Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions

Abstract

Neuromuscular preparations from third instar larvae of Drosophila are not well-maintained in commonly used physiological solutions: vacuoles form in the muscle fibers, and membrane potential declines. These problems may result from the Na:K ratio and total divalent cation content of these physiological solutions being quite different from those of haemolymph. Accordingly haemolymph-like solutions, based upon ion measurements of major cations, were developed and tested. Haemolymph-like solutions maintained the membrane potential at a relatively constant level, and prolonged the physiological life of the preparations. Synaptic transmission was well-maintained in haemolymph-like solutions, but the excitatory synaptic potentials had a slower time course and summated more effectively with repetitive stimulation, than in standard Drosophila solutions. Voltage-clamp experiments suggest that these effects are linked to more pronounced activation of muscle fiber membrane conductances in standard solutions, rather than to differences in passive muscle membrane properties or changes in postsynaptic receptor channel kinetics. Calcium dependence of transmitter release was steep in both standard and haemolymph-like solutions, but higher external calcium concentrations were required for a given level of release in haemolymph-like solutions. Thus, haemolymph-like solutions allow for prolonged, stable recording of synaptic transmission.

Ca2+Dynamics along Identified Synaptic Terminals inDrosophilaLarvae

“…After 40 min, the nerve was removed from the pipette and the preparation was left at room temperature for another hour. The Schneider's solution was then replaced with HL3 saline (Stewart et al, 1994) containing 1 mM Ca 2ϩ for physiological measurements.…”

Genetic Analysis of SolubleN-Ethylmaleimide-Sensitive Factor Attachment Protein Function inDrosophilaReveals Positive and Negative Secretory Roles

“…All larval electrophysiological recordings were made in hemolymph-like solution (HL3) (Stewart et al, 1994) containing 1 mM CaCl 2 . The motor neurons innervating abdominal segment 3 (Budnik et al, 1990) were stimulated with 0.3 msec pulses using a voltage sufficient to reliably elicit a response from both axons 1 and 2 (Lnenicka and Keshishian, 2000).…”

The atypical cadherin flamingo regulates synaptogenesis and helps prevent axonal and synaptic degeneration in Drosophila

“…The larva was bathed in HL-3 solution (Stewart et al, 1994) containing 1 mM calcium (Ca 2+ ) for measuring evoked synaptic potentials and 0.1 mM Ca 2+ and 1 ÎM tetrodotoxin for recording spontaneous synaptic potentials (Zhang et al, 1998;Bao et al, 2005). To preserve dorsal muscles for intracellular recordings, the dissection method was slightly modified.…”