Tongqiao Huoxue Decoction inhibits ferroptosis by facilitating ACSL4 ubiquitination degradation for neuroprotection against cerebral ischemia-reperfusion injury.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) refers to brain tissue injury caused by the temporary interruption of cerebral blood flow ischemia followed by the restoration of reperfusion, which is the main cause of post-stroke brain injury. A traditional Chinese herbal preparation called Tongqiao Huoxue Decoction (TQHX) has shown promise in reducing CIRI in rats. However, the mechanism of this herbal preparation for CIRI remains unclear. PURPOSE: This study aimed to evaluate the therapeutic effect of TQHX extract on rats with CIRI and to further explore the underlying mechanisms. METHODS: The active ingredients of TQHX extract were quantified by the high-performance liquid chromatography (HPLC) condition. We conducted thorough investigations to assess the effects of TQHX on CIRI and ferroptosis using oxygen-glucose deprivation/reperfusion (OGD/R)-treated PC12 cells as an in vitro model and transient middle cerebral artery occlusion (tMCAO) animals as an in vivo model. The neurological score assessment was performed to evaluate the neuroprotective effects of TQHX extract on tMCAO rats. Using histologic methods to study the extent of cerebral infarction, blood-brain barrier, and rat brain tissue. We examined the impact of TQHX on ferroptosis-related markers of Fe2+, superoxide dismutase (SOD), reactive oxygen species (ROS), and malondialdehyde (MDA) in the brain tissue. In addition, the expression of key proteins and markers of ferroptosis, as well as key factors associated with Acyl-CoA synthetase long-chain family member 4 (ACSL4) were detected by Western blot and quantitative real-time PCR (RT-qPCR). RESULTS: TQHX extract could decrease the Longa score and extent of cerebral infarction of tMCAO rats, which exerted the function of neuroprotection. Additionally, TQHX treatment efficiently decreased levels of MDA and ROS while increasing the expression of SOD and ferroptosis-related proteins including ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) at the transcription and translation level. Meanwhile, TQHX provided strong protection against oxidative stress and ferritin accumulation by increasing the ubiquitination and degradation of ACSL4. The injection of OE-ACSL4 reversed the effects of TQHX on neuroprotection and ferroptosis inhibition in PC12 cells. The injection of shACSL4 reversely validate the crucial role of ACSL4 in CIRI rat treatment. CONCLUSION: This work shows that TQHX promotes the ubiquitination-mediated degradation of ACSL4, which improves oxidative stress and inhibits the beginning of ferroptosis in cells. TQHX provides a possible path for additional research in CIRI therapies, advancing translational investigations.

Single cell optogenetics reveals attenuation-by-suppression in visual cortical neurons.

The relationship between neurons' input and spiking output is central to brain computation. Studies in vitro and in anesthetized animals suggest nonlinearities emerge in cells' input-output (activation) functions as network activity increases, yet how neurons transform inputs in vivo has been unclear. Here, we characterize cortical principal neurons' activation functions in awake mice using two-photon optogenetics and imaging. We find responses to fixed optogenetic input are nearly unchanged as neurons are excited, reflecting a linear response regime above neurons' resting point. In contrast, responses are dramatically attenuated by suppression. This attenuation is a powerful means to filter inputs arriving to suppressed cells, privileging other inputs arriving to excited neurons. These data have two major implications: first, neural activation functions in vivo accord with the activation functions used in recent machine learning systems, and second, neurons' IO functions can enhance sensory processing by attenuating some inputs while leaving others unchanged.

Amplified cortical neural responses as animals learn to use novel activity patterns.

Cerebral cortex supports representations of the world in patterns of neural activity, used by the brain to make decisions and guide behavior. Past work has found diverse, or limited, changes in the primary sensory cortex in response to learning, suggesting that the key computations might occur in downstream regions. Alternatively, sensory cortical changes may be central to learning. We studied cortical learning by using controlled inputs we insert: we trained mice to recognize entirely novel, non-sensory patterns of cortical activity in the primary visual cortex (V1) created by optogenetic stimulation. As animals learned to use these novel patterns, we found that their detection abilities improved by an order of magnitude or more. The behavioral change was accompanied by large increases in V1 neural responses to fixed optogenetic input. Neural response amplification to novel optogenetic inputs had little effect on existing visual sensory responses. A recurrent cortical model shows that this amplification can be achieved by a small mean shift in recurrent network synaptic strength. Amplification would seem to be desirable to improve decision-making in a detection task; therefore, these results suggest that adult recurrent cortical plasticity plays a significant role in improving behavioral performance during learning.

Bicistronic Expression of a High-Performance Calcium Indicator and Opsin for All-Optical Stimulation and Imaging at Cellular Resolution.

State-of-the-art all-optical systems promise unprecedented access to neural activity in vivo, using multiphoton optogenetics to allow simultaneous imaging and control of activity in selected neurons at cellular resolution. However, to achieve wide use of all-optical stimulation and imaging, simple strategies are needed to robustly and stably express opsins and indicators in the same cells. Here, we describe a bicistronic adeno-associated virus (AAV) that expresses both the fast and bright calcium indicator jGCaMP8s, and a soma-targeted (st) and two-photon-activatable opsin, ChrimsonR. With this method, stChrimsonR stimulation with two-photon holography in the visual cortex of mice drives robust spiking in targeted cells, and neural responses to visual sensory stimuli and spontaneous activity are strong and stable. Cells expressing this bicistronic construct show responses to both photostimulation and visual stimulation that are similar to responses measured from cells expressing the same opsin and indicator via separate viruses. This approach is a simple and robust way to prepare neurons in vivo for two-photon holography and imaging.

Near-field THz micropolarimetry.

We introduce a method for rapid determination of anisotropic terahertz absorption with sub micron resolution and high spectral integrity in the terahertz range. The method is ideal for microscopic and environmentally sensitive materials such as 2-D materials and protein crystals where the anisotropic absorption is critical to understanding underlying physics. We introduce the idea of using an iso-response relationship between the THz polarization and electro optic probe polarization to enable stationary sample polarization measurements covering a full 2π polarization dependence measurement.