Aim
Isoflurane anesthesia is commonly used for functional imaging studies in animals due to the ease of anesthetic induction and an ability to perform survival experiments. However, it was reported that functional-metabolic coupling in the rat somatosensory cortex induced by rectangular pulse stimulation (0.3-ms pulse duration and 5-Volt intensity at 3 Hz) was achieved only under alpha-chloralose anesthesia (Ueki et al. 1992). This observation can be due to either species variations (rats vs. other animals), and/or unoptimized somatosensory stimulus. In the present study, evoked neural and hemodynamic responses in the rat somatosensory cortex were characterized under isoflurane anesthesia.
Materials and methods
Eight male Sprague-Dawley rats (350–450 g) were used for measurements of neural activity and hemodynamic responses induced by forepaw somatosensory stimulation, and two rats were used for functional MRI (fMRI) studies. End-tidal CO2 and arterial blood pressure were continuously monitored and blood gases were maintained at a normal range. Isoflurane concentration was kept at 1.3–1.4% in a mixture of O2 and air. The skull was thinned to measure 620-nm wavelength intrinsic optical imaging for the localization of the forelimb area. Local field potential (LFP) and cerebral blood flow (CBF) were simultaneously recorded at the activation focus induced by electrical forepaw stimulation using a tungsten microelectrode and laser-Doppler flowmetry, respectively. Parameters of forepaw somatosensory stimulus consisting of ten rectangular pulses were systematically varied by i) pulse width (0.1–5.0 ms) at fixed current (1.0 mA) and frequency (8 Hz), ii) current level (0.2–2.0 mA) at fixed pulse width (1.0 ms) and frequency (8 Hz), and iii) inter-pulse intervals (50–500 ms) at fixed pulse width (1.0 ms) and current (1.0 mA). Finally, blood oxygenation level-dependent (BOLD) fMRI (9.4T/31 cm, Varian System) was performed in isoflurane-anesthetized rats with optimized forepaw stimulation parameters as determined by LDF.
Results and discussion Stimulus optimization
When pulse width and current level increased, the summation of LFP and the amplitude of CBF changes in the forelimb area were similarly augmented, and reached to plateau levels (90% of LFP and CBF plateau at 1.0 ms and 1.4 mA vs. 0.5 ms and 1.0 mA, respectively). On the other hand, the summation of LFP increased monotonically with an increase of inter-pulse intervals (90% of plateau: 250 ms), while the largest CBF change (70 ± 30%, Mean ± SD) was found at stimulus frequency of 12 Hz (an inter-pulse intervals: 82.5 ms). These stimulus conditions did not change blood pressure. Our results indicate that coupling between evoked neural and hemodynamic response is not impaired under isoflurane- anesthetized rats, but that the optimum stimulus frequency is strongly influenced by the anesthesia used. fMRI study: Using BOLD fMRI in isoflurane-anesthetized rats, we detected the focal activation at the primary forelimb somatosensory area. This further indicates that the isoflurane-anesthetized rat model can be also used for hemodynamic-based functional imaging. Volatile isoflurane anesthetic can be used for rodent functional neuroimaging, allowing the repetition of survival studies over days and months for clinical and basic neuroscience research (e.g., development, learning, and plasticity).