No Replication of Direct Neuronal Activity-related (DIANA) fMRI in Anesthetized Mice

Toi et al. (Science, 378, 160–168, 2022) reported direct imaging of neuronal activity (DIANA) by fMRI in anesthetized mice at 9.4 T, which could be a revolutionary approach for advancing systems neuroscience research. There have been no independent replications of this observation to date. We performed fMRI experiments in anesthetized mice at an ultrahigh field of 15.2 T using the identical protocol as in their paper. The BOLD response to whisker stimulation was reliably detected in the primary barrel cortex before and after DIANA experiments; however, no direct neuronal activity-like fMRI peak was observed in individual animals’ data with the 50–300 trials used in the DIANA publication. Extensively averaged data involving 1,050 trials in 6 mice (1,050×54 = 56,700 stimulus events) and having a temporal signal-to-noise ratio of 7,370, showed a flat baseline and no detectable neuronal activity-like fMRI peak. Thus we were unable to replicate the previously reported results using the same methods, despite a much higher number of trials, a much higher temporal signal-to-noise ratio, and a much higher magnetic field strength. We were able to demonstrate spurious, non-replicable peaks when using a small number of trials. It was only when performing the inappropriate approach of excluding outliers not conforming to the expected temporal characteristics of the response did we see a clear signal change; however, these signals were not observed when such a outlier elimination approach was not used.