tetrodotoxin and Necrosis

Excitotoxicity is associated with stroke, brain trauma, and a number of neurodegenerative disorders. In the brain, during excitotoxic insults, neurons undergo rapid swelling in both the soma and dendrites. Focal swellings along the dendrites called varicosities are considered to be a hallmark of acute excitotoxic neuronal injury. However, it is not clear what pathway is involved in the neuronal anion flux that leads to the formation and resolution of excitotoxic varicosities. Here, we assessed the roles of the volume-sensitive outwardly rectifying (VSOR) Cl- channel in excitotoxic responses in mouse cortical neurons. Whole-cell patch-clamp recordings revealed that the VSOR Cl- channel in cultured neurons was activated by NMDA exposure. Moreover, robust expression of this channel on varicosities was confirmed by on-cell and nystatin-perforated vesicle patch techniques. VSOR channel blockers, but not blockers of GABA(A) receptors and Cl- transporters, abolished not only varicosity resolution after sublethal excitotoxic stimulation but also necrotic death after sustained varicosity formation induced by prolonged NMDA exposure in cortical neurons. The present slice-patch experiments demonstrated, for the first time, expression of the VSOR Cl- channels in somatosensory pyramidal neurons. NMDA-induced necrotic neuronal death in slice preparations was largely suppressed by a blocker of the VSOR Cl- channel but not of the GABA(A) receptor. These results indicate that VSOR Cl- channels exert dual, reciprocal actions on neuronal excitotoxicity by serving as major anionic pathways both for varicosity recovery after washout of an excitotoxic stimulant and for persistent varicosity formation under prolonged excitotoxic insults leading to necrosis in cortical neurons.

Topics: 2-Amino-5-phosphonovalerate; 4-Aminopyridine; Animals; Apoptosis; Benzothiadiazines; Bicuculline; Bumetanide; Cell Size; Cells, Cultured; Cerebral Cortex; Chlorides; Dendrites; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; GABA-A Receptor Antagonists; Glycolates; Ion Channels; Mice; Mice, Inbred C57BL; N-Methylaspartate; Necrosis; Neurons; Neurotoxins; Nitrobenzoates; Patch-Clamp Techniques; Phloretin; Picrotoxin; Potassium Channel Blockers; Potassium Channels; Quinine; Receptors, N-Methyl-D-Aspartate; Sodium Chloride Symporter Inhibitors; Sodium Chloride Symporters; Somatosensory Cortex; Tetrodotoxin

High extracellular potassium induces spreading depression-like depolarizations and elevations of extracellular glutamate. Both occur in the penumbra of a focal ischemic infarct, and may be responsible for the spread of cell death from the infarct core to the penumbra. We have modeled this situation with microdialysis of an isotonic high-potassium solution into the normal rat amygdala for 70 min. This elevates extracellular glutamate up to 8-fold or more and produces irreversibly damaged, acidophilic neurons. NMDA-receptor blockade protects neurons and reduces the elevation of extracellular glutamate. Here we investigated the effects of sodium channel blockade with the voltage-sensitive sodium channel blocker tetrodotoxin and the AMPA receptor antagonist 2,3-dihydroxy-6-nitro-1,2,3,4-tetrahydrobenzo(f)quinoxaline-7-sulfonamide disodium (NBQX disodium) on high potassium-induced neuronal death and extracellular glutamate elevations. The acidophilic neurons produced are necrotic by ultrastructural examination. Tetrodotoxin, at dialysate concentrations of 33, 330 and 3300 microM (only a small fraction is extracted by tissue), markedly reduced the elevations of glutamate in rat amygdala at nearly all time points during high-potassium perfusion, but it reduced tissue edema only at the highest concentration, and it was neuroprotective only if dialyzed prior to high-potassium microdialysis (at 330 microM concentration). Although both 250 microM (6.2% is extracted by tissue) and 500 microM NBQX reduced elevations of glutamate, neither was neuroprotective, and neuropil edema was not reduced by either concentration. Our results suggest that in vivo, sodium influx through voltage-sensitive sodium channels but not through ligand-gated AMPA receptor channels contributes to high potassium-induced neuronal necrosis.

Topics: Amygdala; Animals; Cell Death; Dose-Response Relationship, Drug; Electrophysiology; Excitatory Amino Acid Antagonists; Glutamic Acid; Ion Channel Gating; Male; Necrosis; Neurons; Potassium; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

Virus infection of neurons leads to different outcomes ranging from latent and noncytolytic infection to cell death. Viruses kill neurons directly by inducing either apoptosis or necrosis or indirectly as a result of the host immune response. Sindbis virus (SV) is an alphavirus that induces apoptotic cell death both in vitro and in vivo. However, apoptotic changes are not always evident in neurons induced to die by alphavirus infection. Time lapse imaging revealed that SV-infected primary cortical neurons exhibited both apoptotic and necrotic morphological features and that uninfected neurons in the cultures also died. Antagonists of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors protected neurons from SV-induced death without affecting virus replication or SV-induced apoptotic cell death. These results provide evidence that SV infection activates neurotoxic pathways that result in aberrant NMDA receptor stimulation and damage to infected and uninfected neurons.

Topics: 2-Amino-5-phosphonovalerate; Animals; Apoptosis; Calcium; Cell Death; Cell Line; Cells, Cultured; Cricetinae; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Intracellular Fluid; Necrosis; Neurons; Potassium Channel Blockers; Rats; Rats, Long-Evans; Receptors, N-Methyl-D-Aspartate; Sindbis Virus; Tetrodotoxin; Virus Replication

Focal microinjection of tetrodotoxin (TTX), a potent voltage-gated sodium channel blocker, reduces neurological deficits and tissue loss after spinal cord injury (SCI). Significant sparing of white matter (WM) is seen at 8 weeks after injury and is correlated to a reduction in functional deficits. To determine whether TTX exerts an acute effect on WM pathology, Sprague Dawley rats were subjected to a standardized weight-drop contusion at T8 (10 gm x 2.5 cm). TTX (0. 15 nmol) or vehicle solution was injected into the injury site 5 or 15 min later. At 4 and 24 hr, ventromedial WM from the injury epicenter was compared by light and electron microscopy and immunohistochemistry. By 4 hr after SCI, axonal counts revealed reduced numbers of axons and significant loss of large (>/=5 micrometer)-diameter axons. TTX treatment significantly reduced the loss of large-diameter axons. In addition, TTX significantly attenuated axoplasmic pathology at both 4 and 24 hr after injury. In particular, the development of extensive periaxonal spaces in the large-diameter axons was reduced with TTX treatment. In contrast, there was no significant effect of TTX on the loss of WM glia after SCI. Thus, the long-term effects of TTX in reducing WM loss after spinal cord injury appear to be caused by the reduction of acute axonal pathology. These results support the hypothesis that TTX-sensitive sodium channels at axonal nodes of Ranvier play a significant role in the secondary injury of WM after SCI.

Topics: Animals; Axons; Contusions; Female; Glial Fibrillary Acidic Protein; Injections, Spinal; Microinjections; Myelin Sheath; Necrosis; Neuroglia; Oligodendroglia; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Spinal Cord; Spinal Cord Injuries; Tetrodotoxin

1. The myonecrosis induced by guanidine in the mouse phrenic nerve diaphragm preparation was investigated using both light microscopy and myographic recordings. Preparations were incubated with 10 mM guanidine for 60 min in the absence and presence of electrical stimulation. At the end of this period, the drug was washed out and the nutritive medium replaced with fixative solution to prevent morphological artefacts. 2. Guanidine produced a triphasic change in the amplitude of twitch tension evoked indirectly through the motor nerve. This response consisted of an initial facilitation followed by a neuromuscular blockade and a secondary facilitatory effect after removal of the drug. 3. Morphological analysis of the muscle showed various structural alterations of the fibers, including the presence of very dark swollen cells with or without small clear vacuoles, delta lesions with densely or loosely clumped myofibrils, irregular clear spaces, indistinct masses of degraded myofibrils, and, in extreme cases, "ghost" cells. All of these effects were attributed to the presence of high cytosolic calcium concentrations. 4. Pretreatment with tetrodotoxin (TTX, 3.13 microM) diminished but did not prevent the guanidine-induced morphological abnormalities in the muscle cells. This finding suggests that TTX can interfere to a certain extent with the influx of guanidine into muscle fibers through sodium channels. 5. An attempt was made to correlate the myographic findings with the muscle morphological alterations seen after guanidine removal.

Topics: Animals; Diaphragm; Electric Stimulation; Guanidine; Guanidines; In Vitro Techniques; Male; Mice; Muscle Contraction; Necrosis; Neuromuscular Junction; Phrenic Nerve; Sodium Channel Blockers; Tetrodotoxin