Summary Deposition of intracellular tau fibrils has been the focus of research on the mechanisms of neurodegeneration in Alzheimer’s disease (AD) and related tauopathies. Here, we developed a new class of tau ligands, phenyl/pyridinyl-butadienyl-benzothiazoles/benzothiazoliums (PBBs), for visualizing diverse tau inclusions in brains of living patients with AD or non-AD tauopathies and animal models of these disorders. In vivo optical and positron emission tomographic (PET) imaging of a transgenic mouse model demonstrated sensitive detection of tau inclusions by PBBs. A pyridinated PBB, [11C]PBB3 was next applied in a clinical PET study, and its robust signal in the AD hippocampus wherein tau pathology is enriched contrasted strikingly with that of a senile plaque radioligand, [11C]Pittsburgh Compound-B ([11C]PIB). [11C]PBB3-PET data were also consistent with the spreading of tau pathology with AD progression. Furthermore, increased [11C]PBB3 signals were found in a corticobasal syndrome patient negative for [11C]PIB-PET.

Quantifying both arterial cerebral blood volume (CBV a ) changes and total cerebral blood volume (CBV t ) changes during neural activation can provide critical information about vascular control mechanisms, and help to identify the origins of neurovascular responses in conventional blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI). Cerebral blood flow (CBF), CBV a , and CBV t were quantified by MRI at 9.4 T in isoflurane-anesthetized rats during 15-s duration forepaw stimulation. Cerebral blood flow and CBV a were simultaneously determined by modulation of tissue and vessel signals using arterial spin labeling, while CBV t was measured with a susceptibility-based contrast agent. Baseline versus stimulation values in a region centered over the somatosensory cortex were: CBF = 150618 versus 182620 mL/100 g/min, CBV a = 0.8360.21 versus 1.1760.30 mL/100 g, CBV t = 3.1060.55 versus 3.4160.61 mL/100 g, and CBV a /CBV t = 0.2760.05 versus 0.3460.06 (n = 7, mean6s.d.). Neural activity-induced absolute changes in CBV a and CBV t are statistically equivalent and independent of the spatial extent of regional analysis. Under our conditions, increased CBV t during neural activation originates mainly from arterial rather than venous blood volume changes, and therefore a critical implication is that venous blood volume changes may be negligible in BOLD fMRI.

Functional magnetic resonance imaging (fMRI) in anesthetized rodents has been commonly performed with a-chloralose, which can be used only for terminal experiments. To develop a survival fMRI protocol, an isoflurane (ISO) -anesthetized rat model was systematically evaluated by simultaneous measurements of field potential (FP) and cerebral blood flow (CBF) in the somatosensory cortex. A conventional forepaw stimulation paradigm with 0.3 ms pulse width, 1.2 mA current, and 3 Hz frequency induced 54% less evoked FP and 84% less CBF response under ISO than a-chloralose.To improve stimulation-induced responses under ISO, 10-pulse stimulations were performed with variations of width, current, and frequency. For widths of 0.1--5.0 ms and currents of 0.4--2.0 mA, evoked FP and CBF increased similarly and reached a plateau. The evoked FP increased monotonically for intervals from 50 to 500 ms, but the CBF peaked at an interval of 83 ms (~12 Hz frequency). These data suggest that different anesthetics profoundly affect FP and CBF responses in different ways, which requires optimizing stimulation parameters for each anesthetic. With the refined stimulation parameters, fMRI consistently detected a well-localized activation focus at the primary somatosensory cortex in ISOanesthetized rats. Thus, the ISO-anesthetized rat model can be used for cerebrovascular activation studies, allowing repeated noninvasive survival experiments.

Anesthesia has broad actions that include changing neuronal excitability, vascular reactivity, and other baseline physiologies and eventually modifies the neurovascular coupling relationship. Here, we review the effects of anesthesia on the spatial propagation, temporal dynamics, and quantitative relationship between the neural and vascular responses to cortical stimulation. Previous studies have shown that the onset latency of evoked cerebral blood flow (CBF) changes is relatively consistent across anesthesia conditions compared with variations in the time-to-peak. This finding indicates that the mechanism of vasodilation onset is less dependent on anesthesia interference, while vasodilation dynamics are subject to this interference. The quantitative coupling relationship is largely influenced by the type and dosage of anesthesia, including the actions on neural processing, vasoactive signal transmission, and vascular reactivity. The effects of anesthesia on the spatial gap between the neural and vascular response regions are not fully understood and require further attention to elucidate the mechanism of vascular control of CBF supply to the underlying focal and surrounding neural activity. The in-depth understanding of the anesthesia actions on neurovascular elements allows for better decision-making regarding the anesthetics used in specific models for neurovascular experiments and may also help elucidate the signal source issues in hemodynamic-based neuroimaging techniques.

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 the 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 ten 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% to 2.1%), while the CBF response to single-pulse stimulation increased dose-dependently (7 ± 3% to 17 ± 4%). The isoflurane dose did not affect the vascular reactivity induced by a hypercapnic challenge. These findings suggest that the action site of isoflurane affects the neurovascular mechanisms. For ten-pulse stimulation, the sum-FP responses monotonically decreased with an increase in the isoflurane dose, possibly due to an enhancement in the FP adaptation. In contrast, the dose-dependent effect on the CBF response varied depending on the stimulus frequency; a dose-dependent decrease in the CBF response was observed for highfrequency stimulation, whereas a dose-dependent increase was observed for low-frequency stimulation. Further, a linear time-invariant model composed of the single-pulse hemodynamic impulse response convoluted with ten-pulse FP recordings showed that the neurovascular transfer function was altered by the isoflurane dose for high-frequency stimulation. These results indicate that a careful and consistent maintenance of the anesthetic depth is required when comparing fMRI data obtained from different animals or physiological and pharmacological manipulations.

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 = 59-129 μm) and pial veins (resting diameters = 62-361 μm) were observed. The largest increases in vascular PO2 were observed in the small veins (from 33 to 40 mmHg) and small arteries (from 78 to 88 mmHg). 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).

Trial-by-trial variability in local field potential (LFP), tissue partial pressure of oxygen (Po 2 ), cerebral blood flow (CBF) and deoxyhemoglobin-weighted optical imaging of intrinsic signals (OIS) were tested in the rat somatosensory cortex while fixed electrical forepaw stimulation (1.0 ms pulses with amplitude of 1.2 mA at a frequency of 6 Hz) was repeatedly applied. The changes in the cerebral metabolic rate of oxygen (CMRO 2 ) were also evaluated using a hypotension condition established by our group based on the administration of a vasodilator. Under normal conditions, CBF, Po 2 , and OIS showed positive signal changes (48%, 32%, and 0.42%, respectively) following stimulation. Over multiple trials, the CBF responses were well correlated with the integral of the LFP amplitudes (ΣLFP) (R mean = 0.78), whereas a lower correlation was found between Po 2 and ΣLFP (R mean = 0.60) and between OIS and ΣLFP (R mean = 0.54). Under the hypotension condition the LFP responses were preserved, but the CBF responses were suppressed and the Po 2 and OIS changes were negative (−12% and −0.28%, respectively). In this condition, the trial-by-trial variations in Po 2 and OIS were well correlated with the variability in ΣLFPs (R mean = −0.77 and −0.76, respectively), indicating a single trial coupling between CMRO 2 changes and ΣLFP. These findings show that CBF and CMRO 2 signals are more directly correlated with neural activity compared to blood-oxygen sensitive methods such as OIS and BOLD fMRI.

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 (< 10 s) 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.