Neural Stem Cell Grafts Form Extensive Synaptic Networks that Integrate with Host Circuits after Spinal Cord Injury.

Neural stem/progenitor cell (NSPC) grafts can integrate into sites of spinal cord injury (SCI) and generate neuronal relays across lesions that can provide functional benefit. To determine if and how grafts become synaptically organized and connect with host systems, we performed calcium imaging of NSPC grafts in SCI sites in vivo and in adult spinal cord slices. NSPC grafts organize into localized and spontaneously active synaptic networks. Optogenetic stimulation of host corticospinal tract axons regenerating into grafts elicited distinct and segregated neuronal network responses throughout the graft. Moreover, optogenetic stimulation of graft-derived axons extending from the graft into the denervated spinal cord also triggered local host neuronal network responses. In vivo imaging revealed that behavioral stimulation likewise elicited focal synaptic responses within grafts. Thus neural progenitor grafts can form functional synaptic subnetworks whose activity patterns resemble intact spinal cord.

Phosphatidylserine Exposure Controls Viral Innate Immune Responses by Microglia.

Microglia are the intrinsic immune sentinels of the central nervous system. Their activation restricts tissue injury and pathogen spread, but in some settings, including viral infection, this response can contribute to cell death and disease. Identifying mechanisms that control microglial responses is therefore an important objective. Using replication-incompetent adenovirus 5 (Ad5)-based vectors as a model, we investigated the mechanisms through which microglia recognize and respond to viral uptake. Transgenic, immunohistochemical, molecular-genetic, and fluorescence imaging approaches revealed that phosphatidylserine (PtdSer) exposure on the outer leaflet of transduced cells triggers their engulfment by microglia through TAM receptor-dependent mechanisms. We show that inhibition of phospholipid scramblase 1 (PLSCR1) activity reduces intracellular calcium dysregulation, prevents PtdSer externalization, and enables months-long protection of vector-transduced, transgene-expressing cells from microglial phagocytosis. Our study identifies PLSCR1 as a potent target through which the innate immune response to viral vectors, and potentially other stimuli, may be controlled.

Imaging large-scale cellular activity in spinal cord of freely behaving mice.

Sensory information from mechanoreceptors and nociceptors in the skin plays key roles in adaptive and protective motor behaviours. To date, very little is known about how this information is encoded by spinal cord cell types and their activity patterns, particularly under freely behaving conditions. To enable stable measurement of neuronal and glial cell activity in behaving mice, we have developed fluorescence imaging approaches based on two- and miniaturized one-photon microscopy. We show that distinct cutaneous stimuli activate overlapping ensembles of dorsal horn neurons, and that stimulus type and intensity is encoded at the single-cell level. In contrast, astrocytes show large-scale coordinated calcium responses to intense but not weak sensory inputs. Sensory-evoked activity is potently suppressed by anaesthesia. By revealing the cellular and computational logic of spinal cord networks under behaving conditions, our approach holds promise for better understanding of healthy and aberrant spinal cord processes.

Stepwise recruitment of transcellular and paracellular pathways underlies blood-brain barrier breakdown in stroke.

Brain endothelial cells form a paracellular and transcellular barrier to many blood-borne solutes via tight junctions (TJs) and scarce endocytotic vesicles. The blood-brain barrier (BBB) plays a pivotal role in the healthy and diseased CNS. BBB damage after ischemic stroke contributes to increased mortality, yet the contributions of paracellular and transcellular mechanisms to this process in vivo are unknown. We have created a transgenic mouse strain whose endothelial TJs are labeled with eGFP and have imaged dynamic TJ changes and fluorescent tracer leakage across the BBB in vivo, using two-photon microscopy in the t-MCAO stroke model. Although barrier function is impaired as early as 6 hr after stroke, TJs display profound structural defects only after 2 days. Conversely, the number of endothelial caveolae and transcytosis rate increase as early as 6 hr after stroke. Therefore, stepwise impairment of transcellular followed by paracellular barrier mechanisms accounts for the BBB deficits in stroke.