Edited by: Christian Hansel, University of Chicago, United States
Reviewed by: Vassiliy Tsytsarev, University of Maryland, Baltimore County, United States; Arianna Maffei, Stony Brook University, United States
*Correspondence: Mikhail Sintsov
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Optical Intrinsic Signal imaging (OISi) is a powerful technique for optical brain studies. OIS mainly reflects the hemodynamic response (HR) and metabolism, but it may also involve changes in tissue light scattering (LS) caused by transient cellular swelling in the active tissue. Here, we explored the developmental features of sensory-evoked OIS in the rat barrel cortex during the first 3 months after birth. Multispectral OISi revealed that two temporally distinct components contribute to the neonatal OIS: an early phase of LS followed by a late phase of HR. The contribution of LS to the early response was also evidenced by an increase in light transmission through the active barrel. The early OIS phase correlated in time and amplitude with the sensory-evoked electrophysiological response. Application of the Modified Beer-Lambert Law (MBLL) to the OIS data revealed that HR during the early phase involved only a slight decrease in blood oxygenation without any change in blood volume. In contrast, HR during the late phase manifested an adult-like increase in blood volume and oxygenation. During development, the peak time of the delayed HR progressively shortened with age, nearly reaching the stimulus onset and overlapping with the early LS phase by the fourth postnatal week. Thus, LS contributes to the sensory-evoked OIS in the barrel cortex of rats at all ages, and it dominates the early OIS phase in neonatal rats due to delayed HR. Our results are also consistent with the delayed blood oxygen level dependent (BOLD) signal in human preterm infants.
Optical Intrinsic Signal imaging (OISi) is a simple yet powerful technique of minimal invasiveness for studying brain activity. It has been successfully used for studies of sensory functional topography (Grinvald et al.,
In adults, fNIRS and OISi are both considered to be effective substitutes for functional Magnetic Resonance Imaging (fMRI) (Strangman et al.,
In the present study, we aimed to characterize sensory-evoked OIS in the somatosensory cortex of neonatal rats during their development using multispectral OISi and concomitant intracortical recordings of the electrophysiological signals. Newborn rat pups served as a convenient animal model of preterm human infants (Workman et al.,
All animal-use protocols followed the guidelines of the French National Institute of Health and Medical Research (INSERM, provisional approval N007.08.01) and the Kazan Federal University on the use of laboratory animals (ethical approval by the Institutional Animal Care and Use Committee of Kazan State Medical University N9-2013). Wistar rats of both sexes from postnatal days [P] 2–25 and 60–90 were used (P0 was the day of birth). The surgery was performed under isoflurane anesthesia (5% for induction and 1.5% during surgery). The rat skull was cleaned of skin and periosteum and was covered with dental cement (Grip Cement) except for a 5 × 5 mm2 window above the barrel cortex. The metal plate attached to the cement helmet was mounted to the ball joint in a stereotaxic apparatus. Subsequently, the rats were warmed and left for an hour to recover from anesthesia, surrounded by a cotton nest and heated via a thermal pad (35–37°C). All the recordings were made under urethane anesthesia (by intraperitoneal injection; 1 g/kg). To remove visual artifacts skull thinning or a simple skull polishing was performed depending on animal age. The skull was then covered with saline and a coverslip.
Multispectral OIS was recorded using a video acquisition system (Figure
Setup design for the OIS recordings.
Multispectral OISi was performed using diodes with different wavelengths to reflect volumetric and oximetric changes of HR as described in Hillman (
Natural sensory stimulation was performed as described previously (Peterson and Goldreich,
Data preprocessing included per-frame spatial filtering with 2D Gaussian filter (σ = 2
Local field potential (LFP) was recorded using either a silicon probe (NeuraNexus A1x16-5mm-100-413-A16) or a glass pipette filled with artificial cerebrospinal fluid (resistance of 2–6 MOhm). The electrode was inserted into L4 of the principal barrel at a 30° angle to the skull surface which allowed simultaneous recordings of OIS. LFP signals were then amplified and digitized by DigitalLynx (Neuralynx) at 10 kHz. To measure the neuronal activity associated with OIS, LFP was averaged over the stimulation period.
We performed single whisker-evoked OISi in the barrel cortex in
OIS is typically characterized by a low signal-to-noise ratio (SNR). To increase the dynamic range of OIS recordings and to evoke the strongest signal, we first optimized the stimulation protocol. To do so, we modulated stimulation frequency from 0.1 Hz (1 stimulus) to 20 Hz (200 stimuli) and calculated the OIS amplitude recorded in the RED spectrum. Stimulation intensity is known to affect OIS amplitude in a nonlinear fashion (Sheth et al.,
Group data of OIS profiles obtained in the reflectance mode are shown in Figure
Group average OIS in different spectra at different ages. Plots show average OIS recorded in the reflectance mode in different age groups (P4-5, P6-7, P8-10, P11-12, P21-25, P60-90) and different imaging spectra (GREEN, RED, and IR). A solid line depicts a group average and a shaded region—a group's standard deviation of individual OIS traces. Vertical lines indicate the stimulus onset and a horizontal bar above indicates the stimulation period. The group size is shown below each plot.
To quantitatively assess the developmental changes in OIS we calculated OIS peak amplitude and OIS peak position—a time delay from the stimulation onset to a peak (Figures
Developmental profile of the OIS parameters.
The OIS peaks in the GREEN spectrum in the P4-5 (27.5 ± 5.5 s), P6-7 (26.0 ± 7.0 s) and P8-10 (18.5 ± 5.5 s) groups were attained significantly later than the 10 s stimulation train offset (
As mentioned above, OIS in the RED and IR spectra shared similar parameters in neonates (Figure
Comparison of the transmission and the reflectance imaging modes in the neonatal rats.
We further asked how the early OIS phase correlates with the electrophysiological response in neonatal rats. For this, we simultaneously recorded sensory-evoked LFP and OIS in the RED spectrum in P5-7 rats (
Simultaneous recordings of the RED OIS and electrical activity in the barrel cortex of neonatal rats.
We then compared OIS amplitudes and average LFP in the P5-7 group. In spite of the fact that both OIS amplitude and the average LFP exhibited nonlinear dependence on stimulation rate (Figures
The relationship between the RED OIS amplitude, average LFP (<
Intracortical application of CNQX suppresses sensory-evoked LFP and OIS.
Multispectral OIS imaging was shown to reveal the parameters of HR using the modified Beer-Lambert law (MBLL) (Delpy et al.,
where μ
To account for the illumination correction during preprocessing we first, assumed that reference and OIS regions share similar core optical properties and second, that the sensory-evoked changes are small. Therefore the subtraction of reference intensity in the illumination correction
The exact values for
Using Equation (2) we compared the parameters of HR and LS in neonatal (P6-7) and adult (P60-90) rats (Figure
Decomposition of OIS into the hemodynamic response and the light scattering using the modified Beer-Lambert Law (Equation 2) for adult (P60-90) and neonatal (P6-7) rats.
Our results indicate that the early phase of the sensory-evoked OIS response in the neonatal rat barrel cortex is dominated by LS. Changes in LS primarily involved an increase in brain tissue transparency
The LS component of the sensory-evoked OIS in the neonatal rat barrel cortex may involve the same mechanisms as in adults such as cellular swelling, with some developmental differences. We found that LS raises and decays much slower than in adults (Figure
We showed that a positive HR in neonatal rats was considerably delayed from the stimulus onset (Figure
We have found that OIS could be consistently recorded from the barrel cortex of neonatal rats starting from age P4 but not before. This is despite the formation of thalamocortical connections already at birth and the functional segregation of barrels by P3 (Mitrukhina et al.,
We performed multispectral optical intrinsic signal imaging of sensory-evoked responses in the rat barrel cortex starting from age P4 onwards. Applying the MBLL, we found that the optical signal in neonatal rats is organized into two temporally separated processes: an early phase with a change in tissue light scattering (LS) and a late phase with a hemodynamic response (HR). We also found that the HR delay shortens with age and largely overlaps with LS by the end of the first postnatal month. These findings may be of great importance for optical functional neuroimaging in human infants, particularly in preterm babies.
MM, RK: Study concept and supervision. MS, DS: Acquisition of data and analysis. MS: Drafting of the manuscript. RK: Critical revision of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The work was supported by RSF grant 16-15-10174 (MM) and performed in the frame of the Program of competitive growth of Kazan Federal University.
The Supplementary Material for this article can be found online at:
Frame by frame comparison of OIS recorded in a P6 rat using IR reflected, and IR transmitted light. Dashed regions in OIS maps indicate regions corresponding to the D1 barrel. Below are the trial-averaged OIS time courses averaged in the D1 regions (
Optimal stimulation studies for different age groups to maximize OIS.
Diodes for multispectral OISi, their wavelength of maximal emission (λmax) and averaged extinction coefficient of oxy- and deoxy-hemoglobin (ϵHbO and ϵHbR). Calculations are based on Zijlstra et al. (
Parameters values used for MBLL to decompose OIS signals.ϵnap stands for the extinction coefficient corrected for the Napierian base,