tetrodotoxin and Wallerian-Degeneration

Activity and disuse of synapses are thought to influence progression of several neurodegenerative diseases in which synaptic degeneration is an early sign. Here we tested whether stimulation or disuse renders neuromuscular synapses more or less vulnerable to degeneration, using axotomy as a robust trigger. We took advantage of the slow synaptic degeneration phenotype of axotomized neuromuscular junctions in flexor digitorum brevis (FDB) and deep lumbrical (DL) muscles of Wallerian degeneration-Slow (Wld(S)) mutant mice. First, we maintained ex vivo FDB and DL nerve-muscle explants at 32°C for up to 48 h. About 90% of fibers from Wld(S) mice remained innervated, compared with about 36% in wild-type muscles at the 24-h checkpoint. Periodic high-frequency nerve stimulation (100 Hz: 1s/100s) reduced synaptic protection in Wld(S) preparations by about 50%. This effect was abolished in reduced Ca(2+) solutions. Next, we assayed FDB and DL innervation after 7 days of complete tetrodotoxin (TTX)-block of sciatic nerve conduction in vivo, followed by tibial nerve axotomy. Five days later, only about 9% of motor endplates remained innervated in the paralyzed muscles, compared with about 50% in 5 day-axotomized muscles from saline-control-treated Wld(S) mice with no conditioning nerve block. Finally, we gave mice access to running wheels for up to 4 weeks prior to axotomy. Surprisingly, exercising Wld(S) mice ad libitum for 4 weeks increased about twofold the amount of subsequent axotomy-induced synaptic degeneration. Together, the data suggest that vulnerability of mature neuromuscular synapses to axotomy, a potent neurodegenerative trigger, may be enhanced bimodally, either by disuse or by hyperactivity.

Topics: Animals; Axotomy; Calcium; Electric Stimulation Therapy; Female; Male; Mice, Inbred C57BL; Mice, Transgenic; Muscle, Skeletal; Nerve Tissue Proteins; Neuromuscular Junction; Running; Sciatic Nerve; Sodium Channel Blockers; Tetrodotoxin; Tibial Nerve; Tissue Culture Techniques; Wallerian Degeneration

Axons degenerate after injury and in neuropathies and disease via a self-destruction program whose mechanism is poorly understood. Axons that have lost connection to their cell bodies have altered electrical and synaptic activities, but whether such changes play a role in the axonal degeneration process is not clear. We have used a Drosophila model to study the Wallerian degeneration of motoneuron axons and their neuromuscular junction synapses. We found that degeneration of the distal nerve stump after a nerve crush is greatly delayed when there is increased potassium channel activity (by overexpression of two different potassium channels, Kir2.1 and dORKΔ-C) or decreased voltage-gated sodium channel activity (using mutations in the para sodium channel). Conversely, degeneration is accelerated when potassium channel activity is decreased (by expressing a dominant-negative mutation of Shaker). Despite the effect of altering voltage-gated sodium and potassium channel activity, recordings made after nerve crush demonstrated that the distal stump does not fire action potentials. Rather, a variety of lines of evidence suggest that the sodium and potassium channels manifest their effects upon degeneration through changes in the resting membrane potential, which in turn regulates the level of intracellular free calcium within the isolated distal axon.

Topics: Action Potentials; Animals; Axons; Calcium; Drosophila; Electrophysiological Phenomena; Immunohistochemistry; Microscopy, Confocal; Nerve Crush; Neuromuscular Junction; Potassium Channels; Shaker Superfamily of Potassium Channels; Sodium Channel Blockers; Sodium Channels; Synapses; Temperature; Tetrodotoxin; Wallerian Degeneration

Measurements of extracellular space volume and imaging of intrinsic optical signals (IOSs) have shown that neuronal activity increases light transmittance by causing cellular swelling. However, the cellular mechanisms underlying these volume changes and the contribution of astrocyte swelling to the changes in tissue volume are unclear. In this study, we have investigated IOSs in optic nerves to analyze the mechanisms contributing to these signals in a system consisting of only axons and glial cells. We examined both intact optic nerves and enucleated optic nerves, which contained no axons and consisted primarily of astrocytes. Electrical stimulation of intact optic nerves evoked an increase in light transmittance, which was graded with increasing stimulation frequency and was mimicked by raising extracellular K(+) concentration ([K(+)](o)). The stimulation-induced IOS grew in amplitude and had a time course similar to extracellular space shrinkage. Tetrodotoxin (TTX) blocked the electrically induced but not the high K(+)-induced IOS. In enucleated nerves, light transmittance progressively increased in higher [K(+)](o). The high [K(+)](o)-induced IOSs were reversibly depressed by furosemide and bumetanide, antagonists for Na-K-2Cl cotransport, but were unaltered by TTX. We also used a monoclonal antibody to the NKCC1 form of the Na-K-2Cl cotransporter to show that NKCC1 is expressed in optic nerves as shown in Western blotting and is colocalized in GFAP immunopositive astrocytes. In summary, these results indicated that KCl uptake into astrocytes via an Na-K-2Cl cotransporter during raised [K(+)](o) contributes to the generation of cellular swelling and the intrinsic optical signals.

Topics: Action Potentials; Animals; Animals, Newborn; Astrocytes; Axons; Bumetanide; Cell Size; Diuretics; Electric Stimulation; Eye Enucleation; Furosemide; Immunohistochemistry; Light; Optic Nerve; Optics and Photonics; Organ Culture Techniques; Potassium; Rats; Rats, Sprague-Dawley; Signal Transduction; Sodium-Potassium-Chloride Symporters; Solute Carrier Family 12, Member 2; Tetrodotoxin; Vision, Ocular; Wallerian Degeneration

Schwann cells are considered to be electrically silent satellite cells surrounding axons, although they exhibit ionic channels, some of which are similar to those employed by axons for the generation and transmission of nerve impulses. Here, we show that Schwann cells generate, in response to a short and gentle electrical stimulus, a long-lasting depolarizing potential, slowly propagating along the Schwann cell synsitium. This electrical signal, which in situ might be generated by the Schwann cells in response to the axonal electrical activity, constitutes in the peripheral glia a novel form of long-range intercellular signalling, which may be involved in the regulation and modulation of the axonal excitability.

Topics: Animals; Axons; Calcium; Chlorides; Electric Stimulation; Female; Male; Membrane Potentials; Peripheral Nerves; Rabbits; Schwann Cells; Sodium; Tetrodotoxin; Vagus Nerve; Vagus Nerve Injuries; Wallerian Degeneration