Antagonistic postsynaptic and presynaptic actions of cyclohexanol on neuromuscular synaptic transmission and function.

Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Some survivors develop a severe, acute or delayed myasthenic syndrome. In animal models, similar myasthenia has been associated with increasing plasma concentration of one insecticide solvent metabolite, cyclohexanol. We investigated possible mechanisms using voltage and current recordings from mouse neuromuscular junctions (NMJs) and transfected human cell lines. Cyclohexanol (10-25 mM) reduced endplate potential (EPP) amplitudes by 10-40% and enhanced depression during repetitive (2-20 Hz) stimulation by up to 60%. EPP decay was prolonged more than twofold. Miniature EPPs were attenuated by more than 50%. Cyclohexanol inhibited whole-cell currents recorded from CN21 cells expressing human postjunctional acetylcholine receptors (hnAChR) with an IC50 of 3.74 mM. Cyclohexanol (10-20 mM) also caused prolonged episodes of reduced-current, multi-channel bursting in outside-out patch recordings from hnAChRs expressed in transfected HEK293T cells, reducing charge transfer by more than 50%. Molecular modelling indicated cyclohexanol binding (-6 kcal/mol) to a previously identified alcohol binding site on nicotinic AChR α-subunits. Cyclohexanol also increased quantal content of evoked transmitter release by ∼50%. In perineurial recordings, cyclohexanol selectively inhibited presynaptic K+ currents. Modelling indicated cyclohexanol binding (-3.8 kcal/mol) to voltage-sensitive K+ channels at the same site as tetraethylammonium (TEA). TEA (10 mM) blocked K+ channels more effectively than cyclohexanol but EPPs were more prolonged in 20 mM cyclohexanol. The results explain the pattern of neuromuscular dysfunction following ingestion of organophosphorus insecticides containing cyclohexanol precursors and suggest that cyclohexanol may facilitate investigation of mechanisms regulating synaptic strength at NMJs. KEY POINTS: Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Survivors may develop a severe myasthenic syndrome or paralysis, associated with increased plasma levels of cyclohexanol, an insecticide solvent metabolite. Analysis of synaptic transmission at neuromuscular junctions in isolated mouse skeletal muscle, using isometric tension recording and microelectrode recording of endplate voltages and currents, showed that cyclohexanol reduced postsynaptic sensitivity to acetylcholine neurotransmitter (reduced quantal size) while simultaneously enhancing evoked transmitter release (increased quantal content). Patch recording from transfected cell lines, together with molecular modelling, indicated that cyclohexanol causes selective, allosteric antagonism of postsynaptic nicotinic acetylcholine receptors and block of presynaptic K+ -channel function. The data provide insight into the cellular and molecular mechanisms of neuromuscular weakness following intentional ingestion of agricultural organophosphorus insecticides. Our findings also extend understanding of the effects of alcohols on synaptic transmission and homeostatic synaptic function.

Impaired neuromuscular function by conjoint actions of organophosphorus insecticide metabolites omethoate and cyclohexanol with implications for treatment of respiratory failure.

BACKGROUND: Ingestion of agricultural organophosphorus insecticides is a significant cause of death in rural Asia. Patients often show acute respiratory failure and/or delayed, unexplained signs of neuromuscular paralysis, sometimes diagnosed as "Intermediate Syndrome". We tested the hypothesis that omethoate and cyclohexanol, circulating metabolites of one agricultural formulation, cause muscle weakness and paralysis. METHODS: Acetylcholinesterase activity of insecticide components and metabolites was measured using purified enzyme from eel electroplaque or muscle homogenates. Mechanomyographic recording of pelvic limb responses to nerve stimulation was made in anaesthetized pigs and isometric force was recorded from isolated nerve-muscle preparations from mice. Omethoate and cyclohexanol were administered intravenously or added to physiological saline bathing isolated muscle. We also assessed the effect of MgSO4 and cooling on neuromuscular function. RESULTS: Omethoate caused tetanic fade in pig muscles and long-lasting contractions of the motor innervation zone in mouse muscle. Both effects were mitigated, either by i.v. administration of MgSO4 in vivo or by adding 5 mM Mg2+ to the medium bathing isolated preparations. Combination of omethoate and cyclohexanol initially potentiated muscle contractions but then rapidly blocked them. Cyclohexanol alone caused fade and block of muscle contractions in pigs and in isolated preparations. Similar effects were observed ex vivo with cyclohexanone and xylene. Cyclohexanol-induced neuromuscular block was temperature-sensitive and rapidly reversible. CONCLUSIONS: The data indicate a crucial role for organophosphorus and solvent metabolites in muscle weakness following ingestion of agricultural OP insecticide formulations. The metabolites omethoate and cyclohexanol acted conjointly to impair neuromuscular function but their effects were mitigated by elevating extracellular Mg2+ and decreasing core temperature, respectively. Clinical studies of MgSO4 therapy and targeted temperature management in insecticide-poisoned patients are required to determine whether they may be effective adjuncts to treatment.

Reducing sarcolipin expression mitigates Duchenne muscular dystrophy and associated cardiomyopathy in mice.

Sarcolipin (SLN) is an inhibitor of the sarco/endoplasmic reticulum (SR) Ca2+ ATPase (SERCA) and is abnormally elevated in the muscle of Duchenne muscular dystrophy (DMD) patients and animal models. Here we show that reducing SLN levels ameliorates dystrophic pathology in the severe dystrophin/utrophin double mutant (mdx:utr -/-) mouse model of DMD. Germline inactivation of one allele of the SLN gene normalizes SLN expression, restores SERCA function, mitigates skeletal muscle and cardiac pathology, improves muscle regeneration, and extends the lifespan. To translate our findings into a therapeutic strategy, we knock down SLN expression in 1-month old mdx:utr -/- mice via adeno-associated virus (AAV) 9-mediated RNA interference. The AAV treatment markedly reduces SLN expression, attenuates muscle pathology and improves diaphragm, skeletal muscle and cardiac function. Taken together, our findings suggest that SLN reduction is a promising therapeutic approach for DMD.

Perturbation to Cholesterol at the Neuromuscular Junction Confers Botulinum Neurotoxin A Sensitivity to Neonatal Mice.

Botulinum neurotoxin A (BoNT/A) cleaves SNAP25 at the motor nerve terminals and inhibits stimulus evoked acetylcholine release. This causes skeletal muscle paralysis. However, younger neonatal mice (P7) mice. However, neonatal mice younger than 7 days-age remained unaffected by BoNT/A injection. Also, BoNT/A inhibited stimulus evoked acetylcholine release and stimulus-evoked twitch tension of diaphragm nerve muscle preparations (NMPs) of adult mouse and >P7 neonates but not that of P7. However, cholesterol depletion using methyl-ß-cyclodextrin (MßCD) sensitized

Altered active zones, vesicle pools, nerve terminal conductivity, and morphology during experimental MuSK myasthenia gravis.

Recent studies demonstrate reduced motor-nerve function during autoimmune muscle-specific tyrosine kinase (MuSK) myasthenia gravis (MG). To further understand the basis of motor-nerve dysfunction during MuSK-MG, we immunized female C57/B6 mice with purified rat MuSK ectodomain. Nerve-muscle preparations were dissected and neuromuscular junctions (NMJs) studied electrophysiologically, morphologically, and biochemically. While all mice produced antibodies to MuSK, only 40% developed respiratory muscle weakness. In vitro study of respiratory nerve-muscle preparations isolated from these affected mice revealed that 78% of NMJs produced endplate currents (EPCs) with significantly reduced quantal content, although potentiation and depression at 50 Hz remained qualitatively normal. EPC and mEPC amplitude variability indicated significantly reduced number of vesicle-release sites (active zones) and reduced probability of vesicle release. The readily releasable vesicle pool size and the frequency of large amplitude mEPCs also declined. The remaining NMJs had intermittent (4%) or complete (18%) failure of neurotransmitter release in response to 50 Hz nerve stimulation, presumably due to blocked action potential entry into the nerve terminal, which may arise from nerve terminal swelling and thinning. Since MuSK-MG-affected muscles do not express the AChR γ subunit, the observed prolongation of EPC decay time was not due to inactivity-induced expression of embryonic acetylcholine receptor, but rather to reduced catalytic activity of acetylcholinesterase. Muscle protein levels of MuSK did not change. These findings provide novel insight into the pathophysiology of autoimmune MuSK-MG.

Capsaicin modulates acetylcholine release at the myoneural junction.

Transient receptor potential (TRP) proteins are non-selective cation channel proteins that are expressed throughout the body. Previous studies demonstrated the expression of TRP Vanilloid 1 (TRPV1), capsaicin (CAP) receptor, in sensory neurons. Recently, we reported TRPV1 expression in mouse motor nerve terminals [MNTs; (Thyagarajan et al., 2009)], where we observed that CAP protected MNTs from botulinum neurotoxin A (BoNT/A). Phrenic nerve diaphragm nerve muscle preparations (NMP) isolated from isoflurane anesthetized adult mice were analyzed for twitch tension, spontaneous (mEPCs) and nerve stimulus evoked (EPCs) acetylcholine release. When acutely applied to isolated NMP, CAP produced a concentration-dependent decline of twitch tension and produced a significant decline in the amplitude of EPCs and quantal content without any effect on the mEPCs. The suppression of nerve stimulus evoked acetylcholine release by CAP was antagonized by capsazepine (CPZ), a TRPV1 antagonist. CAP did not suppress phrenic nerve stimulus evoked acetylcholine release in TRPV1 knockout mice. Also, CAP treatment, in vitro, interfered with the localization of adapter protein 2 in cholinergic Neuro 2a cells. Wortmannin, (WMN; non-selective phosphoinositol kinase inhibitor), mimicked the effects of CAP by inhibiting the acetylcholine exocytosis. Our data suggest that TRPV1 proteins expressed at the MNT are coupled to the exo-endocytic mechanisms to regulate neuromuscular functions.

Spatial and temporal features of superordinate semantic processing studied with fMRI and EEG.

The relationships between the anatomical representation of semantic knowledge in the human brain and the timing of neurophysiological mechanisms involved in manipulating such information remain unclear. This is the case for superordinate semantic categorization-the extraction of general features shared by broad classes of exemplars (e.g., living vs. non-living semantic categories). We proposed that, because of the abstract nature of this information, input from diverse input modalities (visual or auditory, lexical or non-lexical) should converge and be processed in the same regions of the brain, at similar time scales during superordinate categorization-specifically in a network of heteromodal regions, and late in the course of the categorization process. In order to test this hypothesis, we utilized electroencephalography and event related potentials (EEG/ERP) with functional magnetic resonance imaging (fMRI) to characterize subjects' responses as they made superordinate categorical decisions (living vs. non-living) about objects presented as visual pictures or auditory words. Our results reveal that, consistent with our hypothesis, during the course of superordinate categorization, information provided by these diverse inputs appears to converge in both time and space: fMRI showed that heteromodal areas of the parietal and temporal cortices are active during categorization of both classes of stimuli. The ERP results suggest that superordinate categorization is reflected as a late positive component (LPC) with a parietal distribution and long latencies for both stimulus types. Within the areas and times in which modality independent responses were identified, some differences between living and non-living categories were observed, with a more widespread spatial extent and longer latency responses for categorization of non-living items.

Acetylcholinesterase deficiency contributes to neuromuscular junction dysfunction in type 1 diabetic neuropathy.

Diabetic neuropathy is associated with functional and morphological changes of the neuromuscular junction (NMJ) associated with muscle weakness. This study examines the effect of type 1 diabetes on NMJ function. Swiss Webster mice were made diabetic with three interdaily ip injections of streptozotocin (STZ). Mice were severely hyperglycemic within 7 days after the STZ treatment began. Whereas performance of mice on a rotating rod remained normal, the twitch tension response of the isolated extensor digitorum longus to nerve stimulation was reduced significantly at 4 wk after the onset of STZ-induced hyperglycemia. This mechanical alteration was associated with increased amplitude and prolonged duration of miniature end-plate currents (mEPCs). Prolongation of mEPCs was not due to expression of the embryonic acetylcholine receptor but to reduced muscle expression of acetylcholine esterase (AChE). Greater sensitivity of mEPC decay time to the selective butyrylcholinesterase (BChE) inhibitor PEC suggests that muscle attempts to compensate for reduced AChE levels by increasing expression of BChE. These alterations of AChE are attributed to STZ-induced hyperglycemia since similar mEPC prolongation and reduced AChE expression were found for db/db mice. The reduction of muscle end-plate AChE activity early during the onset of STZ-induced hyperglycemia may contribute to endplate pathology and subsequent muscle weakness during diabetes.

Defective neuromuscular transmission in the SOD1 G93A transgenic mouse improves after administration of human umbilical cord blood cells.

To assess the effect of human umbilical cord blood (hUCB) transplantation on neuromuscular transmission in SOD1(G93A) transgenic mice, we studied the probability of neuromuscular transmission (PNMT), a relevant physiological indicator of motor nerve function, in 3 SOD1(G93A) mice transplanted with hUCB and compared to PNMT in 4 SOD1(G93A) mice without cell transplantation and 3 non-mutant SOD1 transgenic mice. For preparations isolated from non-mutant SOD1 transgenic mice, PNMT was 0.93 and 0.84 during the first 5 s of 70 and 90 Hz trains, respectively. PNMT gradually declined to 0.77 and 0.42 at the end of the trains. In striking contrast, PNMT for preparations from non-treated mutant SOD1(G93A) mice was 0.52 and 0.36 in the first 5 s of 70 and 90 Hz trains, respectively (p<0.05). Treatment with hUCB significantly (p<0.05) improved PNMT in SOD1(G93A) preparations. That is, the initial 5 s PNMT was 0.88 and 0.68 for the 70 and 90 Hz stimuli, respectively. We concluded that hUCB transplantation significantly improved PNMT for muscles removed from SOD1(G93A) mice. Testing PNMT in the SOD1(G93A) mouse model could be used as a simple in vitro protocol to detect a positive cellular response to therapeutic interventions in ALS.

A new mouse model for the slow-channel congenital myasthenic syndrome induced by the AChR εL221F mutation.

We have generated a new mouse model for congenital myasthenic syndromes by inserting the missense mutation L221F into the ε subunit of the acetylcholine receptor by homologous recombination. This mutation has been identified in man to cause a mild form of slow-channel congenital myasthenic syndrome with variable penetrance. In our mouse model we observe as in human patients prolonged endplate currents. The summation of endplate potentials may account for a depolarization block at increasing stimulus frequencies, moderate reduced muscle strength and tetanic fade. Calcium and intracellular vesicle accumulation as well as junctional fold loss and organelle degeneration underlying a typical endplate myopathy, were identified. Moreover, a remodeling of neuromuscular junctions occurs in a muscle-dependent pattern expressing variable phenotypic effects. Altogether, this mouse model provides new insight into the pathophysiology of congenital myasthenia and serves as a new tool for deciphering signaling pathways induced by excitotoxicity at peripheral synapses.

Novel mouse model reveals distinct activity-dependent and -independent contributions to synapse development.

The balanced action of both pre- and postsynaptic organizers regulates the formation of neuromuscular junctions (NMJ). The precise mechanisms that control the regional specialization of acetylcholine receptor (AChR) aggregation, guide ingrowing axons and contribute to correct synaptic patterning are unknown. Synaptic activity is of central importance and to understand synaptogenesis, it is necessary to distinguish between activity-dependent and activity-independent processes. By engineering a mutated fetal AChR subunit, we used homologous recombination to develop a mouse line that expresses AChR with massively reduced open probability during embryonic development. Through histological and immunochemical methods as well as electrophysiological techniques, we observed that endplate anatomy and distribution are severely aberrant and innervation patterns are completely disrupted. Nonetheless, in the absence of activity AChRs form postsynaptic specializations attracting motor axons and permitting generation of multiple nerve/muscle contacts on individual fibers. This process is not restricted to a specialized central zone of the diaphragm and proceeds throughout embryonic development. Phenotypes can be attributed to separate activity-dependent and -independent pathways. The correct patterning of synaptic connections, prevention of multiple contacts and control of nerve growth require AChR-mediated activity. In contrast, myotube survival and acetylcholine-mediated dispersal of AChRs are maintained even in the absence of AChR-mediated activity. Because mouse models in which acetylcholine is entirely absent do not display similar effects, we conclude that acetylcholine binding to the AChR initiates activity-dependent and activity-independent pathways whereby the AChR modulates formation of the NMJ.

Automated Neuropsychological Assessment Metrics: repeated assessment with two military samples.

INTRODUCTION: U.S. military troops deploying to war zones are currently administered the Automated Neuropsychological Assessment Metrics (ANAM4) Traumatic Brain Injury (TBI) Battery to establish individual neurocognitive performance baselines. In part, the utility of the ANAM4 TBI Battery baseline measurement depends on test-retest reliability of this instrument. The purpose of this report was to evaluate performance following multiple administrations of the ANAM4 TBI Battery: does performance in a repeated measures paradigm constitute a stable, interpretable indication of baseline neurocognitive ability? METHODS: The data presented here are from the ANAM4 TBI Battery administered four times to a group of U.S. Marines in Study 1 and eight times to a group of New Zealand Defence Force personnel in Study 2. RESULTS: The results show practice effect in five of six performance subtests in both Study 1 and Study 2. DISCUSSION: Results are consistent with expectations that multiple test sessions are required to reach stable performance on some computerized tasks. These results have implications for taking ANAM4 TBI Battery practice effects into account in test administration and in data interpretation.

Recombinant botulinum neurotoxin A heavy chain-based delivery vehicles for neuronal cell targeting.

The long half-life of botulinum neurotoxin serotype A (BoNT/A) in cells poses a challenge in developing post-exposure therapeutics complementary to existing antitoxin strategies. Delivery vehicles consisting of the toxin heavy chain (HC), including the receptor-binding domain and translocation domain, connected to an inhibitory cargo offer a possible solution for rescuing intoxicated neurons in victims paralyzed from botulism. Here, we report the expression and purification of soluble recombinant prototype green fluorescent protein (GFP) cargo proteins fused to the entire BoNT/A-HC (residues 544-1295) in Escherichia coli with up to a 40 amino acid linker inserted between the cargo and BoNT/A-HC vehicle. We show that these GFP-HC fusion proteins are functionally active and readily taken up by cultured neuronal cells as well as by neuronal cells in mouse motor nerve endings.

Effects of hydroxamate metalloendoprotease inhibitors on botulinum neurotoxin A poisoned mouse neuromuscular junctions.

Currently the only therapy for botulinum neurotoxin A (BoNT/A) poisoning is antitoxin. Antidotes that are effective after BoNT/A has entered the motor nerve terminals would dramatically benefit BoNT/A therapy. Inhibition of proteolytic activity of BoNT/A light chain by metalloendoprotease inhibitors (MEIs) is under development. We tested the effects of MEIs on in vitro as well as in vivo BoNT/A poisoned mouse nerve-muscle preparations (NMPs). The K(i) for inhibition of BoNT/A metalloendoprotease was 0.40 and 0.36 muM, respectively, for 2,4-dichlorocinnamic acid hydroxamate (DCH) and its methyl derivative, ABS 130. Acute treatment of nerve-muscle preparations with 10 pM BoNT/A inhibited nerve-evoked muscle twitches, reduced mean quantal content, and induced failures of endplate currents (EPCs). Bath application of 10 muM DCH or 5 muM ABS 130 reduced failures, increased the quantal content of EPCs, and partially restored muscle twitches after a delay of 40-90 min. The restorative effects of DCH and ABS 130, as well as 3,4 diaminopyridine (DAP) on twitch tension were greater at 22 degrees C compared to 37 degrees C. Unlike DAP, neither DCH nor ABS 130 increased Ca(2+) levels in cholinergic Neuro 2a cells. Injection of MEIs into mouse hind limbs before or after BoNT/A injection neither prevented the toe spread reflex inhibition nor improved muscle functions. We suggest that hydroxamate MEIs partially restore neurotransmission of acutely BoNT/A poisoned nerve-muscle preparations in vitro in a temperature dependent manner without increasing the Ca(2+) levels within motor nerve endings.

Neural aspects of sentence comprehension: syntactic complexity, reversibility, and reanalysis.

Broca's area is preferentially activated by reversible sentences with complex syntax, but various linguistic factors may be responsible for this finding, including syntactic movement, working-memory demands, and post hoc reanalysis. To distinguish between these, we tested the interaction of syntactic complexity and semantic reversibility in a functional magnetic resonance imaging study of sentence-picture matching. During auditory comprehension, semantic reversibility induced selective activation throughout the left perisylvian language network. In contrast, syntactic complexity (object-embedded vs. subject-embedded relative clauses) within reversible sentences engaged only the left inferior frontal gyrus (LIFG) and left precentral gyrus. Within irreversible sentences, only the LIFG was sensitive to syntactic complexity, confirming a unique role for this region in syntactic processing. Nonetheless, larger effects of reversibility itself occurred in the same regions, suggesting that full syntactic parsing may be a nonautomatic process applied as needed. Complex reversible sentences also induced enhanced signals in LIFG and left precentral regions on subsequent picture selection, but with additional recruitment of the right hemisphere homolog area (right inferior frontal gyrus) as well, suggesting that post hoc reanalysis of sentence structure, compared with initial comprehension, engages an overlapping but larger network of brain regions. These dissociable effects may offer a basis for studying the reorganization of receptive language function after brain damage.

Capsaicin protects mouse neuromuscular junctions from the neuroparalytic effects of botulinum neurotoxin a.

Botulinum neurotoxin A (BoNT/A), the most toxic, naturally occurring protein, cleaves synapse-associated protein of 25 kDa and inhibits acetylcholine release from motor nerve endings (MNEs). This leads to paralysis of skeletal muscles. Our study demonstrates that capsaicin protects mouse neuromuscular junctions from the neuroparalytic effects of BoNT/A. Bilateral injection of BoNT/A near the innervation of the Extensor digitorum longus (EDL) muscle of adult Swiss-Webster mice inhibited the toe spread reflex (TSR). However, when capsaicin was coinjected bilaterally, or injected 4 or 8 h before injecting BoNT/A, the TSR remained normal. In animals that were pretreated with capsazepine, capsaicin failed to protect against the neuroparalytic effects of BoNT/A. In vivo analyses demonstrated that capsaicin protected muscle functions and electromygraphic activity from the incapacitating effects of BoNT/A. The twitch response to nerve stimulation was greater for EDL preparations isolated from mice injected with capsaicin before BoNT/A. Capsaicin pretreatment also prevented the inhibitory effects of BoNT/A on end-plate currents. Furthermore, pretreatment of Neuro 2a cells with capsaicin significantly preserved labeling of synaptic vesicles by FM 1-43. This protective effect of capsaicin was observed only in the presence of extracellular Ca(2+) and was inhibited by capsazepine. Immunohistochemistry demonstrated that MNEs express transient receptor potential protein of the vanilloid subfamily, TRPV1, the capsaicin receptor. Capsaicin pretreatment, in vitro, reduced nerve stimulation or KCl-induced uptake of BoNT/A into motor nerve endings and cholinergic Neuro 2a cells. These data demonstrate that capsaicin interacts with TRPV1 receptors on MNEs to reduce BoNT/A uptake via a Ca(2+)-dependent mechanism.

Motor unit number estimate as a predictor of motor dysfunction in an animal model of type 1 diabetes.

Peripheral neuropathy is a common complication of diabetes that leads to severe morbidity. In this study, we investigated the sensitivity of motor unit number estimate (MUNE) to detect early motor axon dysfunction in streptozotocin (STZ)-treated mice. We compared the findings with in vitro changes in the morphology and electrophysiology of the neuromuscular junction. Adult Thy1-YFP and Swiss Webster mice were made diabetic following three interdaily intraperitoneal STZ injections. Splay testing and rotarod performance assessed motor activity for 6 wk. Electromyography was carried out in the same time course, and compound muscle action potential (CMAP) amplitude, latency, and MUNE were estimated. Two-electrode voltage clamp was used to calculate quantal content (QC) of evoked transmitter release. We found that an early reduction in MUNE was evident before a detectable decline of motor activity. CMAP amplitude was not altered. MUNE decrease accompanied a drop of end-plate current amplitude and QC. We also observed small axonal loss, sprouting of nerve endings, and fragmentation of acetylcholine receptor clusters at the motor end plate. Our results suggest an early remodeling of motor units through the course of diabetic neuropathy, which can be readily detected by the MUNE technique. The early detection of MUNE anomalies is significant because it suggests that molecular changes associated with pathology and leading to neurodegeneration might already be occurring at this stage. Therefore, trials of interventions to prevent motor axon dysfunction in diabetic neuropathy should be administered at early stages of the disorder.

Strategies for longitudinal neuroimaging studies of overt language production.

Longitudinal fMRI studies of language production are of interest for evaluating recovery from post-stroke aphasia, but numerous methodological issues remain unresolved, particularly regarding strategies for evaluating single subjects at multiple timepoints. To address these issues, we studied overt picture naming in eleven healthy subjects, scanned four times each at one-month intervals. To evaluate the natural variability present across repeated sessions, repeated scans were directly contrasted in a unified statistical framework on a per-voxel basis. The effect of stimulus familiarity was evaluated using explicitly overtrained pictures, novel pictures, and untrained pictures that were repeated across sessions. For untrained pictures, we found that activation declined across multiple sessions, equally for both novel and repeated stimuli. Thus, no repetition priming for individual stimuli at one-month intervals was found, but rather a general effect of task habituation was present. Using a set of overtrained pictures identical in each session, no decline was found, but activation was minimized and produced less consistent patterns across participants, as measured by intra-class correlation coefficients. Subtraction of a baseline task, in which subjects produced a stereotyped utterance to scrambled pictures, resulted in specific activations in the left inferior frontal gyrus and other language areas for untrained items, while overlearned stimuli relative to pseudo pictures activated only the fusiform gyrus and supplementary motor area. These findings indicate that longitudinal fMRI is an effective means of detecting changes in neural activation magnitude over time, as long as the effect of task habituation is taken into account.

Peptide-toxin tools for probing the expression and function of fetal and adult subtypes of the nicotinic acetylcholine receptor.

Although the neuromuscular nicotinic acetylcholine receptor (nAChR) is one of the most intensively studied ion channels in the nervous system, the differential roles of fetal and adult subtypes of the nAChR under normal and pathological conditions are still incompletely defined. Until recently, no pharmacological tools distinguished between fetal and adult subtypes. Waglerin toxins (from snake venom) and alphaA(S)-conotoxins (from cone-snail venom) have provided such tools. Because these peptides were characterized by different research groups using different methods, we have: 1) more extensively tested their subtype selectivity, and 2) begun to explore how these peptides may be used in concert to elucidate expression patterns and functions of fetal and adult nAChRs. In heterologous expression systems and native tissues, Waglerin-1 and an alphaA(S)-conotoxin analog, alphaA-OIVA[K15N], are high-affinity, highly selective inhibitors of the adult and fetal muscle nAChRs, respectively. We have used the peptides and their fluorescent derivatives to explore the expression and function of the fetal and adult nAChR subtypes. While fluorescent derivatives of these peptides indicated a gradual transition from fetal to adult muscle nAChRs in mice during the first 2 weeks postnatal, we unexpectedly observed a steeper transition in functional expression in the mouse diaphragm muscle using electrophysiology. As a toolkit of pharmacological agents with complementary specificity, alphaA-OIVA[K15N] and Waglerin-1 should have further utility in determining the roles of fetal and adult nAChR subtypes in development, in mature tissues, and under pathological conditions.