[Electives during preclinical medical curriculum: the Geneva experience ]. / Modules à option au cours des études de médecine: l'expérience genevoise.

Electives have come of age in Medical Schools throughout Europe since the Bologna Guidelines were issued. At the Faculty of Medicine of Geneva its importance was recognized early to satisfy the students' curiosity, enlarge their visions, or deepen their knowledge in certain aspects of their curriculum. It was therefore decided to develop a great number of different electives ranging from basic biomedical research to emblematic clinical syndromes and humanitarian medicine for 2nd and 3rd year Bachelor students. The Electives taken have to be validated through specific examinations corresponding to 10% of the yearly ECTS credits. The experience has so far been a success, with high satisfaction as well of the student as of the teacher body. A major challenge for the future will be opening further these Electives to other Faculties and other professions.

Human myoblast fusion requires expression of functional inward rectifier Kir2.1 channels.

Myoblast fusion is essential to skeletal muscle development and repair. We have demonstrated previously that human myoblasts hyperpolarize, before fusion, through the sequential expression of two K+ channels: an ether-à-go-go and an inward rectifier. This hyperpolarization is a prerequisite for fusion, as it sets the resting membrane potential in a range at which Ca2+ can enter myoblasts and thereby trigger fusion via a window current through alpha1H T channels.

Inhibition of N- and L-type calcium channels by muscarinic receptor activation in rat sympathetic neurons.

Modulation of N- and L-type Ca2+ channels by oxotremorine-M (oxo-M) acting on muscarinic receptors and norepinephrine (NE) acting on alpha-adrenergic receptors was studied in superior cervical ganglion neurons. Oxo-M depresses dihydropyridine-augmented tail currents in whole-cell recordings, whereas NE does not. This modulation of L-type Ca2+ channels by oxo-M is abolished by adding 20 mM BAPTA to the pipette solution. Oxo-M, acting via a diffusible messenger, reduces the probability of opening of single N- and L-type channels recorded in cell-attached patches. We conclude that a diffusible messenger signaling pathway activated by oxo-M inhibits both N- and L-type Ca2+ channels, whereas a membrane-delimited pathway activated by oxo-M and NE inhibits only N-type Ca2+ channels.

Pertussis toxin and voltage dependence distinguish multiple pathways modulating calcium channels of rat sympathetic neurons.

Agonist-induced suppression of current in voltage-gated Ca2+ channels was studied in rat sympathetic neurons. We have previously distinguished two intracellular signaling pathways used by muscarinic agonists to suppress neuronal Ca2+ current-one fast and membrane delimited, the other slow and acting via a diffusible second messenger. We now show that the fast pathway is sensitive mainly to pertussis toxin and shifts the gating of Ca2+ channels to more positive voltages (voltage dependent). The slow pathway is pertussis toxin insensitive and depresses currents at all test potentials (voltage independent). Muscarinic agonists may also activate a pertussis toxin-insensitive fast pathway. alpha-Adrenergic agonists use the fast pertussis toxin-sensitive and the fast insensitive pathways, but not the slow one.

A diffusible second messenger mediates one of the pathways coupling receptors to calcium channels in rat sympathetic neurons.

Muscarinic and alpha-adrenergic suppression of current through Ca2+ channels was studied in adult rat superior cervical ganglion neurons using whole-cell and cell-attached configurations of the patch-clamp technique. Oxotremorine methiodide suppressed ICa by both a rapid (much less than 1 s) and a slow (greater than 4 s) process, whereas norepinephrine suppressed ICa only by a rapid process. The slow muscarinic suppression could be prevented by adding 20 mM BAPTA, a Ca2+ chelator, to the recording pipette, whereas the adrenergic suppression was not affected. Muscarinic, but not alpha-adrenergic, receptors can couple to Ca2+ channels by a second messenger capable of diffusing into an on-cell patch. This signal seems not to be carried by intracellular Ca2+, cGMP, cAMP, or protein kinase C.

Potassium current activated by intracellular sodium in quail trigeminal ganglion neurons.

Whole-cell voltage clamp and single-channel recordings were performed on cultured trigeminal ganglion neurons from quail embryos in order to study a sodium-activated potassium current (KNa). When KNa was activated by a step depolarization in voltage clamp, there was a proportionality between KNa and INa at all voltages between the threshold of INa and ENa. Single-channel recordings indicated that KNa could be activated already by 12 mM intracellular sodium and was almost fully activated at 50 mM sodium. 100 mM lithium, 100 mM choline, or 5 microM calcium did not activate KNa. The relationship between the probability for the channel to be open (Po) vs. the sodium concentration and the relationship of KNa open time-distributions vs. the sodium concentration suggest that two to three sodium ions bind cooperatively before KNa channels open. KNa channels were sensitive to depolarization; at 12 mM sodium, a 42-mV depolarization caused an e-fold increase in Po. Under physiological conditions, the conductance of the KNa channel was 50 pS. This conductance increased to 174 pS when the intra- and extracellular potassium concentrations were 75 and 150 mM, respectively.

Cloning of a human ether-a-go-go potassium channel expressed in myoblasts at the onset of fusion.

An early sign of human myoblast commitment to fusion is the expression of a non-inactivating delayed rectifier K+ current, I(K(NI)), and an associated membrane potential hyperpolarization. We have isolated the full-length coding region of a human ether-a-go-go K+ channel (h-eag) from myoblasts undergoing differentiation. The h-eag gene was localized to chromosome 1q32-41, and is expressed as a approximately 9 kb transcript in myogenic cells and in adult brain tissue. Forced expression of h-eag in undifferentiated myoblasts generates a current with remarkable similarity to I(K(NI)) indicating that h-eag constitutes the channel responsible for this current in vivo.

Human skeletal muscle has a voltage-gated proton current.

A voltage-gated proton current, IH, was studied with the whole-cell patch-clamp technique in human myotubes obtained from biopsies of human muscle. 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. This current is similar in many respects, but not identical, to the proton currents already described in other cell types. IH is activated by depolarization and it can be affected by extracellular pH. IH can be blocked by external divalent cations including Ca2+. This block is voltage-dependent, being more efficient at hyperpolarized than at depolarized voltages. The voltage-dependent properties of IH and its ability to be affected by pH and extracellular Ca2+ suggest that IH might be used by muscle cells to extrude protons during action potentials.

Efficient non-viral DNA-mediated gene transfer to human primary myoblasts using electroporation.

Gene transfer of human primary myoblasts with various non-viral methods has been hampered by low yield of transfection. We report here an efficient, simple and reproducible non-viral DNA-mediated gene transfer procedure for transfecting human myoblasts. We found that electroporation promotes a highly efficient DNA uptake by human primary cultures of myogenic cells. Under optimal conditions, 60-70% of human myoblasts transfected with the enhanced green fluorescent gene expressed the enhanced green fluorescent protein. Electroporated myoblasts behaved normally as judged by their ability to synthesize and express developmentally regulated proteins and to undergo terminal differentiation, i.e. to fuse and form myotubes. We showed, in addition, that a subpopulation of cultured human myoblasts with self-renewing properties and equivalent to native muscle satellite cells were as efficiently transfected by electroporation as proliferating myoblasts. Thus, the development of gene therapies based on the engineering and transplantation of human myoblasts may greatly benefit from gene transfer by electroporation.

Role of nicotinic acetylcholine receptors at the vertebrate myotendinous junction: a hypothesis.

It has long been known that nicotinic acetycholine receptors (nAChRs) are present in muscle fibres not only at the end plate region but also at the myotendinous junction (MTJ). Their function at the MTJ, however, is yet unknown. Recent experiments in our laboratory lead us to suggest that nAChRs at this site might be involved in muscle repair. MTJ is subject to high mechanical stress and therefore is easily damaged. We found in pure cultures of human myogenic cells that (1) the density of nAChRs in myoblasts increases markedly just before cell fusion, (2) the fusion of human myoblasts is accelerated by the presence of a cholinergic agonist acting on nAChRs and (3) human myoblasts and myotubes spontaneously release an ACh-like compound. Based on these observations we propose that in damaged muscles the nAChRs at the MTJ and those of myogenic cells are activated by the ACh-like compound these cells release. This leads to fusion of myogenic cells with damaged muscle fibres and hence promotes repair.

Expression of substance P and of a Ca2+-activated Cl- current in quail sensory trigeminal neurons.

A chloride current activated by an increase in intracellular calcium concentration is not present in all neurons of the trigeminal ganglion. It is not known whether the trigeminal neurons expressing calcium-activated chloride current belong to a defined class of neurons or whether they could belong to any class of sensory neurons. An answer to this question would be of importance because the physiological role of calcium-activated chloride current in neurons has not yet been completely established, nonetheless it is clear that this current, when activated, would act to modulate neuronal excitability. The goal of this study was to determine whether there was a difference in the expression of calcium-activated chloride current between neurons with and without substance P. The rationale was that the use of this morphological marker, which is present in a substantial fraction of embryonic trigeminal neurons, may give a first estimate of a possible inhomogeneity in the expression of calcium-activated chloride current among different classes of sensory neurons. The study was done on freshly dissociated neurons in order to minimize the influence of the culture conditions on the expression of the current or of substance P. By recording from large samples of neurons in cultures either enriched or depleted in substance P-containing neurons, we found that neurons with substance P expressed calcium-activated chloride current three times less frequently than neurons without substance P. This observation was confirmed by performing the immunocytochemical labelling for substance P immediately after the electrophysiological assessment of the presence or absence of calcium-activated chloride current. This result indicates that calcium-activated chloride current may not be randomly distributed in neurons of a sensory ganglion. It raises the possibility that neurons belonging to certain sensory modalities may need calcium-activated chloride current for their physiological functioning.

Mibefradil (Ro 40-5967) inhibits several Ca2+ and K+ currents in human fusion-competent myoblasts.

1. The effect of mibefradil (Ro 40-5967), an inhibitor of T-type Ca2+ current (I(Ca)(T)), on myoblast fusion and on several voltage-gated currents expressed by fusion-competent myoblasts was examined. 2. At a concentration of 5 microM, mibefradil decreases myoblast fusion by 57%. At this concentration, the peak amplitudes of I(Ca)(T) and L-type Ca2+ current (I(Ca)(L)) measured in fusion-competent myoblasts are reduced by 95 and 80%, respectively. The IC50 of mibefradil for I(Ca)(T) and I(Ca)(L) are 0.7 and 2 microM, respectively. 3. At low concentrations, mibefradil increased the amplitude of I(Ca)(L) with respect to control. 4. Mibefradil blocked three voltage-gated K+ currents expressed by human fusion-competent myoblasts: a delayed rectifier K+ current, an ether-à-go-go K+ current, and an inward rectifier K+ current, with a respective IC50 of 0.3, 0.7 and 5.6 microM. 5. It is concluded that mibefradil can interfere with myoblast fusion, a mechanism fundamental to muscle growth and repair, and that the interpretation of the effect of mibefradil in a given system should take into account the action of this drug on ionic currents other than Ca2+ currents.

Multiple G-protein-coupled pathways inhibit N-type Ca channels of neurons.

Muscarinic receptors depress Ca2+ currents in superior cervical ganglion neurons by two signaling pathways. One is sensitive to pertussis toxin and acts rapidly by a membrane-delimited pathway on the channels. The other is not sensitive to pertussis toxin and acts more slowly through an unknown second messenger. These pathways are shared with several other agonists.

Development and evaluation of a community immersion program during preclinical medical studies: a 15-year experience at the University of Geneva Medical School.

BACKGROUND: Significant changes in medical education have occurred in recent decades because of new challenges in the health sector and new learning theories and practices. This might have contributed to the decision of medical schools throughout the world to adopt community-based learning activities. The community-based learning approach has been promoted and supported by the World Health Organization and has emerged as an efficient learning strategy. The aim of the present paper is to describe the characteristics of a community immersion clerkship for third-year undergraduate medical students, its evolution over 15 years, and an evaluation of its outcomes. METHODS: A review of the literature and consensus meetings with a multidisciplinary group of health professionals were used to define learning objectives and an educational approach when developing the program. Evaluation of the program addressed students' perception, achievement of learning objectives, interactions between students and the community, and educational innovations over the years. RESULTS: The program and the main learning objectives were defined by consensus meetings among teaching staff and community health workers, which strengthened the community immersion clerkship. Satisfaction, as monitored by a self-administered questionnaire in successive cohorts of students, showed a mean of 4.4 on a five-point scale. Students also mentioned community immersion clerkship as a unique community experience. The learning objectives were reached by a vast majority of students. Behavior evaluation was not assessed per se, but specific testimonies show that students have been marked by their community experience. The evaluation also assessed outcomes such as educational innovations (eg, students teaching other students), new developments in the curriculum (eg, partnership with the University of Applied Health Sciences), and interaction between students and the community (eg, student development of a website for a community health institution). CONCLUSION: The community immersion clerkship trains future doctors to respond to the health problems of individuals in their complexity, and strengthens their ability to work with the community.

Characterization of muscarinic receptor subtypes inhibiting Ca2+ current and M current in rat sympathetic neurons.

Muscarinic receptors mediating suppression of Ca2+ current and of M-type K+ current in rat superior cervical ganglion neurons were subclassified pharmacologically by using the muscarinic receptor antagonists pirenzepine and himbacine. Our voltage clamp experiments previously distinguished fast and slow intracellular signaling pathways coupling muscarinic receptors to calcium channels. We now establish that the fast, pertussis toxin-sensitive suppression of Ca2+ current is mediated primarily by muscarinic receptors of the M4 subtype, whereas the slow, bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetate (BAPTA)-sensitive suppression of Ca2+ current is mediated primarily by muscarinic receptors of the M1 subtype. Both actions on Ca2+ current are blocked by guanosine 5'-[beta-thio]diphosphate. Muscarinic suppression of M current is slow, BAPTA-sensitive, and mediated by receptors of the M1 subtype. Hence the two muscarinic pathways use different receptors and different guanine nucleotide binding proteins to produce different actions on channels.

Development of anomalous rectification (Ih) and of a tetrodotoxin-resistant sodium current in embryonic quail neurones.

1. The developmental expression of an inwardly rectifying current activated by membrane hyperpolarization (Ih) and of a tetrodotoxin (TTX)-resistant Na+ current (INa(TR)) was studied using freshly dissociated ganglionic quail neurones of various embryonic ages. This work was carried out on parasympathetic (ciliary) and sensory (trigeminal and dorsal root) ganglion neurones with the whole-cell configuration of the patch-clamp technique. 2. In sensory and parasympathetic neurones, Ih was activated at potentials more negative than -60 mV and displayed strong inward rectification. No sign of time- or voltage-dependent inactivation was apparent. Ih was carried by both Na+ and K+ ions and was selectively and reversibly blocked by extracellular Cs+. 3. During the development of sensory neurones, Ih was observed for the first time between embryonic day 10 (E10) and E11 and the percentage of neurones expressing the current increased subsequently, reaching a plateau level of about 80% at E14. In the parasympathetic neurones of the ciliary ganglion, Ih was already detected at E10 and the percentage of neurones possessing the current increased until E16, a stage at which all neurones were found to express Ih. 4. In the presence of TTX (1 microM), an inward Na+ current, INa(TR), was recorded in sensory neurones after E12. This current was activated at potentials more depolarized than -30 mV and its amplitude was maximal at +5 mV. INa(TR) showed time- and voltage-dependent inactivation. Half-maximal steady-state inactivation was observed at -40 mV. 5. INa(TR) was observed for the first time after E12 in sensory neurones and the percentage of neurones with INa(TR) increased until E14. Thereafter, 80% of the neurones had the current. In contrast, INa(TR) was never observed in the parasympathetic neurones of the ciliary ganglion during embryonic development. 6. Our results with parasympathetic and sensory neurones suggest that the expression of INa(TR) is linked to the phenotype and not to the embryonic origin of a neurone.

Transient expression of a Ca2+-activated Cl- current during development of quail sensory neurons.

The expression of a calcium-activated chloride current (ICl(Ca)) was studied during the development of the sensory neurons of quail trigeminal ganglia. This current is expressed in 20% of the neurons by the 5th day of embryonic development; it can be found in nearly all neurons by the 7th day and subsequently disappears in half of them. Similar results were obtained with dorsal root ganglion neurons. The disappearance of ICl(Ca) in part of the sensory neurons during development is not due to a selective death of the neurons possessing this current and our results suggest that it is mediated by an interaction of the sensory neurons with their target tissue.

Distinct contributions of high- and low-voltage-activated calcium currents to afterhyperpolarizations in cholinergic nucleus basalis neurons of the guinea pig.

The contributions made by low- (LVA) and high-voltage-activated (HVA) calcium currents to afterhyperpolarizations (AHPs) of nucleus basalis (NB) cholinergic neurons were investigated in dissociated cells. Neurons with somata >25 microM were studied because 80% of them stained positively for choline acetyltransferase and had electrophysiological characteristics identical to those of cholinergic NB neurons previously recorded in basal forebrain slices. Calcium currents of cholinergic NB neurons first were dissected pharmacologically into an amiloride-sensitive LVA and at least five subtypes of HVA currents. Approximately 17% of the total HVA current was sensitive to nifedipine (3 microM), 35% to omega-conotoxin-GVIA (200-400 nM), 10% to omega-Agatoxin-IVA (100 nM), and 20% to omega-Agatoxin-IVA (300-500 nM), suggesting the presence of L-, N-, P-, and Q-type channels, respectively. A remaining current (R-type) resistant to these antagonists was blocked by cadmium (100-200 microM). We then assessed pharmacologically the role that LVA and HVA currents had in activating the apamin-insensitive AHP elicited by a long train of action potentials (sAHP) and the AHP evoked either by a short burst of action potentials or by a single action potential (mAHP) that is known to be apamin-sensitive. During sAHPs, approximately 60% of the hyperpolarization was activated by calcium flowing through N-type channels and approximately 20% through P-type channels, whereas T-, L-, and Q-type channels were not involved significantly. In contrast, during mAHPs, N- and T-type channels played key roles (approximately 60 and 30%, respectively), whereas L-, P-, and Q-type channels were not implicated significantly. It is concluded that in cholinergic NB neurons various subtypes of calcium channels can differentially activate the apamin-sensitive mAHP and the apamin-insensitive sAHP.

Facilitation of N-type calcium current is dependent on the frequency of action potential-like depolarizations in dissociated cholinergic basal forebrain neurons of the guinea pig.

Voltage-dependent inhibition of high voltage-activated (HVA) calcium currents by G-proteins can be transiently relieved (facilitated) by strong depolarizing prepulses. However, with respect to the physiological significance of facilitation, it remains to be established if it can be induced by action potentials (AP) in central neurons. With the use of whole-cell recordings of dissociated cholinergic basal forebrain neurons of the guinea pig, it is shown that the GTPgammaS-inhibited HVA currents that occur through N-ethylmaleimide (NEM)-sensitive Gi-Go subtypes of G-proteins can be facilitated. Furthermore, although different types of HVA channels are present in these neurons, facilitation occurred mostly through disinhibition of the N-type current. On the basis of data indicating that the recovery from facilitation was relatively slow, we tested if more physiological stimuli that crudely mimicked APs (2 msec long depolarizations to 40 mV from a holding of -50 mV) potentially could induce facilitation of HVA currents inhibited by GTPgammaS and cholinergic agonists. Indeed, evidence is provided that the extent of facilitation is dependent on both the number and frequency of AP-like depolarizations. These results suggest that firing rates and patterns of discharge of neurons could influence their responsiveness to transmitters acting on N-type HVA calcium channels.