Regulation of quantal currents determines synaptic strength at neuromuscular synapses in larval Drosophila

Abstract

Studies of synaptic homeostasis during muscle fiber (MF) growth in Drosophila larvae have focused on the regulation of the quantal content of transmitter release. However, early studies in crayfish and frog suggested that regulation of quantal current size may be an integral mechanism in synaptic homeostasis. To examine this further in Drosophila, we compared the electrical properties, miniature excitatory postsynaptic potentials (minEPSPs) and miniature excitatory postsynaptic currents (minEPSCs) in different-sized MFs in third-instar larvae and for a single MF during larval growth. The third-instar MFs showed differences in input resistance due to differences in size and specific membrane resistance. We found that electrical coupling between MFs did not contribute substantially to the electrical properties; however, the electrode leak conductance and a slower developing increase in membrane conductance can influence the electrical recordings from these MFs. Our results demonstrated that larger MFs had larger minEPSCs to compensate for changes in MF electrical properties. This was most clearly seen for MF4 during larval growth from the second to third instar. During a predicted 80 % decrease in MF input resistance, the minEPSCs showed a 35 % increase in amplitude and 165 % increase in duration. Simulations demonstrated that the increase in minEPSC size resulted in a 129 % increase in minEPSP amplitude for third-instar larvae; this was mainly due to the increase in minEPSC duration. We also found that MFs with common innervation had similar-sized minEPSCs suggesting that MF innervation influences minEPSC size. Overall, the results showed that increased quantal content and quantal current size contribute equally to synaptic homeostasis during MF growth.

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“…EPSP amplitude, EPSP decay time constant (decay τ) and the resting membrane potential (RMP) were measured using pCLAMP 10.5 software. To analyze minEPSPs, they were identified using MiniAnalysis (Synaptosoft Inc.) and their amplitude and decay τ were measured from averaged traces as in a previous study (Powers et al, 2016). The decay τs were determined by fitting a single exponential to the decay phase using least-squares regression.…”

“…The amount of transmitter released before and after citral application was compared by estimating the quantal content (m) of the EPSPs using the equation m=(EPSP amp /minEPSP amp )/[1-(fEPSP amp /E)], where: EPSP amp is the average EPSP amplitude; minEPSP amp is the average minEPSP amplitude; f=synaptic current duration/membrane time constant (τ m ); and E is the RMP-(the synaptic reversal potential) (McLachlan and Martin, 1981). The duration of the synaptic current for MF6 was approximately 10 ms (Powers et al, 2016) and the synaptic reversal potential was -1 mV (Jan and Jan, 1976). Our minEPSP decay τ was used for τ m ; this was supported theoretically and experimentally (Powers et al, 2016).…”

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“…The voltage deflection (after subtraction of electrode resistance) was used to calculate input resistance and qualitatively check membrane time constant as indicators of membrane integrity. Recordings from muscles with input resistances less than 1.5 MΩ or time constant less than 5 ms were rejected (Powers et al, ). Spontaneous EJPs (miniEJPs) were recorded in the absence of nerve stimulation over a period of up to 60 s. EJP recordings were analyzed using pClamp 10.6 and miniEJPs were measured using Minianalysis (Synaptosoft, Atlanta).…”

“…Intracellular microelectrode recordings were made from Muscles 6 and 7 in abdominal segments 3 and 4 of filleted third instar larval preparations bathed in HL3 saline, using standard techniques (Powers, Grizzaffi, Ribchester, & Lnenicka, 2016). The concentrations of Ca 21 and Mg 21 in the saline were reduced (to 0.4 and 10 mM, respectively) in order to depress mean quantal content of excitatory junction potential (EJP; evoked transmitter release), thereby increasing sensitivity to differences in synaptic strength between the Drosophila lines we tested (Dodge & Rahamimoff, 1967;Guan et al, 2017).…”

“…Recordings from muscles with input resistances less than 1.5 MX or time constant less than 5 ms were rejected (Powers et al, 2016). Spontaneous EJPs (miniEJPs) were recorded in the absence of nerve stimulation over a period of up to 60 s. EJP recordings were analyzed using pClamp 10.6 and miniEJPs were measured using Minianalysis (Synaptosoft, Atlanta).…”