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Abstract: The expanding number of disease-causing dysfunctions of synaptic proteins illustrates the importance of investigating newly discovered proteins involved in neuronal transmission. The gene Slc10A4 encodes a recently described carrier protein present in pre-synaptic terminals of cholinergic and monoaminergic neurons. The biological significance of this recently described transporter protein is currently unknown. We here investigated whether absence of the Slc10a4 protein has any impact on function of the cholinergic system. We first investigated the sensitivity of Slc10a4 null mice to cholinergic stimulus in vitro. In contrast to wild type mice, gamma oscillations occurred spontaneously in hippocampal slices from Slc10a4 null mice. Furthermore, moderate treatment of Slc10a4 null slices with the cholinergic agonist carbachol induced epileptiform activity. In vivo, 3-channel EEG measurements in freely behaving mice revealed that Slc10a4 null mice had frequent epileptiform spike-activity before treatment, and developed epileptic seizures, detected by EEG and accompanied by observable behavioral components, more rapidly after injection of the cholinergic agonist pilocarpine. Similar results were obtained on non-operated mice, as evaluated by behavioral seizures and post mortem c-Fos immunohistochemistry. Importantly, Slc10a4 null mice and wild type control mice were equally sensitive to the glutamatergic chemoconvulsant kainic acid, demonstrating that absence of Slc10a4 led to a selective cholinergic hypersensitivity. In summary, we report that absence of the recently discovered synaptic vesicle protein Slc10a4 results in increased sensitivity to cholinergic stimulation. [Copyright &y& Elsevier]

Abstract: Dopamine is cytotoxic and may play a role in the development of Parkinson''s disease. However, its interaction with environmental risk factors such as pesticides remains poorly understood. The vesicular monoamine transporter (VMAT) regulates intracellular dopamine content, and we have tested the neuroprotective effects of VMAT in vivo using the model organism Drosophila melanogaster. We find that Drosophila VMAT (dVMAT) mutants contain fewer dopaminergic neurons than wild type, consistent with a developmental effect, and that dopaminergic cell loss in the mutant is exacerbated by the pesticides rotenone and paraquat. Overexpression of DVMAT protein does not increase the survival of animals exposed to rotenone, but blocks the loss of dopaminergic neurons caused by this pesticide. These results are the first to demonstrate an interaction between a VMAT and pesticides in vivo, and provide an important model to investigate the mechanisms by which pesticides and cellular DA may interact to kill dopaminergic cells. [Copyright &y& Elsevier]

Abstract: Taste cells are chemosensory epithelial cells that sense distinct taste qualities. It is the type II taste cell that express G-protein coupled receptors to sense either umami, sweet, or bitter compounds. Whereas several reports have suggested involvement of ATP in taste signal transduction, there is a paucity of molecular information about how ATP is stored and being released. The recent discovery of a novel vesicular nucleotide transporter (VNUT) led us to examine whether VNUT exist in the taste tissue where ATP is to be released for taste signal transmission. Here, we report that VNUT is selectively expressed in type II cell but not in type III taste cell. In addition, we show that during taste bud development VNUT expression is always accompanied by the expression of type II taste cell markers. Our results, together with previous studies, strongly suggest that VNUT plays a role in type II taste cell. [Copyright &y& Elsevier]

Abstract: Loss of motor neurons is the key neuropathological feature of amyotrophic lateral sclerosis (ALS). Although these neurons are targeted by many synapses, little is known about the importance of the pre-synaptic excitatory and inhibitory input for motor neuron degeneration. The present study aimed in determining the composition and abundance of neurochemically defined input on lumbar spinal cord motor neurons in the SOD1 mouse model of ALS by employing immunoreactivity (IR) for vesicular neurotransmitter transporter proteins. The first signs of motor neuron degeneration, visualized by vesicular acetylcholine transporter IR, were already evident in the pre-symptomatic phase at day 80 of life. With the beginning of the symptomatic phase at around day 110 of life, surviving motor neurons showed reductions in the abundances of vesicular glutamate transporter 1 and 2 appositions. This loss of excitatory input was paralleled by an essentially unchanged inhibitory input, visualized by vesicular inhibitory amino acid transporter IR. In addition, loss of excitatory and inhibitory fibers and terminals was also evident in non-motor neuron areas of the spinal cord. These data are indicative of an imbalanced synaptic input on spinal cord motor neurons with over-inhibition rather than over-excitation being a neuropathological feature during disease progression that may contribute to motor neuron death. [Copyright &y& Elsevier]

Constitutive expression of mRNA was seen for the vesicular glutamate transporter brain-specific Na+-dependent inorganic phosphate cotransporter (BNPI), but not differentiation-associated Na+-dependent inorganic phosphate cotransporter, in rat calvarial osteoblasts cultured for 7 and 21 days in vitro (DIV). Three different agonists for ionotropic glutamate receptors (iGluR) at 1 mM, as well as 50 mM KCl, significantly increased the release of endogenous l-glutamate from osteoblasts cultured for 7 DIV when determined 5 min after the addition by using a high performance liquid chromatograph. The inhibitor of desensitization of dl-α-amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA) receptors cyclothiazide significantly potentiated and prolonged the release of endogenous l-glutamate evoked by AMPA in a dose-dependent manner. The release evoked by AMPA was significantly prevented by the addition of an AMPA receptor antagonist as well as by the removal of Ca2+ ions. These results suggest that endogenous l-glutamate could be released from intracellular vesicular constituents associated with BNPI through activation of particular iGluR subtypes expressed in cultured rat calvarial osteoblasts. [Copyright &y& Elsevier]