The hemodynamic initial-dip consists of both volumetric and oxymetric 1 changes correlated to localized spiking activity 2

11 The “initial-dip” is a transient decrease frequently observed in functional neuroimaging signals, immediately after 12 stimulus onset, and is believed to originate from a rise in deoxy-hemoglobin (HbR) caused by local neural activity. It has been 13 shown to be more spatially specific than the hemodynamic response, and is believed to represent focal neuronal activity. 14 However, despite being observed in various neuroimaging modalities (such as fMRI, fNIRS, etc), its origins are disputed and 15 its neuronal correlates unknown. Here, we show that the initial-dip is dominated by a decrease in total-hemoglobin (HbT). 16 We also find a biphasic response in HbR, with an early decrease and later rebound. However, HbT decreases were always large 17 enough to counter spiking-induced increases in HbR. Moreover, the HbT-dip and HbR-rebound were strongly coupled to 18 highly localized spiking activity. Our results suggest that the HbT-dip helps prevent accumulation of spiking-induced HbR- 19 concentration in capillaries by flushing out HbT, probably by active venule dilation. during epoch II (p<10 -3 ; n=125; Š). In contrast, low spiking trials showed no significant modulation of HbR slopes during either epoch I or II (p>0.1; n=122; Š). G) Correlation of mean dips in HbT, HbO and HbR stimulus induced peaks in the power of various LFP frequencies bands and Spiking. Correlations with p>0.05 are greyed. Only high frequency bands showed a significant correlation with initial dips, with spiking activity eliciting the strongest relationship, that were marginally higher for HbT than HbO. H) Strength of the relationship between the HbT dip and spiking activity decreases with distance from the NIRS emitter. Strongest correlations are observed on tetrode closest to emitter (0.55mm away from emitter edge, 1.8mm from emitter center), whereas no correlations are observed on tetrode 2.95mm away (4.2mm from center). For each combination of DPFs, we observed strong dips in the HbO and HbT signals. We also observed an increase in the dip strength for HbT during high spiking trials. Low spiking trials failed to elicit a significant dip. The HbR signal also elicited a strong dip and rebound modulation in each case. For each combination of DPFs, the results were nearly identical and individually significant. Interestingly, the DFP combination 12,6 revealed increases in HbR within the 0-2s of trial onset. However, there was no difference in HbR concentration in high-spiking vs low-spiking trials (p>0.4; n=130/group, Wilcoxon rank-sum test). Furthermore, the strongest difference between high and low-spiking was still oberved in the HbT-dip (p<10 -5 ) than HbO-dip (p<10 -3 ). This reaffirms previous results 1 that, at least in the primary visual cortex, although the choice of DPF might alter the amplitude of the calculated change in concentrations of HbO, HbR and HbT, it does not alter their relationship to underlying spiking activity. Notches represent 95% confidence intervals. optical density for both imaging wavelenghts (760nm and 850nm) reveal an early decrease in chromophore concentration. A) Specific extinction coefficients for HbO and HbR as a function of wavelength. Although both HbO and HbR absorb light at both wavelengths, 760nm is more sensitive to HbR changes whereas 850nm is more sensitive to HbO changes. Changes in the observed optical density are proportional to changes in the chromatophores according to Beer’s law. B) Mean changes in the optical densities at 760nm and 850nm across all 260 trials. Inset represents the changes between 0-3s. A decrease in optical density for both wavelenghts can be observed. C) Mean change in optical density between 0 and 1s for 760 and 850nm. The distributions are significanlty less than zero. D) Optical density changes for trials with high and low spiking activity. Inset represents the changes between 0-3s. Larger decreases in optical density are observed for the high spiking trials. E) Mean changes in optical density between 0-1s reveal significant decreases in optical density for high-spiking trials. The decrease in optical density for both chromatophores implies a decrease in both HbO and HbR concentrations. Since HbT is the sum of changes in HbO and HbR, simultaneous decreases in HbO and HbR would lead to larger decreases in HbT when changes in optical density are converted to changes in concentration. Surprisingly, we found mildly significant decreases in the optical density at 850nm for low-spiking trials. However, after conversion to concentration changes, none of the traces (HbO, HbR or HbT) revealed significant changes between 0-1s.