Increased expression of chondroitin sulfate proteoglycans in dentate gyrus and amygdala causes postinfectious seizures.

Alterations in the extracellular matrix are common in patients with epilepsy and animal models of epilepsy, yet whether they are the cause or consequence of seizures and epilepsy development is unknown. Using Theiler's murine encephalomyelitis virus (TMEV) infection-induced model of acquired epilepsy, we found de novo expression of chondroitin sulfate proteoglycans (CSPGs), a major extracellular matrix component, in dentate gyrus (DG) and amygdala exclusively in mice with acute seizures. Preventing the synthesis of CSPGs specifically in DG and amygdala by deletion of the major CSPG aggrecan reduced seizure burden. Patch-clamp recordings from dentate granule cells revealed enhanced intrinsic and synaptic excitability in seizing mice that was significantly ameliorated by aggrecan deletion. In situ experiments suggested that dentate granule cell hyperexcitability results from negatively charged CSPGs increasing stationary cations on the membrane, thereby depolarizing neurons, increasing their intrinsic and synaptic excitability. These results show increased expression of CSPGs in the DG and amygdala as one of the causal factors for TMEV-induced acute seizures. We also show identical changes in CSPGs in pilocarpine-induced epilepsy, suggesting that enhanced CSPGs in the DG and amygdala may be a common ictogenic factor and potential therapeutic target.

Using Drosophila Two-Choice Assay to Study Optogenetics in Hands-On Neurobiology Laboratory Activities.

Optogenetics has made a significant impact on neuroscience, allowing activation and inhibition of neural activity with exquisite spatiotemporal precision in response to light. In this lab session, we use fruit flies to help students understand the fundamentals of optogenetics through hands-on activities. The CsChrimson channelrhodopsin, a light-activated cation channel, is expressed in sweet and bitter sensory neurons. Sweet sensory neurons guide animals to identify nutrient-rich food and drive appetitive behaviors, while bitter sensory neurons direct animals to avoid potentially toxic substances and guide aversive behavior. Students use two-choice assays to explore the causality between the stimulation activation of these neurons and the appetitive and avoidance behaviors of the fruit flies. To quantify their observations, students calculate preference indices and use the Student's t-test to analyze their data. After this lab session, students are expected to have a basic understanding of optogenetics, fly genetics, sensory perception, and how these relate to sensory-guided behaviors. They will also learn to conduct, quantify, and analyze two-choice behavioral assays.

Vascular amyloidosis impairs the gliovascular unit in a mouse model of Alzheimer's disease.

Reduced cerebral blood flow impairs cognitive function and ultimately causes irreparable damage to brain tissue. The gliovascular unit, composed of neural and vascular cells, assures sufficient blood supply to active brain regions. Astrocytes, vascular smooth muscle cells, and pericytes are important players within the gliovascular unit modulating vessel diameters. While the importance of the gliovascular unit and the signals involved in regulating local blood flow to match neuronal activity is now well recognized, surprisingly little is known about this interface in disease. Alzheimer's disease is associated with reduced cerebral blood flow. Here, we studied how the gliovascular unit is affected in a mouse model of Alzheimer's disease, using a combination of ex vivo and in vivo imaging approaches. We specifically labelled vascular amyloid in living mice using the dye methoxy-XO4. We elicited vessel responses ex vivo using either pharmacological stimuli or cell-specific calcium uncaging in vascular smooth muscle cells or astrocytes. Multi-photon in vivo imaging through a cranial window allowed us to complement our ex vivo data in the presence of blood flow after label-free optical activation of vascular smooth muscle cells in the intact brain. We found that vascular amyloid deposits separated astrocyte end-feet from the endothelial vessel wall. High-resolution 3D images demonstrated that vascular amyloid developed in ring-like structures around the vessel circumference, essentially forming a rigid cast. Where vascular amyloid was present, stimulation of astrocytes or vascular smooth muscle cells via ex vivo Ca(2+) uncaging or in vivo optical activation produced only poor vascular responses. Strikingly, vessel segments that were unaffected by vascular amyloid responded to the same extent as vessels from age-matched control animals. We conclude that while astrocytes can still release vasoactive substances, vascular amyloid deposits render blood vessels rigid and reduce the dynamic range of affected vessel segments. These results demonstrate a mechanism that could account in part for the reduction in cerebral blood flow in patients with Alzheimer's disease.media-1vid110.1093/brain/awv327_video_abstractawv327_video_abstract.

Astrocytes require perineuronal nets to maintain synaptic homeostasis in mice.

Perineuronal nets (PNNs) are densely packed extracellular matrices that cover the cell body of fast-spiking inhibitory neurons. PNNs stabilize synapses inhibiting synaptic plasticity. Here we show that synaptic terminals of fast-spiking interneurons localize to holes in the PNNs in the adult mouse somatosensory cortex. Approximately 95% of holes in the PNNs contain synapses and astrocytic processes expressing Kir4.1, glutamate and GABA transporters. Hence, holes in the PNNs contain tripartite synapses. In the adult mouse brain, PNN degradation causes an expanded astrocytic coverage of the neuronal somata without altering the axon terminals. The loss of PNNs impairs astrocytic transmitter and potassium uptake, resulting in the spillage of glutamate into the extrasynaptic space. Our data show that PNNs and astrocytes cooperate to contain synaptically released signals in physiological conditions. Their combined action is altered in mouse models of Alzheimer's disease and epilepsy where PNNs are disrupted.

Perineuronal nets support astrocytic ion and glutamate homeostasis at tripartite synapses.

Perineuronal nets (PNNs) are dense, negatively charged extracellular matrices that cover the cell body of fast-spiking inhibitory neurons. Synapses can be embedded and stabilized by PNNs believed to prevent synaptic plasticity. We find that in cortical fast-spiking interneurons synaptic terminals localize to perforations in the PNNs, 95% of which contain either excitatory or inhibitory synapses or both. The majority of terminals also colocalize with astrocytic processes expressing Kir4.1 as well as glutamate (Glu) and GABA transporters, hence can be considered tripartite synapses. In the adult brain, degradation of PNNs does not alter axonal terminals but causes expansion of astrocytic coverage of the neuronal somata. However, loss of PNNs impairs astrocytic transmitter and K+ uptake and causes spillage of synaptic Glu into the extrasynaptic space. This data suggests a hitherto unrecognized role of PNNs, to synergize with astrocytes to contain synaptically released signals.

Astrocyte plasticity in mice ensures continued endfoot coverage of cerebral blood vessels following injury and declines with age.

Astrocytes extend endfeet that enwrap the vasculature, and disruptions to this association which may occur in disease coincide with breaches in blood-brain barrier (BBB) integrity. Here we investigate if focal ablation of astrocytes is sufficient to disrupt the BBB in mice. Targeted two-photon chemical apoptotic ablation of astrocytes induced a plasticity response whereby surrounding astrocytes extended processes to cover vascular vacancies. In young animals, replacement processes occur in advance of endfoot retraction, but this is delayed in aged animals. Stimulation of replacement astrocytes results in constriction of pre-capillary arterioles, suggesting that replacement astrocytes are functional. Pharmacological inhibition of pSTAT3, as well as astrocyte specific deletion of pSTAT3, reduces astrocyte replacement post-ablation, without perturbations to BBB integrity. Similar endfoot replacement occurs following astrocyte cell death due to reperfusion in a stroke model. Together, these studies uncover the ability of astrocytes to maintain cerebrovascular coverage via substitution from nearby cells.

Nano-optoelectrodes Integrated with Flexible Multifunctional Fiber Probes by High-Throughput Scalable Fabrication.

Metallic nano-optoelectrode arrays can simultaneously serve as nanoelectrodes to increase the electrochemical surface-to-volume ratio for high-performance electrical recording and optical nanoantennas to achieve nanoscale light concentrations for ultrasensitive optical sensing. However, it remains a challenge to integrate nano-optoelectrodes with a miniaturized multifunctional probing system for combined electrical recording and optical biosensing in vivo. Here, we report that flexible nano-optoelectrode-integrated multifunctional fiber probes can have hybrid optical-electrical sensing multimodalities, including optical refractive index sensing, surface-enhanced Raman spectroscopy, and electrophysiological recording. By physical vapor deposition of thin metal films through free-standing masks of nanohole arrays, we exploit a scalable nanofabrication process to create nano-optoelectrode arrays on the tips of flexible multifunctional fiber probes. We envision that the development of flexible nano-optoelectrode-integrated multifunctional fiber probes can open significant opportunities by allowing for multimodal monitoring of brain activities with combined capabilities for simultaneous electrical neural recording and optical biochemical sensing at the single-cell level.

Spatially expandable fiber-based probes as a multifunctional deep brain interface.

Understanding the cytoarchitecture and wiring of the brain requires improved methods to record and stimulate large groups of neurons with cellular specificity. This requires miniaturized neural interfaces that integrate into brain tissue without altering its properties. Existing neural interface technologies have been shown to provide high-resolution electrophysiological recording with high signal-to-noise ratio. However, with single implantation, the physical properties of these devices limit their access to one, small brain region. To overcome this limitation, we developed a platform that provides three-dimensional coverage of brain tissue through multisite multifunctional fiber-based neural probes guided in a helical scaffold. Chronic recordings from the spatially expandable fiber probes demonstrate the ability of these fiber probes capturing brain activities with a single-unit resolution for long observation times. Furthermore, using Thy1-ChR2-YFP mice we demonstrate the application of our probes in simultaneous recording and optical/chemical modulation of brain activities across distant regions. Similarly, varying electrographic brain activities from different brain regions were detected by our customizable probes in a mouse model of epilepsy, suggesting the potential of using these probes for the investigation of brain disorders such as epilepsy. Ultimately, this technique enables three-dimensional manipulation and mapping of brain activities across distant regions in the deep brain with minimal tissue damage, which can bring new insights for deciphering complex brain functions and dynamics in the near future.

Polymer Composite with Carbon Nanofibers Aligned during Thermal Drawing as a Microelectrode for Chronic Neural Interfaces.

Microelectrodes provide a direct pathway to investigate brain activities electrically from the external world, which has advanced our fundamental understanding of brain functions and has been utilized for rehabilitative applications as brain-machine interfaces. However, minimizing the tissue response and prolonging the functional durations of these devices remain challenging. Therefore, the development of next-generation microelectrodes as neural interfaces is actively progressing from traditional inorganic materials toward biocompatible and functional organic materials with a miniature footprint, good flexibility, and reasonable robustness. In this study, we developed a miniaturized all polymer-based neural probe with carbon nanofiber (CNF) composites as recording electrodes via the scalable thermal drawing process. We demonstrated that in situ CNF unidirectional alignment can be achieved during the thermal drawing, which contributes to a drastic improvement of electrical conductivity by 2 orders of magnitude compared to a conventional polymer electrode, while still maintaining the mechanical compliance with brain tissues. The resulting neural probe has a miniature footprint, including a recording site with a reduced size comparable to a single neuron and maintained impedance that was able to capture neural activities. Its stable functionality as a chronic implant has been demonstrated with the long-term reliable electrophysiological recording with single-spike resolution and the minimal tissue response over the extended period of implantation in wild-type mice. Technology developed here can be applied to basic chronic electrophysiological studies as well as clinical implementation for neuro-rehabilitative applications.

Disruption of astrocyte-vascular coupling and the blood-brain barrier by invading glioma cells.

Astrocytic endfeet cover the entire cerebral vasculature and serve as exchange sites for ions, metabolites and energy substrates from the blood to the brain. They maintain endothelial tight junctions that form the blood-brain barrier (BBB) and release vasoactive molecules that regulate vascular tone. Malignant gliomas are highly invasive tumours that use the perivascular space for invasion and co-opt existing vessels as satellite tumour form. Here we use a clinically relevant mouse model of glioma and find that glioma cells, as they populate the perivascular space of preexisting vessels, displace astrocytic endfeet from endothelial or vascular smooth muscle cells. This causes a focal breach in the BBB. Furthermore, astrocyte-mediated gliovascular coupling is lost, and glioma cells seize control over the regulation of vascular tone through Ca(2+)-dependent release of K(+). These findings have important clinical implications regarding blood flow in the tumour-associated brain and the ability to locally deliver chemotherapeutic drugs in disease.