tetrodotoxin and Brain-Injuries

The sequelae of traumatic brain injury, including posttraumatic epilepsy, represent a major societal problem. Significant resources are required to develop a better understanding of the underlying pathophysiologic mechanisms as targets for potential prophylactic therapies. Posttraumatic epilepsy undoubtedly involves numerous pathogenic factors that develop more or less in parallel. We have highlighted two potential "prime movers": disinhibition and development of new functional excitatory connectivity, which occur in a number of animal models and some forms of epilepsy in humans. Previous experiments have shown that tetrodotoxin (TTX) applied to injured cortex during a critical period early after lesion placement can prevent epileptogenesis in the partial cortical ("undercut") model of posttraumatic epilepsy. Here we show that such treatment markedly attenuates histologic indices of axonal and terminal sprouting and presumably associated aberrant excitatory connectivity. A second finding in the undercut model is a decrease in spontaneous inhibitory events. Current experiments show that this is accompanied by regressive alterations in fast-spiking gamma-aminobutyric acid (GABA)ergic interneurons, including shrinkage of dendrites, marked decreases in axonal length, structural changes in inhibitory boutons, and loss of inhibitory synapses on pyramidal cells. Other data support the hypothesis that these anatomic abnormalities may result from loss of trophic support normally provided to interneurons by brain-derived neurotrophic factor (BDNF). Approaches that prevent these two pathophysiologic mechanisms may offer avenues for prophylaxis for posttraumatic epilepsy. However, major issues such as the role of these processes in functional recovery from injury and the timing of the critical period(s) for application of potential therapies in humans need to be resolved.

Topics: Animals; Anticonvulsants; Brain Injuries; Cerebral Cortex; Disease Models, Animal; Epilepsy, Post-Traumatic; gamma-Aminobutyric Acid; Humans; Interneurons; Nerve Net; Nerve Regeneration; Neural Inhibition; Neuronal Plasticity; Pyramidal Cells; Tetrodotoxin

Each year, approximately 3.8 million people suffer mild to moderate traumatic brain injuries (mTBI) that result in an array of neuropsychological symptoms and disorders. Despite these alarming statistics, the neurological bases of these persistent, debilitating neuropsychological symptoms are currently poorly understood. In this study we examined the effects of mTBI on the amygdala, a brain structure known to be critically involved in the processing of emotional stimuli. Seven days after lateral fluid percussion injury (LFPI), mice underwent a series of physiological and behavioral experiments to assess amygdala function. Brain-injured mice exhibited a decreased threat response in a cued fear conditioning paradigm, congruent with a decrease in amygdala excitability determined with basolateral amygdala (BLA) field excitatory post-synaptic potentials together with voltage-sensitive dye imaging (VSD). Furthermore, beyond exposing a general decrease in the excitability of the primary input of the amygdala, the lateral amygdala (LA), VSD also revealed a decrease in the relative strength or activation of internuclear amygdala circuit projections after LFPI. Thus, not only does activation of the LA require increased stimulation, but the proportion of this activation that is propagated to the primary output of the amygdala, the central amygdala, is also diminished following LFPI. Intracellular recordings revealed no changes in the intrinsic properties of BLA pyramidal neurons after LFPI. This data suggests that mild to moderate TBI has prominent effects on amygdala function and provides a potential neurological substrate for many of the neuropsychological symptoms suffered by TBI patients.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Amygdala; Animals; Brain Injuries; Brain Mapping; Conditioning, Psychological; Cues; Disease Models, Animal; Electric Stimulation; Escape Reaction; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Fear; Male; Mice; Mice, Inbred C57BL; Sodium Channel Blockers; Tetrodotoxin; Voltage-Sensitive Dye Imaging

Patients that suffer mild traumatic brain injuries (mTBI) often develop cognitive impairments, including memory and learning deficits. The hippocampus shows a high susceptibility to mTBI-induced damage due to its anatomical localization and has been implicated in cognitive and neurological impairments after mTBI. However, it remains unknown whether mTBI cognitive impairments are a result of morphological and pathophysiological alterations occurring in the CA1 hippocampal region. We investigated whether mTBI induces morphological and pathophysiological alterations in the CA1 using the controlled cortical impact (CCI) model. Seven days after CCI, animals subjected to mTBI showed cognitive impairment in the passive avoidance test and deficits to long-term potentiation (LTP) of synaptic transmission. Deficiencies in inducing or maintaining LTP were likely due to an observed reduction in the activation of NMDA but not AMPA receptors. Significant reductions in the frequency and amplitude of spontaneous and miniature GABAA-receptor mediated inhibitory postsynaptic currents (IPSCs) were also observed 7 days after CCI. Design-based stereology revealed that although the total number of neurons was unaltered, the number of GABAergic interneurons is significantly reduced in the CA1 region 7 days after CCI. Additionally, the surface expression of α1, ß2/3, and γ2 subunits of the GABAA receptor were reduced, contributing to a reduced mIPSC frequency and amplitude, respectively. Together, these results suggest that mTBI causes a significant reduction in GABAergic inhibitory transmission and deficits to NMDA receptor mediated currents in the CA1, which may contribute to changes in hippocampal excitability and subsequent cognitive impairments after mTBI.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Avoidance Learning; Brain Injuries; CA1 Region, Hippocampal; Disease Models, Animal; Electric Stimulation; Excitatory Amino Acid Antagonists; GABAergic Neurons; Glucose Transporter Type 1; Glutamate Decarboxylase; Inhibitory Postsynaptic Potentials; Interneurons; Male; Memory Disorders; Rats; Rats, Sprague-Dawley; Reaction Time; Receptors, GABA-A; Sodium Channel Blockers; Tetrodotoxin; Time Factors

There is great concern that one mild traumatic brain injury (mTBI) predisposes individuals to an exacerbated response with a subsequent mTBI. Although no mechanism has been identified, mounting evidence suggests traumatic axonal injury (TAI) plays a role in this process. By using a cell culture system, a threshold of mild TAI was found where dynamic stretch of cortical axons at strains lower than 5% induced no overt pathological changes. However, the axons were found to display an increased expression of sodium channels (NaChs) by 24 hr. After a second, identical mild injury, pathologic increases in [Ca(2+)](i) were observed, leading to axon degeneration. The central role of NaChs in this response was demonstrated by blocking NaChs with tetrodotoxin prior to the second injury, which completely abolished postinjury increases in [Ca(2+)](i). These data suggest that mild TAI induces a form of sodium channelopathy on axons that greatly exaggerates the pathophysiologic response to subsequent mild injuries.

Topics: Aniline Compounds; Animals; Axons; Blotting, Western; Brain Injuries; Calcium; Cells, Cultured; Cerebral Cortex; Channelopathies; Fluorescent Dyes; Immunohistochemistry; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Xanthenes

Formation of new recurrent excitatory circuits after brain injuries has been hypothesized as a major factor contributing to epileptogenesis. Increases in total axonal length and the density of synaptic boutons are present in layer V pyramidal neurons of chronic partial isolations of rat neocortex, a model of posttraumatic epileptogenesis. To explore the functional consequences of these changes, we used laser-scanning photostimulation combined with whole-cell patch-clamp recording from neurons in layer V of somatosensory cortex to map changes in excitatory synaptic connectivity after injury. Coronal slices were submerged in artificial CSF (23 degrees C) containing 100 microM caged glutamate, APV (2-amino-5-phosphonovaleric acid), and high divalent cation concentration to block polysynaptic responses. Focal uncaging of glutamate, accomplished by switching a pulsed UV laser to give a 200-400 micros light stimulus, evoked single- or multiple-component composite EPSCs. In neurons of the partially isolated cortex, there were significant increases in the fraction of uncaging sites from which EPSCs could be evoked ("hot spots") and a decrease in the mean amplitude of individual elements in the composite EPSC. When plotted along the cortical depth, the changes in EPSCs took place mainly between 150 and 200 microm above and below the somata, suggesting a specific enhancement of recurrent excitatory connectivity among layer V pyramidal neurons of the undercut neocortex. These changes may shift the balance within cortical circuits toward increased synaptic excitation and contribute to epileptogenesis.

Topics: 2-Amino-5-phosphonovalerate; Analysis of Variance; Anesthetics, Local; Animals; Animals, Newborn; Brain Injuries; Brain Mapping; Cell Count; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamates; In Vitro Techniques; Lysine; Microscopy, Confocal; Patch-Clamp Techniques; Photic Stimulation; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Somatosensory Cortex; Synaptic Transmission; Tetrodotoxin

Microglial cells represent the immune system of the mammalian brain and therefore are critically involved in various injuries and diseases. Little is known about their role in the healthy brain and their immediate reaction to brain damage. By using in vivo two-photon imaging in neocortex, we found that microglial cells are highly active in their presumed resting state, continually surveying their microenvironment with extremely motile processes and protrusions. Furthermore, blood-brain barrier disruption provoked immediate and focal activation of microglia, switching their behavior from patroling to shielding of the injured site. Microglia thus are busy and vigilant housekeepers in the adult brain.

Topics: Animals; Astrocytes; Bicuculline; Blood-Brain Barrier; Brain Injuries; Capillaries; Cell Movement; Cell Surface Extensions; GABA Antagonists; Green Fluorescent Proteins; Lasers; Lipopolysaccharides; Mice; Mice, Transgenic; Microglia; Microscopy, Fluorescence; Neocortex; Pseudopodia; Sodium Channel Blockers; 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

Interneurons innervating dentate granule cells are potent regulators of the entorhino-hippocampal interplay. Traumatic brain injury, a leading cause of death and disability among young adults, is frequently associated with rapid neuropathological changes, seizures, and short-term memory deficits both in humans and experimental animals, indicating significant posttraumatic perturbations of hippocampal circuits. To determine the pathophysiological alterations that affect the posttraumatic functions of dentate neuronal networks within the important early (hours to days) posttraumatic period, whole cell patch-clamp recordings were performed from granule cells and interneurons situated in the granule cell layer of the dentate gyrus of head-injured and age-matched, sham-operated control rats. The data show that a single pressure wave-transient delivered to the neocortex of rats (mimicking moderate concussive head trauma) resulted in a characteristic ( approximately 10 mV), transient (<4 days), selective depolarizing shift in the resting membrane potential of dentate interneurons, but not in neighboring granule cells. The depolarization was not associated with significant changes in action potential characteristics or input resistance, and persisted in the presence of antagonists of ionotropic and metabotropic glutamate, and GABA(A) and muscarinic receptors, as well as blockers of voltage-dependent sodium channels and of the h-current. The differential action of the cardiac glycosides oubain and stophanthidin on interneurons from control versus head-injured rats indicated that the depolarization of interneurons was related to the trauma-induced decrease in the activity of the electrogenic Na(+)/K(+)-ATPase. In contrast, the Na(+)/K(+)-ATPase activity in granule cells did not change. Intracellular injection of Na(+), Ca(2+)-chelator and ATP, as well as ATP alone, abolished the difference between the resting membrane potentials of control and injured interneurons. The selective posttraumatic depolarization increased spontaneous firing in interneurons, enhanced the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) in granule cells, and augmented the efficacy of depolarizing inputs to discharge interneurons. These results demonstrate that mechanical neurotrauma delivered to a remote site has highly selective effects on different cell types even within the same cell layer, and that the electrogenic Na(+)-pump plays a role in setting the

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Benzoates; Bicuculline; Brain Injuries; Dentate Gyrus; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Glycine; In Vitro Techniques; Interneurons; Membrane Potentials; Patch-Clamp Techniques; Pyrimidines; Rats; Rats, Wistar; Sodium-Potassium-Exchanging ATPase; Strophanthidin; Tetrodotoxin; Wounds, Nonpenetrating

Severe cortical trauma frequently causes epilepsy that develops after a long latency. We hypothesized that plastic changes in excitability during this latent period might be initiated or sustained by the level of neuronal activity in the injured cortex. We therefore studied effects of action potential blockade by application of tetrodotoxin (TTX) to areas of cortical injury in a model of chronic epileptogenesis. Partially isolated islands of sensorimotor cortex were made in 28- to 30-day-old male Sprague-Dawley rats and thin sheets of Elvax polymer containing TTX or control vehicle were implanted over lesions. Ten to 15 days later neocortical slices were obtained through isolates for electrophysiological studies. Slices from all animals (n = 12) with lesions contacted by control-Elvax (58% of 36 slices) exhibited evoked epileptiform field potentials, and those from 4 rats had spontaneous epileptiform events. Only 2 of 11 lesioned animals and 6% of slices from cortex exposed to TTX in vivo exhibited evoked epileptiform potentials, and no spontaneous epileptiform events were observed. There was no evidence of residual TTX during recordings. TTX-Elvax was ineffective in reversing epileptogenesis when implanted 11 days after cortical injury. These data suggest that development of antiepileptogenic drugs for humans may be possible.

Topics: Action Potentials; Animals; Brain; Brain Injuries; Epilepsy; Male; Rats; Rats, Sprague-Dawley; Tetrodotoxin; Time Factors

Calcium ion entry following mechanical neurite transection was examined in cultured sympathetic neurons loaded with the Ca2+ indicator fluo-3. Neurite transection produced a rapid [Ca2+]i rise in the cell soma which preceded any [Ca2+]i rise in the neurite (n = 30). Blocking sodium channels with tetrodotoxin had no effect on the Ca2+ rise, but inactivating voltage-sensitive Ca2+ channels by bath-applying 140 mM potassium prior to the transection, and the simultaneous application of nimodipine and omega-conotoxin GVIA, blockers of L-type and N-type Ca2+ channels, respectively, considerably attenuated the Ca2+ rise in the soma and neurites. These data contradict the intuitive hypothesis that Ca2+ entry following mechanical neurite transection occurs via non-specific influx pathways produced by cell-membrane disruption and provide direct evidence in mammalian neurons that immediate, traumatically-induced, increases in neuronal [Ca2+]i are amenable to pharmacological manipulation.

Topics: Animals; Brain Injuries; Calcium; Calcium Channel Blockers; Calcium Channels; Cells, Cultured; Membrane Potentials; Neurites; Neurons; Nimodipine; omega-Conotoxin GVIA; Peptides; Potassium; Rats; Rats, Sprague-Dawley; Superior Cervical Ganglion; Tetrodotoxin

Traumatic brain injury (TBI) induces massive, transient ion flux, after impact. This may be via agonist gated channels, such as the muscarinic, cholinergic or NMDA receptor, or via voltage-dependent channels. Pharmacological blockade of the former, is neuroprotective in most TBI models, but the role of voltage-dependent Na+/K+ channels has not been tested. We have therefore tested the hypothesis that intraventricular tetrodotoxin (TTX) (20 microliters, 5 mM) induced blockade of post-TBI ion flux will prevent cytotoxic cell swelling, Na+ and K+ flux, and behavioral deficit. Microdialysis demonstrated blockade of [K+]d flux in the TTX group compared to controls. Behavioral evaluation of motor (days 1-5) and memory function (days 11-15) after TBI revealed no beneficial effect in the TTX group compared to controls. Thus, although evidence of reduced ionic flux was demonstrated in the TTX group, memory and behavior were unaffected, suggesting that agonist-operated channel-mediated ion flux is more important after TBI.

Topics: Animals; Brain Injuries; Cell Membrane; Cell Size; Electrophysiology; Injections, Intraventricular; Ion Channel Gating; Maze Learning; Microdialysis; Motor Activity; Postural Balance; Potassium Channels; Rats; Rats, Sprague-Dawley; Sodium Channels; Tetrodotoxin

Topics: Animals; Brain Chemistry; Brain Injuries; Cycloheximide; Gene Expression Regulation; Hippocampus; Humans; Kindling, Neurologic; Neuronal Plasticity; Neurons; Proto-Oncogene Proteins; Proto-Oncogenes; Rats; Receptors, N-Methyl-D-Aspartate; Seizures; Signal Transduction; Sodium Channels; Synapses; Tetrodotoxin

Early cellular swelling following fluid-percussion brain injury was demonstrated in vivo by means of microdialysis in the rat. When rapid cellular swelling occurs, water moves from the extracellular space (ECS) into the cells and the extracellular concentration of ECS marker which does not move into the cells increases. Cellular swelling was therefore demonstrated in vivo as an increase in the dialysate concentration of 14C-sucrose ([14C-sucrose]d) pre-perfused as an ECS marker. The increase in [14C-sucrose]d occurred concomitantly with an increase in the dialysate concentration of K+ ([K+]d) representing large ionic fluxes. The increases in [K+]d and [14C-sucrose]d were both inhibited by kynurenic acid, a broad spectrum antagonist of excitatory amino acids (EAAs), which was administered through the dialysis probe. These findings suggest that early cellular swelling following traumatic brain injury is a result of ionic fluxes mediated by EAAs.

Topics: Amino Acids; Animals; Body Water; Brain Injuries; Female; Ions; Kynurenic Acid; Neurons; Potassium; Rats; Rats, Inbred Strains; Tetrodotoxin