Optical manipulation of local cerebral blood flow in the deep brain of freely moving mice.

An artificial tool for manipulating local cerebral blood flow (CBF) is necessary for understanding how CBF controls brain function. Here, we generate vascular optogenetic tools whereby smooth muscle cells and endothelial cells express optical actuators in the brain. The illumination of channelrhodopsin-2 (ChR2)-expressing mice induces a local reduction in CBF. Photoactivated adenylyl cyclase (PAC) is an optical protein that increases intracellular cyclic adenosine monophosphate (cAMP), and the illumination of PAC-expressing mice induces a local increase in CBF. We target the ventral striatum, determine the temporal kinetics of CBF change, and optimize the illumination intensity to confine the effects to the ventral striatum. We demonstrate the utility of this vascular optogenetic manipulation in freely and adaptively behaving mice and validate the task- and actuator-dependent behavioral readouts. The development of vascular optogenetic animal models will help accelerate research linking vasculature, circuits, and behavior to health and disease.

Characteristics of cortical spreading depression and c-Fos expression in transgenic mice having a mutation associated with familial hemiplegic migraine 2.

BACKGROUND: Cortical spreading depression is thought to be the underlying mechanism of migraine aura. In 2006, three relatives having the point mutation E700K in ATP1A2 exon 15 were diagnosed with familial hemiplegic migraine 2 characterized by complicated forms of aura. Here, we generated a transgenic mouse model having the human E700K mutation in the Atp1a2 orthologous gene. OBJECTIVE: To investigate the characteristics of cortical spreading depression in a mouse model with E700K mutation in the Atp1a2. METHODS: Cortical spreading depression was induced by applying stepwise increases of KCl concentration or electrical stimulation intensity to C57BL/6J-Tg(Atp1a2*E700K)9151Kwk mice (Tg, both sexes) and corresponding wild-type animals. Under urethane anesthesia, the responsiveness and threshold to cortical spreading depression were examined and the distribution of c-Fos expression, a neuronal activity marker, was immunohistochemically determined. RESULTS: Overall, Tg mice showed significantly faster propagation velocity (p < 0.01) and longer full-width-at-half-maximum (p < 0.01) than wild-type animals, representing a slower recovery from direct current potential deflection. The cortical spreading depression threshold tended to be lower in Tg, especially in females. c-Fos-positive cells were significantly enhanced in the ipsilateral somatosensory cortex, piriform cortex, amygdala and striatum (each p < 0.05 vs. contralateral side). Numbers of c-Fos positive cells were significantly higher in the ipsilateral amygdala of Tg, as compared with wild-type animals (p < 0.01). CONCLUSION: The effect of cortical spreading depression may be greater in E700K transgenic mice than that in wild-type animals, while the threshold for cortical spreading depression shows little change. Higher c-Fos expression in the amygdala may indicate alterations of the limbic system in Tg, suggesting an enhanced linkage between cortical spreading depression and amygdala connectivity in familial hemiplegic migraine 2 patients.

Enhanced susceptibility to cortical spreading depression in two types of Na+,K+-ATPase α2 subunit-deficient mice as a model of familial hemiplegic migraine 2.

Background Patients with familial hemiplegic migraine type 2 (FHM2) have a mutated ATP1A2 gene (encoding Na+,K+-ATPase α2 subunit) and show prolonged migraine aura. Cortical spreading depression (CSD), which involves mass depolarization of neurons and astrocytes that propagates slowly through the gray matter, is profoundly related to aura. Methods In two types of Atp1a2-defective heterozygous mice, Atp1a2tm1Kwk (C-KO) and Atp1a2tm2Kwk (N-KO), the sensitivity and responsiveness to CSD were examined under urethane anesthesia. Results In both cases, heterozygotes exhibited a low threshold for induction of CSD, faster propagation rate, slower recovery from DC deflection, and profound suppression of the electroencephalogram, compared to wild-type mice. A high dose of KCl elicited repeated CSDs for a longer period, with a tendency for a greater frequency of CSD occurrence in heterozygotes. The difference of every endpoint was slightly greater in N-KO than C-KO. Change of regional cerebral blood flow in response to CSD showed no significant difference. Conclusion Heterozygotes of Atp1a2-defective mice simulating FHM2 demonstrated high susceptibility to CSD rather than cortical vasoreactivity, and these effects may differ depending upon the knockout strategy for the gene disruption. These results suggest that patients with FHM2 may exhibit high susceptibility to CSD, resulting in migraine.

Establishment and evaluation of a new highly metastatic tumor cell line 5a-D-Luc-ZsGreen expressing both luciferase and green fluorescent protein.

Breast cancer is the most common cancer in women. Although advances in diagnostic imaging for early detection, surgical techniques and chemotherapy have improved overall survival, the prognosis of patients with metastatic breast cancer remains poor. Understanding cancer cell dynamics in the metastatic process is important to develop new therapeutic strategies. Experimental animal models and imaging would be powerful tools for understanding of the molecular events of multistep process of metastasis. In the present study, to develop a new cancer cell line that is applicable to bioluminescence and fluorescence imaging, we transfected the expression vector of a green fluorescent protein ZsGreen1 into a metastatic cell line 5a-D-Luc, which is a subclone of the MDA-MB-231 breast cancer cell line expressing luciferase, and established a new tumor cell line 5a-D-Luc-ZsGreen expressing both luciferase and ZsGreen1. The 5a-D-Luc-ZsGreen cells proliferate more rapidly and have a more invasive phenotype compared with 5a-D-Luc cells following intracardiac injection. Metastasis sites were easily detected in the whole body by bioluminescence imaging and in excised tissues by ex vivo fluorescence imaging. The fluorescence of 5a-D-Luc-ZsGreen cells was not lost after formalin fixation and decalcification. It enabled us to easily evaluate tumor spread and localization at the cellular level in microscopic analysis. The strong fluorescence of 5a-D-Luc-ZsGreen cells allowed for real-time imaging of circulating tumor cells in cerebral blood vessels of live animals immediately after intracardiac injection of cells using two-photon laser-scanning microscopy. These findings suggest that the 5a-D-Luc-ZsGreen cells would be a useful tool for research on mechanisms of metastatic process in animal models.

Pelvic axis-based gait analysis for ataxic mice.

BACKGROUND: Although different gait analysis methods such as Walking Track Analysis exist, they cannot be used to demonstrate the physical condition of mice with specific gait disorder characteristic. Therefore, we developed a new method for the gait analysis of such mice to accurately assess hind limb angle based on the pelvic axis. NEW METHOD: We established and verified a gait analysis method capable of pelvic axis-based limb angle measurement by video-recording the gait of a control mice group (C57BL/6J(B6)) and three ataxic mice (ataxic B6-wob/t, Parkinson's disease model (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treated (MPTP)), and cerebellum hypoplasia (cytosine-ß-d-arabinofuranoside treated)) from the ventral side. RESULTS: The assessed hind limb angles of B6-wob/t and MPTP-treated mice were significantly wider than B6 mice (p<0.01). Moreover, we could draw separating lines with slopes of minus one that could separate the data of each group in the scatter plot of the normalized hind limb step width and angle. COMPARISON WITH EXISTING METHODS: We found no significance when we applied the already existing nose-tail method for the analysis of the hind limb angles of B6 and B6-wob/t mice. In the nose-tail method, since the whole body axis of the trunk varies while the trunk of the mouse is laterally bent changing the hind limb angle, B6 and B6-wob/t mice could not be differentiated. However, the two mice groups could be differentiated by the pelvic axis-based gait analysis method. CONCLUSION: The pelvic axis-based gait analysis method is promising and valid for mice with gait disorder.

REM sleep behavior disorder in Japanese patients with Parkinson's disease: a multicenter study using the REM sleep behavior disorder screening questionnaire.

REM sleep behavior disorder (RBD) is known to be observed more frequently in patients with an α-synucleinopathy such as Parkinson's disease (PD) than in the general population. The precise prevalence of RBD in Japanese PD patients is not known. Therefore, we investigated the prevalence and the clinical characteristics of patients with RBD in a large population of Japanese patients with PD. We investigated various clinical features and employed the Japanese version of the RBD screening questionnaire on 469 non-demented Japanese PD patients in this multicenter study. Probable or possible RBD was detected in 146 patients (31.1%) and was significantly associated with longer PD duration, higher Hoehn and Yahr stage, higher Unified Parkinson's Disease Rating Scale part III subscale (7 items), more motor fluctuations, and a higher levodopa-equivalent daily dose (p < 0.01). As to the major autonomic dysfunctions, severe constipation was significantly more frequent in PD patients with RBD than in those without it (p < 0.01). The RBD symptoms of 53 patients (39.0%) preceded the onset of PD motor symptoms. The median interval from the onset of RBD symptoms to PD motor symptoms was 17.5 years, and 3 patients had intervals of over 50 years. This large-scale multicenter study revealed that RBD is a frequent non-motor symptom in Japanese patients with PD, which may precede the onset of motor symptoms. Moreover, RBD that increases with the duration and severity of PD may be associated with autonomic dysfunction.

Orthostatic decrease in cardiac chaos during the head-up tilt test in patients with vasovagal syncope.

BACKGROUND: Autonomic dysfunction contributes to orthostatic intolerance in vasovagal syncope (VVS), but as it has not been identified by spectral analysis of heart rate variability (HRV) in previous studies, the present hypothesis was that nonlinear analysis of HRV would identify the orthostatic intolerance in VVS. METHODS AND RESULTS: Twenty-six patients with VVS and 14 matched controls were subjected to 80-degree head-up tilt test (positive: 13 patients; negative: 13 patients and 14 controls). There were no differences in the orthostatic changes in the indices of spectral analyses of HRV among the 3 groups. The Lyapunov exponent (LE) was calculated from 200 consecutive RR-intervals to investigate chaotic behavior, and cardiac chaos was defined as the incidence of the presence of a positive finite LE. Orthostatic decreases in cardiac chaos were observed in the VVS patients (both the positive and negative groups), although there was no orthostatic decrease in the control group (ANOVA: p = 0.008). The receiver-operator characteristic curve indicated that cardiac chaos during the tilt identified VVS regardless of the results of the tilt (p < 0.001, sensitivity: 85.7%, specificity: 96.2%). CONCLUSIONS: The decrease in cardiac chaos during the tilt test was specific to patients with VVS, even if their response to the test was negative.

In vivo imaging with cellular resolution of bone marrow cells transplanted into the ischemic brain of a mouse.

The aim of the study was to monitor in vivo and noninvasively the fate of single bone marrow cells (BMCs) transplanted into the ischemic brain of unirradiated mice. In vivo imaging was performed through a closed cranial window, throughout the 2 weeks following cell transplantation, using laser-scanning confocal fluorescence microscopy. The window was chronically implanted above the left parieto-occipital cortex in C57BL/6J adult mice. BMC (3 x 10(5) nucleated cells in 0.5 microL medium) from 5-week-old transgenic mice, ubiquitously expressing green fluorescent protein (GFP), was transplanted into the ipsilateral cortex 24 h after the induction of focal ischemia by coagulation of the left middle cerebral artery (n = 15). Three nonischemic mice served as controls. Repeated in vivo imaging, up to a depth of 200 microm, revealed that BMCs survived within the ischemic and peri-ischemic cortex, migrated significantly towards the lesion, proliferated and adopted a microglia-like morphology over 2 weeks. These results were confirmed using ex vivo imaging after appropriate immunocytochemical treatments. This study indicates that confocal fluorescence microscopy is a reliable and unique tool to repeatedly assess with cellular resolution the in vivo dynamic fate of fluorescent cells transplanted into a mouse brain. These results also provide the first in vivo findings on the fate of single BMCs transplanted into the ischemic brain of unirradiated mice.

Role of vagal control in vasovagal syncope.

The vasovagal reaction is thought to be caused by sympathetic withdrawal and vagal augmentation. While measurements of muscle sympathetic nerve activity support sympathetic withdrawal in tilt induced syncope, the results of previous attempts to quantify vagal control using spectral analyses of heart rate variability (HRV) remain controversial. The sampling period used in the HRV studies is related to the discordant results. In the present study, HRV was computed every second using wavelet transformation to clarify the role of vagal control in tilt induced syncope during the 80-degree head-up tilt test (positive: 10 patients with vasovagal syncope; negative: 10 patients with vasovagal syncope, and 10 control subjects). Autonomic modulations were assessed using the absolute power of the low frequency (LF) (0.04-0.15 Hz) and high frequency (HF) (0.15-2.00 Hz) oscillatory components of R-R variability. Although the LF did not change during the tilt procedure, a decrease in the systolic arterial pressure (SAP) and increases in the R-R interval and HF were observed for the last 30 seconds before the tilt induced syncope in the tilt-positive group. Analyzing the hemodynamic measurements and spectral indices for the last 5 minutes preceding the tilt induced syncope, the study found that the SAP, R-R interval, and HF changed simultaneously during the 30-second period immediately before the tilt induced syncope. Further, the HF was positively correlated with the R-R interval and negatively correlated with the SAP. In conclusion, continuous spectral analysis of the R-R interval demonstrated increased vagal influence on the heart in tilt induced syncope.