Abstract
Vision formation is classically based on projections from retinal ganglion cells (RGC) to the lateral geniculate nucleus (LGN) and the primary visual cortex (V1). Neurons in the mouse V1 are tuned to light stimuli. Although the cellular information of the retina and the LGN has been widely studied, the transcriptome profiles of single light-stimulated neuron in V1 remain unknown. In our study, in vivo calcium imaging and whole-cell electrophysiological patch-clamp recording were utilized to identify 53 individual cells from layer 2/3 of V1 as light-sensitive (LS) or non-light-sensitive (NS) by single-cell light-evoked calcium evaluation and action potential spiking. The contents of each cell after functional tests were aspirated in vivo through a patch-clamp pipette for mRNA sequencing. Moreover, the three-dimensional (3-D) morphological characterizations of the neurons were reconstructed in a live mouse after the whole-cell recordings. Our sequencing results indicated that V1 neurons with a high expression of genes related to transmission regulation, such as Rtn4r and Rgs7, and genes involved in membrane transport, such as Na+/K+ ATPase and NMDA-type glutamatergic receptors, preferentially responded to light stimulation. Furthermore, an antagonist that blocks Rtn4r signals could inactivate the neuronal responses to light stimulation in live mice. In conclusion, our findings of the vivo-seq analysis indicate the key role of the strength of synaptic transmission possesses neurons in V1 of light sensory.
Highlights
Visual perception involves the activity of neurons in the cerebral cortex
We developed a method for functional in vivo single cell RNA-seq analysis for combining intracellular calcium imaging, in vivo whole-cell patch clamp recording, and high-quality RNA sequencing of individual neurons at layer 2/3 of the mouse V1 cortex while the mouse was stimulated via light grating under light anesthetization
In our vivo-seq system (Fig. 1A), the intracellular calcium activity from individual neurons at layer 2/3 of the mouse V1 cortex were recorded while the mouse was stimulated via light grating under light anesthetization
Summary
Visual perception involves the activity of neurons in the cerebral cortex. The ‘retino-geniculo-cortical’ pathway indicates the best-known route for visual information (Chalupa, 2003). The physical environment during whole-cell recording on brain slices is substantially different from the in vivo environment and the local circuitry related to acute stimulation is not able to be tested in the brain slices To address these questions, we developed a method for functional in vivo single cell RNA-seq (vivo-seq) analysis for combining intracellular calcium imaging, in vivo whole-cell patch clamp recording, and high-quality RNA sequencing of individual neurons at layer 2/3 of the mouse V1 cortex while the mouse was stimulated via light grating under light anesthetization. The vivo-seq analysis identified the molecular biomarkers that were involved in the signaling pathways of sensing light in V1, and suggested that the transmission strength and plasticity of synapse in V1 played an important role in light sensitivity of V1 neurons
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