Analysis of the MCTP Amino Acid Sequence Reveals the Conservation of Putative Calcium- and Lipid-Binding Pockets Within the C2 Domains In Silico.

MCTPs (Multiple C2 Domains and Transmembrane region Proteins) are evolutionarily and structurally related to other C2 proteins, which are central to exocytosis and membrane trafficking; however, their specific function has been little studied. MCTPs are associated with endosomes and the endoplasmic reticulum and possess three C2 domains (C2A-C2C) and two transmembrane regions (TMRs) well conserved in different species. Here, we generated structural models of the MCTP C2 domains of C. elegans and analyzed their putative function by docking, which revealed that these domains possess Ca2+- and lipid-binding pockets, suggesting that MCTPs play a significant, calcium-dependent role in membrane physiology.

A poly (saccharide-ester-urethane) scaffold for mammalian cell growth.

Chitosan and poly(3-hydroxybutyrate) are non-toxic, biodegradable, and biocompatible polymers extensively used in regenerative medicine. However, it is unknown whether the chemical combination of these polymers can produce a biomaterial that induces an appropriate cellular response in vitro in mammalian cells. This study aimed to test the ability of a novel salt-leached polyurethane scaffold of chitosan grafted with poly(3-hydroxybutyrate) to support the growth of three mammalian cell lines of different origin: a) HEK-293 cells, b) i28 mouse myoblasts, and c) human dermal fibroblasts. The viability of the cells was assessed by either evaluation of their capacity to maintain the expression of the green fluorescent protein by adenoviral transduction or by esterase activity and plasma membrane integrity. The results indicated that the three cell lines attached well to the scaffold; however, when i28 cells were induced to differentiate, they did not produce morphologically distinct myofibers, and cell growth ceased. In conclusion, the findings reveal that, altogether, these observations suggest that this foam scaffold supports cell growth and proliferation but may not apply to all cell types. Hence, one crucial question yet to be resolved is a poly (saccharide-ester-urethane) derivative with a nano-topography that elicits a similar cellular response for different biological environments.

MCTP-1 modulates neurotransmitter release in C. elegans.

Multiple C2 and Transmembrane Domain Proteins (MCTPs) are putative calcium sensors. Proteins that contain C2 domains play essential roles in membrane trafficking and exocytosis; however, MCTPs functions in neurotransmitter release are not known. Here we report that in C. elegans mctp-1 is under the control of two promoters - one active in the nervous system and the second in the spermatheca. We generated and characterized a loss of function amt1 mutant and compared it to a previously published loss of function mutant (av112). Loss of mctp-1 function causes defects in egg-laying, crawling velocity, and thrashing rates. Both amt1 and av112 mutants are hyposensitive to the acetylcholinesterase blocker aldicarb, suggesting that MCTP-1 may play a role in synaptic vesicle release.

Calcium Signaling in the Cerebellar Radial Glia and Its Association with Morphological Changes during Zebrafish Development.

Radial glial cells are a distinct non-neuronal cell type that, during development, span the entire width of the brain walls of the ventricular system. They play a central role in the origin and placement of neurons, since their processes form structural scaffolds that guide and facilitate neuronal migration. Furthermore, glutamatergic signaling in the radial glia of the adult cerebellum (i.e., Bergmann glia), is crucial for precise motor coordination. Radial glial cells exhibit spontaneous calcium activity and functional coupling spread calcium waves. However, the origin of calcium activity in relation to the ontogeny of cerebellar radial glia has not been widely explored, and many questions remain unanswered regarding the role of radial glia in brain development in health and disease. In this study we used a combination of whole mount immunofluorescence and calcium imaging in transgenic (gfap-GCaMP6s) zebrafish to determine how development of calcium activity is related to morphological changes of the cerebellum. We found that the morphological changes in cerebellar radial glia are quite dynamic; the cells are remarkably larger and more elaborate in their soma size, process length and numbers after 7 days post fertilization. Spontaneous calcium events were scarce during the first 3 days of development and calcium waves appeared on day 5, which is associated with the onset of more complex morphologies of radial glia. Blockage of gap junction coupling inhibited the propagation of calcium waves, but not basal local calcium activity. This work establishes crucial clues in radial glia organization, morphology and calcium signaling during development and provides insight into its role in complex behavioral paradigms.

Subterranean life: Behavior, metabolic, and some other adaptations of Astyanax cavefish.

The ability of fishes to adapt to any aquatic environment seems limitless. It is enthralling how new species keep appearing at the deep sea or in subterranean environments. There are close to 230 known species of cavefishes, still today the best-known cavefish is Astyanax mexicanus, a Characid that has become a model organism, and has been studied and scrutinized since 1936. There are two morphotypes for A. mexicanus, a surface fish and a cavefish. The surface fish lives in central and northeastern Mexico and south of the United States, while the cavefish is endemic to the "Sierra del Abra-Tanchipa region" in northeast Mexico. The extensive genetic and genomic analysis depicts a complex origin for Astyanax cavefish, with multiple cave invasions and persistent gene flow among cave populations. The surface founder population prevails in the same region where the caves are. In this review, we focus on both morphotype's main morphological and physiological differences, but mainly in recent discoveries about behavioral and metabolic adaptations for subterranean life. These traits may not be as obvious as the troglomorphic characteristics, but are key to understand how Astyanax cavefish thrives in this environment of perpetual darkness.

HCN2 activation modulation: An electrophysiological and molecular study of the well-preserved LCI sequence in the pore channel.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated potassium (Kv) and cyclic nucleotide-gated (CNG) channels. HCN channels contain the glycine-tyrosine-glycine (GYG) sequence that forms part of the selectivity filter, a similar structure than some potassium channels; however, they permeate both sodium and potassium, giving rise to an inward current. Yet a second amino acid sequence, leucine-cysteine-isoleucine (LCI), next to GYG, is well-preserved in all HCNs but not in the selective potassium channels. In this study we used site-directed mutagenesis and electrophysiology in frog oocytes to determine whether the LCI sequence affects the kinetics of HCN2 currents. Permeability and voltage dependence were evaluated, and we found a role of LCI in the gating mechanism combined with changes in ion permeability. The I residue resulted critical to this function.

Phosphatidylinositol 4,5-bisphosphate, cholesterol, and fatty acids modulate the calcium-activated chloride channel TMEM16A (ANO1).

The TMEM16A-mediated Ca2+-activated Cl- current drives several important physiological functions. Membrane lipids regulate ion channels and transporters but their influence on members of the TMEM16 family is poorly understood. Here we have studied the regulation of TMEM16A by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), cholesterol, and fatty acids using patch clamp, biochemistry and fluorescence microscopy. We found that depletion of membrane PI(4,5)P2 causes a decline in TMEM16A current that is independent of cytoskeleton, but is partially prevented by removing intracellular Ca2+. On the other hand, supplying PI(4,5)P2 to inside-out patches attenuated channel rundown and/or partially rescued activity after channel rundown. Also, depletion (with methyl-ß-cyclodextrin M-ßCD) or restoration (with M-ßCD+cholesterol) of membrane cholesterol slows down the current decay observed after reduction of PI(4,5)P2. Neither depletion nor restoration of cholesterol change PI(4,5)P2 content. However, M-ßCD alone transiently increases TMEM16A activity and dampens rundown whereas M-ßCD+cholesterol increases channel rundown. Thus, PI(4,5)P2 is required for TMEM16A function while cholesterol directly and indirectly via a PI(4,5)P2-independent mechanism regulate channel function. Stearic, arachidonic, oleic, docosahexaenoic, and eicosapentaenoic fatty acids as well as methyl stearate inhibit TMEM16A in a dose- and voltage-dependent manner. Phosphatidylserine, a phospholipid whose hydrocarbon tails contain stearic and oleic acids also inhibits TMEM16A. Finally, we show that TMEM16A remains in the plasma membrane after treatment with M-ßCD, M-ßCD+cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction.

GABAρ subunits confer a bicuculline-insensitive component to GFAP+ cells of cerebellum.

GABA-A receptors mediating synaptic or extrasynaptic transmission are molecularly and functionally distinct, and glial cells are known to express a plethora of GABA-A subunits. Here we demonstrate that GFAP(+) cells of the granular layer of cerebellum express GABAρ subunits during early postnatal development, thereby conferring peculiar pharmacologic characteristics to GABA responses. Electron microscopy revealed the presence of GABAρ in the plasma membrane of GFAP(+) cells. In contrast, expression in the adult was restricted to Purkinje neurons and a subset of ependymal cells. Electrophysiological studies in vitro revealed that astrocytes express functional receptors with an EC50 of 52.2 ± 11.8 µM for GABA. The evoked currents were inhibited by bicuculline (100 µM) and TPMPA (IC50, 5.9 ± 0.6 µM), indicating the presence of a GABAρ component. Coimmunoprecipitation demonstrated protein-protein interactions between GABAρ1 and GABAα1, and double immunofluorescence showed that these subunits colocalize in the plasma membrane. Three populations of GABA-A receptors in astrocytes were identified: classic GABA-A, bicuculline-insensitive GABAρ, and GABA-A-GABAρ hybrids. Clusters of GABA-A receptors were distributed in the perinuclear space and along the processes of GFAP(+) cells. Time-lapse microscopy showed GABAρ2-GFP accumulation in clusters located in the soma and along the processes. The clusters were relatively immobile, with mean displacement of 9.4 ± 0.9 µm and a net distance traveled of 1-2 µm, owing mainly to directional movement or simple diffusion. Modulation of GABAρ dynamics may be a novel mechanism of extrasynaptic transmission regulating GABAergic control of GFAP(+) cells during early postnatal development.

Anorexia Reduces GFAP+ Cell Density in the Rat Hippocampus.

Anorexia nervosa is an eating disorder observed primarily in young women. The neurobiology of the disorder is unknown but recently magnetic resonance imaging showed a volume reduction of the hippocampus in anorexic patients. Dehydration-induced anorexia (DIA) is a murine model that mimics core features of this disorder, including severe weight loss due to voluntary reduction in food intake. The energy supply to the brain is mediated by astrocytes, but whether their density is compromised by anorexia is unknown. Thus, the aim of this study was to estimate GFAP+ cell density in the main regions of the hippocampus (CA1, CA2, CA3, and dentate gyrus) in the DIA model. Our results showed that GFAP+ cell density was significantly reduced (~20%) in all regions of the hippocampus, except in CA1. Interestingly, DIA significantly reduced the GFAP+ cells/nuclei ratio in CA2 (-23%) and dentate gyrus (-48%). The reduction of GFAP+ cell density was in agreement with a lower expression of GFAP protein. Additionally, anorexia increased the expression of the intermediate filaments vimentin and nestin. Accordingly, anorexia increased the number of reactive astrocytes in CA2 and dentate gyrus more than twofold. We conclude that anorexia reduces the hippocampal GFAP+ cell density and increases vimentin and nestin expression.

Dehydration-Induced Anorexia Reduces Astrocyte Density in the Rat Corpus Callosum.

Anorexia nervosa is an eating disorder associated with severe weight loss as a consequence of voluntary food intake avoidance. Animal models such as dehydration-induced anorexia (DIA) mimic core features of the disorder, including voluntary reduction in food intake, which compromises the supply of energy to the brain. Glial cells, the major population of nerve cells in the central nervous system, play a crucial role in supplying energy to the neurons. The corpus callosum (CC) is the largest white matter tract in mammals, and more than 99% of the cell somata correspond to glial cells in rodents. Whether glial cell density is altered in anorexia is unknown. Thus, the aim of this study was to estimate glial cell density in the three main regions of the CC (genu, body, and splenium) in a murine model of DIA. The astrocyte density was significantly reduced (~34%) for the DIA group in the body of the CC, whereas in the genu and the splenium no significant changes were observed. DIA and forced food restriction (FFR) also reduced the ratio of astrocytes to glial cells by 57.5% and 22%, respectively, in the body of CC. Thus, we conclude that DIA reduces astrocyte density only in the body of the rat CC.

Characterization of an outward rectifying chloride current of Xenopus tropicalis oocytes.

Here, we describe an outward rectifying current in Xenopus tropicalis oocytes that we have called xtClC-or. The current has two components; the major component is voltage activated and independent of intracellular or extracellular Ca(2+), whereas the second is a smaller component that is Ca(2+) dependent. The properties of the Ca(2+)-independent current, such as voltage dependence and outward rectification, resemble those of ClC anion channels/transporters. This current is sensitive to NPPB and NFA, insensitive to 9AC and DIDS, and showed a whole-cell conductance sequence of SCN(-)>I(-)>Br(-)>CI(-). RT-PCR revealed the expression in oocytes of ClC-2 to ClC-7, and major reductions of current amplitudes were observed when a ClC-5 antisense oligonucleotide was injected into oocytes. The Ca(2+)-dependent component was abated after injection of 10mM BAPTA or EGTA, whereas 10mMMg(2+) inhibited the current to 26±3.1%. This component was blocked by 9-AC, NFA, and NPPB, whereas DIDS did not elicit any evident effect. The ion sequence selectivity was SCN=I(-)>Br(-)>Cl(-). To try to determine the molecular identity that gives rise to this component we assessed by RT-PCR the expression of the Ca(2+)-dependent Cl(-) channel TMEM16A, which was found to be present in the oocytes. However, injection of antisense TMEM16A oligonucleotides did not inhibit the transient outward current. This result fits well with the electrophysiological data. Together, these results suggest that ClC-5 is a major, but not the sole channel responsible for this outwardly rectifying Cl(-) current.

Modulation of GABA-A receptors of astrocytes and STC-1 cells by taurine structural analogs.

Taurine activates and modulates GABA receptors in vivo as well as those expressed in heterologous systems. This study aimed to determine whether the structural analogs of taurine: homotaurine and hypotaurine, have the ability to activate GABA-A receptors that include GABAρ subunits. The expression of GABA-A receptors containing GABAρ has been reported in the STC-1 cells and astrocytes. In both cell types, taurine, homo-, and hypotaurine gated with low efficiency a picrotoxin-sensitive GABA-A receptor. The known bimodal modulatory effect of taurine on GABAρ receptors was not observed; however, differences between the activation and deactivation rates were detected when they were perfused together with GABA. In silico docking simulations suggested that taurine, hypo-, and homotaurine do not form a cation-π interaction such as that generated by GABA in the agonist-binding site of GABAρ. This observation complements the electrophysiological data suggesting that taurine and its analogs act as partial agonists of GABA-A receptors. All the observations above suggest that the structural analogs of taurine are partial agonists of GABA-A receptors that occupy the agonist-binding site, but their structures do not allow the proper interaction with the receptor to fully gate its Cl(-) channel.

Identification of the minimal promoter for specific expression of the GABAρ1 receptor in retinal bipolar cells.

γ-aminobutyric acid (GABA)ρ receptors regulate rapid synaptic ion currents in the axon end of retinal ON bipolar neurons, acting as a point of control along the visual pathway. In the GABAρ1 subunit knock out mouse, inhibition mediated by this receptor is totally eliminated, showing its role in neural transmission in retina. GABAρ1 mRNA is expressed in mouse retina after post-natal day 7, but little is known about its transcriptional regulation. To identify the GABAρ1 promoter, in silico analyses were performed and indicated that a 0.290-kb fragment, flanking the 5'-end of the GABAρ1 gene, includes putative transcription factor-binding sites, two Inr elements, and lacks a TATA-box. A rapid amplification of cDNA ends (RACE) assay showed three transcription start sites (TSS) clustered in the first exon. Luciferase reporter assays indicated that a 0.232-kb fragment upstream from the ATG is the minimal promoter in transfected cell lines and in vitro electroporated retinae. The second Inr and AP1 site are important to activate transcription in secretin tumor cells (STC-1) and retina. Finally, the 0.232-kb fragment drives green fluorescent protein (GFP) expression to the inner nuclear layer, where bipolar cells are present. This first work paves the way for further studies of molecular elements that control GABAρ1 transcription and regulate its expression during retinal development.

γ-Aminobutyric acid-ρ expression in ependymal glial cells of the mouse cerebellum.

The ependymal glial cells (EGCs) from the periventricular zone of the cerebellum were studied to determine their distribution and the functional properties of their γ-aminobutyric acid type A (GABA(A) ) receptors. EGCs were identified by the presence of ciliated structures on their ventricular surface and their expression of glial fibrillary acidic protein (GFAP). Interestingly, diverse cell types, including neurons, astrocytes, and other types of glia, were identified in the subventricular zone by their current profiles. Electron microscopy showed ciliated cells and myelinated axons in this zone, but we found no collateral connections to suggest the presence of functional synapses. GABA-mediated currents were recorded from EGCs in cerebellar slices from postnatal days 13 to 35 (PN13-PN35). These currents were blocked by TPMPA (a highly specific GABA(A) ρ subunit antagonist) and bicuculline (a selective antagonist for classic GABA(A) receptors). Pentobarbital failed to modulate GABA(A)-mediated currents despite the expression of GABAα1 and GABAγ2 subunits. In situ hybridization, RT-PCR, and immunofluorescence studies confirmed GABAρ1 expression in EGCs of the cerebellum. We conclude that cerebellar EGCs express GABAρ1, which is functionally involved in GABA(A) receptor-mediated responses that are unique among glial cells of the brain.

Structure-function study of the fourth transmembrane segment of the GABAρ1 receptor.

The Cys-loop family of receptors mediates synaptic neurotransmission in the central nervous system of vertebrates. These receptors share several structural characteristics and assemble in the plasma membrane as multimers with fivefold symmetry. Of these, the ionotropic GABA receptors are key players in the pathogenesis of diseases like epilepsy, anxiety, and schizophrenia. Different experimental approaches have shed some light on the mechanisms behind the function of these receptors; but little is known about their structure at high resolution. Sequence homology with the nicotinic acetylcholine receptor predicts that ionotropic GABA receptors possess four transmembrane segments (TM1-4) and that TM2 forms the wall of the ion channel. However, the role of the other three segments is unclear. The GABAρ1 receptor plays a fundamental role in the regulation of neurotransmission along the visual pathway, is highly sensitive to GABA, and exhibits little desensitization. In our recent investigations of the role of TM4 in receptor function, a key residue in this domain (W475) was found to be involved in activation of the receptor. Here we have generated a structural model of the GABAρ1 receptor in silico and assessed its validity by electrophysiologically testing nine amino acid substitutions of W475 and deletions of the neighboring residues (Y474 and S476). The results identify a critical linkage between the ligand-binding domain and the TM4 domain and provide a framework for more detailed structure-function analyses of ionotropic GABA receptors.

Striatal synaptic changes and behavior in adult mouse upon prenatal exposure to valproic acid.

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders characterized by persistent deficits in social communication and social interaction. Altered synaptogenesis and aberrant connectivity responsible for social behavior and communication have been reported in autism pathogenesis. Autism has a strong genetic and heritable component; however, environmental factors including toxins, pesticides, infection and in utero exposure to drugs such as VPA have also been implicated in ASD. Administration of VPA during pregnancy has been used as a rodent model to study pathophysiological mechanisms involved in ASD, and in this study, we used the mouse model of prenatal exposure to VPA to assess the effects on striatal and dorsal hippocampus function in adult mice. Alterations in repetitive behaviors and shift habits were observed in mice prenatally exposed to VPA. In particular, such mice presented a better performance in learned motor skills and cognitive deficits in Y-maze learning frequently associated with striatal and hippocampal function. These behavioral changes were associated with a decreased level of proteins involved in the formation and maintenance of excitatory synapses, such as Nlgn-1 and PSD-95. In conclusion, motor skill abilities, repetitive behaviors, and impaired flexibility to shift habits are associated with reduced striatal excitatory synaptic function in the adult mouse prenatally exposed to VPA.

Morphological characteristics of astrocytes of the fastigial nucleus.

Astrocytes are a diverse and morphologically complex class of glial cells restricted to the central nervous system which have been implicated in the modulation of neuronal activity. The cerebellum is involved in planning movements and motor learning. Within the cerebellum three deep cerebellar nuclei (dentate, interposed and fastigial) provide the sole neuronal output. The fastigial nucleus participates in saccadic and vestibular function, and recent evidence disclosed neuronal projections to cognitive, affective, and motor areas. However, thus far there are no reliable descriptions of the distribution and morphological classifications of astrocytes in this nucleus. This work aims to describe the characteristics of astrocytes of the fastigial nucleus based on the expression of GFP in a transgenic mouse model.

Analysis of a cell niche with proliferative potential at the roof of the aqueduct of Sylvius.

The aqueduct of Sylvius connects the third with the fourth ventricle and is surrounded by the Periaqueductal Grey. Here, we report a novel niche of cells in the dorsal section of the aqueduct, hereby named dorsal aqueduct niche or DAN, by applying a battery of selective markers and transgenic mouse lines. The somata of DAN cells are located toward the lumen of the ventricle forming multiple layers in close association with the cerebrospinal fluid (CSF). A single process emerges from the soma and run with the blood vessels. Cells of the DAN express radial glia/stem cell markers such as GFAP, vimentin and nestin, and the glutamate transporter GLAST or the oligodendrocyte precursor/pericyte marker NG2, thereby suggesting their potential for the generation of new cells. Morphologically, DAN cells resemble tanycytes of the third ventricle, which transfer biochemical signals from the CSF to the central nervous system and display proliferative capacity. The aqueduct ependymal lining can proliferate as observed by the integration of BrdU and expression of Ki67. Thus, the dorsal section of the aqueduct of Sylvius possesses cells that may act a niche of new glial cells in the adult mouse brain.

Expression of GABAρ receptors in the neostriatum: localization in aspiny, medium spiny neurons and GFAP-positive cells.

GABAergic transmission in the neostriatum plays a central role in motor coordination, in which a plethora of GABA-A receptor subunits combine to modulate neural inhibition. GABAρ receptors were originally described in the mammalian retina. These receptors possess special electrophysiological and pharmacological properties, forming a characteristic class of ionotropic receptors. In previous studies, we suggested that GABAρ receptors are expressed in the neostriatum, and in this report we show that they are indeed present in all the calretinin-positive interneurons of the neostriatum. In addition, they are located in calbindin-positive interneurons and projection neurons that express the dopamine D(2) receptor. GABAρ receptors were also located in 30% of the glial fibrillary acidic protein-positive cells, and may therefore also contribute to gliotransmission. Quantitative reverse transcription-PCR suggested that the mRNAs of this receptor do not express as much as in the retina, and that GABAρ2 is more abundant than GABAρ1. Electrophysiological recordings in brain slices provided evidence of neurons expressing a cis-4-aminocrotonic acid-activated, 1,2,5,6-tetrahydropyridine-4-yl methylphosphinic acid-sensitive ionotropic GABA receptor, indicating the presence of functional GABAρ receptors in the neostriatum. Finally, electron-microscopy and immunogold located the receptors mainly in perisynaptic as well as in extrasynaptic sites. All these observations reinforce the importance of GABAρ receptors in the neostriatum and contribute to the diversity of inhibitory regulation in this area.

Organization of the ventricular zone of the cerebellum.

The roof of the fourth ventricle (4V) is located on the ventral part of the cerebellum, a region with abundant vascularization and cell heterogeneity that includes tanycyte-like cells that define a peculiar glial niche known as ventromedial cord. This cord is composed of a group of biciliated cells that run along the midline, contacting the ventricular lumen and the subventricular zone. Although the complex morphology of the glial cells composing the cord resembles to tanycytes, cells which are known for its proliferative capacity, scarce or non-proliferative activity has been evidenced in this area. The subventricular zone of the cerebellum includes astrocytes, oligodendrocytes, and neurons whose function has not been extensively studied. This review describes to some extent the phenotypic, morphological, and functional characteristics of the cells that integrate the roof of the 4V, primarily from rodent brains.