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First published online January 17, 2007
Journal of Experimental Biology 210, 454-464 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02667

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Excitable properties of adult skeletal muscle fibres from the honeybee Apis mellifera

Claude Collet^* and Luc Belzunces

Ecologie des invertébrés, INRA, Institut National de la Recherche Agronomique, UMR406, Domaine St Paul, Site Agroparc, F-84914 Avignon cedex 9, France

View larger version (114K): [in this window] [in a new window]   Fig. 1. Single skeletal muscle fibres from adult honeybee leg muscle. (A) Micrographs of two cells bathed in Tyrode's solution taken using phase contrast (transmitted light) microscopy. (B) Part of a cell at a highest magnification, (in horizontal orientation). (C–F) Laser confocal micrographs of T-tubule system in isolated fibres. Single fibres bathed in Tyrode's solution were stained with the lipophilic fluorescent dye di-8 ANEPPS (10 µmol l–1) for 20 min in order to reveal the transverse tubule network. (C) In longitudinal axial section, a central chain of nuclei interrupting T-tubules (double arrow) and tracheoles (arrow) are visible. (D) Longitudinal paraxial section, shows longitudinal connections between T-tubules (arrows) within the same or adjacent sarcomeres. The dotted line (i) shows the position at which the transverse fibre reconstruction shown in F was taken. (E) Continuity of T-tubules with the surface membrane. Two T-tubules per sarcomere penetrate the fibre volume, as emphasized by the profiles of pixel intensity (below) taken at positions ii and iii. (F) Transverse confocal section showing the shape of the fibre. (G) Another cylindrical fibre stained with the calcium fluorophore Fluo-3-AM. Scale bars, 100 µm (A); 20 µm (B); 20 µm (C,D,F,G); 4 µm (E).

View larger version (5K): [in this window] [in a new window]   Fig. 2. Glutamate-induced whole-cell currents in voltage-clamped muscle fibres from honeybee. (A) Fast application of L-glutamate (Glu; 1 mmol l–1 in Tyrode's solution) through a pressure-driven perfusion system induces a whole-cell inward current consisting of an inactivating initial peak followed by a steady component in one muscle fibre voltage-clamped at –80 mV. The peak and steady component (measured 3 s after the onset of the current) were respectively –7.4 and –3.6 A/F. On average, those values were –7.2±2.0 and –2.2±0.5 A/F (N=23 fibres). (B) The mean relative amplitude of the steady component was 49±8% of the initial peak (N=23).

View larger version (10K): [in this window] [in a new window]   Fig. 3. Current–voltage relationship of the glutamate-induced current in muscle fibres from honeybee. (A) Protocol of voltage ramps running from –130 mV to +30 mV applied before (ramp 1), during (ramps 2 and 3) and after (ramp 4) pressure application of L-glutamate (1 mmol l–1 in Tyrode's solution) for 8 s in one muscle fibre otherwise voltage-clamped at –80 mV. Voltage ramps induced activation of voltage-dependent currents and only those with weak voltage-dependent inward calcium current were used for analysis. Application of glutamate induced a current with a sustained inward component (with slow inactivation) at –80 mV. (B) Current–voltage relationships of the whole-cell current obtained in response to the voltage ramps shown in A in the absence (1 and 4, black) or in the presence (2 and 3, grey) of glutamate, show that glutamate elicits an inward component at negative potentials. (C) The mean current–voltage relationship of the glutamate-induced current (N=4) appears linear below approx. –20 mV and presents an inward rectification for more positive potential. On the voltage scale considered, the glutamate-induced steady component was null for membrane potentials above approx. 0 mV. The grey shading indicates s.e.m. at each potential explored. Em, voltage command.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Action potentials in muscle fibres from honeybee. (A) Three superimposed current steps of 10 ms duration and of increasing amplitude (i) and membrane voltage responses (ii) in a current-clamped muscle fibre bathed in Tyrode's solution (2 mmol l–1 CaCl2). The membrane potential was held at –80 mV by passing a constant negative current (minus sign at the beginning of the voltage recordings). Both of the lowest stimulating current steps (a, thin traces) only elicited electrotonic responses. The highest current amplitude (i, thick trace) elicited an action potential overshooting 0 mV (broken line marks the 0 mV level). (B) Three superimposed current steps of 1000 ms duration and of increasing amplitude (i) and the three corresponding membrane voltage responses from bottom to top (ii) in another current-clamped muscle fibre bathed in Tyrode's solution. The membrane potential was held at –80 mV by passing a constant negative current. Whereas the first current step only elicited an electrotonus (ii, bottom trace), the intermediate current step triggered a single action potential (ii, middle trace). The highest current step triggered a train of action potentials (ii, upper trace). (C) In current-clamp, tetrodotoxin (TTX) had no effect on the action potential (upper panel) whereas the calcium channel blockers Cd2+ and La3+ converted the regenerative action potential response (black line, lower panel) into an electrotonic response (broken line). Em, voltage command.

View larger version (5K): [in this window] [in a new window]   Fig. 5. Whole cell voltage-dependent currents in muscle fibres from honeybee. (A) In voltage-clamp mode, in the presence of Tyrode's solution (2 mmol l–1 Ca2+) as the extracellular solution, a series of depolarizations bringing the membrane potential from –80 mV to –30, –10, +10 and +30 mV over a period of 200 ms activated both inward and outward currents. (B) In a fibre depolarized to +10 mV for 100 ms, tetrodotoxin (TTX) had no obvious blocking effect on the inward current (middle trace). However, Cd2+ and La3+ completely blocked the inward component of the voltage-activated currents (lower trace).

View larger version (14K): [in this window] [in a new window]   Fig. 6. Calcium currents and corresponding current–voltage relationships in muscle fibres from honeybee. (A) Inward calcium currents were evoked with 100 ms long depolarizing pulses in a voltage-clamped single fibre in the presence of potassium channel blockers, from a holding potential of –80 mV. Open circle, peak of the current; closed circle, end of the depolarization pulse. (B) Mean membrane–current relationship established at the peak of the current (open circles, N=14) and at the end of the depolarization pulses (closed circles). The mean curves were separately fitted using Eqn 1 (see Materials and methods) with values of Gmax, Vrev, V0.5 and k of 210 S/F, +45 mV, –10.9 mV and 5.6 mV (upper panel) and 267 S/F, +32 mV, –10 mV and 6 mV (lower panel). Em, voltage command.