Automatic processing of self-regulation of slow cortical potentials: evidence from brain-computer communication in paralysed patients.

OBJECTIVE: Direct brain-computer communication utilizes self-regulation of brain potentials to select letters, words or symbols from a computer menu. Selection of letters or words with brain potentials requires simultaneous processing of several tasks such as production of certain brain potentials at predefined time points simultaneously with processing of presented letter strings. This study addresses the question of whether the self-regulation of slow cortical potentials (SCP) automatizes with practice and can thus be considered as a skill comparable to motor or cognitive skills. METHODS: Two nearly completely paralysed patients learned over several months to produce electrocortically negative and positive SCP by means of visual feedback. Improved performance and a reduction in performance variability were regarded as behavioural indicators for automaticity, while the topographic focalization of cortical activation was considered as a neurophysiological indicator for automaticity. Different indicators of automaticity were expected to covary along with practice. RESULTS: In patient 1, performance measured as the percentage of correct SCP shifts increased simultaneously with the topographic focalization of cortical activation. His performance became more stable with practice. For this patient the criteria for automaticity were all met. In patient 2, performance also improved, but his cortical activity became topographically less focal. His performance was less stable than that of patient 1. CONCLUSIONS: The present findings, albeit on only two subjects, provide preliminary evidence that SCP self-regulation may automatize with long-term practice and can therefore be considered a skill.

A non-invasive communication device for the paralyzed.

An EEG-based communication system has been developed to re-establish communication in severely paralyzed patients who operate the device by generating shifts of their slow cortical potentials. Training to gain control over slow cortical potentials was based on visual feedback and operant conditioning strategies. The vertical movement of a graphic signal on a computer screen informs the patients about the course of their slow cortical potential amplitude. Positive slow cortical potential shifts move the cursor up, negative shifts move it down. These shifts are then translated into binary responses. When a patient has achieved reliable control over his/her slow cortical potential shifts, these responses can be used to select or reject items presented at the bottom of the screen. As learning processes and applications differ considerably between patients, the present paper describes the data from one patient with amyotrophic lateral sclerosis. After about three months of training, this patient gained stable, near-perfect control over his slow cortical potentials. This skill enabled him to operate a specially designed program to communicate messages to his caregivers.

Brain-computer communication: self-regulation of slow cortical potentials for verbal communication.

OBJECTIVE: To test a training procedure designed to enable severely paralyzed patients to communicate by means of self-regulation of slow cortical potentials. DESIGN: Application of the Thought Translation Device to evaluate the procedure in patients with late-stage amyotrophic lateral sclerosis (ALS). SETTING: Training sessions in the patients' homes. PARTICIPANTS: Two male patients with late-stage ALS. INTERVENTIONS: Patients learned voluntary control of their slow cortical potentials by means of an interface between the brain and a computer. Training was based on visual feedback of slow cortical potentials shifts and operant learning principles. The learning process was divided into small steps of increasing difficulty. MAIN OUTCOME MEASURES: Accuracy of self-control of slow cortical potentials (percentage of correct responses). Learning progress calculated as a function of training session. RESULTS: Within 3 to 8 weeks, both patients learned to self-regulate their slow cortical potentials and to use this skill to select letters or words in the Language Support Program. CONCLUSIONS: This training schedule is the first to enable severely paralyzed patients to communicate without any voluntary muscle control by using self-regulation of an electroencephalogram potential only. The protocol could be a model for training patients in other brain-computer interface techniques.

Self-initiation of EEG-based communication in paralyzed patients.

OBJECTIVES: Severely paralyzed patients could learn to voluntarily generate slow cortical potential (SCP) shifts in their electroencephalogram and to use these signals to operate a communication device. To enhance the patients' autonomy, the present study describes the development of a permanently available communication system that can be turned on and off by locked-in patients without external assistance. A skill necessary for turning the system on is the ability to regulate one's slow potentials in the absence of continuous feedback. METHODS: A stepwise learning approach was employed to train two paralyzed patients to regulate their SCPs without continuous feedback. Elements of the original communication system were gradually removed and elements of the new stand-by mode were introduced. RESULTS: At the end of the learning procedure, both patients achieved correct response rates of above 84% in training sessions without continuous feedback. This skill enabled them to turn the communication device on and off without assistance from others. CONCLUSIONS: These findings suggest that severely paralyzed individuals can learn to operate an EEG-based communication device autonomously.

The thought translation device (TTD) for completely paralyzed patients.

The thought translation device trains locked-in patients to self-regulate slow cortical potentials (SCP's) of their electroencephalogram (EEG). After operant learning of SCP self-control, patients select letters, words or pictograms in a computerized language support program. Results of five respirated, locked-in-patients are described, demonstrating the usefulness of the thought translation device as an alternative communication channel in motivated totally paralyzed patients with amyotrophic lateral sclerosis.

The thought translation device: a neurophysiological approach to communication in total motor paralysis.

A thought translation device (TTD) for brain-computer communication is described. Three patients diagnosed with amyotrophic lateral sclerosis (ALS), with total motor paralysis, were trained for several months. In order to enable such patients to communicate without any motor activity, a technique was developed where subjects learn to control their slow cortical potentials (SCP) in a 2-s rhythm, producing either cortical negativity or positivity according to the task requirement. SCP differences between a baseline interval and an active control interval are transformed into vertical or horizontal cursor movements on a computer screen. Learning SCP self regulation followed an operant-conditioning paradigm with individualized shaping procedures. After prolonged training over more than 100 sessions, all patients achieved self-control, leading to a 70-80% accuracy for two patients. The learned cortical skill enabled the patients to select letters or words in a language-supporting program (LSP) developed for inter-personal communication. The results demonstrate that the fast and stable SCP self-control can be achieved with operant training and without mediation of any muscle activity. The acquired skill allows communication even in total locked-in states.

Distal regulatory regions of the rat MRF4 gene.

MRF4 is a muscle-specific transcription factor that is expressed both in embryonic somites and later in fetal and adult muscle fibers. Cis-regulatory elements of the MRF4 gene responsible for its complex expression pattern have not yet been identified, although previous studies of the rat MRF4 gene have demonstrated the presence of enhancer activity located several kilobases 5' to the transcription start site. Using cell transfection assays in vitro, we have now localized one of the regulatory regions of MRF4 to a 590-base-pair sequence between 4 and 5 kilobases upstream from the start site. This sequence region functioned as an enhancer in combination either with the MRF4 promoter or with the viral thymidine kinase (tk) promoter. Deletion analysis of MRF4 indicated the existence of another regulatory region, closer to the promoter, which functioned as an enhancer in combination with the MRF4 promoter but not with the tk promoter.

Myogenin and MEF2 function synergistically to activate the MRF4 promoter during myogenesis.

The basic helix-loop-helix muscle regulatory factor (MRF) gene family encodes four distinct muscle-specific transcription factors known as MyoD, myogenin, Myf-5, and MRF4. These proteins represent key regulatory factors that control many aspects of skeletal myogenesis. Although the MRFs often exhibit overlapping functional activities, their distinct expression patterns during embryogenesis suggest that each protein plays a unique role in controlling aspects of muscle development. As a first step in determining how MRF4 gene expression is developmentally regulated, we examined the ability of the MRF4 gene to be expressed in a muscle-specific fashion in vitro. Our studies show that the proximal MRF4 promoter contains sufficient information to direct muscle-specific expression. Located within the proximal promoter are a single MEF2 site and E box that are required for maximum MRF4 expression. Mutation of the MEF2 site or E box severely impairs the ability of this promoter to produce a muscle-specific response. In addition, the MEF2 site and E box function in concert to synergistically activate the MRF4 gene in nonmuscle cells coexpressing MEF2 and myogenin proteins. Thus, the MRF4 promoter is regulated by the MEF2 and basic helix-loop-helix MRF protein family through a cross-regulatory circuitry. Surprisingly, the MRF4 promoter itself is not transactivated by MRF4, suggesting that this MRF gene is not subject to an autoregulatory pathway as previously implied by other studies. Understanding the molecular mechanisms regulating expression of each MRF gene is central to fully understanding how these factors control developmental events.

Structure and myofiber-specific expression of the rat muscle regulatory gene MRF4.

We have cloned an 11.3-kb rat genomic DNA fragment encompassing the muscle regulatory factor 4 (MRF4) protein-coding sequence, 8.5 kb of 5'-flanking sequence, and 1.0 kb of 3'-flanking sequence. In order to study MRF4 gene expression, the rat myogenic cell line, L6J1-C, which expresses the endogenous MRF4 gene only in differentiated myofibers, was transfected stably with the full-length genomic clone and various 5' deletions. RNase protection assays demonstrated that MRF4 genes containing as little as 430 bp of 5'-flanking sequence exhibited an increase in expression as the cells differentiated into myofibers, indicating that elements responsible for fiber-specific expression are contained within this cloned DNA fragment. Similar up-regulation was observed with genes containing 1.5 kb of 5'-flanking sequence. Interestingly, MRF4 genes containing 5.0 kb and 8.5 kb of 5'-flanking sequence were up-regulated to even higher levels, suggesting that additional myofiber-specific regulatory elements located between 1.5 and 5.0 kb upstream from the coding region play a role in regulating the expression of this muscle-specific gene.

Expression of the muscle regulatory factor MRF4 during somite and skeletal myofiber development.

The muscle regulatory factors MRF4, myogenin, myf-5, and MyoD constitute a family of proteins that can function as muscle-specific transcriptional activators. Although this gene family has been extensively studied, a specific role for each factor during myogenesis remains to be determined. Understanding how these factors function requires a detailed analysis of their expression patterns during development. Toward this goal, we examined the temporal pattern of expression of MRF4 and the other factors in the rat myogenic cell line L6J1-C, in newborn rat primary muscle cell cultures and in fetal and postnatal rat limb muscle. Our results demonstrate that MyoD, myogenin, and myf-5 transcripts accumulate maximally at various stages of myoblast differentiation and decline to low expression levels in adult muscle tissue. In contrast, MRF4 transcript accumulation is restricted to cell cultures containing multinucleate myofibers, and its expression in vivo increases sharply during late fetal muscle development. This level of MRF4 expression is maintained in the adult which, together with decreased expression of the other three muscle regulatory factors, makes MRF4 the predominant factor in adult muscle. In situ hybridization of mouse embryo tissue sections indicates that MRF4 transcripts accumulate in the limb beginning 13.5 days post coitum, which is 2 days later than the initial appearance of myogenin and MyoD transcripts. Hybridization to earlier stages of development reveals, however, that MRF4 mRNA initially is present in the myotomal compartment of the somites, just after myogenin but 2 days prior to MyoD expression. Unlike myogenin and MyoD, MRF4 expression declines in the myotomes at the time that multinucleate axial muscles begin to form in this region, although during later development MRF4 is expressed in the myofibers of axial muscles at levels comparable to those in the limb. Differences in the expression patterns for MRF4, myogenin, myf-5 and MyoD between myotomal and other skeletal muscle development suggest that the relative timing of expression for each muscle regulatory factor may control the distinct phenotypes associated with myotomal myocytes and multinucleate myofibers.

[The role of the myoblasts and connective tissue in the regeneration of the limbs in amphibians]. / Rol' mioblastov i soedinitel'noi tkani v regeneratsii konechnostei u amfibii.

A possibility of tissue metaplasia (transformation of one cell type into another) during limb regeneration in lower vertebrates has been a matter of vivid arguments over the last decades. These discussions are rather irreconcilable in character mainly due to the lack of reliable cell markers which permit to follow all the stages of cell transformation during metaplasia. The final conclusions can be made only if any artifacts of cell labelling are excluded. Latest findings obtained using nuclear and cytoplasmic markers are presented which suggest that many data interpreted previously as a convincing proof of metaplasia may be a consequence of the involvement of nondifferentiated cells in regeneration. Molecular biological approaches are believed to be most promising for the solution of disputable problems of tissue metaplasia. However, recent findings about actin gene hypomethylation are insufficient for any final conclusions about the possibility of metaplasia during limb regeneration. The answer to many unsolved questions of developmental biology can be made only when combined use is made of modern methods of cell and molecular biology.

Fusion between myoblasts and adult muscle fibers promotes remodeling of fibers into myotubes in vitro.

Muscle satellite cells are residual embryonic myoblast precursors responsible for muscle growth and regeneration. In order to examine the role of satellite cells in the initial events of muscle regeneration, we placed individual mature rat muscle fibers in vitro along with their satellite cells. When the satellite cells were allowed to proliferate, they produced populations of myoblasts that fused together to form myotubes on the laminin substrate. These myoblasts and myotubes also fused with the adult fibers. When they did so, the fibers lost their adult morphology, and by 8 days in vitro, essentially all of them were remodeled into structures resembling embryonic myotubes. However, when proliferating satellite cells were eliminated by exposure to cytosine arabinoside (araC), the vast majority of fibers retained their adult shape. Addition of C2C12 cells (a myoblast line derived from adult mouse satellite cells) to araC-treated fiber cultures resulted in their fusion with the rat muscle fibers and restored the ability of the fibers to remodel, whereas addition of either a fibroblast cell line or a transformed, non-fusing variant of C2C12 cells, or addition of conditioned medium from C2C12 cells, failed to do so. These results imply that myoblast fusion is responsible for triggering adult fiber remodeling in vitro.

Muscle and cartilage differentiation in axolotl limb regeneration blastema cultures.

A tissue culture system is described for explants of mesenchyme from Ambystoma mexicanum limb regeneration blastemas. Explants were cultured on collagen substrate for 3 weeks in minimal essential medium supplemented with the hormones insulin, thyroxine, somatotropin, and hydrocortisone, plus beef embryo extract (EE), 2%. This medium supported extensive cell migration onto the substrate followed by cell proliferation and differentiation of both cartilage matrix and myotubes. Cultures on plastic substrate, rather than on collagen, displayed similar cell outgrowth and cartilage formation, but relatively little myotube formation. Differentiation in EE-supplemented medium was compared with that in two defined media: Explants in medium containing only the hormones showed little outgrowth or cartilage development and never formed myotubes; medium containing the hormones plus fibroblast growth factor, 50 ng/ml, supported an intermediate degree of outgrowth and cartilage development and occasional myotube formation. Explant size was also a factor: Smaller explants survived and formed myotubes less frequently, even when on collagen in EE-supplemented medium.