Synopsis

Diffusion fMRI (DfMRI) is a powerful functional imaging method to investigate neural activity and network without BOLD confounding hemodynamic effects. In this study, we applied DfMRI to evaluate brain activity and network of neuropsychiatric disease model, obsessive–compulsive disorder (OCD). Our DfMRI study revealed that the cortex was excited and the hippocampus was inhibited. Functional connectivity analysis of DfMRI detected alterations of cortico-striatal-thalamic networks, in line with the previous study with OCD patients. Additionally, we found that interesting network alterations of the hippocampal networks in this OCD model mice.

Purpose

Diffusion fMRI (DfMRI)¹ has been shown to reflect neural activity, as DfMRI signals are not of vascular origin², suggesting that DfMRI has an advantage to evaluate neural activity regardless of the status of hemodynamic responses. Previous study has suggested that water apparent diffusion coefficients (ADCs) obtained from the resting state DfMRI (rsDfMRI) closely reflected basal neural activity although hemodynamics was disrupted by anesthesia³. In addition, the study has proposed the method yielding DfMRI-guided cell swelling-composed networks³.

To show the applicability of rsDfMRI, we selected a well conceptualized neuropsychiatric animal model, an obsessive–compulsive disorder (OCD) model mouse, in which the cortico-striatal projection activated⁴ and the functional connectivity (FC) of cortico-striatal-thalamic networks altered⁵. There were two primary outcomes in this study. One was to detect the expected neural activity in the cortex. The other was to detect the expected alterations of cortico-striatal-thalamic networks. The secondary outcome was to find unexplored brain regions where previous BOLD-fMRI failed to extract.

Methods

We employed astrocytic glutamate transporter (GLT1) knockdown mice, which is known as OCD model⁶. The OCD model mouse was homozygous GLT1^flox/flox with heterozygous inducible Cre recombinase (CreERT2) in GLAST locus (GLAST^CreERT2/+) (GLAST^CreERT2/+;GLT1 ^flox/flox, n=15) (Fig. 1A). GLAST^+/+;GLT1^flox/flox mice were used as the control mice (ctl, n=14). Both groups were intraperitoneally injected with 100 mg/kg tamoxifen (TAM) at postnatal days 17-21 for consecutive five days. We conducted a surgery to put an acclimation bar on the skull at 8 weeks of age, started the MRI acclimation⁷ at 10 weeks of age, and finished the acclimation at 12 weeks of age.

MRI were conducted under awake condition at 11.7T equipped with cryoprobe (BioSpec, Bruker). rsDfMRI images were acquired with the following parameters: diffusion-sensitized double SE-EPI sequence; TR/TE=2000/37 ms, FOV=16×16 mm², Matrix=80×80, Slice thickness=0.8 mm. Slice number=10, Repetition number=150, Scan time=10 min, and b-values=1000 and 1800 mm²/s along 1 directions; [X=1,Y=1,Z=1]. Breathing and body temperature were monitored during scanning.

Pre-processing (realignment, slice timing, normalization) was performed using SPM12 and spatial smoothing was conducted with 0.4 mm of full width at half maximum. ADCs were calculated from time series data of diffusion weighted signal acquired with b=1000 and 1800 mm²/s (S_b1000, S_b1800) for each time point as ADC=ln(S_b1000/S_b1800)/800. Statistical analysis of ADCs was performed two-way repeated ANOVA over time and genotype.

Time-series data of S_b1800 were detrended and temporally filtered with 0.008-0.1 Hz. Seed-based FC analysis was performed using time-series data of pre-processed S_b1800. Regions of interest (ROIs) of 157 brain loci were defined with the Allen Mouse Brain Atlas. Pearson’s correlation coefficients between the time course of a predetermined ROI and all other ROIs were estimated after de-noising and de-spiking using functional connectivity toolbox (CONN) in order to generate a 157×157 connectivity matrix. Global mean signal was regressed out from the pre-processed time-series data in order to reduce non-neural signal correlations. Regions of white matter and cerebrospinal fluid were masked out.

Results and Discussion

GLT1 protein expression was significantly knocked down in the cortex (the cingulate cortex and the somatosensory cortex) and the hippocampus, confirming TAM-induced Cre mediated GLT1 deletion (Fig. 1B, C). ADCs were significantly decreased at the cortex in the OCD model compared with the ctl (Fig. 2), reflecting increased basal activity in the cortex in the OCD model. In FC analysis derived from rsDfMRI, we found altered cortico-striatal-thalamic functional connectivity in the OCD model (Fig. 3 and Table 1) as seen in the human studies from BOLD-fMRI⁵. These data demonstrated that rsDfMRI was able to detect expected alterations in OCD model mouse, showing the applicability of rsDfMRI.

We also found that ADC increased at the hippocampus (Fig. 2), reflecting decreased neural activity in the OCD model. This hippocampal inhibition was due to the decrease in hippocampal GLT1 level (Fig. 2) or due to the increase of an input from the entorhinal cortex (Table 2). FC of the hippocampal networks decreased in the OCD model (Fig. 5). The conventional BOLD-fMRI study has not reported this hippocampal alteration in the OCD patients. Our study proposed that the hippocampus was a new target region in OCD which was supported by a glutamate hypothesis.

Conclusion

Acknowledgements

References

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