In order to investigate the major sources of the calcium signal from this technique, we investigated the degree of overlap between the calcium signal and the VSD signal. The VSD signal is known to represent presynaptic subthreshold activity, predominantly from dendrites.13–15 For this experiment, we performed simultaneous calcium and VSD imaging of epileptiform IIS. As previously described in our laboratory,34,35 IIS were induced with local intracortical bicuculline application. As is apparent (Fig. 5), the waveforms of VSD and calcium signals are qualitatively very similar. Both showed identical onset times and monophasic changes during IIS and were temporally locked to the LFP recorded at the same location [Fig. 5(b), ROI 1). The calcium waveforms were found to be significantly broader () than the VSD waveforms () (, IIS from four rats), indicating longer decay kinetics. We fitted the decay of the fluorescence transient with a single-exponential function, yielding a decay time constant of for calcium () and for VSD (). The normalized fast Fourier transform of LFP, VSD, and calcium signals were also highly comparable, with the exception of low-frequency () power in calcium signals being greater than that of VSD, likely due to greater contributions from slower glial activity in the calcium data [Fig. 5(c)]. Very little low-frequency power was observed in LFP due to the fact that a 1-Hz pass filter was built into the amplifier. In the higher-frequency range (), the LFP spectrum is almost identical to VSD and calcium signals. Cross-correlation between the LFP spectrum () and the VSD/calcium spectra showed strong correlation (LFP versus VSD: , , LFP versus Ca: , ). In the low-frequency range (), there is no correlation between the LFP spectrum and the VSD/calcium spectra (LFP versus VSD: , , LFP versus Ca: , ). The normalized calcium and VSD waveforms became approximately equivalent following the application of a 1-Hz high-pass filter [Fig. 5(d), 1 Hz, first-order Butterworth high-pass filter, using MATLAB®]. The amplitudes of VSD and calcium signal during 30 IISs in four rats are shown in Fig. 5(e). The data points followed a linear distribution, with , indicating that the amplitude of VSD and calcium signals were highly correlated (). Our data suggest that the majority of calcium signals closely reflects subthreshold synaptic activity recorded from the neuropil, which was very similar to the VSD signal but with a higher amplitude and higher s/n as well as a slower component, likely reflecting the slow dynamic of calcium clearance and slower glial waves.