1. Overview & Scope Chemistry The resting membrane potential of Drosophila melanogaster larval muscle depends strongly on the calcium gradient. Physiology Increasing available extracellular [Ca++] yields a substantial hyperpolarization of RMP (in SSA: ~8 mV / 1 mM [Ca++]). This effect appears to be mediated by the muscle, rather than the presynaptic motoneurons. Dr. Robin Cooper’s laboratory has reported that the pathological change in presynaptic [Ca++] that is characteristic of cacophony mutants (cac TS2 ) has little or no effect on muscle RMP (Xing et al. 2005). Non-voltage clamp recordings of larval muscle RMP and EJP amplitude should be considered relative to saline [Ca++], regardless of the saline chosen. One means for such consideration for EJPs comes from the McLachlan and Martin equation (1981). Results from this study suggest that saline divalent concentration must be considered carefully when characterizing the physiology of Drosophila mutants, particularly those with neuromuscular excitability phenotypes. This work was supported by a Professor Award to RRH from HHMI and faculty support from Mt. Holyoke College (JLK) and Pomona College (KDP). Jacob Krans 1, Karen Parfitt 2, Patricia Rivlin 3, David Deitcher 3, Ron Hoy 3 1 Biological Sciences, Mount Holyoke College, South Hadley, MA. 2 Department of Biology, Pomona College, Claremont, CA. 3 Department of Neurobiology and Behavior, Cornell University, Ithaca, NY. 650.13 ABOVE: Membrane potential of larval muscle 6 is plotted versus time. Saline containing 0.5 mM [Ca++] is replaced with 3.0 mM [Ca++] saline at ~25 s. At ~150 s, the preparation is returned to 3.0 mM [Ca++] saline. Two commonly used salines are shown: BLUE: Standard Saline A (SSA: Jan and Jan 1976), and RED: Hemolymph-Like saline #3 (HL-3: Stewart et. al. 1994). Mechanical artifacts indicate the exchange of solution. Each exchange was accomplished over several washouts. Introduction Discussion The neuromuscular junction (NMJ) of larval fruit fly, Drosophila melanogaster, has the unique qualities of being a recognized model of synaptic transmission. The preparation illustrates concepts across several disciplines and gives investigators unprecedented control of the expression of a myriad of well-understood genes and their products. Over the course of unrelated studies, we noticed a trend of surprisingly depolarized resting membrane potentials (RMPs) when using salines containing low calcium. This was noted in earlier work by Jan and Jan (1976), but has not been addressed methodically since. Moreover, although the trend is present in many preparations, it’s mechanism is widely variable and its magnitude rarely comparable to that in fruit fly. We have characterized the dependency of RMP upon calcium concentration ([Ca++]). As saline [Ca++] increases, so does the absolute magnitude of the RMP, yielding more hyperpolarized potentials. The strength of the relationship between RMP and [Ca++] led us to question the natural extracellular environment at the NMJ. We thus analyzed, and report here, the ionic composition of larval hemolymph with particular emphasis on our use of voltammetric techniques to measure divalent cations (Ca++ and Mg++). The larval NMJ preparation of fruit fly is simple, anatomically repetitive, and provides large EJPs suitable for quantitative analysis. FAR RIGHT: A single segment showing the segmental nerves and their specific innervation. By severing the nerves, spontaneous activity is abolished and single potentials can be evoked by electrical stimulation of the nerves via suction electrode. The muscles recorded from in this study are 6, 7, 12, and 13. In many preparations, a RMP in the range of -50 to -70 mV is one of several metrics used to determine the viability of a particular recording. Though this is somewhat of an unspoken axiom, the occasional reference is made in reports (Xing et. al. 2005). An RMP of -35 mV might be considered too depolarized in most common NMJ preparations. In larval fruit fly, however, data from these muscles – in very low calcium – may be physiologically normal. The effect of calcium on membrane potential has been documented in a diverse phylogeny of preparations, but the extreme magnitude of the effect is somewhat unique in D. melanogaster. In fruit fly and many organisms (some are listed below), the sign of this effect is opposite of that predicted by permeability equations (e.g. Goldman, 1943; Hodgkin and Katz, 1949). As RMP shifts with [Ca++], so does EJP amplitude. However, the functions these relationships follow are different, which has important implications to analyses of evoked potentials. ABOVE: EJP amplitude (voltage difference) is plotted as a function of saline [Ca++]. Once again, the left panel provides a linear scale, and the right, a logarithmic scale. Data are well fit by a sigmoid (not shown) function based on a 3 variable Hill equation. SSA: 50% EJP amplitude [Ca++] is achieved – using this model – at 0.51 mM. Although the 50% saturation point observed in HL-3 (open squares) is unchanged, the slope of the fit is greater, suggesting increased sensitivity to [Ca++]. ABOVE: RMP vs. saline [Ca++]. Data are equivalent on both sides, but are plotted on a linear scale on the left and logarithmic scale on the right. Data collected in SSA: A logarithmic model is used to fit the data and gives an r 2 value of 0.97. The slope of the relationship, when using either a logarithmic or linear model (r 2 = 0.88), is 7.9 mV RMP / 1 mM [Ca++]. Use of HL-3 saline (open squares) yields more hyperpolarized potentials than SSA and an increased slope (9.5 mV / mM). The relationship between [Ca++] and RMP, in HL-3, is not as well fit by a logarithm (r 2 = 0.91). Muscle: Rat: Thelsleff and Ward, 1975 Rabbit; Guinea-Pig: Pholpramool and Korppaibool, 1977; Bolton, 1972 Sheep and Cow: Reuter and Schultz, 1967 Frog: Bulbring et. al. 1956; Luttgau, 1963; Apter and Koketsu, 1960 Worm: Brading and Caldwell, 1971 Neurons:Squid: Frankenhauser and Hodgkin, 1957 RIGHT: Membrane potential is recorded from a single muscle as saline [Ca++] increases from 0.5 to 1.5 mM. RMP hyperpolarizes about 5 mV, but the peak of the EJP shifts less than half that: 2.3 mV. This phenomenon supports the use of scaling equations such as McLachlan and Martin’s (1981). RIGHT: The bodywall musculature of a larva. Anterior is shown at top, nerves are black The various effects of divalents on membrane physiology underscore the importance of accurate measures of their presence in fruit fly hemolymph. Physiologists have exploited the anesthetic effect of high extracellular / saline magnesium ([Mg++]) – in crustacean preparations – for decades. This is particularly interesting in light of the high divalent concentrations typical of many insects including several Lepidoptera, Coleoptera, and Hymenoptera. [Duchateau et. al. 1953 offers an exhaustive review of hemolymph ion concentrations.] It has been reported that laboratory strains of D. melanogaster – which consume primarily yeast – have high divalent ion concentrations (Begg and Cruickshank, 1963). Additionally, the ratio of [Ca++] to [Mg++] has also been implicated in “membrane stabilizing” effects on resting membrane potential (Stefani and Steinbach, 1969). The ionic composition of larval hemolymph has never been measured in non-diluted / pure form, and only rarely in dilution. Also, we have measured [Mg++] in larval hemolymph, which has not been reported using modern voltammetric techniques. We analyzed two types of hemolymph sample: (1) Non-diluted, “pure”, samples were gathered from thousands of larvae. (2) Hemolymph was diluted to final concentrations between 1:10 to 1:20. Method 1: Centrifugation of lesioned larvae (left, top). A superficial cut was made in the bodywall of the third abdominal segment. Lesioned larvae were placed on a filter within a centrifuge tube. Samples were spun at ~3000 G. Filtered (10 nm CO) hemolymph was analyzed using ion selective, voltammetric equipment. Method 2: Microcapillary collection (left, bottom) has been described (Stewart et. al., 1994). Briefly, microcapillary tubes were placed against small incisions made in the bodywall of 3rd instar larvae, as above. Between 0.5 and 1 ul of hemolymph was gathered from each larva using this technique, which was immediately ejected into an iso-osmotic volume of dilutant.