The mammalian brainstem is particularly vulnerable to mild traumatic brain injury (mTBI), which is associated with prolonged autonomic dysfunction and coma. The spatial cellular connections within the brainstem or the mechanisms underlying its response to injury have been underestimated. Here, we performed single-nucleus RNA sequencing with spatial transcriptome sequencing in both normal and mTBI brainstems in male mice, revealing thirty-five neuron and non-neuron clusters. Typically, we identified subtypes of neurons that co-release multiple neurotransmitters, especially in the sagittal midline of the brainstem. Spatially adjacent neurons sharing similar gene expression patterns. The brainstem's response to mTBI has two features: (1) Oligodendrocytes around the fourth ventricle exhibit widespread disconnection at 1-h post-injury, and (2) Injury-related noradrenergic neurons, particularly in their interaction with neurons located in theIRt and the Sol. These findings provides a reference for further integrative investigations of cellular and circuit functions of brainstem.