Technical Reference #1619

1619.

Direct Enhancement of Presynaptic Calcium Influx in Presynaptic Facilitation at Aplysia Sensorimotor Synapses Karina Leal; Marc Klein, University of California Los Angeles, Molecular and Cellular Neuroscience, 41(1619), (2009)

Abstract:
Regulation of synaptic transmission by modulation of the calcium influx that triggers transmitter releaseunderlies different forms of synaptic plasticity and thus could contribute to learning. In the mollusk Aplysiathe neuromodulator serotonin (5-HT

Materials & Methods:
Culture preparation Cultures were prepared following published procedures (Klein 1993; Schacher and Proshansky 1983). Adult Aplysia californica (76– 100 g; RSMAS University of Miami Miami FL) were anesthetized by injection of 50–100 ml of 400 mM(isotonic) MgCl2. Pleural-pedal and abdominal ganglia were removed and digested in artificial seawater (ASW; see below for composition) containing 1% protease type IX (Sigma). Tail sensory neurons and siphon (LFS) motor neurons were isolated and plated in modified L15 containing 10% Aplysia hemolymph on dishes (Glass Bottom Mircowell Dish; MatTek Corporation) pretreated with poly-L-lysine (molecular weight N300000; Sigma). Dishes were prepared with isolated sensory neurons or co-cultures of sensory neurons and motor neurons for synapse formation. Electrophysiology An Axoclamp 2A or 2B amplifier (Molecular Devices Palo Alto CA) and glass micropipettes (tip resistance 10–20 MΩ) filled with 0.5 M potassium chloride and 2 M potassium acetate were used for intracellular recordings. All recordings were done in ASW (in mM: 460 NaCl 10 KCl 11 CaCl2 55 MgCl2 and 10 HEPES pH 7.5). The membrane potential of sensory neurons was held at −55 mV. In cocultures the motor neuron was impaled first and its membrane potential was maintained at −80 mV. To elicit a postsynaptic potential (PSP) in the motor neuron an action potential was evoked in the sensory neuron with a short (5 ms) depolarizing current pulse at an interstimulus interval of 30 or 60 s. In order to ensure accurate measurements of action potentials the amplitude of the depolarizing current pulse was adjusted so that the action potential was triggered after the end of the current pulse. Action potentials and PSPs were recorded and measured with Axograph X software (AxoGraph Scientific Sydney Australia). Action potential duration was measured from the peak of the action potential to the point at which the falling limb crossed−10 mV at approximately 40% of the peak amplitude of the spike which is at about the peak of the current-voltage curve for calcium current in the sensory neurons (Klein 1994). We chose to measure duration at a fixed membrane potential rather than at a fraction of the peak amplitude because the variable of interest is the calcium influx triggered by the action potential and the calcium current is determined by the absolute membrane potential not by a fraction of the peak amplitude of the spike. If the peak amplitude of the action potential does not change it would make no difference which way the duration was measured. However any change in the peak amplitude of the action potential would change the point at which the membrane potential reached any given fraction of the peak without any actual broadening or narrowing of the spike. Measurement of the duration at a fraction of the peak could nonetheless indicate a change in duration whereas measurement of duration at a fixed membrane potential far from the peak would show no change. Peak PSP amplitudes were measured for PSPs smaller than 20– 30 mV while for larger PSPs the maximal slope of the PSP corresponding to the peak of the underlying synaptic current was recorded in order to avoid the complications of non-linear summation and the recruitment of voltage-gated conductances by large PSPs. The maximal slope and the amplitude of the PSP are closely correlated and are linearly related over a large range of amplitudes (unpublished). Rise times of PSPs were measured from 20% to 50% of the peak amplitude except for larger PSPs that triggered action potentials. For these PSPs the time from 20% to 80% of the peak of the derivative of the PSP was taken as the rise time (i.e. from 20% to 80% of the peak synaptic current). In any given experiment all parameters were measured in the same manner. Facilitation of the PSP and the calcium transient was calculated as the average of the 2 responses immediately following drug application divided by the average of the 2 responses immediately prior to the application except where noted otherwise. Serotonin (creatinine sulfate; Sigma) was delivered as a bolus of 100 μl (1 μM 5 μM 10 μM or 100 μM in ASW) by a hand-held pipette directly into the bath ∼5 mm from the cells. The concentration to which the cells were exposed was estimated to be about half the concentration in the pipette (Klein 1994). The potassium channel blocker 34-diaminopyridine (34-DAP; 1 mM) and the calcium channel blocker nitrendipine (100 μM in DMSO 0.5%) (both from Sigma) were applied in the same way except for some experiments in which 34-DAP (500 μM) was perfused into the bath. Microinjection of fluo-4 To monitor calcium transients sensory neurons were loaded with the fluorescent calcium indicator fluo-4 (Molecular Probes Eugene OR; 10 mM in HEPES 100 mM pH 7.6) by microinjection from backfilled glass micropipettes. The tip of the micropipettewas inserted into the cell body and short pressure pulses (10 ms duration 20 psi) were delivered until the cell body became uniformly fluorescent. The cells were incubated in the dark at room temperature until the indicator diffused into neurites usually about 10 min. Fluorescence microscopy Cultures were viewed at 20× or 40× with a Nikon Diaphot microscope attached to a xenon lamp for fluorescence excitation. A combination of optical filters and dichroic mirror (Omega Optical filter set XF23) was used for excitation centered at 485 nm; emission measurements were centered at 535 nm. Images were acquired with a cooled CCD camera (Retiga EXi) controlled by IPLab software (version 3.65; Scanalytics Fairfax VA). Image acquisition was begun just prior to triggering of an action potential in the sensory neuron. Twenty frames were captured at exposures of 50 or 100 ms. Values for peak calcium transients (dF) were obtained by subtracting the average of the 2 or 3 frames immediately preceding the transient from the average of 2 or 3 frames at the peak. In some experiments the dF values were divided by the baseline fluorescence (with background subtracted) to adjust for bleaching; these values are given as dF/F. Relative changes in fluorescence with experimental treatments within an experiment were generally unchanged by this correction so either dF or dF/F values were used. Saturation of the indicator by high calcium concentrations would be detected as a relative flattening of the relationship between the PSPs and the calcium transients with increasing values. As this was not the case saturation of the indicator was not considered to be a confounding factor in the experiments. The relationship between changes in fluorescence and in calcium concentration was presumed to be linear because the measured changes in fluorescence were on the order of 2-fold or less which is less than 5% of the dynamic range of fluo-4 reported by the manufacturer. Linear regressions for log–log plots of PSP vs. calcium transients started from the 4th PSP in all experiments because PSPs undergo homosynaptic depression which is not reflected in a decrease in the calcium transients (Armitage and Siegelbaum 1998). In control experiments strong depolarization and firing of the postsynaptic neuron had no effect on the fluorescence of the presynaptic neuron confirming that the observed changes in fluorescence were not due to postsynaptic depolarization being transmitted to the presynaptic cell by electrical coupling or to a change in fluorescence in the postsynaptic neuron resulting from leakage of fluo-4 into the postsynaptic neuron. All measurements are given as means±SEM. All experiments were performed at room temperature (20–23 °C).

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
Tail sensory neurons