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.

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.

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.

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.

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.

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.