Synopsis

Keywords: fMRI (task based), Animals

The fMRI mapping with selective modulation of local neural population at the manipulated region is a powerful approach that can causally link circuit-specific interactions to behavioral performance. However, most fMRI studies combined with chemogenetics were conducted in the resting state, which is difficult to elucidate whether and how differences in functional activity by neuromodulation induce behavioral changes. Here, we demonstrated the effects of fMRI-guided focal chemogenetic modulation on both somatosensory-evoked network and its relevant behaviors in mice.

Purpose

Concurrent cell type/circuit-specific modulations and fMRI in animals can provide valuable insights into understanding the causal relationships between neuronal activity and brain-wide network. The neural modulation can be achieved via optogenetics¹ and chemogenetics². These strategies are employed to light-sensitive ion channels or designer receptors exclusively by designer drugs (DREADDs), respectively. fMRI combined with optogenetics typically required chronic intracranial fiber implants to deliver light pulses, which can often cause image artifacts of distortion and signal drops in fMRI studies³. Alternatively, chemogenetics offers the advantage of not requiring fiber implants, and enabling sustained neural modulation with a single drug administration. Most chemogenetic fMRI studies were conducted at rest^4,5. However, since resting-state fMRI does not measure functional afferent/efferent circuit processes⁶, it is difficult to reflect how differences in functional activity due to neuromodulation induce behavioral changes. Therefore, we investigated the effects of fMRI-guided chemogenetic modulation on both somatosensory-evoked network and its relevant behaviors in mice.

Materials & Methods

To causally link brain-wide networks to behavioral performance, three different studies were designed: 1) fMRI-guided detection on aberrant somatosensory circuit, 2) sensory-evoked fMRI combined with chemogenetic silencing in the aberrant functional region, and 3) behavior tests.
All BOLD-fMRI experiments were performed on 15.2T using single-shot GE-EPI sequence (TR/TE=1000/11.5ms, spatial resolution=132×132×500μm³) under dexmedetomidine-isoflurane anesthesia⁷. Sensory-sensitized transgenic mice (HT) with autism spectrum disorder (ASD)-risk C456Y heterozygous mutation in the Grin2b gene⁸ and naïve C57BL/6 mice (WT) were used.
First, we asked which brain regions exhibit abnormal functional activity in HT mice (n=11) compared to WT controls (n=10). For somatosensory activation, the right whisker-pad was electrically stimulated with current intensity of 0.9-mA, pulse width of 0.5-ms, and frequency of 4-Hz. Functional trial consisted of a 30-s prestimulus, 20-s stimulus, 30-s interstimulus, 20-s stimulus, and 30-s post stimulus period, and 15 fMRI trials were obtained for signal averaging (Fig.1A). Next, we investigated whether functional normalization or rescue in HT mice using chemogenetic modulation of the aberrant brain region could be observed in somatosensory-evoked fMRI. To inhibit the excitatory neuronal activity, AAV-CaMKIIα-hM4D(Gi)-mCherry (DREAD virus, ≥ 8.6×10¹²vg/mL) were injected into the relevant brain region determined from the fMRI-guided experiments. Three-four weeks after the DREAD virus injection, somatosensory-evoked fMRI was repeated before and after 10-mins of clozapine-n-oxide (CNO, 5mg/kg, n=11 each group) or dimethyl sulfoxide (DMSO, as control for CNO) injection intraperitoneally (Fig.2A). Finally, to examine whether fMRI findings directly reflect the somatosensory behaviors, three different behavioral tests were performed; Von Frey Test (VFT), thermal place preference test (TPT), and electric foot shock test (EFS) respectively to the evaluate mechanical thresholds for somatosensory stimuli, degrees of thermal place avoidance response, and freezing responses to electrical stimuli.
To estimate the regional specificity of sensory-evoked BOLD changes, fMRI data were spatially normalized onto the mouse brain atlas space⁹. Time-series data were extracted based on the somatosensory-related ROIs¹⁰ (Fig.1B.i) and then the signal changes over the stimulus duration were averaged.

Results

Animal-wise averaged fMRI maps (Fig.1B.ii-iii) and ROI-based fMRI signals (Fig.1C) show that stimulation of the whisker-pad showed stable positive response in the somatosensory pathway, whereas hyperactive fMRI responses were clearly observed in four regions including ACC, PAG, ipsilateral S1, and M1 in sensory-sensitized HT mice (Fig.1D). Among them, ACC was found to be the most aberrant region determined by a minimum redundancy maximum relevance (MRMR)-based feature selection (Fig.1E).
With this fMRI-guided regional detection, somatosensory-evoked fMRI was combined with chemogenetic silencing of ACC (Fig.2A). Before CNO injection, the hyperactive regions including ACC were reproducibly observed in HT mice (Fig.2B.i-ii and 2D.i). However, after ACC silencing, HT mice showed a significant DREADD-mediated normalization of fMRI activities within the somatosensory circuit, similar to those levels of WT mice (Fig.2B.iii-iv, 2C, and 2D.ii). We did not observe the similar effects with DMSO injection, confirming that the normalization of fMRI response was well driven by DREADD treatment (Fig.2E).
In the behavioral tests (Fig.3), most somatosensory performances including mechanical thresholds for stimuli (VFT) and freezing responses to electrical stimuli (EFS) were significantly returned to a normal level during chemogenetic silencing of ACC in HT mice. The degree of thermal place avoidance response (TPT) was not significant, but slightly changed.
These two independent experiments, fMRI mapping and behavioral tests, indicate that functional circuit-evoked fMRI combined with chemogenetics can measure the behavior-modulating network changes.

Discussion & Conclusion

We examined the effects of chemogenetic circuit-specific silencing on the hyperactivities of the somatosensory network and relevant behaviors using ASD-risk mutant HT mice. The ACC, known for vast top-down modulatory function¹⁰, was determined as a potential hub involved in the hyperactivities in the genetic model. Chemogenetic inhibition of ACC lead to normalization of the responses in both somatosensory fMRI and behavior experiments in HT mice. Sensory fMRI with chemogenetic manipulation is a valuable tool for monitoring the modulation of aberrant brain networks leading to change the behavioral performance. The main benefit of the tonic activation of DREADD receptors via chemogenetics over optogenetics is to perform prolonged inactivation of ACC, which circumvents the need for optic fiber placement and possible heating issues¹¹. Therefore, our findings underscore the potential utility of task-fMRI-guided chemogenetic approaches to observe changes in the functional network directly linking to behaviors and furthermore to facilitate translatable manipulation for disease treatment.