Anne Baroffio

Activation of nicotinic acetylcholine receptors increases the rate of fusion of cultured human myoblasts.

1. Fusion of myogenic cells is important for muscle growth and repair. The aim of this study was to examine the possible involvement of nicotinic acetylcholine receptors (nAChR) in the fusion process of myoblasts derived from postnatal human satellite cells. 2. Acetylcholine‐activated currents (ACh currents) were characterized in pure preparations of freshly isolated satellite cells, proliferating myoblasts, myoblasts triggered to fuse and myotubes, using whole‐cell and single‐channel voltage clamp recordings. Also, the effect of cholinergic agonists on myoblast fusion was tested. 3. No nAChR were observed in freshly isolated satellite cells. nAChR were first observed in proliferating myoblasts, but ACh current densities increased markedly only just before fusion. At that time most mononucleated myoblasts had ACh current densities similar to those of myotubes. ACh channels had similar properties at all stages of myoblast maturation. 4. The fraction of myoblasts that did not fuse under fusion‐promoting conditions had no ACh current and thus resembled freshly isolated satellite cells. 5. The rate of myoblast fusion was increased by carbachol, an effect antagonized by alpha‐bungarotoxin, curare and decamethonium, but not by atropine, indicating that nAChR were involved. Even though a prolonged exposure to carbachol led to desensitization, a residual ACh current persisted after several days of exposure to the nicotinic agonist. 6. Our observations suggest that nAChR play a role in myoblast fusion and that part of this role is mediated by the flow of ions through open ACh channels.

A voltage-dependent proton current in cultured human skeletal muscle myotubes.

1. A voltage‐dependent proton current, IH, was studied in cultured myotubes obtained from biopsies of human muscle, using whole‐cell recording with the patch‐clamp technique. 2. With a pHo of 8.0 and a calculated pHi of 6.3, IH was activated at voltages more depolarized than ‐50 mV and its conductance reached its maximum value at voltages more depolarized than +10 mV. 3. Studies of the reversal potential of IH during substitution of K+, Na+, Ca2+, Cl‐, Cs+ and H+ in the extracellular solution indicated that protons were the major charge carriers of IH. 4. IH was also activated during a voltage step to +22 mV with a pHo of 7.3 and a calculated pHi of 7.3. 5. Acidification of the extracellular solution led to a shift towards depolarized voltages of the conductance‐voltage relationship. 6. Stationary noise analysis of IH suggested that the elementary event underlying IH was very small with a conductance of less than 0.09 pS. 7. Extracellular application of various divalent cations blocked IH. The block by divalent cations was voltage dependent, being more efficient at hyperpolarized than at depolarized voltages. For Cd2+, the Michaelis‐Menten constant (Km) for the block was 0.6 microM at ‐28 mV and 10.4 microM at +12 mV. 8. Ca2+ was a less efficient blocker than Cd2+ but could block IH at physiological concentrations (the Km values for the block were 0.9 mM at ‐38 mV and 7.3 mM at ‐8 mV). 9. The voltage‐dependent properties of IH and its ability to be affected by pH and Ca2+ suggest that IH might be used by skeletal muscle cells to extrude protons during action potentials. 10. A model of IH activation suggests that under extreme conditions, the conductance of IH can reach 40% of its maximum value after less than ten action potentials.

Needle muscle biopsy in the investigation of neuromuscular disorders.

We have evaluated needle muscle biopsies in 220 patients with various neuromuscular disorders, using a method developed previously at Tufts University. The method uses a 14‐gauge needle propelled by an automatic device. An average of 3.5 samplings were taken per patient. Muscle samples were used for histological and molecular genetic analysis, and for the isolation of muscle satellite cells for in vitro cultures. The biopsy is well tolerated by the patients who never declined multiple samplings. Complications were few and minor, with no sequelae. In most cases the small size of the muscle specimen (ca. 15 mg per sampling) was sufficient to perform the various procedures and to yield a diagnosis. Specimens were considered insufficient for histological results in 9 patients (4%), due to technical artifacts or insufficient material. We now routinely use this method, which has several advantages over the surgical technique for most muscle biopsies.

Synthesis and release of an acetylcholine-like compound by human myoblasts and myotubes.

1. Exogenously applied acetylcholine (ACh) is a modulator of human myoblast fusion. Using a chemiluminescent method, we examined whether an endogenous ACh‐like compound (ACh‐lc) was present in, and released by, pure human myogenic cells. 2. Single, freshly isolated satellite cells and proliferating myoblasts contained 15 and 0.5 fmol ACh‐lc, respectively. Cultured myotubes contained ACh‐lc as well. Also, ACh‐like immunoreactivity was detected in all myogenic cells. 3. Part of the ACh‐lc was synthesized by choline acetyltransferase (ChAT), as indicated by the reduction of ACh‐lc content when bromoACh was present in the culture medium, and by direct measurements of ChAT activity. Also, ChAT‐like immunoreactivity was observed in all myogenic cells. 4. Myoblasts and myotubes released ACh‐lc spontaneously by a partially Ca(2+)‐dependent mechanism. 5. The application by microperfusion of medium conditioned beforehand by myoblasts (thus presumably containing ACh‐lc) onto a voltage‐clamped myotube induced inward currents resembling ACh‐induced currents in their kinetics, reversal potential, and sensitivity to nicotinic antagonists. 6. In vitro, the spontaneously released ACh‐lc promoted myoblast fusion but only in the presence of an anticholinesterase. 7. Our observations indicate that human myogenic cells synthesize and release an ACh‐lc and thereby promote the fusion process that occurs in muscle during growth or regeneration.

Sodium and potassium currents in freshly isolated and in proliferating human muscle satellite cells.

1. Human muscle satellite cells (SC) were studied either immediately after dissociation of muscle biopsies or later, as they proliferated in culture. A purification procedure combined with clonal cultures ensured that electrophysiological recordings were done in myogenic cells. Hoechst staining for the DNA attested that cells were mononucleated. 2. The goals of this study were to examine (i) whether the electrophysiological properties of freshly isolated SC resembled those of SC that proliferated in culture for several weeks, (ii) whether freezing and thawing affected these properties, and (iii) whether SC constituted a homogeneous population. 3. We found that there were only subtle differences between the electrophysiological results obtained in freshly isolated SC and in proliferating SC with or without previous freezing and thawing. Most SC expressed two voltage‐gated currents, a TTX‐resistant Na+ current and a calcium‐activated potassium current (IK, Ca). 4. The level of expression of the Na+ current and of IK, Ca was affected in a different way by cellular proliferation; the normalized Na+ conductance (pS pF‐1) of proliferating cells resembled that of freshly isolated SC, whereas the IK, Ca conductance increased 10 times. The analysis of the amplitude distributions of the Na+ current and of IK, Ca in the various SC preparations suggested that there was only one class of SC.

Effect of teaching context and tutor workshop on tutorial skills

Effective faculty development workshops are essential to develop and sustain the quality of facultys teaching. In an integrated problem-based curriculum, tutors expressed the needs to further develop their skills in facilitating students’ content learning and small-group functioning. Based on the authors’ prior observations that tutors’ performance depends on their teaching context, a workshop was designed not only tailored to the tutors’ needs but also organized within their respective teaching unit. The purposes of this study are (1) to evaluate whether this workshop is effective and improves tutors’ teaching skills, and (2) to assess whether workshop effectiveness depends on tutors’ performance before the workshop and on their teaching unit environment. Workshop effectiveness was assessed using (a) tutors’ perception of workshop usefulness and of their improvement in tutorial skills, and (b) students’ ratings of tutor performance before and after the workshop. In addition, an analysis of variance model was designed to analyse how tutors’ performance before the workshop and their teaching unit influence workshop effectiveness. Tutors judged the workshop as helpful in providing them with new teaching strategies and reported having improved their tutorial skills. Workshop attendance enhanced students’ ratings of tutors’ knowledge of problem content and ability to guide their learning. This improvement was also long-lasting. The workshop effect on tutor performance was relative: it varied across teaching units and was higher for tutors with low scores before the workshop. A workshop tailored to tutors’ needs and adapted to their teaching unit improves their tutorial skills. Its effectiveness is, however, influenced by tutors’ level of performance before the workshop and by the environment of their teaching unit. Thus, to be efficient, the design of a workshop should consider not only individual tutors’ needs, but also the background of their teaching units, with special attention to their internal organization and tutor group functioning.

Effectiveness of a training program in supervisors' ability to provide feedback on residents' communication skills.

Teaching communication skills (CS) to residents during clinical practice remains problematic. Direct observation followed by feedback is a powerful way to teach CS in clinical practice. However, little is known about the effect of training on feedback skills in this field. Controlled studies are scarce as well as studies that go beyond self-reported data. The aim of the study was to develop and assess the effectiveness of a training program for clinical supervisors on how to give feedback on residents’ CS in clinical practice. The authors designed a pretest–posttest controlled study in which clinical supervisors working in two different medical services were invited to attend a sequenced and multifaceted program in teaching CS over a period of 6–9xa0months. Outcome measures were self-perceived and observed feedback skills collected during questionnaires and three videotaped objective structured teaching encounters. The videotaped feedbacks made by the supervisors were analysed using a 20-item feedback rating instrument. Forty-eight clinical supervisors participated (28 in the intervention, 20 in the control group). After training, a higher percentage of trained participants self-reported and demonstrated statistically significant improvement in making residents more active by exploring residents’ needs, stimulating self-assessment, and using role playing to test strategies and checking understanding, with effect sizes ranging from 0.93 to 4.94. A training program on how to give feedback on residents’ communication skills was successful in improving clinical supervisors’ feedback skills and in helping them operate a shift from a teacher-centered to a more learner-centered approach.

Effects of item and rater characteristics on checklist recording: what should we look for?

Objectiveu2002 Examinations based on using standardised patients (SPs) commonly use checklist recordings to evaluate students clinical performance. This paper examines whether and to what extent item and rater characteristics affect the reliability of history checklist recording in an SP‐based assessment.

The New Preclinical Medical Curriculum at the University of Geneva: Processes of Selecting Basic Medical Concepts and Problems for the PBL Learning Units

On October 1995, the medical faculty of the University of Geneva has started a new second and third years preclinical curriculum. It consists of 16 integrated “Problem-Based Learning (PBL) Units”. The first “Introduction” Unit of 2 weeks is aimed at introducing students with the PBL technique. Eleven PBL Units each of one month duration, comprise of 7 to 8 problems with themes related to important body functions or organ. Finally 4 Synthesis Units, each of 2 weeks duration, and following 2 to 3 PBL Units, comprise 4 problems aimed at integrating concepts common to preceding Units. The elaboration of such a new PBL curriculum requires to address two main issues, namely 1) how to select the important basic medical concepts and relevant problems leading to the learning objectives of each learning Unit and 2) how to verify the quality and relevance of the designed problems. This paper is aimed mainly at describing the steps followed in Geneva to solve these issues. First, it will present the process of selecting the basic science concepts, designing the problems and establishing their sequence. Second, it will describe the process of verifying whether problems fit to the identified concepts, lead to learning objectives and reach the appropriate level of learning for the students.

Evolutionary trends of problem-based learning practices throughout a two-year preclinical program: a comparison of students’ and teachers’ perceptions

Implementation of a pedagogical approach is a continuous and evolving process. As an institution with more than 15xa0years problem-based learning (PBL), we studied how the learning and teaching processes are currently practiced in a 2-year preclinical basic sciences program to assess whether they still match the intended objectives. Using both students’ and tutors’ evaluations, we analyzed and compared their perceptions on the program content and its organization, on tutors’ functioning and on the duration of PBL sessions throughout 11 instructional units of the second and third-year of a 6xa0years medical curriculum. Whereas both tutors and students indicated that the content and problems selected for the curriculum were well adapted to the PBL process, they felt the references as well as the self-study time as moderately appropriate. Over the course of the 2-year program, tutorial sessions got linearly shorter, whereas reporting sessions got longer. While tutors knew well the PBL approach and were suitably prepared to their tutorials, they however, became less regular in providing feedback and in evaluating group functioning over the 2xa0years. Our results suggest that the practice of the PBL process evolves within and throughout a 2-year preclinical program and moves away from the original intentions. Possible underlying reasons and their implications are discussed within the context of tutors’ and students’ concepts of teaching and learning, the medical schools’ learning environment and teaching practices and the difficulty of developing and maintaining in the long term a deep and self-directed learning approach.