Na+, K+-ATPase inhibition induces neuronal cell death in rat hippocampal slice cultures: Association with GLAST and glial cell abnormalities.

Na+, K+-ATPase is a highly expressed membrane protein. Dysfunction of Na+, K+-ATPase has been implicated in the pathophysiology of several neurodegenerative and psychiatric disorders, however, the underlying mechanism of neuronal cell death resulting from Na+, K+-ATPase dysfunction is poorly understood. Here, we investigated the mechanism of neurotoxicity due to Na+, K+-ATPase inhibition using rat organotypic hippocampal slice cultures. Treatment with ouabain, a Na+, K+-ATPase inhibitor, increased the ratio of propidium iodide-positive cells among NeuN-positive cells in the hippocampal CA1 region, which was prevented by MK-801 and d-AP5, specific blockers of the N-methyl-d-aspartate (NMDA) receptor. EGTA, a Ca2+-chelating agent, also protected neurons from ouabain-induced injury. We observed that astrocytes expressed the glutamate aspartate transporter (GLAST), and ouabain changed the immunoreactive area of GFAP-positive astrocytes as well as GLAST. We also observed that ouabain increased the number of Iba1-positive microglial cells in a time-dependent manner. Furthermore, lithium carbonate, a mood-stabilizing drug, protected hippocampal neurons and reduced disturbances of astrocytes and microglia after ouabain treatment. Notably, lithium carbonate improved ouabain-induced decreases in GLAST intensity in astrocytes. These results suggest that glial cell abnormalities resulting in excessive extracellular concentrations of glutamate contribute to neurotoxicity due to Na+, K+-ATPase dysfunction in the hippocampal CA1 region.

Effects of triphenyltin on glycinergic transmission on rat spinal neurons.

Glycine is a fast inhibitory transmitter like γ-aminobutyric acid in the mammalian spinal cord and brainstem, and it is involved in motor reflex, nociception, and neuronal development. Triphenyltin (TPT) is an organometallic compound causing environmental hazard to many wild creatures. Our previous findings show that TPT ultimately induces a drain and/or exhaustion of glutamate in excitatory presynaptic nerve terminals, resulted in blockage of neurotransmission as well as methylmercury. Therefore, we have investigated the neurotoxic mechanism how TPT modulates inhibitory glycinergic transmission in the synaptic bouton preparation of rat isolated spinal neurons using a patch clamp technique. TPT at environmentally relevant concentrations (3-300 nM) significantly increased the number of frequency of glycinergic spontaneous and miniature inhibitory postsynaptic currents (sIPSC and mIPSC) without affecting the current amplitude and decay time. The TPT effects were also observed in external Ca2+-free solution containing tetrodotoxin (TTX) but removed in Ca2+-free solution with both TTX and BAPTA-AM (Ca2+ chelator). On the other hand, the amplitude of glycinergic evoked inhibitory postsynaptic currents (eIPSCs) increased with decreasing failure rate (Rf) and paired pulse ratio (PPR) in the presence of 300 nM TPT. At a high concentration (1 µM), TPT completely blocked eIPSCs after a transient facilitation. Overall, these results suggest that TPT directly acts transmitter-releasing machinery in glycinergic nerve terminals. Effects of TPT on the nerve terminals releasing fast transmitters were greater in the order of glycinergic > glutamatergic > GABAergic ones. Thus, TPT is supposed to cause a strong synaptic modulations on glycinergic neurotransmission in wild creatures.

Intra-axonal Ca2+ mobilization contributes to triphenyltin-induced facilitation in glycinergic transmission of rat spinal neurons.

Triphenyltin (TPT) is an organotin compound causing environmental hazard to many wild creatures. Our previous findings show that TPT increases of the frequency of spontaneous glycinergic inhibitory postsynaptic currents (sIPSCs) in rat spinal neurons without changing the amplitude and 1/e decay time. In our study, the effects of 2-aminoethoxydiphenyl borate (2-APB), dantrolene sodium, and thapsigargin on sIPSC frequency were examined to reveal the contribution of intra-axonal Ca2+ mobilization by adding TPT. 2-APB considerably attenuated the TPT-induced facilitation of sIPSC frequency while dantrolene almost completely masked the TPT effects, suggesting that the TPT-induced synaptic facilitation results from the activation of both IP3 and ryanodine receptors on endoplasmic reticulum (ER) membrane, though inositol triphosphate (IP3) receptor is less sensitive to TPT. Thapsigargin itself significantly increased the sIPSC frequency without affecting the current amplitude and decay time. Successive addition of TPT could not further increase the sIPSC frequency in the presence of thapsigargin, indicating that thapsigargin completely masked the facilitatory action of TPT. Results suggest that TPT activates the IP3 and ryanodine receptors while TPT inhibits the Ca2+-pump of ER membranes, resulting in the elevation of intra-axonal Ca2+ levels, leading to the increase of spontaneous glycine release from synaptic vesicles.