tetrodotoxin and Nerve-Degeneration

Topics: Animals; Botulinum Toxins; Bungarotoxins; Electromyography; Fatty Acids; Humans; Mice; Motor Neurons; Muscle Denervation; Muscles; Myasthenia Gravis; Nerve Degeneration; Nerve Regeneration; Neuromuscular Diseases; Neuromuscular Junction; Peripheral Nerve Injuries; Receptors, Cholinergic; Schwann Cells; Tetrodotoxin

KCNQ5/Kv7.5, a low-threshold noninactivating voltage-gated potassium channel, is preferentially targeted to excitatory endings of auditory neurons in the adult rat brainstem. Endbulds of Held from auditory nerve axons on the bushy cells of the ventral cochlear nucleus (VCN) and calyces of Held around the principal neurons in the medial nucleus of the trapezoid body (MNTB) are rich in KCNQ5 immunoreactivity. We have previously shown that this synaptic distribution occurs at about the time of hearing onset. The current study tests whether this localization in excitatory endings depends on the peripheral activity carried by the auditory nerve. Auditory nerve activity was abolished by cochlear removal or intracochlear injection of tetrodotoxin (TTX). Presence of KCNQ5 was analyzed by immunocytochemistry, Western blotting, and quantitative reverse transcription polymerase chain reaction. After cochlear removal, KCNQ5 immunoreactivity was virtually undetectable at its usual location in endbulbs and calyces of Held in the anteroventral CN and in the MNTB, respectively, although it was found in cell bodies in the VCN. The results were comparable after intracochlear TTX injection, which drastically reduced KCNQ5 immunostaining in MNTB calyces and increased immunolabeling in VCN cell bodies. Endbulbs of Held in the VCN also showed diminished KCNQ5 labeling after intracochlear TTX injection. These results show that peripheral activity from auditory nerve afferents is necessary to maintain the subcellular distribution of KCNQ5 in synaptic endings of the auditory brainstem. This may contribute to adaptations in the excitability and neurotransmitter release properties of these presynaptic endings under altered input conditions.

Topics: Anesthetics, Local; Animals; Auditory Diseases, Central; Brain Stem; Calbindin 2; Cochlear Diseases; Disease Models, Animal; Evoked Potentials, Auditory, Brain Stem; Female; Fluoresceins; Gene Expression Regulation; KCNQ Potassium Channels; Male; Nerve Degeneration; Neurons; Rats; Rats, Wistar; RNA, Messenger; Tetrodotoxin; Time Factors

Neuronal gene expression is modulated by activity via calcium-permeable receptors such as NMDA receptors (NMDARs). While gene expression changes downstream of evoked NMDAR activity have been well studied, much less is known about gene expression changes that occur under conditions of basal neuronal activity. In mouse dissociated hippocampal neuronal cultures, we found that a broad NMDAR antagonist, AP5, induced robust gene expression changes under basal activity, but subtype-specific antagonists did not. While some of the gene expression changes are also known to be downstream of stimulated NMDAR activity, others appear specific to basal NMDAR activity. The genes altered by AP5 treatment of basal cultures were enriched for pathways related to class IIa histone deacetylases (HDACs), apoptosis, and synapse-related signaling. Specifically, AP5 altered the expression of all three class IIa HDACs that are highly expressed in the brain, HDAC4, HDAC5, and HDAC9, and also induced nuclear accumulation of HDAC4. HDAC4 knockdown abolished a subset of the gene expression changes induced by AP5, and led to neuronal death under long-term tetrodotoxin or AP5 treatment in rat hippocampal organotypic slice cultures. These data suggest that basal, but not evoked, NMDAR activity regulates gene expression in part through HDAC4, and, that HDAC4 has neuroprotective functions under conditions of low NMDAR activity.

Topics: Animals; Cell Survival; Cells, Cultured; Female; Gene Expression Regulation; Gene Knockdown Techniques; Hippocampus; Histone Deacetylases; Male; Mice; Nerve Degeneration; Neurons; Rats; Receptors, N-Methyl-D-Aspartate; Tetrodotoxin; Valine

The mouse Dach1 gene, involved in the development of the neocortex and the hippocampus, is expressed by neural stem cells (NSCs) during early neurogenesis, and its expression also continues in a subpopulation of cells in the dorsal part of the lateral ventricles (LV) of the adult mouse brain. In this study we aimed to elucidate the role of Dach1-expressing cells in adult neurogenesis/gliogenesis under physiological as well as post-ischemic conditions, employing transgenic mice in which the expression of green fluorescent protein (GFP) is controlled by the D6 promotor of the mouse Dach1 gene. A neurosphere-forming assay of GFP⁺ cells isolated from the dorsal part of the LV was carried out with subsequent differentiation in vitro. To elucidate the neurogenic/gliogenic potential of GFP⁺ cells in the dorsal part of the LV, in situ immunohistochemical/electrophysiological analyses of GFP⁺ cells in adult sham-operated brains (controls) and those after middle cerebral artery occlusion (MCAo) were performed. The GFP⁺ cells isolated from the dorsal part of the LV of controls formed neurospheres and differentiated solely into a glial phenotype, while those isolated after MCAo also gave rise to cells with the properties of neuronal precursors. In situ analyses revealed that GFP⁺ cells express the phenotype of adult NSCs or neuroblasts in controls as well as following ischemia. Following MCAo we found a significantly increased number of GFP⁺ cells expressing doublecortin as well as a number of GFP⁺ cells migrating through the rostral migratory stream into the olfactory bulb, where they probably differentiated into calretinin⁺ interneurons. Collectively, our results suggest the involvement of the mouse Dach1 gene in adult neurogenesis; cells expressing this gene exhibit the properties of adult NSCs or neuroblasts and respond to MCAo by enhanced neurogenesis.

Topics: 4-Aminopyridine; Adult Stem Cells; Animals; Cell Count; Cell Differentiation; Disease Models, Animal; Eye Proteins; Green Fluorescent Proteins; In Vitro Techniques; Infarction, Middle Cerebral Artery; Lateral Ventricles; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nerve Degeneration; Nerve Tissue Proteins; Neurogenesis; Neurons; Patch-Clamp Techniques; Sodium Channel Blockers; Tetraethylammonium; Tetrodotoxin

Axonal degeneration occurs in multiple neurodegenerative disorders of the central and peripheral nervous system. Although the underlying molecular pathways leading to axonal degeneration are incompletely understood, accumulating evidence suggests contributions of impaired mitochondrial function, disrupted axonal transport, and/or dysfunctional intracellular Ca(2+)-homeostasis in the injurious cascade associated with axonal degeneration. Utilizing an in vitro model of axonal degeneration, we studied a subset of mouse peripheral sensory neurons in which neurites were exposed selectively to conditions associated with the pathogenesis of axonal neuropathies in vivo. Rotenone-induced mitochondrial dysfunction resulted in neurite degeneration accompanied by reduced ATP levels and increased ROS levels in neurites. Blockade of voltage-gated sodium channels with TTX and reverse (Ca(2+)-importing) mode of the sodium-calcium exchanger (NCX) with KB-R7943 partially protected rotenone-treated neurites from degeneration, suggesting a contribution of sodium channels and reverse NCX activity to the degeneration of neurites resulting from impaired mitochondrial function. Pharmacological inhibition of the Na(+)/K(+)-ATPase with ouabain induced neurite degeneration, which was attenuated by TTX and KB-R7943, supporting a contribution of sodium channels in axonal degenerative pathways accompanying impaired Na(+)/K(+)-ATPase activity. Conversely, oxidant stress (H2O2)-induced neurite degeneration was not attenuated by TTX. Our results demonstrate that both energetic and oxidative stress targeted selectively to neurites induces neurite degeneration and that blockade of sodium channels and of reverse NCX activity blockade partially protects neurites from injury due to energetic stress, but not from oxidative stress induced by H2O2.

Topics: Animals; Axons; Axotomy; Cell Death; Cells, Cultured; Ganglia, Spinal; Humans; Hydrogen Peroxide; Immunohistochemistry; Mice; Mice, Transgenic; Microtubules; Mitochondrial Diseases; Nerve Degeneration; Neurites; Oxidants; Rotenone; Sodium Channel Blockers; Sodium Channels; Sodium-Calcium Exchanger; Sodium-Potassium-Exchanging ATPase; Tetrodotoxin; Thiourea; Uncoupling Agents

Alterations in mitochondrial dynamics (fission, fusion, and movement) are implicated in many neurodegenerative diseases, from rare genetic disorders such as Charcot-Marie-Tooth disease, to common conditions including Alzheimer's disease. However, the relationship between altered mitochondrial dynamics and neurodegeneration is incompletely understood. Here we show that disease associated MFN2 proteins suppressed both mitochondrial fusion and transport, and produced classic features of segmental axonal degeneration without cell body death, including neurofilament filled swellings, loss of calcium homeostasis, and accumulation of reactive oxygen species. By contrast, depletion of Opa1 suppressed mitochondrial fusion while sparing transport, and did not induce axonal degeneration. Axon degeneration induced by mutant MFN2 proteins correlated with the disruption of the proper mitochondrial positioning within axons, rather than loss of overall mitochondrial movement, or global mitochondrial dysfunction. We also found that augmenting expression of MFN1 rescued the axonal degeneration caused by MFN2 mutants, suggesting a possible therapeutic strategy for Charcot-Marie-Tooth disease. These experiments provide evidence that the ability of mitochondria to sense energy requirements and localize properly within axons is key to maintaining axonal integrity, and may be a common pathway by which disruptions in axonal transport contribute to neurodegeneration.

Topics: Adaptor Proteins, Vesicular Transport; Animals; Arginine; Axons; Calcium; Deoxyglucose; Embryo, Mammalian; Fluoresceins; Ganglia, Spinal; Gene Expression Regulation; Glutamine; Green Fluorescent Proteins; GTP Phosphohydrolases; Humans; Mitochondria; Mitochondrial Proteins; Nerve Degeneration; Nerve Tissue Proteins; Neurons; Point Mutation; Rats; Reactive Oxygen Species; RNA, Small Interfering; Sodium Channel Blockers; Tetrodotoxin; Transfection

The neuron-astrocyte synaptic complex is a fundamental operational unit of the nervous system. Astroglia regulate synaptic glutamate, via neurotransmitter transport by GLT1/EAAT2. Astroglial mechanisms underlying this essential neuron-glial communication are not known. We now show that presynaptic terminals regulate astroglial synaptic functions, GLT1/EAAT2, via kappa B-motif binding phosphoprotein (KBBP), the mouse homolog of human heterogeneous nuclear ribonucleoprotein K (hnRNP K), which binds the GLT1/EAAT2 promoter. Neuron-stimulated KBBP is required for GLT1/EAAT2 transcriptional activation and is responsible for astroglial alterations in neural injury. Denervation of neuron-astrocyte signaling by corticospinal tract transection, ricin-induced motor neuron death, or neurodegeneration in amyotrophic lateral sclerosis all result in reduced astroglial KBBP expression and transcriptional dysfunction of astroglial transporter expression. Presynaptic elements dynamically coordinate normal astroglial function and also provide a fundamental signaling mechanism by which altered neuronal function and injury leads to dysregulated astroglia in CNS disease.

Topics: Analysis of Variance; Animals; Animals, Newborn; Astrocytes; Cerebral Cortex; Coculture Techniques; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Embryo, Mammalian; Excitatory Amino Acid Agents; Excitatory Amino Acid Transporter 2; Green Fluorescent Proteins; Humans; Kainic Acid; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microfluidic Analytical Techniques; Mutagenesis; Nerve Degeneration; Neurons; NF-kappa B; Presynaptic Terminals; Pyramidal Tracts; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Spinal Cord Injuries; Superoxide Dismutase; Synapses; Tetrodotoxin; Transfection; Up-Regulation

Networks of neurons express persistent spontaneous network activity when maintained in dissociated cultures. Prolonged blockade of the spontaneous activity with tetrodotoxin (TTX) causes the eventual death of the neurons. In this study, we investigated some molecular mechanisms that may underlie the activity-suppressed slow degeneration of cortical neurons in culture. Already after 3-4 days of exposure to TTX, well before the neurons die, they began to express markers that lead to their eventual death, 7-10 days later. There was a reduction in glutamate receptor (GluR2) expression, a persistent increase in intracellular calcium concentration, activation of calpain, and an increase in spectrin breakdown products. At this point, blockade of GluR2-lacking GluR1 or calpain (either with a selective antagonist or through the natural regulator of calpain, calpastatin), protected cells from the toxic action of TTX. Subsequently, mitochondria lost their normal elongated shape as well as their membrane potential. Eventually, neurons activated caspase 3 and PUMA (p53 up-regulated modulator of apoptosis), hallmarks of neuronal apoptosis, and died. These experiments will lead to a better understanding of slow neuronal death, typical of neurodegenerative diseases.

Topics: Action Potentials; Animals; Animals, Newborn; Apoptosis; Calcium; Calcium Signaling; Calpain; Cells, Cultured; Cerebral Cortex; Energy Metabolism; Mitochondria; Mitochondrial Diseases; Nerve Degeneration; Nerve Net; Neural Pathways; Neurons; Rats; Rats, Wistar; Signal Transduction; Sodium Channel Blockers; Synaptic Transmission; Tetrodotoxin

Central neurons express persistent spontaneous electrical network activity both in the developing brain in vivo as well as in dissociated cultures. This electrical activity is important for the formation of connections among neurons, and for their survival. Prolonged suppression of the spontaneous activity using the sodium channel blocker tetrodotoxin (TTX) causes the death of the cultured neurons. In the present study, we investigated molecular mechanisms that may underlie the activity-suppressed slow degeneration of cortical neurons in culture. Already after 6-7 days of exposure to TTX, neurons begin to express apoptotic vacuoles and shrunken dendrites. Eventually, neurons activate p53, caspase-3 and BAX, hallmarks of neuronal apoptosis, before they die. This death is restricted to neurons, and no parallel process is seen in glial cells that co-exist in the culture. These experiments may lead to a better understanding of slow neuronal death, akin to that found in neurodegenerative diseases of the brain.

Topics: Action Potentials; Animals; Animals, Newborn; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Survival; Cells, Cultured; Cerebral Cortex; Dendrites; Models, Neurological; Nerve Degeneration; Neurodegenerative Diseases; Neurons; Rats; Rats, Wistar; Sodium Channel Blockers; Tetrodotoxin; Tumor Suppressor Protein p53; Vacuoles

When deprived of spontaneous ongoing network activity by chronic exposure to tetrodotoxin (TTX), cultured cortical neurons retract their dendrites, lose dendritic spines, and degenerate over a period of 1-2 weeks. Electrophysiological properties of these slowly degenerating neurons prior to their death are normal, but they express very large miniature excitatory postsynaptic currents (mEPSCs). Chronic blockade of these mEPSCs by the alpha-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist 6,7-Dinitroquinoxaline-2,3-dione (DNQX) had no effect of its own on cell survival, yet, paradoxically, it protected the TTX-silenced neurons from degenerating. TTX-treated neurons also exhibited deficient Ca(2+) clearance mechanisms. Thus, upscaled mEPSCs are sufficient to trigger apoptotic processes in otherwise chronically silenced neurons.

Topics: Action Potentials; Anesthetics, Local; Animals; Apoptosis; Calcium; Cells, Cultured; Dendritic Spines; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Nerve Degeneration; Neurons; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Synaptic Transmission; Tetrodotoxin

Loss of axons is a major contributor to nonremitting deficits in the inflammatory demyelinating disease multiple sclerosis (MS). Based on biophysical studies showing that activity of axonal sodium channels can trigger axonal degeneration, recent studies have tested sodium channel-blocking drugs in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and have demonstrated a protective effect on axons. However, it is possible that, in addition to a direct effect on axons, sodium channel blockers may also interfere with inflammatory mechanisms. We therefore examined the novel hypothesis that sodium channels contribute to activation of microglia and macrophages in EAE and acute MS lesions. In this study, we demonstrate a robust increase of sodium channel Nav1.6 expression in activated microglia and macrophages in EAE and MS. We further demonstrate that treatment with the sodium channel blocker phenytoin ameliorates the inflammatory cell infiltrate in EAE by 75%. Supporting a role for sodium channels in microglial activation, we show that tetrodotoxin, a specific sodium channel blocker, reduces the phagocytic function of activated rat microglia by 40%. To further confirm a role of Nav1.6 in microglial activation, we examined the phagocytic capacity of microglia from med mice, which lack Nav1.6 channels, and show a 65% reduction in phagocytic capacity compared with microglia from wildtype mice. Our findings indicate that sodium channels are important for activation and phagocytosis of microglia and macrophages in EAE and MS and suggest that, in addition to a direct neuroprotective effect on axons, sodium channel blockade may ameliorate neuroinflammatory disorders via anti-inflammatory mechanisms.

Topics: Animals; Axons; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gliosis; Macrophages; Male; Mice; Mice, Inbred C57BL; Microglia; Multiple Sclerosis; NAV1.6 Voltage-Gated Sodium Channel; Nerve Degeneration; Nerve Tissue Proteins; Neuroprotective Agents; Phagocytosis; Phenytoin; RNA, Messenger; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Up-Regulation

In this study, we investigated whether changes in the vestibular neuronal activity per se influence the pattern of astrocytes morphology, glial fibrillary acidic protein (GFAP) expression and ultimately their activation within the vestibular nuclei after unilateral transtympanic tetrodotoxin (TTX) injections and after unilateral inner ear lesion. The rationale was that, theoretically the noninvasive pharmacological functional blockade of peripheral vestibular inputs with TTX, allowed us to dissociate the signals exclusively related to the shutdown of the resting activity of the first-order vestibular neurons and from neuronal signals associated with trans-ganglionic changes in first order vestibular neurons induced by unilateral labyrinthectomy (UL). Since the cochlea was removed during the surgical procedure, we also studied the astrocytic reaction within the deafferented cochlear nuclei. No significant changes in the distribution or relative levels of GFAP mRNA expression, relative levels of GFAP protein or immunoreactivity for GFAP were found in the ipsilateral vestibular nuclei at any post-TTX injection times studied. In addition, no sign of microglia activation was observed. In contrast, a robust increase of the distribution and relative levels of GFAP mRNA expression, protein levels and immunoreactivity was observed in the deafferented vestibular and cochlear nuclei beginning at 1 day after inner ear lesion. GFAP mRNA expression and immunoreactivity in the cochlear nucleus was qualitatively stronger than in the ipsilateral vestibular nuclei. The results suggest that astrocyte activation in the vestibular nuclei is not related to drastic changes of vestibular nuclei neuronal activity per se. Early trans-ganglionic changes due to vestibular nerve dendrites lesion provoked by the mechanical destruction of vestibular receptors, most probably induced the glial reaction. Its functional role in the vestibular compensation process remains to be elucidated.

Topics: Afferent Pathways; Animals; Astrocytes; Cochlear Nucleus; Denervation; Ear, Inner; Functional Laterality; Glial Fibrillary Acidic Protein; Gliosis; Immunohistochemistry; Male; Nerve Degeneration; Rats; Rats, Long-Evans; RNA, Messenger; Sensory Deprivation; Sodium Channel Blockers; Synaptic Transmission; Tetrodotoxin; Up-Regulation; Vestibular Nerve; Vestibular Nuclei

Axotomy typically leads to retrograde neuronal degeneration in the CNS. Studies in the hypothalamo-neurohypophysial system (HNS) have suggested that neural activity is supportive of magnocellular neuronal (MCN) survival after axotomy. In this study, we directly test this hypothesis by inhibiting neural activity in the HNS, both in vivo and in vitro, by the use of tetrodotoxin (TTX). After median eminence compression to produce axonal injury, unilateral superfusion of 3 microM TTX into the rat supraoptic nucleus (SON), delivered with the use of a miniature osmotic pump for 2 weeks in vivo, produced a decrease in the number of surviving MCNs in the TTX-treated SON, compared with the contralateral untreated side of the SON. In vitro application of 2.5 microM TTX for 2 weeks to the SON in organotypic culture produced a 73% decrease in the surviving MCNs, compared with untreated control cultures. Raising the extracellular KCl in the culture medium to 25 mM rescued the MCNs from the axotomy- and TTX-induced cell death. These data support the proposal that after axotomy, neural activity is neuroprotective in the HNS.

Topics: Animals; Apoptosis; Axotomy; Cell Survival; Male; Median Eminence; Nerve Degeneration; Neurites; Neurons; Organ Culture Techniques; Potassium Chloride; Rats; Rats, Sprague-Dawley; Supraoptic Nucleus; Synaptic Transmission; Tetrodotoxin

To assess the structural and functional consequences of local ganglion cell (GC) loss in an experimental model of a retinal nerve fiber layer (NFL) bundle defect. To evaluate and compare three commonly used multifocal electroretinogram (mfERG) stimuli, as well as the standard transient pattern-reversal ERG (pERG) and the photopic full-field ERG, for detection of local GC damage.. Intraretinal axotomy was achieved by multiple treatments with a diode laser adapted to a slit lamp biomicroscope. Retinal laser burns were applied along an arc, subtending approximately 60 degrees, about one disc diameter superotemporal to the optic nerve. Functional measures were acquired before laser application and at numerous time points thereafter. These included mfERGs for three different stimuli: a standard fast m-sequence flicker, a global-field flash paradigm (MOFO), and a slowed m-sequence (with seven dark frames inserted to each m-step [7F]). pERGs were measured for a 24 degrees x 32 degrees checkerboard stimulus (0.56 cyc/deg, 90% contrast, 75 cd/m(2), 5 reversals/s). Photopic full-field ERGs were measured for red flashes (0.42 log photopic cd-s/m(2)) on a blue rod-saturating background (30 scotopic cd/m(2)). Retinal photography, fluorescein angiography and postmortem histologic evaluation of the optic nerve, NFL, and retinal tissues were performed.. After six laser sessions, the NFL bundle defect appeared to be complete and contiguous and was visible both proximal to and distal to the site of the photoablation by clinical examination of the fundus and stereoscopic photographs. Histologic evaluation demonstrated localized loss of GC axons, confirmed at the level of the retrobulbar optic nerve. Retinal cross sections in the temporal retina (distal to the axotomy) showed loss of GC soma and NFL degeneration, whereas all other layers appeared intact. mfERGs showed loss of high-frequency components (HFCs) for responses located within the arcuate region corresponding to the NFL defect. Local GC damage was most easily detected using the slowed 7F m-sequence stimulus. This stimulus elicited relatively large HFCs that were significantly reduced from local responses after axotomy and that were tetrodotoxin (TTX)-sensitive in a control experiment. Low-frequency component loss with the 7F stimulus did not reach statistical significance. The photopic full-field ERG was not significantly affected. pERG amplitudes declined significantly from baseline but remained within normal limits.. Focal loss of GC function in the macaque retina is most easily detected using the slowed-sequence mfERG. Local 7F HFCs depend on intact GC function.

Topics: Animals; Axons; Axotomy; Electroretinography; Female; Fluorescein Angiography; Fluorescent Antibody Technique, Indirect; Immunoenzyme Techniques; Laser Coagulation; Macaca mulatta; Nerve Degeneration; Optic Nerve; Photic Stimulation; Retinal Ganglion Cells; Tetrodotoxin

Neurofilaments are key structural components of white matter axons. The effect of in vitro anoxia or oxygen-glucose deprivation (OGD) on the integrity of the 160 and 200 kDa neurofilament isoforms was studied by immunoblot, and correlated with physiological function. Adult rat optic nerves were exposed to 60 min of either anoxia or OGD. Compound action potential area recovered to 22+/-6% of control after 60 min of anoxia, and to 4+/-1% after 60 min of OGD. Ca(2+)-free (+EGTA) perfusate allowed complete recovery after OGD (108+/-42%). Tetrodotoxin (TTX, 1 microM) was less protective (45+/-6%). Both anoxia and OGD induced breakdown of neurofilament 160 (NF160) and NF200 revealed by the appearance of multiple lower molecular weight bands mainly in the 75-100 kDa range. Zero-Ca(2+)/EGTA completely prevented NF breakdown. TTX only partially reduced NF160 degradation. Non-phosphorylated NF200 appeared after reperfusion post-anoxia or OGD, and was also greatly reduced by zero-Ca(2+) or TTX. Calpain inhibitors (10 microM calpain inhibitor I or 50 microM MDL 28,170) significantly reduced NF160 and NF200 breakdown/dephosphorylation, but did not improve electrophysiological recovery. Significant calpain-mediated breakdown of NF160 and NF200 indicates structural damage to the axonal cytoskeleton, which was completely Ca(2+)-dependent. While pharmacological inhibition of calpain alone greatly reduced NF proteolysis, there was no concomitant improvement in function. These results imply that calpain inhibition is necessary but not sufficient for white matter protection, and emphasize the existence of multiple Ca(2+)-dependent degradative pathways activated in injured white matter.

Topics: Action Potentials; Animals; Axons; Calcium; Calcium Signaling; Calpain; Central Nervous System; Chelating Agents; Enzyme Inhibitors; Hypoxia-Ischemia, Brain; Male; Nerve Degeneration; Nerve Fibers, Myelinated; Neurofilament Proteins; Optic Nerve; Organ Culture Techniques; Rats; Rats, Long-Evans; Recovery of Function; Tetrodotoxin

Excessive nitric oxide formation may contribute to the pathology occurring in diseases affecting central white matter, such as multiple sclerosis. The rat isolated optic nerve preparation was used to investigate the potential toxicity of the molecule towards such tissue. The nerves were exposed to a range of concentrations of different classes of nitric oxide donor for up to 23 h, with or without a subsequent period of recovery, and the damage assessed by quantitative histological methods. Degeneration of axons and macroglia occurred in a time- and concentration-dependent manner, the order of susceptibility being: axons>oligodendrocytes>astrocytes. Use of NONOate donors differing in half-life indicated that nitric oxide delivered in an enduring manner at relatively low concentration was more toxic than the same amount supplied rapidly at high concentration. The mechanism by which nitric oxide affects axons was studied using a donor [3-(n-propylamino)propylamine/NO adduct, PAPA/NO] with an intermediate half-life that produced selective axonopathy after a 2-h exposure (plus 2 h recovery). Axon damage was abolished if, during the exposure, Na(+) or Ca(2+) was removed from the bathing medium or the sodium channel inhibitors tetrodotoxin or BW619C89 (sipatrigine) were added. In electrophysiological experiments, the donor elicited a biphasic depolarisation. The second, larger component (occurring after 7-10 min) was associated with a block of nerve conduction and could be inhibited by tetrodotoxin. Coincident with the secondary depolarisation was a reduction in ATP levels by about 50%, an effect that was also inhibited by tetrodotoxin. It is concluded that nitric oxide, in submicromolar concentrations, can kill axons and macroglia in white matter. The findings lend support to the hypothesis that nitric oxide may be of importance to white matter pathologies, particularly those in which inducible nitric oxide synthase is expressed. The axonopathy, at least when elicited over relatively short time intervals, is likely to be caused by metabolic inhibition. As in anoxia and anoxia/aglycaemia, nitric oxide-induced destruction of axons is likely to be caused by the Ca(2+) overload that follows a reduction in ATP levels in the face of continued influx of Na(+) through voltage-dependent channels.

Topics: Adenosine Triphosphate; Animals; Axons; Calcium; Central Nervous System; Demyelinating Diseases; Disease Models, Animal; Dose-Response Relationship, Drug; Membrane Potentials; Nerve Degeneration; Nerve Fibers, Myelinated; Neuroglia; Neurotoxins; Nitric Oxide; Nitric Oxide Donors; Optic Nerve; Organ Culture Techniques; Rats; Rats, Wistar; Sodium; Tetrodotoxin

The objective of the present study was to assess the contribution of sodium influx to development of dark cell degeneration (DCD) in Purkinje neurons (PNs) following AMPA (DL-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid) receptor activation. During the course of these experiments, we observed inconsistent protection against DCD by Na(+) ion substitutes leading us to consider other potential mechanisms. A 30 min application of AMPA (30 microM, induction or trigger phase) followed by a 90-120 min AMPA-free expression period produced DCD in the majority of PNs. Substitution of NaCl with choline chloride (120 mM) produced a marked suppression of AMPA-induced toxicity. Suppression of DCD by choline was concentration dependent. Concentrations of choline as low as 10 mM effectively attenuated DCD when substituted on an equimolar basis for NaCl in the artificial cerebrospinal fluid (ACSF). Unlike choline, substitution of NMDG for NaCl failed to suppress AMPA-induced DCD. Lidocaine and TTX (tetrodotoxin), two agents that inhibit Na(+) influx failed to significantly alter DCD. Because choline is a prototypical alpha7 nicotinic receptor selective agonist, methyllycaconitine (MLA), an alpha7 receptor antagonist was tested and significantly attenuated the protective effects of choline in a concentration-dependent manner. Nicotine (100 microM) added to normal ACSF was effective in attenuating AMPA-induced toxicity. These findings suggest that DCD is not heavily dependent on Na(+)-mediated phenomena and that nicotinic alpha7 receptor activation may be neuroprotective against some types of excitotoxicity that are mediated by active cellular programs.

Topics: Aconitine; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; alpha7 Nicotinic Acetylcholine Receptor; Animals; Choline; Excitatory Amino Acid Agonists; Extracellular Space; Female; Gluconates; Insecticides; Lidocaine; Male; Meglumine; Nerve Degeneration; Neurotoxins; Nicotine; Nootropic Agents; Organ Culture Techniques; Purkinje Cells; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Nicotinic; Sodium; Sodium Channels; Sodium Chloride; Tetrodotoxin

We investigated the pathophysiological mechanisms of glutamate-induced delayed neuronal damage in rat hippocampal slice cultures [Stoppini et al. (1991) J. Neurosci. Methods 37, 173-182], with propidium iodide as a marker of cell death. Exposure of the cultures to growth medium containing 10 mM glutamate for 30 min resulted in a slowly developing degeneration of hippocampal principal cells, starting from the medial end of the CA1 region and reaching the dentate gyrus by 48 h. By 24 h, most pyramidal cells in CA1 were damaged. An acute phase of degeneration preceded the delayed damage at 2-6 h, affecting cells in a spatially diffuse manner. When tetrodotoxin (0.5 microM) was present during the glutamate insult, a marked protection (mean 57%, P<0.001) of the CA1 damage was observed. Rather strikingly, when tetrodotoxin was applied immediately following or even with a delay of 30 min after the insult, a similar amount of protection was achieved. In field recordings carried out after the insult, the glutamate-treated slices exhibited spontaneously occurring negative shifts with a duration of 1-10 s and an amplitude of up to 400 microV in the CA3 region, whereas the control slices were always quiescent. Taken together, the results suggest that post-insult neuronal network activity, rather than the direct action of exogenous glutamate, is a major cause of delayed CA1 pyramidal cell death in the organotypic slices. These observations may have implications in the design of neuroprotective strategies for the treatment of brain traumas which are accompanied by delayed and/or distal neuronal damage.

Topics: Action Potentials; Animals; Brain Injuries; Brain Ischemia; Cell Death; Epilepsy; Glutamic Acid; Hippocampus; Nerve Degeneration; Nerve Net; Neurotoxins; Organ Culture Techniques; Pyramidal Cells; Rats; Tetrodotoxin; Time Factors

Light close to and in the near-infrared range has documented benefits for promoting wound healing in human and animals. However, mechanisms of its action on cells are poorly understood. We hypothesized that light treatment with a light-emitting diode array at 670 nm (LED) is therapeutic in stimulating cellular events involving increases in cytochrome oxidase activity. LED was administered to cultured primary neurons whose voltage-dependent sodium channels were blocked by tetrodotoxin. The down-regulation of cytochrome oxidase activity by TTX was reverted to control levels by LED. LED alone also up-regulated enzyme activity. Thus, the results are consistent with our hypothesis that LED has a stimulating effect on cytochrome oxidase in neurons, even when they have been functionally silenced by TTX.

Topics: Aging; Animals; Animals, Newborn; Cells, Cultured; Dose-Response Relationship, Drug; Down-Regulation; Electron Transport Complex IV; Infrared Rays; Nerve Degeneration; Neurons; Photic Stimulation; Rats; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Visual Cortex; Wound Healing

In normal rats maintained in the dark, very few cells in the primary visual centers, including the superior colliculus, show Fos-like immunoreactivity. By contrast, in rats presented with flashing lights many Fos-like immunoreactivity cells are observed distributed throughout the visual centers. In the dystrophic Royal College of Surgeons rat, in which there is major loss of photoreceptors over the first 3 months of life, similar numbers of Fos-like immunoreactivity cells are seen on light presentation, but in marked contrast, cell densities in the rats maintained in the dark are many times higher than in non-dystrophic rats maintained under similar conditions. Here we show that this elevated dark response can be abolished by intravitreal injection of the sodium channel blocker tetrodotoxin, indicating that this effect results from changed retinal activity, rather than being centrally generated. We suggest that since Fos-like immunoreactivity is not usually elicited by steady state conditions, the elevated levels in the superior colliculus in these animals reflect the return of waves of activity, first seen in development coursing across the retina, but lost with photoreceptor maturation.

Topics: Action Potentials; Animals; Cell Count; Darkness; Disease Models, Animal; Female; Functional Laterality; Immunohistochemistry; Male; Nerve Degeneration; Neuronal Plasticity; Photoreceptor Cells, Vertebrate; Proto-Oncogene Proteins c-fos; Rats; Rats, Mutant Strains; Retinal Degeneration; Retinal Ganglion Cells; Sodium Channel Blockers; Sodium Channels; Superior Colliculi; Tetrodotoxin; Up-Regulation; Visual Pathways

The differential behavioral and neurochemical effects of exogenous L-DOPA in animals with intact versus dopamine (DA)-denervated striata raise questions regarding the role of DA terminals in the regulation of dopaminergic neurotransmission after administration of exogenous L-DOPA. In vivo microdialysis was used to monitor the effect of exogenous L-DOPA on extracellular DA in intact and DA-denervated striata of awake rats. In intact striatum, a small increase in extracellular DA was observed after administration of L-DOPA (50 mg/kg i.p.) but in DA-denervated striatum a much larger increase in extracellular DA was elicited. Additional experiments assessed the role of high-affinity DA uptake and impulse-dependent neurotransmitter release in the effect of exogenous L-DOPA on extracellular DA in striatum. Pretreatment with GBR-12909 (20 mg/kg i.p.), a selective DA uptake inhibitor, enhanced the ability of L-DOPA to increase extracellular DA in intact striatum. However, in DA-denervated striatum, inhibition of DA uptake did not alter the extracellular DA response to L-DOPA. Impulse-dependent neurotransmitter release was blocked by the infusion of tetrodotoxin (TTX; 1 microM), an inhibitor of fast sodium channels, through the dialysis probe. Application of TTX significantly attenuated the L-DOPA-induced increase in extracellular DA observed in striatum of intact rats pretreated with GBR-12909. In a similar manner, TTX infusion significantly attenuated the increase in extracellular DA typically observed in striatum of 6-OHDA-lesioned rats after the administration of L-DOPA. The present results indicate that DA terminals, via high-affinity uptake, play a crucial role in the clearance of extracellular DA formed from exogenous L-DOPA in intact striatum. This regulatory mechanism is absent in the DA-denervated striatum. In addition, this study has shown that DA synthesized from exogenous L-DOPA primarily is released by an impulse-dependent mechanism in both intact and DA-denervated striatum. The latter result suggests an important role for a nondopaminergic neuronal element in striatum that serves as the primary source of extracellular DA formed from exogenous L-DOPA.

Topics: Animals; Biological Transport; Corpus Striatum; Dopamine; Dopamine Agents; Dopamine Uptake Inhibitors; Extracellular Space; Levodopa; Male; Microdialysis; Nerve Degeneration; Oxidopamine; Piperazines; Rats; Rats, Sprague-Dawley; Sympathomimetics; Tetrodotoxin

The mechanisms by which neurons die after cerebral ischemia and related conditions in vivo are unclear, but they are thought to involve voltage-dependent Na+ channels, glutamate receptors, and nitric oxide (NO) formation because selective inhibition of each provides neuroprotection. It is not known precisely what their roles are, nor whether they interact within a single cascade or in parallel pathways. These questions were investigated using an in vitro primary cell culture model in which striatal neurons undergo a gradual and delayed neurodegeneration after a brief (5 min) challenge with the glutamate receptor agonist NMDA. Unexpectedly, NO was generated continuously by the cultures for up to 16 hr after the NMDA exposure. Neuronal death followed the same general time course except that its start was delayed by approximately 4 hr. Application of the NO synthase inhibitor nitroarginine after, but not during, the NMDA exposure inhibited NO formation and protected against delayed neuronal death. Blockade of NMDA receptors or of voltage-sensitive Na+ channels [with tetrodotoxin (TTX)] during the postexposure period also inhibited both NO formation and cell death. The NMDA exposure resulted in a selective accumulation of glutamate in the culture medium during the period preceding cell death. This glutamate release could be inhibited by NMDA antagonism or by TTX, but not by nitroarginine. These data suggest that Na+ channels, glutamate receptors, and NO operate interdependently and sequentially to cause neurodegeneration. At the core of the mechanism is a vicious cycle in which NMDA receptor stimulation causes activation of TTX-sensitive Na+ channels, leading to glutamate release and further NMDA receptor stimulation. The output of the cycle is an enduring production of NO from neuronal sources, and this is responsible for delayed neuronal death. The same neurons, however, could be induced to undergo more rapid NMDA receptor-dependent death that required neither TTX-sensitive Na+ channels nor NO.

Topics: Amino Acids; Animals; Glutamic Acid; Immunohistochemistry; Kinetics; Nerve Degeneration; Nitric Oxide; Nitric Oxide Synthase; Rats; Receptors, N-Methyl-D-Aspartate; Sodium Channels; Tetrodotoxin; Time Factors

1. Do motoneurons regulate muscle extrajunctional membrane properties through chemical (trophic) factors in addition to evoked activity? We addressed this question by comparing the effects of denervation and nerve conduction block by tetrodotoxin (TTX) on extrajunctional acetylcholine (ACh) sensitivity and action potential resistance to TTX in adult rats. 2. We applied TTX to sciatic or tibial nerves for up to 5 weeks using an improved blocking technique which completely suppresses conduction but avoids nerve damage. 3. Reinnervation by TTX-blocked axons had no effect on the high ACh sensitivity and TTX resistance induced by nerve crush. 4. Long-lasting block of intact nerves (up to 38 days) induced extrajunctional changes as pronounced as after denervation. At shorter times (3 days), however, denervation induced much larger changes than TTX block; such a difference is thus only transiently present in muscle. 5. The effects of long-lasting block were dose dependent. Dose levels (6.6 micrograms day-1) corresponding to those used in the literature to block the rat sciatic nerve induced muscle effects much smaller than those induced by denervation, confirming published data. Our novel finding is that equal effects are obtained using doses substantially higher (up to 10.5 micrograms day-1). For the soleus it was necessary in addition to apply the TTX directly to the smaller tibial nerve. 6. The TTX-blocked nerves were normal in their histological appearance and capacity to transport anterogradely 3H-labelled proteins, to release ACh in quantal and non-quantal form or cluster ACh receptors and induce functional ectopic junctions on denervated soleus muscles. 7. We conclude that muscle evoked activity is the physiological regulator of extrajunctional membrane properties. Chemical factors from the nerve do not appear to participate in this regulation. The stronger response to denervation at short times only is best accounted for by factors produced by degenerating nerves.

Topics: Acetylcholine; Animals; Axonal Transport; Axons; Dose-Response Relationship, Drug; Electrophysiology; Fluorescent Dyes; Male; Membrane Potentials; Motor Neurons; Muscle, Skeletal; Nerve Degeneration; Nerve Endings; Nerve Regeneration; Neural Conduction; Neuromuscular Junction; Paralysis; Rats; Rats, Wistar; Receptors, Cholinergic; Sciatic Nerve; Sodium Channels; Tetrodotoxin; Time Factors; Tritium

We examined the role of extracellular calcium entry, the possible involvement of axonal calcium channels, and the potential protective effect of calcium channel and calpain antagonists in axotomy-induced axonal degeneration using murine dorsal root ganglia in cell culture. We found that calcium entry is both necessary and sufficient to induce axonal degeneration after axotomy, and may be inhibited by cobalt, manganese, dihydropyridines, and bepridil. Tetrodotoxin and omega-conotoxin are ineffective in preventing axonal degeneration. The activation of calpains also appears to be necessary and sufficient for axonal degeneration to proceed, and can be blocked with membrane-permeant leupeptin analogs and the oxirane aloxistatin. Although other calcium-activated events may occur, it appears that inhibition of calpain is sufficient to preserve the axon at the light microscope level, and to prevent axonal cytoskeleton degradation as detected by immunofluorescent staining. Our results suggest that axonal degeneration after axotomy involves the following sequence of events: (1) a lag-period after axotomy prior to the onset of axonal degeneration, (2) entry of calcium into the axon through an intact axolemma via a calcium-specific ion transport mechanism, (3) activation of calcium-dependent effector molecules such as calpains, (4) degradation of the axonal cytoskeleton. The details of the second step require further elucidation, and are of particular interest because this step is a potential target for therapies directed towards peripheral neuropathies.

Topics: Animals; Axons; Bepridil; Calcium; Calcium Channel Blockers; Calcium Channels; Calpain; Culture Techniques; Ganglia, Spinal; Mice; Nerve Degeneration; omega-Conotoxin GVIA; Peptides; Tetrodotoxin

To investigate whether local anesthetic neurotoxicity results from sodium channel blockade, we compared the effects of intrathecally administered lidocaine, bupivacaine, and tetrodotoxin (TTX), the latter a highly selective sodium channel blocker, on sensory function and spinal cord morphology in a rat model. First, to determine relative anesthetic potency, 25 rats implanted with intrathecal catheters were subjected to infusions of lidocaine (n = 8), bupivacaine (n = 8), or TTX (n = 9). The three drugs produced parallel dose-effect curves that differed significantly from one another: the EC50 values for lidocaine, bupivacaine, and TTX were 28.2 mM (0.66%), 6.6 mM (0.19%), and 462 nM, respectively. Twenty-five additional rats were then given intrathecal lidocaine (n = 8), bupivacaine (n = 8), or TTX (n = 9) at concentrations 10 times the calculated EC50 for sensory block. Lidocaine and bupivacaine induced persistent sensory impairment, whereas TTX did not. Finally, 28 rats were given either intrathecal bupivacaine (n = 10) or TTX (n = 9) at 10 times the EC50, or normal saline (n = 9). Significant sensory impairment again occurred after infusion of bupivacaine, but not after infusion of TTX or saline. Neuropathologic evaluation revealed moderate to severe nerve root injury in bupivacaine-treated animals; histologic changes in TTX- and saline-treated animals were minimal, similar, and restricted to the area adjacent to the catheter. These results indicate that local anesthetic neurotoxicity does not result from blockade of the sodium channel, and suggest that development of a safer anesthetic is a realistic goal.

Topics: Animals; Bupivacaine; Demyelinating Diseases; Dose-Response Relationship, Drug; Injections, Spinal; Ion Channel Gating; Lidocaine; Male; Nerve Block; Nerve Degeneration; Rats; Rats, Sprague-Dawley; Reaction Time; Sensation Disorders; Sodium Channel Blockers; Sodium Channels; Spinal Cord; Spinal Nerve Roots; Tetrodotoxin

HIV-1-associated cognitive/motor complex is a frequent neurological complication of the acquired immunodeficiency syndrome (AIDS). The pathogenesis of this syndrome implicates immunopathological and toxic events such as the production of cytokines. The HIV envelope glycoprotein gp120 seems also to play a major role in this process. Gp120 could produce a slow neuronal death probably via the release of neurotoxic factors by CNS macrophages/monocytes. NMDA antagonists and Ca2+ channel blockers in vitro have a powerful neuroprotective effect against gp120 neurotoxicity. The purpose of the present work is to determine whether gp120-induced neurotoxicity is associated with an abnormal neuronal depolarization induced by putative neurotoxins. We have compared in vitro the neuroprotective effects of Tetrodotoxin a Na+ channel blocker, the Ca2+ channel blocker nifedipine and the NMDA antagonist MK-801 in primary cortical neurons taken from embryonic rat and intoxicated with gp120. We observed comparable neuroprotective effects with the 3 precited compounds suggesting that gp120-induced neurotoxic factors act on Na+ channels, NMDA receptors and Ca2+ channels in a cascade of cellular events. We confirmed that the presence of macrophages is needed to trigger a marked gp120-induced neurotoxicity. These results underline the fact that depolarization is an important component of gp120 neurotoxicity in primary neuronal cultures.

Topics: Animals; Cell Survival; Cells, Cultured; Cerebral Cortex; Dizocilpine Maleate; HIV Envelope Protein gp120; Macrophages, Alveolar; Nerve Degeneration; Neurons; Nifedipine; Rats; Receptors, N-Methyl-D-Aspartate; Tetrodotoxin

Local cerebral glucose utilization was assessed during whisker stimulation by 2-deoxyglucose autoradiography. Whisker stimulation increased local cerebral glucose utilization in brainstem, thalamus and whisker sensory cortex in normal rats. Whereas whisker stimulation increased glucose metabolism in brainstem, whisker stimulation failed to increase glucose metabolism in thalamus of rats that had whisker sensory cortex ablated 5 h to five weeks previously. The failure of whisker stimulation to activate thalamus after cortical ablations was probably not due to decreased cortical input to thalamus because whisker stimulation activated thalamus after large cortical tetrodotoxin injections. Failure of whisker stimulation to activate thalamus at early times (5 h and one day) after cortical ablations was not due to thalamic neuronal death, since it takes days to weeks for axotomized thalamic neurons to die. The failure of whisker stimulation to activate thalamus at early times after cortical ablations was likely due to the failure of trigeminal brainstem neurons that project to thalamus to activate axotomized thalamic neurons. This might occur because of synaptic retraction, glial stripping or inhibition of trigeminal brainstem synapses onto thalamic neurons. The thalamic neuronal death that occurs over the days and weeks following cortical ablations was associated with thalamic hypometabolism. This is consistent with the idea that the thalamic neurons die because of the absence of a cortically derived trophic factor, since the excitotoxic thalamic cell death that occurs following cortical kainate injections is associated with thalamic hypermetabolism. The glucose metabolism of parts of the host thalamus was higher and the glucose metabolism in surrounding nuclei lower than the normal side of thalamus in rats that sat quietly and had fetal cortex transplants placed into cavities in whisker sensory cortex five to 16 weeks previously. Whisker stimulation in these subjects activated the contralateral host thalamus and fetal cortical transplants. This was accomplished using a double-label 2-deoxyglucose method to assess brain glucose metabolism in the same rat while it was resting and during whisker stimulation. The high glucose metabolism of parts of host thalamus ipsilateral to the fetal cortical transplants is consistent with prolonged survival of some axotomized thalamic neurons. The finding that whisker stimulation activates portions of host thalamus further su

Topics: Animals; Brain Stem; Brain Tissue Transplantation; Cell Death; Cerebral Decortication; Deoxyglucose; Energy Metabolism; Female; Fetal Tissue Transplantation; Nerve Degeneration; Rats; Rats, Sprague-Dawley; Somatosensory Cortex; Stress, Mechanical; Tetrodotoxin; Thalamus; Trigeminal Nerve; Trigeminal Nuclei; Vibrissae

Previous studies indicate that tuberculoventral and cartwheel cells in the dorsal cochlear nucleus as well as a group of stellate cells in the ventral cochlear nucleus are likely to be glycinergic. To test whether these neurons contain higher levels of free glycine than cells that are probably not glycinergic, immunocytochemical studies with antibodies against glycine conjugates were undertaken on slices of the murine cochlear nuclear complex. Present results show that the cell bodies of all three groups of neurons are immunolabeled. However, the somatic labeling of the tuberculoventral and cartwheel cells can be modulated by experimental conditions. In slices fixed immediately after cutting, many cell bodies in the deep layer of the dorsal cochlear nucleus (DCN), presumably tuberculoventral neurons, are labeled. As a slice is incubated in vitro, cell bodies in the deep layer of the DCN lose their glycine-like immunoreactivity. After 7 hours in vitro, labeled cells are absent in the deep DCN, but the immunoreactivity can be regained by electrically stimulating the auditory nerve for 20 minutes. The loss of immunoreactivity is prevented by electrical stimulation, by axotomy, and by inclusion of 0.8 microM tetrodotoxin, or 1 microM strychnine, or 50 microM colchicine or 50 microM beta-lumicolchicine in the bathing saline. Cartwheel cells retain their immunoreactivity during incubation in vitro without electrical stimulation, but lose it under two conditions. One is following a cut across the ventral cochlear nucleus (VCN) that severs most of their granule cell input, and the other is the inclusion of tetrodotoxin in the bathing saline. The labeling of cell bodies in the ventral cochlear nucleus and of puncta and processes is not changed by any of these experimental manipulations.

Topics: Animals; Brain Chemistry; Cochlear Nucleus; Colchicine; Electric Stimulation; Glycine; Immunohistochemistry; Mice; Mice, Inbred CBA; Mice, Inbred ICR; Nerve Degeneration; Neurons; Strychnine; Tetrodotoxin; Vestibulocochlear Nerve

The effect of action potentials on elimination of mouse neuromuscular junctions (NMJ) was studied in a three-compartment cell culture preparation. Axons from superior cervical ganglion or ventral spinal cord neurons in two lateral compartments formed multiple neuromuscular junctions with muscle cells in a central compartment. The loss of synapses over a 2-7-day period was determined by serial electrophysiological recording and a functional assay. Electrical stimulation of axons from one side compartment during this period, using 30-Hz bursts of 2-s duration, repeated at 10-s intervals, caused a significant increase in synapse elimination compared to unstimulated cultures (p < 0.001). The extent of homosynaptic and heterosynaptic elimination was comparable, i.e., of the 226 functional synapses of each type studied, 111 (49%) of the synapses that had been stimulated were eliminated, and 87 (39%) of unstimulated synapses on the same muscle cells were eliminated. Also, simultaneous bilateral stimulation caused significantly greater elimination of synapses than unilateral stimulation (p < 0.005). These observations are contrary to the Hebbian hypothesis of synaptic plasticity. A spatial effect of stimulus-induced synapse elimination was also evident following simultaneous bilateral stimulation. Prior to stimulation, most muscle cells were innervated by axons from both side compartments, but after bilateral stimulation, muscle cells were predominantly unilaterally innervated by axons from the closer compartment. These experiments suggest that synapse elimination at the NMJ is an activity-dependent process, but it does not follow Hebbian or anti-Hebbian rules of synaptic plasticity. Rather, elimination is a consequence of postsynaptic activation and a function of location of the muscle cell relative to the neuron. An interaction between spatial and activity-dependent effects on synapse elimination could help produce optimal refinement of synaptic connections during postnatal development.

Topics: Action Potentials; Animals; Axons; Cells, Cultured; Electric Stimulation; Mice; Motor Endplate; Muscles; Nerve Degeneration; Neuromuscular Junction; Neuronal Plasticity; Parasympathetic Nervous System; Superior Cervical Ganglion; Synapses; Tetrodotoxin

This study evaluates whether the rapid transient increases in glial fibrillary acidic protein (GFAP) mRNA in the hippocampus after electrolytic lesions of the entorhinal cortex (EC) are triggered by lesion-induced changes in hippocampal neuronal activity (either the decreases that result from loss of afferent drive or transient increases that occur during lesion production). To evaluate the role of activity, we carried out four experiments: (1) tetrodotoxin (TTX) was injected into the EC to mimic the decreases in afferent drive that occur after lesion; (2) TTX was injected into the EC or hippocampus before producing electrolytic lesions to block any abnormal activity induced during lesion production; (3) the EC was destroyed by aspiration, thus creating a lesion comparable in size to the electrolytic lesion, without passing direct current; (4) seizures were elicited by stimulating the EC of anesthetized rats, to examine whether electrographic seizures alone can induce the same type of increases in GFAP mRNA as lesions. Our results demonstrated that: (1) TTX injections into the EC did not induce the same increases in GFAP mRNA levels that occurred after EC lesions; (2) animals that received TTX injections into the EC prior to lesions exhibited increases in hippocampal GFAP mRNA that were nearly as great as following EC lesions alone; (3) aspiration lesions of the EC resulted in increases in GFAP mRNA that were comparable to those observed after electrolytic lesions; and (4) seizure-inducing stimulation of the EC resulted in 2-fold increases in GFAP mRNA in the hippocampus 24 hr after stimulation rather than the 5-13-fold increases observed after lesions. These results suggest that lesion-induced changes in hippocampal neuronal activity are not solely responsible for inducing the rapid transient increases in GFAP mRNA levels in the hippocampus ipsilateral to EC lesions.

Topics: Animals; Cerebral Cortex; Electric Stimulation; Evoked Potentials; Glial Fibrillary Acidic Protein; Hippocampus; Kindling, Neurologic; Male; Nerve Degeneration; Neurons; Nucleic Acid Hybridization; Rats; Rats, Sprague-Dawley; RNA Probes; RNA, Messenger; Seizures; Tetrodotoxin; Up-Regulation

The surface of the frog optic nerve consists of astrocytic processes separated by narrow extracellular clefts underlying a pial sheath of loose connective tissue. Macroscopic voltage dependent currents can be recorded from this surface using the loose patch-clamp technique. In this study the changes in ultrastructure and voltage dependent Na currents have been studied for up to 1 year following removal of the retina. During the first 1-4 weeks, many of the myelinated and unmyelinated axons of the retinal ganglion cells degenerate, and the debris is phagocytosed by macrophages and glial cells. However, some morphologically intact axons remain even 12 weeks after surgery. Finally, after 16 weeks all the axons have disappeared, leaving a nerve consisting only of glial cells, some of which contain phagosomes. At 40-52 weeks after enucleation, the nerve persists, at 20-40% of the normal diameter, consisting mostly of normal looking astrocytes. The amplitude of the voltage dependent Na currents recorded from nerves during the first 1-4 weeks after enucleation, with the pial sheath intact, decreases by about 50%. After 8 weeks, the Na current recorded from the surface is about 30% of control. At 16-52 weeks after removal of the retina, when there are no intact axons, the Na current is reduced by 90%. If, however, the pial sheath is stripped away, the Na currents recorded from the glial surface are 40-50% of control during this same 16- to 52-week period, suggesting that in the all-glia nerve, the currents are shunted by the relatively thicker pial sheath.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Action Potentials; Animals; Astrocytes; Electric Stimulation; Extracellular Matrix; Eye Enucleation; Microscopy, Electron; Nerve Degeneration; Optic Nerve; Rana pipiens; Retina; Sodium Channels; Tetrodotoxin

Close or distant denervation of the rat soleus muscle indicated that (1) longer soleus nerve stumps delay the onset of axon terminal degeneration and of muscle membrane changes (spike resistance to TTX) by strictly comparable times, and (2) the stump-induced delay of the muscle effect is independent of synaptic connections, because it is also obtained (RMP fall and TTX-resistance development) when sectioning a foreign nerve previously transplanted on the soleus surface but not making synaptic contacts. Both lines of evidence are consistent with the interpretation that, as far as the extrajunctional membrane properties are concerned, the effect of the length of the nerve stump on muscle is mediated by nerve terminal breakdown.

Topics: Animals; Axons; Male; Membrane Potentials; Muscle Denervation; Muscles; Nerve Degeneration; Nerve Endings; Neurons; Rats; Rats, Wistar; Synapses; Tetrodotoxin

In light of the evidence that calcium plays a critical role in excitotoxic neuronal death, it has been speculated that the metabotropic glutamate receptor may also contribute to excitotoxic damage through the mobilization of Ca2+ from intracellular stores. In the present study we examined this possibility by studying the neurotoxicity of trans-1-amino-cyclopentyl-1,3-dicarboxylate (trans-ACPD), a selective agonist of the metabotropic glutamate receptor. Exposure of cortical neurons to 100 microM trans-ACPD substantially increased phosphoinositide hydrolysis and intraneuronal free calcium in the presence of CPP and CNQX. Despite the presence of functional metabotropic receptors on cultured neurons, however, exposure of cultures to as high as 1 mM trans-ACPD for 24 h failed to produce any morphological or chemical signs of neuronal damage. Furthermore, trans-ACPD did not potentiate submaximal neurotoxicity produced by other non-N-methyl-D-aspartate (NMDA) agonists, kainate and D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA).

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Cells, Cultured; Cerebral Cortex; Cycloleucine; Embryo, Mammalian; Glutamates; Ibotenic Acid; Kainic Acid; L-Lactate Dehydrogenase; Mice; N-Methylaspartate; Nerve Degeneration; Neurons; Neurotoxins; Receptors, Glutamate; Receptors, Neurotransmitter; Tetrodotoxin

Topics: Animals; Anura; Axons; Electrophysiology; Nerve Degeneration; Neuroglia; Optic Nerve; Quaternary Ammonium Compounds; Sodium; Tetrodotoxin

The long-term excitability and the ionic currents in the nodes of Ranvier were studied in the severed frog sciatic nerve and the unoperated contralateral control nerve. After unilateral nerve section, compound action potentials of the nerve bundles and action potentials of single myelinated nerve fibres remained normal in amplitude and duration in either sciatic nerve for more than 38 days when frogs were kept at 11 degrees C. During this period the resting potentials averaged about -75 mV. Under voltage-clamp conditions Na currents and K currents in transected single myelinated nerve fibres also appeared normal in their kinetics, and in their peak amplitudes. These results indicate that the Na- and K-channel densities are quantitatively unchanged after the nerve transection, up to several weeks. The excitability of the severed sciatic nerve, expectedly, depends strongly on the time course of Wallerian degeneration. When frogs were kept at room temperature, the nerve excitability remained normal for only about 8-10 days, due to the faster Wallerian degeneration; whereas at 4 degrees C it was maintained for more than 84 days, as long as the myelinated nerve fibres did not degenerate. Together, these findings demonstrate that Na channels, K channels, and Na-K pumps are continuously present for several weeks in the transected nerve before nerve degeneration. It is surmised that either, (a) these proteins are extremely stable in the transected myelinated nerve fibres, or (b) they are supplied locally by Schwann cells, an open question recently posed by Chiu, Schrager & Ritchie (1984). In either event, the myelinated nerve fibres do not require cell bodies to provide a significant amount of new channels and pumps in order to retain their long-term excitability.

Topics: Action Potentials; Animals; Axons; Denervation; Ion Channels; Nerve Degeneration; Potassium; Rana pipiens; Ranvier's Nodes; Saxitoxin; Sciatic Nerve; Sodium; Tetraethylammonium; Tetraethylammonium Compounds; Tetrodotoxin; Time Factors; Veratridine

The action of nerve breakdown products on innervated fibres of soleus and extensor digitorum longus muscles was investigated with the following procedures: partial denervation, sensory or sympathetic denervation, section of a previously transplanted foreign nerve. Each procedure was performed either in isolation or combined with chronic muscle inactivity obtained by blocking impulse conduction along the sciatic nerve. Silastic cuffs containing tetrodotoxin (TTX) and sodium chloride were utilized for the block. Partial denervation induced extrajunctional sensitivity to acetylcholine (ACh) and resistance to tetrodotoxin not only in the denervated but also in the innervated fibres. The effects in the innervated fibres were equal in magnitude to those in the denervated fibres, provided they were paralysed. The onset of the membrane changes was synchronous in the two classes of fibres and their amount correlated with the extent of partial denervation. If the innervated fibres were normally active, the membrane changes were still detectable, but considerably smaller than in the denervated fibres. Sensory denervation (removal of dorsal root ganglia L4 and L5) was followed by the development of moderate ACh supersensitivity and TTX resistance in chronically paralysed muscles. Furthermore, section of radicular nerves (total denervation, i.e. efferent plus afferent) induced larger membrane changes than those observed following section of ventral roots alone (efferent denervation). Sympathetic denervation was ineffective even when associated with chronic muscle paralysis. Section of a previously transplanted mixed nerve (superficial fibular) was ineffective if the soleus muscle was normally active, while it induced marked extrajunctional ACh sensitivity and TTX resistance when combined with chronic paralysis of the muscle. Section of a transplanted sensory nerve (sural) also induced extrajunctional membrane changes in paralysed soleus muscles, but their magnitude was much smaller than after section of mixed nerves. We conclude that products of nerve destruction, especially those of motor axons, induce membrane changes of striking magnitude when potentiated by muscle inactivity. Such an action may also explain the greater efficacy of denervation vs. pure inactivity, at least at early times after their onset.

Topics: Acetylcholine; Action Potentials; Animals; Axons; Dexamethasone; Male; Membrane Potentials; Muscle Denervation; Muscles; Nerve Degeneration; Neural Conduction; Neurons; Neurons, Afferent; Neurons, Efferent; Rats; Sympathectomy; Tetrodotoxin

After extirpation of the superior cervical ganglion in the rat a degeneration secretion of amylase occurs in an in vitro preparation of the parotid gland. It can be detected about 14 h after the sympathectomy, reaches a maximum after about 17 h and then slowly subsides. It is abolished by atenolol, but not by dihydroergotamine or atropine, nor by tetrodotoxin. From this it is concluded that the phenomenon is due to an action mainly on beta 1-adrenoceptors exerted by noradrenaline, which is released from the degenerating sympathetic nerves at the neuro-glandular junctions, independently of propagated nerve impulses.

Topics: Amylases; Animals; Atenolol; Atropine; Culture Techniques; Dihydroergotamine; Ganglia, Sympathetic; Isoproterenol; Male; Nerve Degeneration; Parotid Gland; Rats; Rats, Inbred Strains; Sympathectomy; Tetrodotoxin; Time Factors

Possible causal factors of denervation-induced changes in muscle include inactivity, products of nerve degeneration and lack of a nerve-borne trophic agent. We now show that if the innervated fibres in a partially denervated rat muscle are rendered inactive, they undergo a reaction as intense as that of the denervated fibres. This provides further support for the view that the effects of denervation on the extrajunctional muscle membrane result from a combination of muscle inactivity and of nerve breakdown products acting diffusely throughout the muscle.

Topics: Action Potentials; Animals; Motor Endplate; Muscle Denervation; Muscles; Nerve Degeneration; Rats; Tetrodotoxin

1. Nerve impulses in the rat sciatic nerve were blocked for long periods by tetrodotoxin (TTX) released from capillary implants. The TTX capillaries did not block axonal transport, nor did they cause any sign of nerve degeneration. 2. A comparison of the effects of TTX paralysis and denervation was made on both extensor digitorium longus (e.d.l.) and soleus muscles over 21 days, a time when the products of nerve degeneration were unlikely to contribute to the changes associated with denervation. The resting membrane potential of TTX-paralysed muscles was significantly different (P less than 0.005) from that of the denervated muscles at all periods and at 21 days the decrease that can be attributed to inactivity was 61% (e.d.l.) and 49% (soleus) of that which follows denervation. This disparity was even more pronounced for the ACh receptor density where the increase in receptors due to inactivity was only 34% (e.d.l.) and 21% (soleus) of that due to denervation. 3. A similar comparison was made on muscles which had been reinnervated by TTX-inactive nerves. These muscles were found to have a significantly higher resting membrane potential and lower ACh receptor density than the denervated muscles (P less than 0.05). 4. The experiments on reinnervated muscles preclude the possibility that nerve degeneration products are solely responsible for the difference between the TTX-paralysed and denervated muscles and suggest that the difference can be attributed to the trophic influence of the nerve. 5. An observed increase in the m.e.p.p. frequency of the TTX-paralysed muscles indicated that nerve action potentials play a role in regulating the spontaneous release from nerve terminals.

Topics: Acetylcholine; Animals; Axonal Transport; Female; Male; Membrane Potentials; Muscle Denervation; Muscle Proteins; Muscles; Nerve Degeneration; Nerve Regeneration; Rats; Receptors, Cholinergic; Sciatic Nerve; Tetrodotoxin

The origin of the membrane changes induced in skeletal muscle by denervation has been investigated by examining partially denervated rat hindlimb muscles rendered inactive for 2-3 days by a chronic conduction block in the sciatic nerve. Extra-junctional sensitivity to acetylcholine and spike resistance to tetrodotoxin developed to the same extent in the denervated and the adjacent innervated but inactive fibres. On the other hand, impulse-blocked fibres of control muscles not containing denervated fibres showed, at this early time, little membrane changes. These results are interpreted as indicating that the response of muscle to denervation is due to the combined action of inactivity and products of nerve degeneration.

Topics: Acetylcholine; Animals; Muscle Denervation; Muscles; Nerve Block; Nerve Degeneration; Rats; Receptors, Cholinergic; Sciatic Nerve; Synaptic Membranes; Tetrodotoxin

Degeneration of afferent nerve fibres was induced in rats in order to observe its effects on the properties of the extra-junctional membrane of soleus muscle fibres. In one approach, removal of dorsal root ganglia L4 and L5 was accomplished in preparations with intact or impulse-blocked (with tetrodotoxin containing cuffs around the sciatic nerve) efferent innervation. Spike resistance to tetrodotoxin developed in the inactive deafferented preparations earlier and to a greater extent than in control, that is only impulse-blocked, preparations. In another series of experiments, efferent denervation alone proved to be less effective than the association of efferent and afferent denervation. On the other hand, section of the afferent fibres central to the dorsal root ganglia was without effect. These results are consistent with the interpretation that products of nerve degeneration contribute together with inactivity to the development of the extrajunctional membrane changes observed in skeletal muscle after denervation.

Topics: Acetylcholine; Afferent Pathways; Animals; Efferent Pathways; Muscle Denervation; Muscles; Nerve Degeneration; Rats; Sciatic Nerve; Spinal Nerve Roots; Tetrodotoxin

Partial denervation of the rat extensor digitorum longus muscle was performed by sectioning only one of the sciatic nerve roots. Measurements of spike resistance to tetrodotoxin in individual muscle fibers revealed denervation changes not only in the denervated fibers but also in the adjacent innervated ones. The results support the concept that products of nerve degeneration play a role in the origin of muscle changes induced by denervation.

Topics: Action Potentials; Animals; Drug Resistance; Membrane Potentials; Motor Endplate; Muscle Denervation; Muscles; Nerve Degeneration; Neuromuscular Junction; Rats; Tetrodotoxin

Topics: Animals; Axonal Transport; Bungarotoxins; Female; Muscle Denervation; Muscles; Nerve Degeneration; Paralysis; Rats; Receptors, Cholinergic; Tetrodotoxin

1. After a local lesion of the diaphragm muscle, which produced a segment of innervated muscle fibres connected with an intact nerve-free segment by a region of crushed muscle fibres, the sensitivity to ACh, the presence of tetrodotoxin resistant action potentials (AP) and the transmission of AP along the muscle fibres were studied. 2. Three days after local injury of the diaphragm muscle ACh sensitivity and TTX resistance appeared in the crushed and nerve-free segments between the place of injury and the tendineous attachment. 5-7 days after injury transmission of action potentials through the damaged to the undamaged ("decentralized"), nerve-free part of the fibres is restored. High ACh sensitivity and TTX resistance of the latter segment, however, are completely lost only 20 days after the local injury. During this period contractility of the muscle remains practically unchanged. Enzymatic activities (SDH, ATPase and phosphorylase) of the damaged part were lost 3 days after crushing and recovered slowly between 7-10 days of regeneration of the diaphragm muscle fibres. 3. The experiments suggest that during regeneration of damaged muscle fibres supersensitivity to ACh remains high inspite of normal AP activity and that intracellular mechanisms may be involved in the induction and disappearance of ACh hypersensitivity.

Topics: Acetylcholine; Action Potentials; Animals; Diaphragm; Muscle Contraction; Muscle Denervation; Nerve Degeneration; Neuromuscular Junction; Rats; Synaptic Transmission; Tetrodotoxin

Topics: Acetylcholine; Action Potentials; Adenosine Triphosphatases; Animals; Caffeine; Cell Membrane; Colchicine; Cycloheximide; Electrophysiology; Iontophoresis; Membrane Potentials; Microtubules; Muscle Contraction; Muscles; Nerve Degeneration; Neuromuscular Junction; Peripheral Nerves; Rats; Sarcoplasmic Reticulum; Tetrodotoxin; Time Factors; Vinblastine

Topics: Action Potentials; Animals; Disease Models, Animal; Electric Stimulation; Female; In Vitro Techniques; Male; Membrane Potentials; Mice; Muscle Denervation; Muscles; Muscular Dystrophies; Nerve Degeneration; Tetrodotoxin