Sensory cortices contain extensive descending (corticofugal) projections, yet the impact of cortical input on brainstem processing remains poorly understood. In the central auditory system, the auditory midbrain nuclei, called the inferior colliculus (IC), receive projections from both the auditory (AC)¹ and visual (VC)^{2, 3} cortices. Previous studies probing the functional influences of the corticofugal projections were mostly through electrically activating or cryogenically inactivating the cortex whilst examining the activities of single IC neurons⁴. Such manipulations likely affect all cortical neuron types, though the corticofugal projections arise mostly from the excitatory neurons in deep layers⁵. Furthermore, with single neuron recordings, the large-scale effects of the corticofugal input are not easily extrapolated. To date, the VC descending influences on auditory midbrain processing have not been comprehensively investigated. Our recent fMRI study found that the gain of IC response decreased after bilateral ablation of the VC⁶. Although this study provided important functional evidence for the crossmodal descending influences of the VC on auditory midbrain processing, a more detailed investigation, e.g. the contribution of excitatory neurons, is hindered by the cortical ablation model. In this study, a new method, combined auditory and optogenetic fMRI, was developed to investigate the corticofugal effects on IC auditory processing. Large-view fMRI was used to characterize the auditory midbrain responses to noise stimulation during cell-type and spatiotemporally specific optogenetic stimulation of the VC. The results demonstrate the feasibility of this novel approach in investigating the corticofugal effects and show that VC activation normally facilitates auditory midbrain processing.

Animal preparation AAV5-CaMKIIa::ChR2(H134R)-mCherry was injected to the VC of adult rats (n=4, 200-250g, male, SD strain) to express ChR2 in layer VI CaMKIIa excitatory neurons, which is a major source of corticofugal projections⁵. Four weeks after injection, a plastic optic fiber (diameter 450μm) was implanted at the injection site (Figure 1) to deliver light stimulation. During MRI scanning, the animals were mechanically ventilated and maintained stable with mixed air and 1.0% isoflurane. Auditory stimulation was delivered using a custom-built system⁶.

Auditory and optogenetic stimulation First, the effects of optogenetically stimulating the VC were examined by applying blue (473nm) light pulses (10Hz pulse rate, 10% duty cycle, 50mW/mm² light power) in a block-design paradigm (20s on and 60s off), without presenting sound stimulation. Subsequently, the effects of activating the VC on auditory processing within the IC were investigated. Monaural (left) noise stimulation was presented in a block-design paradigm (20s on and 50s off) while the 10Hz optogenetic stimulation was presented to the contralateral (right) VC from 10s before to 10s after every second sound-on period (Figure 2).

fMRI acquisition and analysis All fMRI data was acquired on a 7T Bruker scanner using GE-EPI (FOV=32×32mm2, matrix=64×64, α=56°, TE/TR=20/1000ms, 12 contiguous slices with 1mm thickness). Data were first realigned, co-registered, in-plane smoothed and high-pass filtered before the standard GLM analysis was applied to identify significant BOLD responses (p<0.05, corrected for FWE).

Figure 3 shows that optogenetically activating the VC evokes strong BOLD responses in local VC and the ipsilateral hippocampus (HP), likely through cortical-hippocampal projections 7. Notably, no BOLD signaling was induced in the IC.

Figure 4 presents the IC BOLD responses to noise stimulation. The response magnitude within the IC was higher during optogenetic stimulation of the VC (p<0.05, paired t-test on averaged β value), indicating that VC activation facilitates IC auditory processing.

Our results clearly demonstrate that combined auditory and optogenetic fMRI is a feasible approach for investigating the cortical descending influences on auditory midbrain processing. Optogenetically activating the VC does not directly induce BOLD signaling in the IC, yet it enhances the IC BOLD responses to noise stimulation. The results are consistent with our recent auditory fMRI study that found the IC noise response decreased after bilateral ablation of the VC⁶. Together, our results present direct evidence that VC normally provides large-scale facilitatory influence for auditory midbrain processing, and the excitatory neurons in the VC play an important role in this process. Such crossmodal influence is likely through the direct VC-to-IC projections, which are innervated by the excitatory neurons^{2, 3}. The hippocampus may also play a role in mediating such influences, as it was robustly activated by the optogenetic stimulation of the VC. This possibility might be associated with the hippocampal-IC interactions reported by earlier studies⁸. Nevertheless, future studies are necessary to further elucidate the underlying neural mechanisms. Presently, our ongoing experiments are expanding the sample size and elaborating the analysis protocol for the current results, in purpose to examine the VC influences in different IC subnuclei. Our future studies will apply this novel method to systematically examine the cortical descending influences from both auditory and visual cortices (and their sub-nuclei) on different aspects of auditory processing in the midbrain.