Blockade Of Action Potentials Research Articles

Pattern-Dependent Role of NMDA Receptors in Action Potential Generation: Consequences on Extracellular Signal-Regulated Kinase Activation

Synaptic long-term potentiation is maintained through gene transcription, but how the nucleus is recruited remains controversial. Activation of extracellular signal-regulated kinases (ERKs) 1 and 2 with synaptic stimulation has been shown to require NMDA receptors (NMDARs), yet stimulation intensities sufficient to recruit action potentials (APs) also appear to be required. This has led us to ask the question of whether NMDARs are necessary for AP generation as they relate to ERK activation. To test this, we examined the effects of NMDAR blockade on APs induced with synaptic stimulation using whole-cell current-clamp recordings from CA1 pyramidal cells in hippocampal slices. NMDAR antagonists were found to potently inhibit APs generated with 5 and 100 Hz synaptic stimulation. Blockade of APs and ERK activation could be overcome with the addition of the GABAA antagonist bicuculline, indicating that APs are sufficient to activate signals such as ERK in the nucleus and throughout the neuron in the continued presence of NMDAR antagonists. Interestingly, no effects of the NMDAR antagonists were observed when theta-burst stimulation (TBS) was used. This resistance to the antagonists is conferred by temporal summation during the bursts. These results clarify findings from a previous study showing that ERK activation induced with TBS is resistant to 2-amino-5-phosphonovalerate, in contrast to that induced with 5 or 100 Hz stimulation, which is sensitive. By showing that NMDAR blockade inhibits AP generation, we demonstrate that a major role that NMDARs play in cell-wide and nuclear ERK activation is through their contribution to action potential generation.

Modulation of GABA receptor subunits in rat facial motoneurons after axotomy

Facial nerve axotomy is a good model for studying neuronal plasticity and regeneration in the peripheral nervous system. In the present study, we investigated the effect of axotomy on the different subunits of GABA A and GABA B receptors of facial motoneurons. The facial nerve trunk was unilaterally sectioned and operated rats were sacrificed at 1, 3, 8, 30, and 60 days later. mRNAs coding for α1, β2, and γ2 of GABA A receptors and for GABA 1B and GABA B2 receptors were down-regulated by axotomy. This decrease began as soon as 1 or 3 days after axotomy, and the minimum was 8 days post-lesion; the mRNA levels remained lower than normal at day post-lesion 60. The abundance of mRNAs coding for the three other α2, β1, and β3 facial subunits of GABA A receptors and for the pre-synaptic GABA B1A subunit remained unchanged during the period 1–8 days post-lesion. Immunohistochemistry using specific antibodies against α1, γ2 subunits of GABA A and against GABA B2 subunits confirmed this down-regulation. Colchicine treatment and blockade of action potential by tetrodotoxin significantly decreased GABA Aα1 immunoreactivity in the axotomized facial nucleus after 7 days. Finally, muscle destruction by cardiotoxin or facial palsy induced by botulinum toxin failed to change GABA Aα1 subunit expression. Our data demonstrate that axotomy strongly reduced the amounts of α1, β2, and γ2 subunits of GABA A receptors and B 1B and B 2 subunits of GABA B receptors in the axotomized facial motoneurons. The loss of GABA Aα1 subunit was most probably induced by both the loss of trophic factors transported from the periphery and a positive injury signal. It also seems to be dependent on activity disruption.

Selective deletion of the α5 subunit differentially affects somatic–dendritic versus axonally targeted nicotinic ACh receptors in mouse

We have compared the functional properties of nicotinic acetylcholine receptors (nAChRs) within both somatic and presynaptic domains of superior cervical ganglion (SCG) neurones from wild-type (WT) mice with those expressed by SCG neurones from mice with a targeted deletion of the gene for the alpha5-subunit. The functional profile of somatic nAChRs was assayed by direct macroscopic current recording and from measurements of nicotinic agonist-induced calcium transients with fura-2 imaging. The profile of nAChRs at presynaptic sites was assayed by measurement of nicotinic agonist-induced transmitter release (as preloaded [3H]noradrenaline) under conditions of action potential blockade. We have examined the responses to the nicotinic agonists acetylcholine, nicotine, cytisine, dimethylphenylpiperazinium iodide (DMPP) and epibatidine. Macroscopic current and calcium imaging assays revealed several differences in the functional profile of somatic nAChRs in WT SCG neurones compared with those from mice with the alpha5 subunit deleted. Somatic nAChRs in control animals were more potently activated by cytisine as compared to DMPP. In contrast, DMPP was consistently more potent than cytisine in mice lacking the alpha5 nAChR subunit. Differences in the somatic nAChR rank order of potency were most prominent after a least 1 day in vitro. The magnitude of somatic nAChR responses to nicotinic agonists was not substantially different in control mice compared with those of alpha5 subunit-deleted animals. Comparison of presynaptic nAChR-mediated responses in WT versus alpha5 subunit-deleted animals revealed a very different set of changes in the functional profile of prejunctional nAChRs compared with somatic nAChRs. In contrast to somatic nAChRs, the responses of prejunctional receptors were markedly enhanced in alpha5 knockout animals compared with control. Furthermore, all prejunctional receptor responses were most potently activated by DMPP in both control and in alpha5 subunit-deleted mice. Hence, the presence or absence of the alpha5 subunit did not affect the rank order of potency of agonists at preterminal sites but greatly affected the magnitude of presynaptic nAChR-mediated responses. The enhanced efficacy of nicotine at presynaptic receptors was corroborated in an acute atrium preparation from postnatal alpha5 subunit-deleted mice. These results confirm and significantly extend our previous observation that in the sympathetic nervous system, somatic and prejunctional receptors are different and rely on the presence of the alpha5 subunit in a distinct manner.

Modulation of the glutamatergic receptors (AMPA and NMDA) and of glutamate vesicular transporter 2 in the rat facial nucleus after axotomy

Facial nerve axotomy is a good model for studying neuronal plasticity and regeneration in the peripheral nervous system. We investigated in the rat the effect of axotomy on the different subunits of excitatory glutamatergic AMPA (GLuR1-4), NMDA (NR1, NR2A-D) receptors, post-synaptic density 95, vesicular glutamate transporter 2, β catenin and cadherin. mRNA levels and/or protein production were analyzed 1, 3, 8, 30 and 60 days after facial nerve axotomy by in situ hybridization and immunohistofluorescence. mRNAs coding for the GLuR2-4, NR1, NR2A, B, D subunits of glutamatergic receptors and for post-synaptic density 95, were less abundant after axotomy. The decrease began as early as 1 or 3 days after axotomy; the mRNAs levels were lowest 8 days post-lesion, and returned to normal or near normal 60 days after the lesion. The NR2C subunit mRNAs were not detected in either lesioned or intact facial nuclei. Immunohistochemistry using specific antibodies against GLuR2-3 subunits and against NR1 confirmed this down-regulation. There was also a large decrease in vesicular glutamate transporter 2 immunostaining in the axotomized facial nuclei at early stages following facial nerve section. In contrast, no decrease of NR2A subunit and of post-synaptic density 95 could be detected at any time following the lesion. β Catenin and cadherin immunoreactivity pattern changed around the cell body of facial motoneuron by day 3 after axotomy, and then, tends to recover at day post-lesion 60 days. Therefore, our results suggest a high correlation between restoration of nerve/muscle synaptic contact, synaptic structure and function in facial nuclei. To investigate the mechanisms involved in the change of expression of these proteins following axotomy, the facial nerve was perfused with tetrodotoxin for 8 days. The blockade of action potential significantly decreased GLuR2-3, NR1and NR2A mRNAs in the ipsilateral facial nuclei. Thus, axotomy-induced changes in mRNA abundance seemed to depend partly on disruption of activity.

Presynaptic Activity and Ca2+ Entry Are Required for the Maintenance of NMDA Receptor–Independent LTP at Visual Cortical Excitatory Synapses

We have shown that some neural activity is required for the maintenance of long-term potentiation (LTP) at visual cortical inhibitory synapses. We tested whether this was also the case in N-methyl-d-aspartate (NMDA) receptor-independent LTP of excitatory connections in layer 2/3 cells of developing rat visual cortex. This LTP occurred after 2-Hz stimulation was applied for 15 min and always persisted for several hours while test stimulation was continued at 0.1 Hz. When test stimulation was stopped for 1 h after LTP induction, only one-third of the LTP instances disappeared, but most did disappear under a pharmacological suppression of spontaneous firing, indicating that LTP maintenance requires either evoked or spontaneous activities. LTP was totally abolished by a temporary blockade of action potentials with lidocaine or the removal of extracellular Ca(2+) after LTP induction, but it persisted under a voltage clamp of postsynaptic cells or after a temporary blockade of postsynaptic activity with the glutamate receptor antagonist kynurenate, suggesting that LTP maintenance requires presynaptic, but not postsynaptic, firing and Ca(2+) entry. More than one-half of the LTP instances were abolished after a pharmacological blockade of P-type Ca(2+) channels, whereas it persisted after either L-type or Ni(2+)-sensitive Ca(2+) channel blockades. These results show that the maintenance of NMDA receptor-independent excitatory LTP requires presynaptic firing and Ca(2+) channel activation as inhibitory LTP, although the necessary level of firing and Ca(2+) entry seems lower for the former than the latter and the Ca(2+) channel types involved are only partly the same.

Regulation of dendritic protein synthesis by miniature synaptic events.

We examined dendritic protein synthesis after a prolonged blockade of action potentials alone and after a blockade of both action potentials and miniature excitatory synaptic events (minis). Relative to controls, dendrites exposed to a prolonged blockade of action potentials showed diminished protein synthesis. Dendrites in which both action potentials and minis were blocked showed enhanced protein synthesis, suggesting that minis inhibit dendritic translation. When minis were acutely blocked or stimulated, an immediate increase or decrease, respectively, in dendritic translation was observed. Taken together, these results reveal a role for miniature synaptic events in the acute regulation of dendritic protein synthesis in neurons.

Pentobarbital modulates intrinsic and gaba-receptor conductances in thalamocortical inhibition

We investigated interactions of an anesthetic barbiturate, pentobarbital, with non-ligand gated channels and identified inhibitory synaptic transmission in thalamic neurons. Using whole cell voltage-clamp, current-clamp and single channel recording techniques in rat ventrobasal neurons of slices and dispersed preparations, we determined the mechanisms of pentobarbital actions on ionic currents and inhibitory postsynaptic currents (IPSCs), mediated by aminobutyric acid (GABA). We investigated pentobarbital effects on intrinsic currents using hyperpolarizing voltage commands from rest and tetrodotoxin blockade of action potentials. At concentrations near 8 μM, pentobarbital increased input conductance and induced net outward current, I PB , at potentials near action potential threshold. The reversal potential of I PB was −75 mV, implicating K + and other ions. Cs + (3 mM) which inhibits both K + currents and inward rectifier (I h ), completely blocked I PB , whereas the selective I h blocker, ZD-7288 (25 μM), or Ba 2+ (2 mM) which suppresses only K + currents, reduced I PB . Pentobarbital inhibited the I h , consistent with a ZD-7288-induced shift in reversal potential for I PB toward K + equilibrium potential. Pentobarbital increased the inward K + rectifier, I Kir , and leak current, I leak . We determined the susceptibility of IPSCs, evoked by reticular stimulation, to antagonism by bicuculline, picrotoxinin and 2-hydroxysaclofen and identified their receptor subclass components. At EC 50 =53 μM, pentobarbital increased the duration of IPSCs. The prolonged IPSC duration during pentobarbital was attributable to enhanced open probability of GABA A channels, because combined with GABA, pentobarbital application increased mean channel open time without affecting channel conductance. At concentrations up to 100 μM, pentobarbital did not directly activate GABA A receptors. The concentration-response relationships for pentobarbital effects on the intrinsic currents and IPSCs overlapped, implying multiple sites of action and possible redundancy in anesthetic mechanisms. This is the first study to show that an i.v. anesthetic modulates the intrinsic currents, I h , I Kir , and I leak , as well as IPSC time course in the same neurons. These effects likely underlie inhibition in thalamocortical neurons during pentobarbital anesthesia.

Pentobarbital modulates intrinsic and GABA-receptor conductances in thalamocortical inhibition

We investigated interactions of an anesthetic barbiturate, pentobarbital, with non-ligand gated channels and identified inhibitory synaptic transmission in thalamic neurons. Using whole cell voltage-clamp, current-clamp and single channel recording techniques in rat ventrobasal neurons of slices and dispersed preparations, we determined the mechanisms of pentobarbital actions on ionic currents and inhibitory postsynaptic currents (IPSCs), mediated by aminobutyric acid (GABA). We investigated pentobarbital effects on intrinsic currents using hyperpolarizing voltage commands from rest and tetrodotoxin blockade of action potentials. At concentrations near 8 μM, pentobarbital increased input conductance and induced net outward current, I PB, at potentials near action potential threshold. The reversal potential of I PB was −75 mV, implicating K + and other ions. Cs + (3 mM) which inhibits both K + currents and inward rectifier (I h), completely blocked I PB, whereas the selective I h blocker, ZD-7288 (25 μM), or Ba 2+ (2 mM) which suppresses only K + currents, reduced I PB. Pentobarbital inhibited the I h, consistent with a ZD-7288-induced shift in reversal potential for I PB toward K + equilibrium potential. Pentobarbital increased the inward K + rectifier, I Kir, and leak current, I leak. We determined the susceptibility of IPSCs, evoked by reticular stimulation, to antagonism by bicuculline, picrotoxinin and 2-hydroxysaclofen and identified their receptor subclass components. At EC 50=53 μM, pentobarbital increased the duration of IPSCs. The prolonged IPSC duration during pentobarbital was attributable to enhanced open probability of GABA A channels, because combined with GABA, pentobarbital application increased mean channel open time without affecting channel conductance. At concentrations up to 100 μM, pentobarbital did not directly activate GABA A receptors. The concentration-response relationships for pentobarbital effects on the intrinsic currents and IPSCs overlapped, implying multiple sites of action and possible redundancy in anesthetic mechanisms. This is the first study to show that an i.v. anesthetic modulates the intrinsic currents, I h, I Kir, and I leak, as well as IPSC time course in the same neurons. These effects likely underlie inhibition in thalamocortical neurons during pentobarbital anesthesia.

Distribution of Fos labeling in the inferior olive following transient blockade of the VIIIth cranial nerve

The sodium channel blocker, tetrodotoxin (TTX), is an effective tool for blockade of action potentials in neurons. Unilateral transtympanic administration of 3 mM TTX produced behavioral symptoms paralleling those previously reported following unilateral vestibular ablation. Behavioral symptoms were evident as early as 15 min post-TTX. Fos immunocytochemistry revealed an initial bilateral distribution of Fos in the inferior olive (IO) followed by an almost exclusively unilateral distribution of Fos. By 1 h, Fos was predominantly localized in subdivisions of the IO contralateral to TTX treatment. Fos labeling in the IO was most pronounced at 2- and 6-h survival times and was localized in the contralateral IOA, IOB, IOC, IOBe, and IOK subdivisions and bilaterally in the IOM and IODM. Other regions of the brainstem including the vestibular nuclei, prepositus hypoglossi, dorsal paragigantocellular reticular nucleus, nucleus of the tractus solitarius and locus coeruleus also exhibited altered patterns of Fos labeling following TTX. The finding that Fos activity in the IO is initially bilateral and then rapidly becomes unilateral has not been reported for the traditional vestibular ablation models and may be unique to the TTX model. In addition, since altered Fos activity is readily detected in the IO at time-points prior to detectable changes in Fos in the central vestibular complex it is possible that the IO is particularly sensitive to events precipitated by unilateral vestibular disturbance.

Tetrodotoxin prevents motor unit enlargement after partial denervation in rat hindlimb muscles.

Findings that increased neuromuscular activity significantly reduced sprouting in partially denervated muscles prompted this present study to determine if the converse is true, namely that reduced activity promotes sprouting and motor unit (MU) enlargement. Partial denervation of rat hindlimb muscles by either the L4 or L5 spinal root avulsion resulted in extensive denervation (> 80 %) in tibialis anterior (TA) and medial gastrocnemius (MG) muscles, and moderate denervation (~50 %) in soleus (SOL) and plantaris (PL) muscles. The partially denervated muscles were then subjected to a 4 week programme of normal caged activity or TTX-induced neuromuscular inactivity. At 1 month, measurement of MU enlargement and quantification of sprouting were evaluated, respectively, by electrophysiological and histochemical means. Analysis of electrophysiological data showed that MU forces were significantly increased in both extensively and moderately denervated muscles 1 month after partial denervation and normal cage activity and that neuromuscular activity blockade by TTX completely abolished the MU enlargement in these partially denervated muscles. Histochemical analysis of sprouting revealed that the number of sprouts was significantly increased after partial denervation and normal cage activity, particularly after extensive denervation. TTX-induced neuromuscular inactivity dramatically reduced the number of sprouts and increased the number of free endplates in the extensively but not the moderately denervated muscles. These data demonstrate that a reduction in neuromuscular activity mediated by presynaptic blockade of neural action potentials reduces MU enlargement in partially denervated muscles by reducing axonal sprouting.

Adrenomedullin influences magnocellular and parvocellular neurons of paraventricular nucleus via separate mechanisms.

We previously reported that adrenomedullin (AM) decreases blood pressure following microinjection into the paraventricular nucleus of the hypothalamus (PVN) of the rat. With the use of whole cell recordings in rat hypothalamic slice preparations, we characterized the effects of AM on electrophysiologically identified PVN neurons and described the membrane events underlying such actions. AM hyperpolarized magnocellular (type I) neurons in a dose-dependent manner, a response associated with an increase in the frequency and amplitude of inhibitory postsynaptic potentials. Blockade of action potentials with tetrodotoxin (TTX) abolished AM effects on membrane potential and synaptic activity in magnocellular neurons, suggesting direct actions on inhibitory interneurons. Furthermore, blockade of inhibitory synaptic transmission with the GABA(A) receptor antagonist bicuculline methiodide also abolished AM effects on membrane potential in magnocellular neurons. In contrast, parvocellular (type II) neurons depolarized following AM receptor activation. AM effects on parvocellular neurons were dose dependent and were maintained in the presence of TTX, indicating direct effects on this population of neurons. Voltage-clamp recordings from parvocellular neurons showed AM enhances a nonselective cationic conductance, suggesting a potential mechanism through which AM influences membrane potential. These observations show clear population-specific actions of AM on separate identified groups of PVN neurons. Such effects on magnocellular neurons likely contribute to the hypotensive actions of this peptide in PVN. Although the effects on parvocellular neurons may also contribute to such cardiovascular effects of AM, it is more likely that actions on this population of PVN neurons underlie the previously demonstrated activational effects of AM on the hypothalamic-pituitary-adrenal axis.

Extended Plasticity of Visual Cortex in Dark-Reared Animals May Result from Prolonged Expression ofcpg15-Like Genes

cpg15 is an activity-regulated gene that encodes a membrane-bound ligand that coordinately regulates growth of apposing dendritic and axonal arbors and the maturation of their synapses. These properties make it an attractive candidate for participating in plasticity of the mammalian visual system. Here we compare cpg15 expression during normal development of the rat visual system with that seen in response to dark rearing, monocular blockade of retinal action potentials, or monocular deprivation. Our results show that the onset of cpg15 expression in the visual cortex is coincident with eye opening, and it increases until the peak of the critical period at postnatal day 28 (P28). This early expression is independent of both retinal activity and visual experience. After P28, a component of cpg15 expression in the visual cortex, lateral geniculate nucleus (LGN), and superior colliculus (SC) develops a progressively stronger dependence on retinally driven action potentials. Dark rearing does not affect cpg15 mRNA expression in the LGN and SC at any age, but it does significantly affect its expression in the visual cortex from the peak of the critical period and into adulthood. In dark-reared rats, the peak level of cpg15 expression in the visual cortex at P28 is lower than in controls. Rather than showing the normal decline with maturation, these levels are maintained in dark-reared animals. We suggest that the prolonged plasticity in the visual cortex that is seen in dark-reared animals may result from failure to downregulate genes such as cpg15 that could promote structural remodeling and synaptic maturation.

Transtympanic tetrodotoxin alters the VOR and Fos labeling in the vestibular complex

The sodium channel blocker tetrodotoxin (TTX) is an effective tool for blockade of action potentials. Unilateral transtympanic administration of 3 mM TTX produced behavioral symptoms similar to those following unilateral peripheral vestibular ablation. Complete resolution of visible symptoms occurred between 48 and 72 h post-TTX. Eye-coil recordings indicated a spontaneous nystagmus and a decrease in the VOR in TTX-treated animals. Neuronal activity in the central vestibular complex (VC), as monitored with Fos immunocytochemistry, revealed an asymmetric pattern of Fos labeling in the medial, inferior and superior vestibular nuclei and the prepositus hypoglossal nucleus. Although the spatio-temporal pattern of Fos labeling was consistent and reproducible at each time-point, changes were noted among time-points. Transient blockade with TTX may be useful for studying the central vestibular response to recurrent or episodic vestibular disruption in the intact system.

GABA Itself Promotes the Developmental Switch of Neuronal GABAergic Responses from Excitation to Inhibition

GABA is the main inhibitory neurotransmitter in the adult brain. Early in development, however, GABAergic synaptic transmission is excitatory and can exert widespread trophic effects. During the postnatal period, GABAergic responses undergo a switch from being excitatory to inhibitory. Here, we show that the switch is delayed by chronic blockade of GABA A receptors, and accelerated by increased GABA A receptor activation. In contrast, blockade of glutamatergic transmission or action potentials has no effect. Furthermore, GABAergic activity modulated the mRNA levels of KCC2, a K +-Cl − cotransporter whose expression correlates with the switch. Finally, we report that GABA can alter the properties of depolarization-induced Ca 2+ influx. Thus, GABA acts as a self-limiting trophic factor during neural development.

Apolipoprotein E and ϐA4-amyloid: signals and effects

In humans, the apolipoprotein E gene (APOE) is polymorphic with the alleles APOE epsilon 2, 3 and 4 coding for apolipoproteins (Apo) E2, 3 and 4. Apart from age, the APOE epsilon 4 allele represents the most important risk factor in sporadic Alzheimer's disease (AD). Compared to APOE epsilon 3 homozygotes, the histopathological onset of tau pathology is found 1-2 decades earlier but progresses with the same speed. ApoE dose-dependently and specifically increases free intraneuronal calcium levels in the order ApoE4 > ApoE3 > ApoE2. This effect is amplified in the presence of beta A4-peptide. The ApoE effects on calcium are not affected by the blockade of action potentials with tetrodotoxin, or by inhibition of common ApoE binding sites. The calcium channel involved has been identified as a P/Q-type-like channel. Brain tissue ApoE levels differ with respect to APOE alleles and Braak-stage for Alzheimer-histopathology. The production of ApoE in astrocytes is controlled by several receptor/effector systems such as adrenoceptors and cAMP. In the presence of beta A4-peptide fragments, astrocytes stop their synthesis of ApoE resulting in a massive reduction in the bioavailability of ApoE. In the periphery, ApoE directs cholesterol transport and thereby influences its cellular concentrations. In neurons, changes in the concentration of cholesterol influence the phosphorylation status of the microtubule-associated protein tau at sites known to be altered in AD.

Block of neuronal tetrodotoxin-resistant Na+ currents by stereoisomers of piperidine local anesthetics.

Tetrodotoxin (TTX)-sensitive Na(+) channels in the peripheral nervous system are the major targets for local anesthetics. In the peripheral nociceptive system, a Na(+) channel subtype resistant to TTX and with distinct electrophysiological properties seems to be of importance for impulse generation and conduction. A current through TTX-resistant Na(+) channels displays slower activation and inactivation kinetics and has an increased activation threshold compared with TTX-sensitive Na(+) currents and may have different pharmacological properties. We studied the effects of stereoisomers of piperidine local anesthetics on neuronal TTX-resistant Na(+) currents recorded with the whole-cell configuration of the patch clamp method in enzymatically dissociated dorsal root ganglion neurons of adult rats. Stereoisomers of mepivacaine, ropivacaine, and bupivacaine reversibly inhibited TTX-resistant Na(+) currents in a concentration and use-dependent manner. All drugs accelerated time course of inactivation. Half-maximal blocking concentrations were determined from concentration-inhibition relationships. Potencies for tonic and for use-dependent block increased with rising lipid solubilities of the drugs. Stereoselective action was not observed. We conclude that block of TTX-resistant Na(+) currents may lead to blockade of TTX-resistant action potentials in nociceptive fibers and consequently may be responsible for pain suppression during local anesthesia.

Studies on the prevention of nigericin action in neuroblastoma X glioma hybrid (NG108-15) cells.

Electrophysiological analysis of neuroblastoma X glioma hybrid (NG108-15) cells was used as an in vitro neuronal model system to evaluate antagonists of the K+-selective carboxylic ionophore, nigericin. Changes in membrane electrical characteristics induced by nigericin with and without the simultaneous administration of antagonists were measured using intracellular microelectrode techniques. Bath application of nigericin (3 microM) produced a severe hyperpolarization and blocked the generation of action potentials in response to electrical stimulation. Simultaneous administration of nigericin plus the Na+-K+ pump inhibitor ouabain or drugs known to influence Ca++ signaling in cells, i.e., quinidine, compound R24571, verapamil or haloperidol, was able to significantly attenuate the hyperpolarization. All antagonists acted in a concentration-dependent manner. However, nigericin plus maximally effective concentrations of ouabain (1 microM), verapamil (3 microM) and haloperidol (3 and 10 microM) resulted in moderate-to-severe depolarization by the end of 24 min. superfusions, suggesting that the concentrations of antagonists were excessive and that NG108-15 cell damage had occurred. In addition, none of the compounds studied was able to effectively prevent nigericin-induced blockade of action potentials. Thus, none of these antagonists appears suitable for transition to in vivo antidotal protection studies.

The mlenapts RNA Helicase Mutation in Drosophila Results in a Splicing Catastrophe of the para Na+ Channel Transcript in a Region of RNA Editing

The mlenapts mutation causes temperature-dependent blockade of action potentials resulting from decreased abundance of para-encoded Na+ channels. Although maleless (mle) encodes a double-stranded RNA (dsRNA) helicase, exactly how mlenapts affects para expression remained uncertain. Here, we show that para transcripts undergo adenosine-to-inosine (A-to-I) RNA editing via a mechanism that apparently requires dsRNA secondary structure formation encompassing the edited exon and the downstream intron. In an mlenapts background, >80% of para transcripts are aberrant, owing to internal deletions that include the edited exon. We propose that the Mle helicase is required to resolve the dsRNA structure and that failure to do so in an mlenapts background causes exon skipping because the normal splice donor is occluded. These results explain how mlenapts affects Na+ channel expression and provide new insights into the mechanism of RNA editing.

Dynamic Regulation ofcpg15during Activity-Dependent Synaptic Development in the Mammalian Visual System

During visual system development, neural activity regulates structural changes in connectivity including axonal branching and dendritic growth. Here we have examined a role for the candidate plasticity gene 15 (cpg15), which encodes an activity-regulated molecule that can promote dendritic growth, in this process. We report that cpg15 is expressed in the cat visual system at relatively high levels in the lateral geniculate nucleus (LGN) but at very low levels in its synaptic target, layer 4 of the visual cortex. Prenatally, when cpg15 mRNA in the LGN is most abundant, expression is insensitive to action potential blockade by tetrodotoxin. Postnatally, activity regulation of cpg15 emerges in the LGN coincident with development of ocular dominance columns in the visual cortex. cpg15 can be detected in layers 2/3 and 5/6 of visual cortex postnatally, and expression in layers 2/3 is activity-regulated during known periods of activity-dependent plasticity for these layers. Localization and regulation of cpg15 expression in the visual system are consistent with a presynaptic role for CPG15 in shaping dendritic arbors of target neurons during activity-dependent synaptic rearrangements, both in development and adulthood.

Tetrodotoxin prevents posttraumatic epileptogenesis in rats.

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.