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Negative BOLD responses in the rat visual pathway
Rita Gil1, Francisca Fernandes1, Clémence Ligneul1, and Noam Shemesh1

1Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown, Lisbon, Portugal

Synopsis

Negative BOLD responses (NBRs) in the rat visual cortex (VC) are reported, for the first time, upon high frequency visual stimulation. So far, in rats, only an attenuation of the positive BOLD response (PBR) in VC had been reported with increase of the stimulus frequency up to 10-12Hz1,2. Here, experiments with very high sensitivity thanks to a cryoprobe operating at 9.4T, reveal NBRs in VC and how they are modulated by hyperoxia (already reported for PBRs3,4). Results suggest the possibility that the neurovascular couplings operating under PBRs and NBRs might not be the same.

Introduction

Previous rodent visual system studies5-11show strong Positive BOLD Responses (PBRs) in superficial layers of the superior colliculus (SC) and lateral geniculate nucleus (LGN). Negative BOLD Responses (NBRs) in Visual Cortex (VC) have been reported for 10 Hz visual stimulation in mice11; however, insofar high frequency stimulation evidenced only a decrease in cortical PBRs in rats1,2,7. Here we report, for the first time, NBRs in VC of the rat when a 15Hz stimulus is presented, and study how they are modulated by hyperoxia. Our findings suggest that the neurovascular couplings operating under PBRs and NBRs might not be the same.

Methods

All animal experiments were preapproved by the institutional and national authorities and were carried out according to European Directive 2010/63.

Animal preparation. Adult Female Long Evan rats (n=5) were kept under medetomidine sedation17 while temperature and respiration rate were continuously monitored and remained stable.

MRI experiments. Images were acquired using a 9.4T BioSpec scanner (Bruker, Karlsruhe, Germany) with an 86mm quadrature resonator for transmittance and a 4-element array cryoprobe20,21 (Bruker, Fallanden, Switzerland) for signal reception. For fMRI, a SE-EPI sequence was used: TE/TR=42.5/1500msec, partial Fourier coefficient 1.5, FOV=18x16.1 mm2, resolution=269x268 μm2, slice thickness=1.5mm, tacq=7min 30sec.

Paradigm design. A 470nm LED (8.1x10-1 W/m2) was used for binocular visual stimulation delivered using two optic fibers placed near the rat eyes. The paradigm consisted of 15sec stimulation (frequency=15Hz; pulse width=670μsec) and 45sec rest, repeated six times (Fig.1A). The paradigm was repeated twice for each condition in a pseudo-random order (except for one animal where only one run per condition was performed) with 7 min of rest between runs.

Hyperoxia challenge. Oxygen percentage was varied between 22%, 28% and 95% and the same experiments as above were repeated after the animals stabilized in breathing rate.

Data analysis. Data was first denoised22, and then analysed using Statistical Parametric Mapping in Matlab®. Slice-timing was corrected using sinc-interpolation. Data were spatially smoothed (3D Gaussian kernel, FWHM=0.2685mm isotropic), realigned to the mean volume and co-registered to an anatomical reference. An HRF (peaking at 1.39 sec) was convolved with the stimulation paradigm prior to General Linear Model (GLM) analysis.The p-value and minimum cluster size thresholds considered for statistical significance were 0.001 and 8, respectively. ROI analysis was performed using anatomically-defined regions.

To avoid assumptions on the HRF, we additionally performed a data-driven spectral analysis as in [23]. The area under the paradigm’s fundamental frequency and first harmonic (Fig.1A) were mapped for every pixel’s Fourier spectrum.

Statistical test were performed using the Kruskal-Wallis test.

Results

The SNR in the fMRI experiments was rather high: 144.4, 263 and 170.6 in SC, LGN and V1 respectively. Fig.2 shows activation maps derived from GLM analysis, where strong PBRs for SC and LGN can be seen along with NBRs in the VC for all oxygen concentrations. The t-values of NBRs as well as their area increased with increasing oxygen concentration, despite that the stimulus remained constant. Data-driven spectral analysis (Fig.3) revealed very similar patterns as GLM (Fig.2). NBR areas clearly increased with increasing O2 levels. Averaged cycles for different structures are plotted in Fig.4. Higher response magnitudes were observed for PBRs and NBRs with increasing %O2. NBR temporal profiles evidenced oscillations at lower O2 percentage and flatter (yet stronger) responses with increasing oxygen fraction. The NBRs varied significantly (p<0.001) with O2, whereas PBRs varied only between the highest and lowest oxygen concentration (Fig.5).

Discussion

We report NBRs in rat VC for the first time (to our knowledge), consistent with previous reports of NBRs in mice11. Previous rat studies showed that high-frequency stimulation attenuated PBRs in rat VC1. However, electrical recordings revealed that multiunit activity remain elevated1,8,12 which leads to the hypothesis that NBRs in rat VC may reflect inhibitory activity.

Since our visual stimulation was identical for all O2 concentrations and no significant oxygen consumption changes upon hyperoxia were reported in humans24, it can be assumed that hyperoxia does not affect neural activity per-se. Therefore, NBR modulations with O2 likely reflect a vascular component. Interestingly, the underlying neurovascular couplings for PBRs and NBRs seem not be mirroring one another. As PBRs were shown to increase with hyperoxia due to increases in oxygenated blood4,11, the same mechanism would decrease the amplitude of NBRs, which contradicts our observations. This suggests potentially interesting differences in neurovascular coupling mechanisms governing PBRs and NBRs

Conclusions

We observed NBRs in the rat VC upon visual stimulation using highly sensitive SE-BOLD experiments. These NBRs were modulated in different ways than PBRs upon oxygen challenges, suggesting different underlying neurovascular coupling mechanisms.

Acknowledgements

This study was supported by funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Starting Grant, agreement No. 679058) and Fundação da Ciência e Tecnologia (FCT), Portugal (PD/BD/128297/2017). RG would like to thank Dr. Daniel Nunes for the help during the preparation of MRI experiments and animal monitoring and Dr. Rui Simões for the helpful discussions.

References

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Figures

Figure 1: A: On the left the stimulation paradigm of 15sec stimulation and 45sec rest period repeated six time is represented. On the right side the FFT spectrum of the stimulation paradigm is shown with fundamental frequency and first harmonic used in the spectral analysis marked. B: Raw anatomical and functional (left and right) data is shown for the three slices.

Figure 2: BOLD activation maps for the different O2 regimes using General Linear Model SPM12 analysis. Strong PBRs can be seen in SC and LGN and NBRs can be seen in VC for all conditions. As the O2 concentrations increases we can observe and increase in the contrast of the responses to the stimulus and also a broadening of the PBR in the VC.

Figure 3: Spectral analysis maps showing pixels which frequency spectrum correlates with the stimulation paradigm fundamental frequency and first harmonic (superimposed on a mean functional image). Top and bottom rows show pixels that showed positive and negative correlation with the stimulation paradigm, respectively. Yellow, red and blue areas correspond to pixels that correlated with the paradigm at 22%, 28% and 95% O2. As the O2 concentration increases, a broadening of activation areas can be observed for both positive and negative responses with a more pronounced effect seen for the NBRs.

Figure 4: Averaged cycle (mean±sem) for different anatomical based ROIs of structures from the visual pathway. Top row: VC; Bottom row: SC and LGN. Yellow, red and blue curves correspond to 22%, 28% and 95% O2 respectively. Dashed grey lines indicate beginning and ending of stimulation. On the right top corner of each plot an example of the applied mask can be seen. For the top row an absolute increase in the NBRs can be seen with a shape change when hyperoxia state is imposed. For the PBRs only the increase in magnitude response can be observed.

Figure 5: Mean rank plots from a Kruskal-Wallis test for the different structures, showing which regimes are significantly different (p<0.001). VC responses were inverted for an easier comparison with SC and LGN. Responses in SC and LGN are quite similar with only a significant difference between the lower and higher O2 concentrations. However, for the VC, where NBRs are seen, responses in the hyperoxia state are significantly different from the other two oxygen regimes.

Proc. Intl. Soc. Mag. Reson. Med. 27 (2019)
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