Monte Carlo simulations results
In the well configuration, at a 1.0-cm SD separation, we obtained ac changes of 0.5%, 2.1% and 4% for 1%, 5%, 10 % V1 scattering changes (P values <10−4), respectively. We verified the linear dependence of the ac changes with respect to the scattering changes and estimated an ac change of 0.19% for a 0.4% scattering change and 0.12% for a 0.2% scattering change. We obtained similar results at 1.5-cm SD separation, with slightly higher P values. These changes in ac are well above our measurement noise level when averaging hundreds of stimuli (ac standard errors ~0.005–0.01%).
For the phase shift, at a 1-cm SD separation, we obtained changes of 0.006 and 0.014 deg for 5and 10 % V1 scattering changes, respectively (P values 2−5 and 0.04). We did not achieve any statistical significant phase change for a 1% scattering change. Considering a linear scattering dependence, the extrapolated phase changes due to a 1% scattering change will be 0.001 deg, and only 0.0003 deg for a 0.4% scattering change, both below our experimental errors even after averaging thousand of stimuli (phase standard error ~ 0.001–0.002 deg).
On the surface, by changing the scattering from 1 to 1.1 cm−1 (i.e. a 10% change) in V1 we obtained ac changes of 2–2.4% at 2–3 cm SD separations (P values 3−4–0.04). These ac changes are 40% smaller than the ac changes measured in the well. This is due to the smaller partial volume effect for the well which overcomes the smaller path-length from the smaller SD separation (1–1.5 cm for the well vs. 2–3 cm for the surface). Another advantage of the well is that, with shorter SD separations, a larger number of photons reach the detector, thus decreasing the noise, which in the Monte Carlo simulations translates to much lower P values for the well ac changes with respect to the surface ones. With the large 10% scattering changes, phase changes on the surface were 0.008 deg at 1.5-cm SD separation, and 0.06 deg at 3-cm separation with high but significant P values (0.05); at 2- and 2.5-cm SD separations we did not reach statistically significant changes.
For the surface measurements, by extrapolating the ac results at 10% scattering to the 0.4% scattering changes of the fast signal, we estimated an ac change of less than 0.1% at 2–3 cm SD separations. Such a change is 50% smaller than the change in the well, and the higher noise level expected at the larger distances would make the fast signal measurement from the surface much more challenging than in our experimental setting with an exposed dura.
Visual evoked potential results
shows representative VEP results for the electrode on the well for M2 during a session in which all three stimulation paradigms were employed. The three panels of show the VEP for the 4-Hz (panel a), 7.5-Hz (panel b) and random stimulation sequences (panel c) obtained by averaging all of the black-to-white reversing epochs on from representative single 6-min runs. The error bars are standard errors. For the 4-Hz stimulation run shown in the figure we averaged 350 reversals; for the 7.5-Hz stimulation run, 675 reversals; and for the random stimulation, 240 reversals. Reversal onsets are indicated by gray vertical bars. For each reversal the VEP signal is composed of three major peaks: a positive peak at ~ 50 ms, a negative peak at ~90 ms and a large positive peak at ~200 ms, which is not present at 7.5-Hz stimulation because of the onset of a new reversal at time 133 ms. The peak latencies of these VEP responses’ were consistent across runs, across sessions, and across monkeys. These responses are similar to VEP on macaque monkeys for similar stimuli reported in the literature (Previc, 1986
; Schmid et al., 2006
Figure 5 VEP responses for the 4-Hz (panel a), 7.5-Hz (panel b) and random stimulation sequences (panel c) obtained by averaging representative single 6-min runs during a session on M2. The error bars are standard errors. Please note that the time scale for the (more ...)
shows the power spectra of the EEG signal from the electrode on the recording well during the same runs as shown in . The two traces correspond to the FFT for the intervals when the checkerboard was on (black lines) and off (gray lines). For the on intervals, the EEG response at the stimulation frequency is 15–20 dB higher than for the off intervals and has strong harmonics.
Hemodynamic response results
in the Methods section shows examples of the slow hemodynamic response for each stimulation block for the CW and FD measurements at the 4-Hz stimulation. shows the results for CW and FD, averaging all of the runs and measurement sessions for the two monkeys. For the CW measurements, results at the two wavelengths (ac amplitudes) are shown for the 4-Hz and 7.5-Hz reversal stimulation (panels a and b, respectively). Panel c shows the results for ac and phase at 830 nm obtained with the FD measurements at the 4-Hz stimulation. The error bars are standard errors. For the 4-Hz CW we averaged 260 blocks; for the 7.5-Hz CW, and for the FD, we averaged 200 blocks.
Figure 7 Grand average of the slow optical responses (hemodynamic) for the CW measurements at 4Hz (panel a) and at 7.5Hz (panel b), and for the FD measurements at 4Hz (panel c). The error bars are standard errors. Please note that the y axis for the FD measurements (more ...)
The ac signal decreases 2–3% at 830 nm and increases ~1% at 690 nm, which, converted to hemodynamic changes, corresponds to a 2–3 μM×cm (not pathlength corrected) increase of HbO and a ~0.5 μM×cm decrease of HbR. The slow response starts ~1 s after the stimulation onset, increases for the 20 s of stimulation and rapidly decreases 1–2 s after the end of stimulation. The ac results with the FD are noisier than with the CW, because of the higher frequency of modulation of the lasers (110 MHz vs. 4 KHz) and the lower emitted laser power (2 mW vs. 10 mW). The phase shift increases 0.06 deg with increased absorption (in the figure the phase shift is multiplied by 50 for convenience, to be of similar magnitude to the ac changes), and the changes are above the noise level.
Fast signal results
shows the grand averages of the ac data obtained with the CW instrument for the three stimulation protocols, averaging all of the black-to-white reversing epochs. The error bars are standard errors. For the 4-Hz stimulation, we averaged ~10,000 reversal epochs; for the 7.5-Hz stimulation, ~15,000 reversal epochs; and for the random stimulation, 3,300 reversal epochs. Left panels are the results at 830 nm, and the right panels, at 690 nm. Black lines are the results after applying the adaptive heart filter to remove the arterial pulsations; dashed gray lines are the results without applying the heart filter. In panels a, b, c and d the arterial pulsation is reduced by the large average of stimuli, while in panels e and f, where we average only 3,300 stimuli, the arterial pulsation is not completely averaged out. In all cases, applying the adaptive filter reduces the error bars, which for the filtered signals are ≤0.005%. While we expected to see an ac change of 0.1–0.2% from the Monte Carlo results, we do not see any significant fast signal change. In particular, we do not see any changes with periodicity similar to the VEP response ().
Figure 8 Grand average of the fast amplitude responses for the CW measurements at 4Hz (panel a and b), at 7.5Hz (panel c and d), and for the random stimulation (panel e and f). The error bars are standard errors. The left panels show results at 830 nm, the right (more ...)
shows the results for the frequency-domain measurements where, again, the fast signal is not visible in either the ac or the phase measurements. The unfiltered ac responses have a strong arterial component despite the large number of stimuli averaged (10,000). In most of our experiments the arterial pulsations are larger and have a larger harmonic content for the FD measurements than for the CW measurements (20 dB for FD ac vs. 25 dB for CW ac in , and in general 0.02–0.05% arterial pulsation magnitude in the FD ac vs. 0.01–0.03% magnitude in the CW ac). A possible explanation may be the different dates on which the FD and CW experiments were done, since the dura grew new vessels over time. The phase in shows the arterial component in both the filtered and unfiltered data, because of the lower success in removing the arterial pulsation from the phase data.
Figure 9 Grand average of the fast amplitude (panel a) and phase shift (panel b) responses for the FD measurements at 4Hz. The error bars are standard errors. Black traces data after adaptive heart filter, dashed gray traces data without heart filter correction. (more ...)
With the FFT analysis averaging sessions and animals, as with and , we noted the absence of any peak at the stimulation frequencies due to a fast signal response (figures not shown since grand average results are very similar to the one reported in for single session).