Protocol for highly selective transgene expression through the flip-excision switch system by using a unilateral spacer sequence in rodents

Summary: We present a protocol to induce Cre-dependent transgene expression in specific cell types in the rat brain, suppressing a leak expression in off-target cells, by using a flip-excision switch system with a unilateral spacer sequence. We describe steps for construction of transfer plasmids, preparation of adeno-associated viral vectors, intracranial injection, and detection of transgene expression. Our protocol provides a useful strategy for a better understanding of the structure and function of specific cell types in the complex neural circuit.For complete details on the use and execution of this protocol, please refer to Matsushita et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Motor skills mediated through cerebellothalamic tracts projecting to the central lateral nucleus

Abstract The cerebellum regulates complex animal behaviors, such as motor control and spatial recognition, through communication with many other brain regions. The major targets of the cerebellar projections are the thalamic regions including the ventroanterior nucleus (VA) and ventrolateral nucleus (VL). Another thalamic target is the central lateral nucleus (CL), which receives the innervations mainly from the dentate nucleus (DN) in the cerebellum. Although previous electrophysiological studies suggest the role of the CL as the relay of cerebellar functions, the kinds of behavioral functions mediated by cerebellothalamic tracts projecting to the CL remain unknown. Here, we used immunotoxin (IT) targeting technology combined with a neuron-specific retrograde labeling technique, and selectively eliminated the cerebellothalamic tracts of mice. We confirmed that the number of neurons in the DN was selectively decreased by the IT treatment. These IT-treated mice showed normal overground locomotion with no ataxic behavior. However, elimination of these neurons impaired motor coordination in the rotarod test and forelimb movement in the reaching test. These mice showed intact acquisition and flexible change of spatial information processing in the place discrimination, Morris water maze, and T-maze tests. Although the tract labeling indicated the existence of axonal collaterals of the DN-CL pathway to the rostral part of the VA/VL complex, excitatory lesion of the rostral VA/VL did not show any significant alterations in motor coordination or forelimb reaching, suggesting no requirement of axonal branches connecting to the VL/VA complex for motor skill function. Taken together, our data highlight that the cerebellothalamic tracts projecting to the CL play a key role in the control of motor skills, including motor coordination and forelimb reaching, but not spatial recognition and its flexibility

Intake of Radionuclides in the Trees of Fukushima Forests 1. Field Study

The earthquake and tsunami on 11 March 2011 led to a meltdown followed by a hydrogen explosion at the Fukushima–Daiichi nuclear power plant in Japan, causing the dispersal of abundant radionuclides into the atmosphere and ocean. The radionuclides were deposited onto trees and local residences in aerosol or gaseous forms that were partly absorbed by rain or melting snow. Here, we show that the radionuclides attached to the surfaces of trees, in which some radiocesium was incorporated into the xylem through ray cells and through symplastic pathways. The level of incorporated radiocesium varied based on tree species and age because of the ability of radiocesium to attach to the surface of the outer bark. After four years, the radiocesium level in the forest has been decreasing as it is washed out with rainwater into the sea and as it decays over time due to its half-life, but it can also be continuously recycled through leaf tissue, litter, mulch, and soil. As a result, the level of radiocesium was relatively increased in the heartwood and roots of trees at four years after the event. In private forest fields, most trees were left as afforested trees without being used for timber, although some trees were cut down. We discuss an interdisciplinary field study on the immediate effects of high radiation levels upon afforested trees in private forest fields

Action Selection and Flexible Switching Controlled by the Intralaminar Thalamic Neurons

Summary: Learning processes contributing to appropriate selection and flexible switching of behaviors are mediated through the dorsal striatum, a key structure of the basal ganglia circuit. The major inputs to striatal subdivisions are provided from the intralaminar thalamic nuclei, including the central lateral nucleus (CL) and parafascicular nucleus (PF). Thalamostriatal neurons in the PF modulate the acquisition and performance of stimulus-response learning. Here, we address the roles of the CL thalamostriatal neurons in learning processes by using a selective neural pathway targeting technique. We show that the CL neurons are essential for the performance of stimulus-response learning and for behavioral flexibility, including reversal and attentional set-shifting of learned responses. In addition, chemogenetic suppression of neural activity supports the requirements of these neurons for behavioral flexibility. Our results suggest that the main contribution of the CL thalamostriatal neurons is functional control of the basal ganglia circuit linked to the prefrontal cortex. : Intralaminar thalamic nuclei provide the major inputs to the cortico-basal ganglia circuit. Kato et al. describe the essential roles of the thalamostriatal neurons in the CL in the performance of stimulus-response learning and in reversal and set-shifting of learned behaviors, forming a pivotal route that affects the frontostriatal circuit functions. Keywords: action selection, behavioral flexibility, intralaminar thalamic nucleus, central lateral nucleus, retrograde gene transfer, lentiviral vector, immunotoxin targeting, chemogenetic

Efficacy of the Continuous Resuscitation Training with the Gap Period Due to the COVID-19 Pandemic

(1) Objective: This study evaluates the effects of simulation education at our institute on cardiac arrest resuscitation regarding knowledge, attitude, and practice (KAP) over a five-year period (2016–2020). (2) Subjects: Staff responded to the annual survey questionnaires followed by monthly training in Basic Life Support/Advanced Cardiovascular Life Support (BLS/ACLS) and Immediate Cardiac Life Support (ICLS) of the Japanese Association for Acute Medicine (JAAM) in 2016, 2017, and 2018. Additionally, in-house training was implemented in 2019 without post-assessment followed by training suspension in 2020 due to the COVID-19 pandemic. The last delivery of the survey questionnaires was in late 2020 for KAP retention measurement. (3) Measurements and Results: The self-efficacy level of BLS/ACLS/ICLS KAP of the survey respondents was analyzed using a five-point Likert scale. The mean self-efficacy level of BLS/ACLS/ICLS KAP increased over time, and that of the trained people was three-fold that of the untrained people. Trainees that had no cardiopulmonary resuscitation (CPR) experience gained the BLS/ACLS/ICLS key-point self-efficacy level, which we call the “Grip 14” in this study, as high as their untrained counterparts who had three-time CPR experience. Training suspension lessened the BLS/ACLS/ICLS KAP in both groups. (4) Conclusions: Continuous training enhances not only the BLS/ACLS/ICLS KAP of trainees but also of their untrained colleagues. The training likely had the same efficacy as the CPR experience

National trends in the outcomes of subarachnoid haemorrhage and the prognostic influence of stroke centre capability in Japan: retrospective cohort study

Objectives To examine the national, 6-year trends in in-hospital clinical outcomes of patients with subarachnoid haemorrhage (SAH) who underwent clipping or coiling and the prognostic influence of temporal trends in the Comprehensive Stroke Center (CSC) capabilities on patient outcomes in Japan.Design Retrospective study.Setting Six hundred and thirty-one primary care institutions in Japan.Participants Forty-five thousand and eleven patients with SAH who were urgently hospitalised, identified using the J-ASPECT Diagnosis Procedure Combination database.Primary and secondary outcome measures Annual number of patients with SAH who remained untreated, or who received clipping or coiling, in-hospital mortality and poor functional outcomes (modified Rankin Scale: 3–6) at discharge. Each CSC was assessed using a validated scoring system (CSC score: 1–25 points).Results In the overall cohort, in-hospital mortality decreased (year for trend, OR (95% CI): 0.97 (0.96 to 0.99)), while the proportion of poor functional outcomes remained unchanged (1.00 (0.98 to 1.02)). The proportion of patients who underwent clipping gradually decreased from 46.6% to 38.5%, while that of those who received coiling and those left untreated gradually increased from 16.9% to 22.6% and 35.4% to 38%, respectively. In-hospital mortality of coiled (0.94 (0.89 to 0.98)) and untreated (0.93 (0.90 to 0.96)) patients decreased, whereas that of clipped patients remained stable. CSC score improvement was associated with increased use of coiling (per 1-point increase, 1.14 (1.08 to 1.20)) but not with short-term patient outcomes regardless of treatment modality.Conclusions The 6-year trends indicated lower in-hospital mortality for patients with SAH (attributable to better outcomes), increased use of coiling and multidisciplinary care for untreated patients. Further increasing CSC capabilities may improve overall outcomes, mainly by increasing the use of coiling. Additional studies are necessary to determine the effect of confounders such as aneurysm complexity on outcomes of clipped patients in the modern endovascular era