Transcription Factor (REST) is a repressor whose association with Htt in the cytoplasm is disrupted in HD, leading to increased nuclear REST and concomitant repression of several neuronal-specific genes, including brain-derived neurotrophic factor (Bdnf). Here, we explored a wide set of HD dysregulated genes to identify direct REST targets whose expression is altered in a cellular model of HD but that can be rescued by knock-down of REST activity. We found many direct REST target genes encoding proteins important for nervous system development, including a cohort involved in synaptic transmission, at least two of which can be rescued at the protein level by REST knock-down. We also identified several microRNAs (miRNAs) whose aberrant repression is directly mediated by REST, including miR-137, which has not previously been shown to be a direct REST target in mouse. These data provide evidence of the contribution of inappropriate REST-mediated transcriptional repression to the widespread changes in coding and non-coding gene expression in a cellular model of HD that may affect normal neuronal function and survival.

Interaction between the lateral preoptic area and the subfornical organ in the control of water ingestion caused by cellular dehydration, hypotension, hypovolemia, and deprivation

Water intake was studied in albino rats with lesions in the lateral preoptic area, in the subfornical organ, and in both the lateral preoptic area and the subfornical organ. Drinking was induced by cellular dehydration, hypovolemia, hypotension (isoproterenol or caval ligation), and water deprivation. The animals with lesions in both areas showed a significant reduction in their water intake in response to cellular dehydration. Drinking due to extracellular dehydration was reduced in the animals that received only subfornical organ lesions, and was reduced even further in the animals with both areas ablated. The lesions in the subfornical organ were sufficient to reduce the thirst induced by caval ligation. The lesions in both areas inhibit water intake induced by caval ligation. Water intake induced by deprivation was reduced when both areas were destroyed. These findings demonstrate that both the lateral preoptic area and the subfornical organ are necessary for normal drinking in response to cellular dehydration, hypovolemia, and hypotension. There is further evidence that the lateral preoptic area and subfornical organ interact in the control of water intake induced by a variety of thirst challenges.

Bovine herpesvirus-5 infection in a rabbit experimental model: Immunohistochemical study of the cellular response in the CNS

Since little information is available regarding cellular antigen mapping and the involvement of non-neuronal cells in the pathogenesis of bovine herpesvirus type 5 (BHV-5) infection, it were determined the BHV-5 distribution, the astrocytic reactivity, the involvement of lymphocytes and the presence of matrix metalloproteinase (MMP)-9 in the brain of rabbits experimentally infected with BHV-5. Twelve New Zealand rabbits that were seronegative for BHV-5 were used for virus inoculation, and five rabbits were used as mock-infected controls. The rabbits were kept in separate areas and were inoculated intranasally with 500 μl of virus suspension (EVI 88 Brazilian isolate) into each nostril (virus titer, 107.5 TCID50). Control rabbits were inoculated with the same volume of minimum essential medium. Five days before virus inoculation, the rabbits were submitted to daily administration of dexamethasone. After virus inoculation, the rabbits were monitored clinically on a daily basis. Seven rabbits showed respiratory symptoms and four animals exhibited neurological symptoms. Tissue sections were collected for histological examination and immunohistochemistry to examine BHV-5 antigens, astrocytes, T and B lymphocytes and MMP-9. By means of immunohistochemical and PCR methods, BHV-5 was detected in the entire brain of the animals which presented with neurological symptoms, especially in the trigeminal ganglion and cerebral cortices. Furthermore, BHV-5 antigens were detected in neurons and/or other non-neural cells. In addition to the neurons, most infiltrating CD3 T lymphocytes observed in these areas were positive for MMP-9 and also for BHV-5 antigen. These infected cells might contribute to the spread of the virus to the rabbit brain along the trigeminal ganglia and olfactory nerve pathways. © 2013 Elsevier Ltd.

Different approaches, one target: understanding cellular mechanisms of Parkinson's and Alzheimer's diseases

Neurodegenerative disorders are undoubtedly an increasing problem in the health sciences, given the increase of life expectancy and occasional vicious life style. Despite the fact that the mechanisms of such diseases are far from being completely understood, a large number of studies; that derive from both the basic science and clinical approaches have contributed substantial data in that direction. In this review, it is discussed several frontiers of basic research on Parkinson's and Alzheimer's diseases, in which research groups from three departments of the Institute of Biomedical Sciences of the University of Sao Paulo have been involved in a multidisciplinary effort. The main focus of the review involves the animal models that have been developed to study cellular and molecular aspects of those neurodegenerative diseases, including oxidative stress, insulin signaling and proteomic analyses, among others. We anticipate that this review will help the group determine future directions of joint research in the field and, more importantly, set the level of cooperation we plan to develop in collaboration with colleagues of the Nucleus for Applied Neuroscience Research that are mostly involved with clinical research in the same field.

Differential cellular FGF-2 upregulation in the rat facial nucleus following axotomy, functional electrical stimulation and corticosterone: a possible therapeutic target to Bell's palsy

Abstract Background The etiology of Bell's palsy can vary but anterograde axonal degeneration may delay spontaneous functional recovery leading the necessity of therapeutic interventions. Corticotherapy and/or complementary rehabilitation interventions have been employed. Thus the natural history of the disease reports to a neurotrophic resistance of adult facial motoneurons leading a favorable evolution however the related molecular mechanisms that might be therapeutically addressed in the resistant cases are not known. Fibroblast growth factor-2 (FGF-2) pathway signaling is a potential candidate for therapeutic development because its role on wound repair and autocrine/paracrine trophic mechanisms in the lesioned nervous system. Methods Adult rats received unilateral facial nerve crush, transection with amputation of nerve branches, or sham operation. Other group of unlesioned rats received a daily functional electrical stimulation in the levator labii superioris muscle (1 mA, 30 Hz, square wave) or systemic corticosterone (10 mgkg-1). Animals were sacrificed seven days later. Results Crush and transection lesions promoted no changes in the number of neurons but increased the neurofilament in the neuronal neuropil of axotomized facial nuclei. Axotomy also elevated the number of GFAP astrocytes (143% after crush; 277% after transection) and nuclear FGF-2 (57% after transection) in astrocytes (confirmed by two-color immunoperoxidase) in the ipsilateral facial nucleus. Image analysis reveled that a seven days functional electrical stimulation or corticosterone led to elevations of FGF-2 in the cytoplasm of neurons and in the nucleus of reactive astrocytes, respectively, without astrocytic reaction. Conclusion FGF-2 may exert paracrine/autocrine trophic actions in the facial nucleus and may be relevant as a therapeutic target to Bell's palsy.

Convergent and divergent modulatory pathways in {\it Aplysia\/}: Cellular and network studies

Neuromodulation is essential to many functions of the nervous system. In the simple gastropod mollusk Aplysia californica, neuromodulation of the circuits for the defensive withdrawal reflexes has been associated with several forms of learning. In the present work, the neurotransmitters and neural circuitry which contribute to the modulation of the tail-siphon withdrawal reflex were examined.^ A recently-identified neuropeptide transmitter, buccalin A was found to modulate the biophysical properties of the sensory neurons that mediate the reflex. The actions of buccalin A on the sensory neurons were compared with those of the well-characterized modulatory transmitter serotonin, and convergence and divergence in the actions of these two transmitters were evaluated. Buccalin A dramatically increased the excitability of sensory neurons and occluded further enhancement of excitability by serotonin. Buccalin A produced no significant change in spike duration, and it did not block serotonin-induced spike broadening. Voltage-clamp analysis revealed the currents that may be involved in the effects on spike duration and excitability. Buccalin A decreased an outward current similar to the S-K$\sp+$ current (I$\sb{\rm K,S}$). Buccalin A appeared to occlude further modulation of I$\sb{\rm K,S}$ by serotonin, but did not block serotonin-induced modulation of the voltage-dependent delayed rectifier K$\sp+$ current (I$\sb{\rm K,V}$). These results suggest that buccalin A converges on some, but not all, of the same subcellular modulatory pathways as serotonin.^ In order to begin to understand neuromodulation in a more physiological context for the tail-siphon withdrawal reflex, the modulatory circuitry for the tail-withdrawal circuit was examined. Mechanoafferent neurons in the J cluster of the cerebral ganglion were identified as elements of a modulatory circuit for the reflex. Excitatory and inhibitory connections were observed between the J cells and the pleural sensory neurons, the tail motor neurons, and several classes of interneurons for the tail-siphon withdrawal circuit. The J cells produced both fast and slow PSPs in these neurons. Of particular interest was the ability of the J cells to produce slow EPSPs in the pleural sensory neurons. These slow EPSPs were associated with an increase in the excitability of the sensory neurons. The J cells appear to mediate both sensory and modulatory inputs to the circuit for the tail-siphon withdrawal reflex from the anterior part of the animal. ^