Mitsuhiro Fukuda

Dose-dependent effect of isoflurane on neurovascular coupling in rat cerebral cortex.

Neurovascular coupling studies are widely conducted in anesthetized animals using functional magnetic resonance imaging (fMRI). In this study, the dose‐dependent effects of isoflurane on neurovascular coupling were examined with concurrent recordings of the local field potential (FP) and cerebral blood flow (CBF) in the rat somatosensory cortex. Electrical forepaw stimulation was used, and consisted of either a single pulse or 10 pulses at various frequencies. We observed that the FP response to single‐pulse stimulation remained unaffected across the different levels of isoflurane tested (1.1–2.1%), whereas the CBF response to single‐pulse stimulation increased dose‐dependently (7u2003±u20033% to 17u2003±u20034%). The isoflurane dose did not affect the vascular reactivity induced by a hypercapnic challenge. These findings suggest that the action of isoflurane affects the neurovascular mechanisms. For 10‐pulse stimulation, the summation of the evoked FP responses monotonically decreased with an increase in the isoflurane dose, possibly due to enhancement of the neural adaptation. In contrast, the dose‐dependent effect on the CBF response varied with the stimulus frequency; a dose‐dependent decrease in the CBF response was observed for high‐frequency stimulation, whereas a dose‐dependent increase was observed for low‐frequency stimulation. Furthermore, a linear time‐invariant model consisting of the single‐pulse hemodynamic impulse response convoluted with 10‐pulse FP recordings showed that the neurovascular transfer function was altered by the isoflurane dose for high‐frequency stimulation. These results indicate that careful and consistent maintenance of the depth of anesthesia is required when comparing fMRI data obtained from different animals or physiological and pharmacological manipulations.

Effects of the α2-adrenergic receptor agonist dexmedetomidine on neural, vascular and BOLD fMRI responses in the somatosensory cortex

This article describes the effects of dexmedetomidine (DEX) – the active ingredient of medetomidine, which is the latest popular sedative for functional magnetic resonance imaging (fMRI) in rodents – on multiple unit activity, local field potential (LFP), cerebral blood flow (CBF), pial vessel diameter [indicative of cerebral blood volume (CBV)], and blood oxygenation level‐dependent (BOLD) fMRI. These measurements were obtained from the rat somatosensory cortex during 10 s of forepaw stimulation. We found that the continuous intravascular systemic infusion of DEX (50 μg/kg/h, doses typically used in fMRI studies) caused epileptic activities, and that supplemental isoflurane (ISO) administration of ~0.3% helped to suppress the development of epileptic activities and maintained robust neuronal and hemodynamic responses for up to 3 h. Supplemental administration of N2O in addition to DEX nearly abolished hemodynamic responses even if neuronal activity remained. Under DEX + ISO anesthesia, spike firing rate and the delta power of LFP increased, whereas beta and gamma power decreased, as compared with ISO‐only anesthesia. DEX administration caused pial arteries and veins to constrict nearly equally, resulting in decreases in baseline CBF and CBV. Evoked LFP and CBF responses to forepaw stimulation were largest at a frequency of 8–10 Hz, and a non‐linear relationship was observed. Similarly, BOLD fMRI responses measured at 9.4 T were largest at a frequency of 10 Hz. Both pial arteries and veins dilated rapidly (artery, 32.2%; vein, 5.8%), and venous diameter returned to baseline slower than arterial diameter. These results will be useful for designing, conducting and interpreting fMRI experiments under DEX sedation.

Changes in Cerebral Arterial, Tissue and Venous Oxygenation with Evoked Neural Stimulation: Implications for Hemoglobin-Based Functional Neuroimaging

Little is known regarding the changes in blood oxygen tension (PO2) with changes in brain function. This work aimed to measure the blood PO2 in surface arteries and veins as well as tissue with evoked somato-sensory stimulation in the anesthetized rat. Electrical stimulation of the forepaw induced average increases in blood flow of 44% as well as increases in the tissue PO2 of 28%. More importantly, increases in PO2 throughout pial arteries (resting diameters=59 to 129 μm) and pial veins (resting diameters=62 to 361 μm) were observed. The largest increases in vascular PO2 were observed in the small veins (from 33 to 40 mm Hg) and small arteries (from 78 to 88 mm Hg). The changes in oxygen saturation (SO2) were calculated and the largest increases were observed in small veins (Δ=+11%) while its increase in small arteries was small (Δ=+4%). The average diameter of arterial vessels was observed to increase by 4 to 6% while that of veins was not observed to change with evoked stimulation. These findings show that the increases in arterial PO2 contribute to the hyper-oxygenation of tissue and, mostly likely, also to the signal changes in hemoglobin-based functional imaging methods (e.g. BOLD fMRI).

Frequency-dependent Neural Activity, CBF, and BOLD fMRI to Somatosensory Stimuli in Isoflurane-anesthetized Rats

Inhalation anesthetics (e.g. isoflurane) are preferable for longitudinal fMRI experiments in the same animals. We previously implemented isoflurane anesthesia for rodent forepaw stimulation studies, and optimized the stimulus parameters with short stimuli (1-3-s long stimulation with ten electric pulses). These parameters, however, may not be applicable for long periods of stimulation because repetitive stimuli induce neural adaptation. Here we evaluated frequency-dependent responses (pulse width of 1.0 ms and current of 1.5 mA) for 30-s long stimulation under 1.3-1.5% isoflurane anesthesia. The cerebral blood flow (CBF) response (using laser Doppler flowmetry: CBF(LDF)) and field potential (FP) changes were simultaneously measured for nine stimulus frequencies (1-24 Hz). CBF (using arterial spin labeling: CBF(ASL)) and blood oxygenation level dependent (BOLD) fMRI responses were measured at 9.4 T for four stimulus frequencies (1.5-12 Hz). Higher stimulus frequencies (12-24 Hz) produced a larger FP per unit time initially, but decreased more rapidly later due to neural adaptation effects. On the other hand, lower stimulus frequencies (1-3 Hz) induced smaller, but sustained FP activities over the entire stimulus period. Similar frequency-dependencies were observed in CBF(LDF), CBF(ASL) and BOLD responses. A linear relationship between FP and CBF(LDF) was observed for all stimulus frequencies. Stimulation frequency for the maximal cumulative neural and hemodynamic changes is dependent on stimulus duration; 8-12 Hz for short stimulus durations (<10s) and 6-8 Hz for 30-s stimulation. Our findings suggest that neural adaptation should be considered in determining the somatosensory stimulation frequency and duration under isoflurane anesthesia.

Spatial specificity of the enhanced dip inherently induced by prolonged oxygen consumption in cat visual cortex: Implication for columnar resolution functional MRI

Since changes in oxygen consumption induced by active neurons are specific to cortical columns, the small and transient dip of deoxyhemoglobin signal, which indicates an increase in oxygen consumption, has been of great interest. In this study, we succeeded in enhancing and sustaining the dip in the deoxyhemoglobin-weighted 620-nm intrinsic optical imaging signals from a 10-s orientation-selective stimulation in cat visual cortex by reducing arterial blood pressure with sodium nitroprusside (a vasodilator) to mitigate the contribution of stimulus-induced blood supply. During this condition, intact spiking activity and a significant reduction of stimulus-induced blood volume changes (570-nm intrinsic signals) were confirmed. The deoxyhemoglobin signal from the prolonged dip was highly localized to iso-orientation domains only during the initial approximately 2 s; the signal specificity weakened over time although the domains were still resolvable after 2 s. The most plausible explanation for this time-dependent spatial specificity is that deoxyhemoglobin induced by oxygen consumption drains from active sites, where spiking activity occurs, to spatially non-specific downstream vessels over time. Our results suggest that the draining effect of pial and intracortical veins in dHb-based imaging techniques, such as blood oxygenation-level dependent (BOLD) functional MRI, is intrinsically unavoidable and reduces its spatial specificity of dHb signal regardless of whether the stimulus-induced blood supply is spatially specific.

Neural and Hemodynamic Responses to Optogenetic and Sensory Stimulation in the Rat Somatosensory Cortex

Introducing optogenetics into neurovascular research can provide novel insights into the cell-specific control of the hemodynamic response. To generalize findings from molecular approaches, it is crucial to determine whether light-activated circuits have the same effect on the vasculature as sensory-activated ones. For that purpose, rats expressing channelrhodopsin (ChR2) specific to excitatory glutamatergic neurons were used to measure neural activity, blood flow, hemoglobin-based optical intrinsic signal, and blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) during optogenetic and sensory stimulation. The magnitude of the evoked hemodynamic responses was monotonically correlated with optogenetic stimulus strength. The BOLD hemodynamic response function was consistent for optogenetic and sensory stimuli. The relationship between electrical activities and hemodynamic responses was comparable for optogenetic and sensory stimuli, and better explained by the local field potential (LFP) than the firing rate. The LFP was well correlated with cerebral blood flow, moderately with cerebral blood volume, and less with deoxyhemoglobin (dHb) level. The presynaptic firing rate had little impact on evoking vascular response. Contribution of the postsynaptic LFP to the blood flow response induced by optogenetic stimulus was further confirmed by the application of glutamate receptor antagonists. Overall, neurovascular coupling during optogenetic control of glutamatergic neurons largely conforms to that of a sensory stimulus.

Cerebral oxygen delivery and consumption during evoked neural activity

Increases in neural activity evoke increases in the delivery and consumption of oxygen. Beyond observations of cerebral tissue and blood oxygen, the role and properties of cerebral oxygen delivery and consumption during changes in brain function are not well understood. This work overviews the current knowledge of functional oxygen delivery and consumption and introduces recent and preliminary findings to explore the mechanisms by which oxygen is delivered to tissue as well as the temporal dynamics of oxygen metabolism. Vascular oxygen tension measurements have shown that a relatively large amount of oxygen exits pial arterioles prior to capillaries. Additionally, increases in cerebral blood flow (CBF) induced by evoked neural activation are accompanied by arterial vasodilation and also by increases in arteriolar oxygenation. This increase contributes not only to the down-stream delivery of oxygen to tissue, but also to delivery of additional oxygen to extra-vascular spaces surrounding the arterioles. On the other hand, the changes in tissue oxygen tension due to functional increases in oxygen consumption have been investigated using a method to suppress the evoked CBF response. The functional decreases in tissue oxygen tension induced by increases in oxygen consumption are slow to evoked changes in CBF under control conditions. Preliminary findings obtained using flavoprotein autofluorescence imaging suggest cellular oxidative metabolism changes at a faster rate than the average changes in tissue oxygen. These issues are important in the determination of the dynamic changes in tissue oxygen metabolism from hemoglobin-based imaging techniques such as blood oxygenation-level dependent functional magnetic resonance imaging (fMRI).

Evolution of the dynamic changes in functional cerebral oxidative metabolism from tissue mitochondria to blood oxygen.

The dynamic properties of the cerebral metabolic rate of oxygen consumption (CMRO2) during changes in brain activity remain unclear. Therefore, the spatial and temporal evolution of functional increases in CMRO2 was investigated in the rat somato-sensory cortex during forelimb stimulation under a suppressed blood flow response condition. Temporally, stimulation elicited a fast increase in tissue mitochondria CMRO2 described by a time constant of ~ 1 second measured using flavoprotein autofluorescence imaging. CMRO2-driven changes in the tissue oxygen tension measured using an oxygen electrode and blood oxygenation measured using optical imaging of intrinsic signal followed; however, these changes were slow with time constants of ~ 5 and ~ 10 seconds, respectively. This slow change in CMRO2-driven blood oxygenation partly explains the commonly observed post-stimulus blood oxygen level-dependent (BOLD) undershoot. Spatially, the changes in mitochondria CMRO2 were similar to the changes in blood oxygenation. Finally, the increases in CMRO2 were well correlated with the evoked multi-unit spiking activity. These findings show that dynamic CMRO2 calculations made using only blood oxygenation data (e.g., BOLD functional magnetic resonance imaging (fMRI)) do not directly reflect the temporal changes in the tissues mitochondria metabolic rate; however, the findings presented can bridge the gap between the changes in cellular oxidative rate and blood oxygenation.

BOLD responses to different temporal frequency stimuli in the lateral geniculate nucleus and visual cortex: Insights into the neural basis of fMRI

The neural basis of the blood oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI) remains largely unknown after decades of research. To investigate this issue, the unique property of the temporal frequency tuning that could separate neural input and output in the primary visual cortex was used as a model. During moving grating stimuli of 1, 2, 10 and 20Hz temporal frequencies, we measured 9.4-T BOLD fMRI responses simultaneously in the primary visual cortex of area 17 (A17) and area 18 (A18), and the lateral geniculate nucleus (LGN) of isoflurane-anesthetized cat. Our results showed that preferred temporal frequencies of the BOLD responses for A17, A18 and LGN were 3.1Hz, 4.5Hz and 6.0Hz, respectively, which were comparable to the previously reported electrophysiological data. Additionally, the difference of BOLD response onset time between LGN and A17 was 0.5s, which is 18 times larger than the difference of neural activity onset time between these areas. We then compared the frequency-dependent BOLD fMRI response of A17 with tissue partial pressure of oxygen (pO(2)) and electrophysiological data of the same animal model reported by Viswanathan and Freeman (Nature Neuroscience, 2007). The BOLD tuning curve resembled the low frequency band (<12Hz) of local field potential (LFP) tuning curve rather than spiking activity, gamma band (25-90Hz) of LFP, and tissue pO(2) tuning curves, suggesting that the BOLD fMRI signal relates closer to low frequency LFP.

Spatiotemporal characteristics and vascular sources of neural-specific and -nonspecific fMRI signals at submillimeter columnar resolution

The neural specificity of hemodynamic-based functional magnetic resonance imaging (fMRI) signals is dependent on both the vascular regulation and the sensitivity of the applied fMRI technique to different types and sizes of blood vessels. In order to examine the specificity of MRI-detectable hemodynamic responses, submillimeter blood oxygenation level-dependent (BOLD) and cerebral blood volume (CBV) fMRI studies were performed in a well-established cat orientation column model at 9.4 T. Neural-nonspecific and -specific signals were separated by comparing the fMRI responses of orthogonal orientation stimuli. The BOLD response was dominantly neural-nonspecific, mostly originating from pial and intracortical emerging veins, and thus was highly correlated with baseline blood volume. Uneven baseline CBV may displace or distort small functional domains in high-resolution BOLD maps. The CBV response in the parenchyma exhibited dual spatiotemporal characteristics, a fast and early neural-nonspecific response (with 4.3-s time constant) and a slightly slower and delayed neural-specific response (with 9.4-s time constant). The nonspecific CBV signal originates from early-responding arteries and arterioles, while the specific CBV response, which is not correlated with baseline blood volume, arises from late-responding microvessels including small pre-capillary arterioles and capillaries. Our data indicate that although the neural specificity of CBV fMRI signals is dependent on stimulation duration, high-resolution functional maps can be obtained from steady-state CBV studies.