The chemogenetic technology designer receptors exclusively activated by designer drugs (DREADDs) afford remotely reversible control of cellular signaling, neuronal activity and behavior. Although the ...combination of muscarinic-based DREADDs with clozapine-N-oxide (CNO) has been widely used, sluggish kinetics, metabolic liabilities and potential off-target effects of CNO represent areas for improvement. Here, we provide a new high-affinity and selective agonist deschloroclozapine (DCZ) for muscarinic-based DREADDs. Positron emission tomography revealed that DCZ selectively bound to and occupied DREADDs in both mice and monkeys. Systemic delivery of low doses of DCZ (1 or 3 μg per kg) enhanced neuronal activity via hM3Dq within minutes in mice and monkeys. Intramuscular injections of DCZ (100 μg per kg) reversibly induced spatial working memory deficits in monkeys expressing hM4Di in the prefrontal cortex. DCZ represents a potent, selective, metabolically stable and fast-acting DREADD agonist with utility in both mice and nonhuman primates for a variety of applications.
A variety of brain cells participates in neurovascular coupling by transmitting and modulating vasoactive signals. The present study aimed to probe cell type-dependent cerebrovascular (i.e., pial and ...penetrating arterial) responses with optogenetics in the cortex of anesthetized mice. Two lines of the transgenic mice expressing a step function type of light-gated cation channel (channelrhodopsine-2; ChR2) in either cortical neurons (muscarinic acetylcholine receptors) or astrocytes (Mlc1-positive) were used in the experiments. Photo-activation of ChR2-expressing astrocytes resulted in a widespread increase in cerebral blood flow (CBF), extending to the nonstimulated periphery. In contrast, photo-activation of ChR2-expressing neurons led to a relatively localized increase in CBF. The differences in the spatial extent of the CBF responses are potentially explained by differences in the involvement of the vascular compartments. In vivo imaging of the cerebrovascular responses revealed that ChR2-expressing astrocyte activation led to the dilation of both pial and penetrating arteries, whereas ChR2-expressing neuron activation predominantly caused dilation of the penetrating arterioles. Pharmacological studies showed that cell type-specific signaling mechanisms participate in the optogenetically induced cerebrovascular responses. In conclusion, pial and penetrating arterial vasodilation were differentially evoked by ChR2-expressing astrocytes and neurons.
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•No infarct was detected in mice without SD during experimental ischemia.•The infarct size tended to be larger in repeated SD-occurred mice.•The specific blood flow response depending ...on ischemic level were summarized.•Ischemia-induced SD may trigger infarct formation and/or exacerbate the damage.
Clinical and experimental evidence suggests that spreading depolarizations (SD) usually occur in patients with ischemic or hemorrhagic stroke when the gray matter of the brain is affected. In this study, we evaluated spatiotemporal changes of cerebral blood flow (CBF) during middle cerebral artery (MCA) occlusion and examined the relationship between SD occurrence and cerebral infarct development. In male isoflurane-anesthetized C57BL/6J mice, CBF changes over the ipsilateral parietal bone were recorded by laser speckle flowgraphy during and after transient (45 min, n = 22) or permanent occlusion (n = 22) of the distal MCA. Infarct volume was evaluated 24 hr after the operation. Upon MCA occlusion, CBF decreased by −55.6 ± 8.5 % in the lowest CBF and linearly recovered with increasing distance from the region. At 1–10 min after onset of occlusion, SD occurred and concentrically propagated from the core region, showing a decrease of CBF in the whole observed area along with a transient hyperemia and oligemia in the normal region. SD spontaneously re-occurred and propagated around the ischemic area in 37 % of mice, accompanied with a marked decrease of CBF in the core or a marked increase of CBF in the normal region. The CBF response to SDs gradually changed from the core to the normal area, depending upon the distance from the core region. Infarction was not observed in transiently (n = 2) or permanently (n = 4) occluded mice without SD. The infarct area tended to be larger with increasing number of SDs in transiently occluded mice. In conclusion, our findings suggest that the occurrence of SD during ischemia might elicit infarct formation and/or influence infarct development.
To assess if magnetic resonance spectroscopy (MRS)-measured Glutamate (Glu) and GABA reflect excitatory and inhibitory neural activities, respectively, we conducted MRS measurements along with ...two-photon mesoscopic imaging of calcium signals in excitatory and inhibitory neurons of living, unanesthetized mice. For monitoring stimulus-driven activations of a brain region, MRS signals and mesoscopic neural activities were measured during two consecutive sessions of 15-min prolonged sensory stimulations. In the first session, putative excitatory neuronal activities were increased, while inhibitory neuronal activities remained at the baseline level. In the second half, while excitatory neuronal activities remained elevated, inhibitory neuronal activities were significantly enhanced. We assessed regional neurochemical statuses by measuring MRS signals, which were overall in accordance with the neural activities, and neuronal activities and neurochemical statuses in a mouse model of Dravet syndrome under resting condition. Mesoscopic assessments showed that activities of inhibitory neurons in the cortex were diminished relative to wild-type mice in contrast to spared activities of excitatory neurons. Consistent with these observations, the Dravet model exhibited lower concentrations of GABA than wild-type controls. Collectively, the current investigations demonstrate that MRS-measured Glu and GABA can reflect spontaneous and stimulated activities of neurons producing and releasing these neurotransmitters in an awake condition.
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.
Abstract Employing high-speed camera laser-scanning confocal microscopy with RBC-tracking software, we previously showed that RBC velocities in intraparenchymal capillaries of rat cerebral cortex are ...distributed over a wide range. In the present work, we measured RBC velocities in mice, whose body weights are less than one-tenth of that of rats. In an isoflurane-anesthetized mouse, a cranial window was opened in the left temporo-parietal region. Intravenously administered FITC-labeled RBCs were automatically recognized and tracked frame-by-frame at 500 fps, and the velocities of all RBCs recognized were calculated with our Matlab-domain software, KEIO-IS2. Among 15 241 RBCs detected in the ROI in 21 mice, 1655 were identified as flowing in capillaries. The velocities of these RBCs ranged from 0.15 to 8.6 mm/s, with a mean of 2.03 ± 1.42 mm/s. A frequency distribution plot showed that RBC velocities were clustered at around 1.0 mm/s, tailing up to 8.6 mm/s, and 59% of the RBCs in capillaries showed velocities within the range of 0.5 to 2.0 mm/s. Unexpectedly, these characteristics of RBC velocities in mice were very similar to those of rats, despite differences in RBC diameter (6.0 vs. 6.5 μm), body size (25 vs. 327 g), heart rate (461 vs. 319 bpm) and arterial blood pressure (86 vs. 84 mm Hg). We speculate that physical factors relating to oxygen exchange may constrain general RBC velocity in capillaries to a certain range for optimum oxygen exchange, regardless of species.
Cortical spreading depression (CSD) induces marked hyperemia with a transient decrease of regional cerebral blood flow (rCBF), followed by sustained oligemia. To further understand the ...microcirculatory mechanisms associated with CSD, we examined the temporal changes of diameter of intraparenchymal penetrating arteries during CSD. In urethane-anesthetized mice, the diameter of single penetrating arteries at three depths was measured using two-photon microscopy during passage of repeated CSD, with continuous recordings of direct current potential and rCBF. The first CSD elicited marked constriction superimposed on the upstrokes of profound dilation throughout each depth of the penetrating artery, and the vasoreaction temporally corresponded to the change of rCBF. Second or later CSD elicited marked dilation with little or no constriction phase throughout each depth, and the vasodilation also temporally corresponded to the increase of rCBF. Furthermore, the peak dilation showed good negative correlations with basal diameter and increase of rCBF. Vasodilation induced by 5% CO2 inhalation was significantly suppressed after CSD passage at any depth as well as hyperperfusion. These results may indicate that CSD-induced rCBF changes mainly reflect the diametric changes of the intraparenchymal arteries, despite the elimination of responsiveness to hypercapnia.
Proper brain function is maintained by an integrated system called the neurovascular unit (NVU) comprised cellular and acellular elements. Although the individual features of specific neurovascular ...components are understood, it is unknown how they respond to ischemic stress as a functional unit. Therefore, we established an in vivo imaging method and clarified the NVU response to chronic cerebral hypoperfusion.
Green mice (b-act-EGFP) with SR101 plasma labeling were used in this experiment. A closed cranial window was made over the left somatosensory cortex. To mimic chronic cerebral hypoperfusion, mice were subjected to bilateral common carotid artery stenosis operations using microcoils. In vivo real-time imaging was performed using 2-photon laser-scanning microscopy during the preoperative period, and after 1 day and 1 and 2 weeks of bilateral common carotid artery stenosis or sham operations.
Our method allowed 3-dimensional observation of most of the components of the NVU, as well as dynamic capillary microcirculation. Under chronic cerebral hypoperfusion, we did not detect any structural changes of each cellular component in the NVU; however, impairment of microcirculation was detected over a prolonged period. In the pial small arteries and veins, rolling and adhesion of leukocyte were detected, more prominently in the latter. In the deep cortical capillaries, flow stagnation because of leukocyte plugging was frequently observed.
We established an in vivo imaging method for real-time visualization of the NVU. It seems that under chronic cerebral hypoperfusion, leukocyte activation has a critical role in microcirculation disturbance.
As humans, we constantly change our movement strategies to adapt to changes in physical functions and the external environment. We have to walk very slowly in situations with a high risk of falling, ...such as walking on slippery ice, carrying an overflowing cup of water, or muscle weakness owing to aging or motor deficit. However, previous studies have shown that a normal gait pattern at low speeds results in reduced efficiency and stability in comparison with those at a normal speed. Another possible strategy is to change the gait pattern from normal to step-to gait, in which the other foot is aligned with the first swing foot. However, the efficiency and stability of the step-to gait pattern at low speeds have not been investigated yet. Therefore, in this study, we compared the efficiency and stability of the normal and step-to gait patterns at intermediate, low, and very low speeds. Eleven healthy participants were asked to walk with a normal gait and step-to gait on a treadmill at five different speeds (i.e., 10, 20, 30, 40, and 60 m/min), ranging from very low to normal walking speed. The efficiency parameters (percent recovery and walk ratio) and stability parameters (center of mass lateral displacement) were analyzed from the motion capture data and then compared for the two gait patterns. The results suggested that step-to gait had a more efficient gait pattern at very low speeds of 10-30 m/min, with a larger percent recovery, and was more stable at 10-60 m/min in comparison with a normal gait. However, the efficiency of the normal gait was better than that of the step-to gait pattern at 60 m/min. Therefore, step-to gait is effective in improving gait efficiency and stability when faced with situations that force us to walk slowly or hinder quick walking because of muscle weakness owing to aging or motor deficit along with a high risk of falling.
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.
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•The Pvalb promoter is active in arterial smooth muscle cells and endothelial cells•ChR2 and PAC are CBF-reducing and -increasing actuators, respectively•Understanding spatiotemporal CBF change is key to interpreting behavioral outcomes•Vascular optogenetics in awake animals facilitates behavioral neurovascular studies
Abe et al. generate vascular optogenetic animal models and demonstrate the local cerebral blood flow (CBF) manipulation in freely moving animals. They provide detailed information about the spatiotemporal titration of optogenetics-mediated CBF changes and exemplify the behavior- and circuitry-level outcomes by defined vascular optogenetics.
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