Blood oxygenation level-dependent (BOLD) fMRI is widely-used to investigate brain activity. However, BOLD fMRI, relying on the neurovascular coupling, fails in some conditions which prevent neurovascular coupling, i.e., anesthesia and alcohol intoxication¹. In contrast, diffusion fMRI² is directly linked to neuronal activation showing neuronal responses even when neurovascular coupling is abolished³. Hence, we compared BOLD fMRI, diffusion fMRI and ADC changes with local field potentials (LFPs) in the nucleus accumbens (NAc) by alcohol stimulation (vasodilator and dopamine secretion facilitator in NAc) under medetomidine anesthesia (vasoconstrictor and dopamine secretion inhibitor in NAc).

Animals

Male Wistar rats (200-300 g) (n = 6 – 10 / group) were anesthetized under a high (0.25 mg/kg/h) or a low (0.1 mg/kg/h) medetomidine anesthesia (i.v.). As shown in Fig. 1A, following EtOH (4 mg/kg) or saline (0.9% NaCl) injection (i.p.) 10 min after the start of the data acquisition, it was then continued for 20 min.

FMRI

FMRI acquisition was conducted at 7T using a volume coil (Pharmascan, Bruker, Germany). FMRI images were acquired using a diffusion-sensitized double spin-echo EPI sequence, TR/TE = 9,000/35 ms, 32 x 32 mm (100 x 100 pixels), 1.2 mm thickness, 6 slices, b = 200, 1000 and 1800 s/mm². The images with b = 200 s/mm² were used as the BOLD (spin-echo) fMRI signal. The ADC was calculated as ADC= ln[S(1000) / S(1800)] / 800 to eliminate IVIM and T2-weighting residual effects. For t-contrast, the regressor was constructed using the time-course of averaged LFPs gamma bands in NAc. Time-courses were extracted using region of interest of NAc (Fig. 2B) and then calculated as percent changes to averaged baseline (within 5 min before injection)

LFP

Separated from fMRI, LFPs in the NAc were recorded with a single tungsten microelectrode as shown in Fig. 1B, at 1 kHz sampling rate using data acquisition software (Power Lab, ADInstruments, New Zealand). The reference electrode was positioned on the scalp. Then powers of frequency bands (alpha, 8–12 Hz; beta, 18–30 Hz; gamma, 60–80 Hz) were analyzed.The power of the gamma oscillation increased to reach a peak around 5 min after EtOH infusion under high medetomidine, then decreased below baseline level. Under low medetomidine, it marked a biphasic response, and then fluctuated and gradually decreased (Fig. 2). The powers of alpha and beta bands decreased slightly. The ADC time-course in NAc mirrored, but in opposite direction, the LFP gamma oscillation power, showing a fast negative response at high dose of medetomidine followed by ADC increase (Fig. 3). Under low dosage of medetomidine, the ADC response was biphasic, and then fluctuated slightly. Overall, there was a significant negative correlation between the ADC and the power of LFPs gamma oscillation time-courses at both medetomidine concentrations (p < 0.05; regression analysis) (Fig. 3B). While, the BOLD fMRI signals decreased following EtOH infusion and the decrease with low medetomidine was greater than that with high medetomidine. There was no significant correlation between BOLD and the power of gamma band (p > 0.05; regression analysis) (Fig. 3B). The ADC and BOLD fMRI time-courses remain flat by a saline infusion as negative control (Fig. 4).As both medetomidine and EtOH oppositely regulate dopamine secretion in NAc^{4, 5}, the neuronal activity pattern differed according to medetomidine dose, which was well reflected by the ADC response and not the BOLD response. Those results confirm that water diffusion more closely reflects neural events without interference with the neurovascular coupling upon which BOLD fMRI relies. It is important to notice that diffusion-weighted signals are necessarily T2-weighted and, thus, include a BOLD fraction⁶. This residual BOLD effect can be removed using ADCs.Diffusion fMRI, through the ADC, appears a suitable biomarker of neural activation, reflecting LPFs responses and avoiding confounds from varying conditions of neurovascular coupling, with potential for pharmacological fMRI studies.