Prolong intermittent injection of human parathyroid hormone in rats modulates the expression of several proteins in the neuropil of the cerebellar cortex.

INTRODUCTION: The intermittent use of recombinant human parathyroid hormone (iPTH) alters calcium metabolism and induces osteogenesis in experimental models. However, the real effects of iPTH in excitable cells and neurons that require membrane receptors to undergo membrane depolarization/repolarization (Na+K+ATPase) to generate ATP, voltage-gated calcium channel (calcium-IP3R-calponin) as well as GABAergic (GABAA) signaling remains unclear. OBJECTIVES: In this study, the expression of IP3R, Na+K+-ATPase, GABAA and calmodulin proteins were evaluated in histological sections of the cerebellum of rats following prolonged injection of iPTH. METHODS: Twenty Wistar rats were used in this study and randomly assigned as either or control group. The test group were subcutaneously injected with 20 µg/kg of iPTH, 3×/week for 8 weeks, while the control group received 1 ml/kg of 0.9% saline solution. The rats were euthanized on the 60th day after the first administration, and their cerebellar vermis was removed and submitted to histological and immunohistochemical evaluation for detection of IP3R, Na+K+-ATPase, GABAA and calmodulin proteins. The expression of proteins was evaluated in the areas corresponding to the Purkinje cells as well as in neuropil of molecular layer of cerebellum. All results were transformed into a percentage for each area analyzed to verify significance between groups. RESULTS: Rats that received iPTH demonstrated significant reduction of IP3R, calmodulin and GABAA in Purkinje cells and neuropil of molecular layer while the expression of Na+K+-ATPase was similar. CONCLUSION: It was concluded that iPTH decreased the expression of IP3R and calmodulin while it did not alter the expression of Na+K+-ATPase. These changes insinuate the ionic activity of calcium and sodium/potassium. Yet, the iPTH alters GABAergic signaling in Purkinje cells, suggesting neurotransmission activity changes in the cerebellum.

A Balancing Act: The Immune System Supports Neurodegeneration and Neurogenesis.

Decapod crustaceans, like mammals, retain the ability to make new neurons throughout life. In mammals, immune cells are closely associated with stem cells that generate adult-born neurons. In crayfish, evidence suggests that immune cells (hemocytes) originating in the immune system travel to neurogenic regions and transform into neural progenitor cells. This nontraditional immune activity takes place continuously under normal physiological conditions, but little is known under pathological conditions (neurodegeneration). In this study, the immune system and its relationship with neurogenesis were investigated during neurodegeneration (unilateral antennular ablation) in adult crayfish. Our experiments show that after ablation (1) Proliferating cells decrease in neurogenic areas of the adult crayfish brain; (2) The immune response, but not neurogenesis, is ablation-side dependent; (3) Inducible nitric oxide synthase (iNOS) plays a crucial role in the neurogenic niche containing neural progenitors during the immune response; (4) Brain areas targeted by antennular projections respond acutely (15 min) to the lesion, increasing the number of local immune cells; (5) Immune cells are recruited to the area surrounding the ipsilateral neurogenic niche; and (6) The vasculature in the niche responds acutely by dilation and possibly also neovascularization. We conclude that immune cells are important in both neurodegeneration and neurogenesis by contributing in physiological conditions to the maintenance of the number of neural precursor cells in the neurogenic niche (neurogenesis), and in pathological conditions (neurodegeneration) by coordinating NO release and vascular responses associated with the neurogenic niche. Our data suggest that neural damage and recovery participate in a balance between these competing immune cell roles.

MALDI-imaging analyses of honeybee brains exposed to a neonicotinoid insecticide.

BACKGROUND: Toxicological studies evaluating the possible harmful effects of pesticides on bees are important and allow the emergence of protection and pollinator conservation strategies. This study aimed to evaluate the effects of exposure to a sublethal concentration of imidacloprid (LC50/100 : 0.014651 ng imidacloprid µL-1 diet) on the distribution of certain proteins identified in the brain of Apis mellifera worker bees using a MALDI-imaging approach. This technique enables proteomic analysis of tissues in situ by monitoring the spatiotemporal dynamics of the biochemical processes occurring at a specific time in specific brain neuropils. For this purpose, foraging bees were exposed to an 8-day diet containing a sublethal concentration of imidacloprid corresponding to the LC50/100 . Bees were collected on day 8 of exposure, and their brains analyzed using protein density maps. RESULTS: The results showed that exposure to imidacloprid led to a series of biochemical changes, including alterations in synapse regulation, apoptosis regulation and oxidative stress, which may adversely impair the physiology of these colony bees. CONCLUSION: Worker bee contact with even tiny amounts of imidacloprid had potent effects leading to the overexpression of a series of proteins related to important cellular processes that were possibly damaged by the insecticide. © 2018 Society of Chemical Industry.

The transcription factor SoxD controls neuronal guidance in the Drosophila visual system.

Precise control of neurite guidance during development is essential to ensure proper formation of neuronal networks and correct function of the central nervous system (CNS). How neuronal projections find their targets to generate appropriate synapses is not entirely understood. Although transcription factors are key molecules during neurogenesis, we do not know their entire function during the formation of networks in the CNS. Here, we used the Drosophila melanogaster optic lobe as a model for understanding neurite guidance during development. We assessed the function of Sox102F/SoxD, the unique Drosophila orthologue of the vertebrate SoxD family of transcription factors. SoxD is expressed in immature and mature neurons in the larval and adult lobula plate ganglia (one of the optic lobe neuropils), but is absent from glial cells, neural stem cells and progenitors of the lobula plate. SoxD RNAi knockdown in all neurons results in a reduction of the lobula plate neuropil, without affecting neuronal fate. This morphological defect is associated with an impaired optomotor response of adult flies. Moreover, knocking down SoxD only in T4/T5 neuronal types, which control motion vision, affects proper neurite guidance into the medulla and lobula. Our findings suggest that SoxD regulates neurite guidance, without affecting neuronal fate.

The Plastic Glial-Synaptic Dynamics within the Neuropil: A Self-Organizing System Composed of Polyelectrolytes in Phase Transition.

Several explanations have been proposed to account for the mechanisms of neuroglial interactions involved in neural plasticity. We review experimental results addressing plastic nonlinear interactions between glial membranes and synaptic terminals. These results indicate the necessity of elaborating on a model based on the dynamics of hydroionic waves within the neuropil. These waves have been detected in a small scale experimental model of the central nervous system, the in vitro retina. We suggest that the brain, as the heart and kidney, is a system for which the state of water is functional. The use of nonlinear thermodynamics supports experiments at convenient biological spatiotemporal scales, while an understanding of the properties of ions and their interactions with water requires explanations based on quantum theories. In our approach, neural plasticity is seen as part of a larger process that encompasses higher brain functions; in this regard, hydroionic waves within the neuropil are considered to carry both physiological and cognitive functions.

A role for the membrane protein M6 in the Drosophila visual system.

BACKGROUND: Members of the proteolipid protein family, including the four-transmembrane glycoprotein M6a, are involved in neuronal plasticity in mammals. Results from our group previously demonstrated that M6, the only proteolipid protein expressed in Drosophila, localizes to the cell membrane in follicle cells. M6 loss triggers female sterility, which suggests a role for M6 in follicular cell remodeling. These results were the basis of the present study, which focused on the function and requirements of M6 in the fly nervous system. RESULTS: The present study identified two novel, tissue-regulated M6 isoforms with variable N- and C- termini, and showed that M6 is the functional fly ortholog of Gpm6a. In the adult brain, the protein was localized to several neuropils, such as the optic lobe, the central complex, and the mushroom bodies. Interestingly, although reduced M6 levels triggered a mild rough-eye phenotype, hypomorphic M6 mutants exhibited a defective response to light. CONCLUSIONS: Based on its ability to induce filopodium formation we propose that M6 is key in cell remodeling processes underlying visual system function. These results bring further insight into the role of M6/M6a in biological processes involving neuronal plasticity and behavior in flies and mammals.

Changes in carbohydrate expression in the cervical spinal cord of rats during aging.

Aging is a process where histochemical changes occur. Some of these may consist of age-dependent loss of expression of some cell markers. Conversely, cell markers not expressed in young animals may be detectable in their older counterparts. Histochemical age changes in carbohydrate profiles in the spinal cord have not been documented. In order to fill this information gap lectin histochemistry and image analysis were used to characterize the histochemical age changes occurring in the cervical segments of the rat spinal cord. From a battery of 11 lectins, the more important age changes were detected with Glicine maximus (SBA)-lectin. Thus, SBA-lectin neuronal staining which was moderately positive in the cervical segments of young animals was negative in old rats. In contrast the same lectin which did not react with the ependyma of young animals strongly bound to the ependyma of senescent rats. None of the tested lectins bound to glial cells, either in young or old animals. In no case the senile animals evidenced anatomopathological changes. We conclude that although in the aged spinal cord changes in lectin histochemical binding patterns occur, they do not reflect a pathologic situation.

Distribution of calbindin-28kD and parvalbumin in V1 in normal adult Cebus apella monkeys and in monkeys with retinal lesions.

Several proteins have their normal patterns of distributions altered by monocular visual deprivation. We studied the distribution of the calcium-binding proteins calbindin-28kD (Cb) and parvalbumin (Pv) in V1 in normal adult Cebus apella monkeys and in monkeys with monocular retinal lesions. In normal monkeys, the interblobs regions in layers 2/3 and the layer 4B are intensely labeled for Cb, while Pv reaction showed a complementary labeling pattern with a stronger staining in layers 4A, 4C and in the blob regions in layers 2/3. In monkeys with monocular retinal lesion, the laminar distribution of these proteins was differentially affected, although both reactions resulted in stronger labeling in non-deprived ocular dominance columns. While Cb reaction resulted in stronger labeling in layers 1 through 5, Pv labeling was heavier in layers 2/3, 4A and 4C. There was a clear reduction in the intensity of neuropil staining for both Pv and Cb in deprived ocular dominance columns with little or no reduction in number of labeled cells. This reduction could thus be attributed to activity-dependent changes at synapses level.

Regionally specific distribution of the binding of anti-glutamine synthetase and anti-S100 antibodies and of Datura stramonium lectin in glial domains of the optic lobe of the giant prawn.

We previously characterized some crustacean glial cells by markers such as 2',3'-cyclic nucleotide 3'-phosphodiesterase and glial fibrillary acidic protein. Here we use antibodies against glutamine synthetase full-length molecule (anti-GS/FL), a GS C-terminal peptide (anti-GS/20aa-C), and brain S100 (anti-S100), as well as the binding of the insect glia and rat astrocytic marker Datura stramonium lectin (DSL), in the optic lobe of the prawn Macrobrachium rosenbergii. All markers label the lamina ganglionaris cartridge region (lighter: anti-GS/FL; heavier: DSL). In addition, anti-GS/FL labels superficial somata of external and internal medullas and internal chiasm cells. Both anti-GS/20aa-C and anti-S100 label heavily the glial sheaths of the lamina ganglionaris. In addition, anti-S100 binds to the perineurial glia of medullary parenchymal vessels. Western blot analyses show that both anti-GS/FL and anti-GS/20aa-C bind mostly to a band of 50-55 kDa, compatible with a long isoform of vertebrate GS, and accessorily to a possible dimer and, in the case of anti-GS/20aa-C, to an ill-defined band of intermediate mass. Binding of anti-S100 is selective for a single band of about 68 kDa but shows no protein in the weight range of the canonical S100 protein superfamily. DSL reveals two bands of about 75 and about 120 kDa, thus within the range of maximal recognition for rat astrocytes. Our results suggest that phenotype protein markers of the optic lobe glia share antigenic determinants with S100 and (a long form of) GS and that, similarly to vertebrate and insect glia, crustacean glia protein and N-glycan residue markers display regional heterogeneity.

FMRFamide-like immunocytochemistry in the brain and subesophageal ganglion of Triatoma infestans (Insecta: Heteroptera). Coexpression with beta-pigment-dispersing hormone and small cardioactive peptide B.

The distribution of FMRFamide (FMRFa)-like immunoreactivity (LI) was studied in the brain and subesophageal ganglion of Triatoma infestans, the insect vector of Chagas' disease. The neuropeptide displayed a widespread distribution with immunostained somata in the optic lobe, in the anterior, lateral, and posterior soma rinds of the protocerebrum, and around the antennal sensory and mechanosensory and motor neuropils of the deutocerebrum. FMRFa-immunoreactive profiles of the subesophageal ganglion were seen in the mandibular, maxillary, and labial neuromeres. Immunostained neurites were detected in the medulla and lobula of the optic lobe, the lateral protocerebral neuropil, the median bundle, the calyces and the stalk of the mushroom bodies, and the central body. In the deutocerebrum, the sensory glomeruli showed a higher density of immunoreactive processes than the mechanosensory and motor neuropil, whereas the neuropils of each neuromere of the subesophageal ganglion displayed a moderate density of immunoreactive neurites. Colocalization of FMRFa-LI and crustacean pigment-dispersing hormone-LI was found in perikarya of the proximal optic lobe, the lobula, the sensory deutocerebrum, and the labial neuromere of the subesophageal ganglion. The distribution pattern of small cardioactive peptide B (SCP(B))-LI was also widespread, with immunolabeled somata surrounding every neuropil region of the brain and subesophageal ganglion, except for the optic lobe. FMRFa- and SCP(B)-LIs showed extensive colocalization in the brain of this triatomine species. The presence of immunolabeled perikarya displaying either FMRFa- or SCP(B)-LI confirmed that each antisera identified different peptide molecules. The distribution of FMRFa immunostaining in T. infestans raises the possibility that FMRFa plays a role in the regulation of circadian rhythmicity. The finding of immunolabeling in neurosecretory somata of the protocerebrum suggests that this neuropeptide may also act as a neurohormone.

Neuropil and neuronal changes in hippocampal NADPH-diaphorase histochemistry in the ME7 model of murine prion disease.

Nitric oxide (NO) has been implicated in neurotoxicity and cerebral blood flow changes in chronic neurodegeneration, but its activity in the mammalian prion diseases has not been studied in detail. Nicotine adenine dinucleotide phosphate (NADPH)-diaphorase (NADPH-d) histochemistry is a simple and robust histochemical procedure that allows localization of the tissue distribution of NO synthases. The aim of the present study is to assess whether NADPH-d histochemical activity is altered in the hippocampus in the ME7 model of prion disease in C57BL/6J mice. At early and late stages after the initiation of the disease we assessed features of the NADPH-d positive cells and the neuropil histochemical activity in CA1 and dentate gyrus using densitometric analysis. In C57BL/6J mice 13 weeks postinjection of the prion agent ME7, when behavioural changes first become apparent, neuropil NADPH-d histochemical staining increases, whereas at late stages it decreases dramatically. Both type I and type II NADPH-d positive cells were found to survive throughout the hippocampal formation into the late stages of the disease, but diaphorase activity was reduced in dendritic branches and abnormal varicosities were present in both dendritic and axonal processes of NADPH-d positive type I cells. The pathophysiological implications of the results remain to be investigated but both blood flow alteration and NO neurotoxicity may be features of the disease.

Distribution of serotonin in the central nervous system of the blood-feeding heteropteran, Triatoma infestans (Heteroptera: Reduviidae).

The distribution of serotonin was studied in the Triatoma infestans central nervous system by using immunocytochemistry. Serotonin immunoreactive cell bodies and fibers were observed in the brain, subesophageal ganglion, and thoracic ganglia. In the brain, serotonin-like immunoreactivity was detected in a limited number of somata, which gave rise to an extensive network of labeled neurites in patterned as well as in nonglomerular neuropils. Immunolabeled perikarya were observed in the optic lobe and in the anteromedial and caudolateral soma rinds of the protocerebrum. Deutocerebral immunoreactive somata were mainly found in the medial layer surrounding the antennal lobe glomeruli, as well as in relationship to the antennal mechanosensory and motor center. The subesophageal ganglion contained serotonin immunoreactive perikarya of variable sizes and moderate to low density of positive fibers. In the prothoracic ganglion, immunoreactive somata were detected near the cephalic connectives as well as in its caudal end. Serotonin immunoreactive somata and fibers were observed in the posterior ganglion of the thorax, with the abdominal neuromeres harboring the highest number of immunolabeled perikarya. These results show that there is a widespread unique serotonergic system in the CNS of Triatoma infestans and suggest that the indolamine could act as a neuromodulator or as a neurohormone.

Distribution of NADPH-diaphorase in the superior colliculus of Cebus monkeys, and co-localization with calcium-binding proteins.

We examined the distribution of the enzyme dihydronicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in the superior colliculus (SC) of the New World monkey Cebus apella, and the co-localization of this enzyme with the calcium-binding proteins (CaBPs) calbindin-D28K, parvalbumin and calretinin. Despite the intensely labeled neuropil, rare NADPH-d-positive cells were observed in the stratum griseum superficiale (SGS). Most of the labeled cells in the SC were found in the intermediate layers, with a great number also in the deeper layers. This pattern is very similar to that described in the opossum (Didelphis marsupialis) and in the cat, and different from the pattern found in the rat, which shows labeled cells mainly in the SGS. Cells doubly stained for NADPH-d and CaBPs were observed throughout the SC, although in a small number. Of the NADPH-d-positive cells, 20.3% were doubly labeled for NADPH-d and parvalbumin, 10.2% revealed co-localization with calretinin, and 5.6% with calbindin. The low number of double-stained cells for NADPH-d and the CaBPs indicates that these molecules must participate in different functional circuits within the SC.