Long-range Connections Of Cholinergic Neurons In The Mice Pontomesencephalic Tegmentum

The pontomesencephalic tegmentum(PMT)forms complex functional circuits with different nuclei in the central nervous system through abundant fibers and greatly contributes to various functions including locomotion and arousal.Previous studies have divided the PMT into the pedunculopontine nucleus(PPN)and laterodorsal tegmental nucleus(LDT)according to the distribution of cholinergic neurons,and analyzed the connections and functions of those cholinergic neurons.However,systematic analysis of the organization logic of their input and output circuits is still limited,especially of the morphological characteristics on the single-neuron level.Combining virus tracing with the fluorescent micro-optical sectional tomography(fMOST),I mapped three dimensional continuous long-range input and output atlas,and single-neuron morphology atlas of cholinergic neurons in the PPN and LDT in the whole brain,and investigated the functions and connections of cholinergic circuits projecting to the thalamus preliminarily.Combining fMOST with monosynaptic retrograde tracing,I acquired the input atlas of the PMT cholinergic neurons and found that the PPN and LDT received wide input ranging from the cortical areas to the spinal cord.They received inputs from similar regions but with quantitative differences that the PPN received stronger input from some nuclei related to locomotion while the LDT acquired richer input from the pons and prelimbic cortex.Furthermore,the immunostaining showed that PPN and LDT cholinergic neurons interconnected with each other directly and both received monosynaptic input from neuromodulatory neurons in the dorsal raphe nucleus and locus ceruleus.Combining fMOST with anterograde tracing,I obtained the whole-brain output atlas of the PMT cholinergic neurons and found that the PPN and LDT cholinergic neurons widely projected to various regions from the cortical areas to the spinal cord with different strengths.The PPN preferred some ascending areas including the thalamus while the LDT preferentially projected to the midbrain and pons.Furthermore,combining sparse-labeling virus with fMOST,I acquired and reconstructed the refined morphology of PMT cholinergic neurons and found that individual cholinergic neurons projected complex axonal branches to various areas in ascending and descending circuits simultaneously.In the ascending circuits,individual neurons innervated the thalamus with three different patterns and projected to the cortical areas with two separated pathways.The PPN and LDT cholinergic neurons had similar projection patterns at the single-cell level and could be divided into four types according to diverse axonal fibers.Meanwhile,axons of the PPN cholinergic neurons presented more dispersively while LDT owned convergent fibers but richer axonal branches.Comparison of input and output atlas suggested the PMT cholinergic neurons projected to most thalamic nuclei uniaxially.Transgenic mice combining with retrograde tracers revealed the cholinergic afferent information of the thalamus and certified that the PPN and LDT were the main source of acetylcholine for the thalamus.Furthermore,optogenetics regulation of unidirectional cholinergic fibers projecting from the PMT to the ventral posterior complex of thalamus(VP)suggested that activating the terminal of PPN→VP circuits promoted the locomotion of mice while the modulation of LDT→VP circuits affected levels of anxiety.Finally,I analyzed the morphology and input of PMT→VP cholinergic neurons.The PPN→VP neurons projected richer co-projecting fibers to locomotion-related nuclei and received richer input from some locomotion-related nuclei;the LDT→VP neurons preferentially co-projected to some nuclei related to anxiety and received stronger input from some of them.These results hinted that the functional differences of the PPN→VP and LDT→VP might result from their diverse morphology and input.In conclusion,I developed the input,output and morphology atlas of cholinergic neurons in the PPN and LDT and preliminarily investigated their differences in functions and connections of the circuits projecting to the thalamus.This study will lead to deep insights into understanding the organization and functions of the pontine-tegmental cholinergic system and treating some neurodegenerative diseases with functional impaired cholinergic neurons.