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As the relay centre for processing sensory information, the thalamus may involve in the abnormal sensory procedure caused by cortical spreading depression (CSD). However, few studies have focused on the transient response of thalamus during CSD. Our study aimed to investigate the neuronal activity of mouse thalamus ventral posteromedial nucleus (VPM) during CSD by in vivo micro-endoscopic fluorescence imaging of the genetic calcium probe GCaMP6s expressed in excitatory glutamatergic neurons.