| Cholinergic neurons in basal forebrain regulate the plasticity of neurons in neocortex and play an important role in multiple brain functions such as cognition,sensory information processing,learning and attention,by releasing acetylcholine as neurotransmitter.Base on neurotomy and electrophysiology,the projection and function of basal forebrain cholinergic neurons in neocortex have been studied.However,the mechanism of the neural circuit for coordinating motor and sensory cortical functional areas is still unclear.In this study,we analysed the co-projection pattern of basal forebrain cholinergic neurons in the neocortex by a case of the neurons that project to primary motor area(MOp)in mice.Firstly,we obtained the distribution of cholinergic neurons that afferent to MOp by utilizing the strategy of retrograde viral tracer in fluorescent reporter mouse line.We found that the majority of afferents to the MOp originated from somatic sensorimotor subnetworks,thalamus,basal forebrain.And the cholinergic neurons that project to MOp were mainly distribute in the globus pallidus(GP)and Substantia innominate(SI)from basal forebrain(95.3 %).From rostral to caudal side of GP / SI,the MOp projecting cholinergic and noncholinergic neurons showed a special relationship in proportional variation and spatial distribution.Subsequently,we explored the morphological labeling strategy that is suitable for the reconstruction of cholinergic neurons with specific projection targets.By comparing the axonal fiber density and specific projection between various viral strategies,a combined strategy of recombinant adeno-associated virus(AAV)and rabies virus(RV)was selected.Cholinergic neurons labeled by this strategy have the characteristics of clearly discernible axons and specific projection targets.The fine morphology of 52 cholinergic neurons that project to MOp was reconstructed via viral labeling and fluorescent micro-optical sectioning tomography(f MOST).We found that the cholinergic neurons exhibited extensive projections by sending axons to multiple cortical areas.According to the analysis of co-projection cortical areas,we discovered that the downstream cortical areas of single cholinergic neuron are not randomly combined but follow the rigid hierarchical organization of the cortical networks.These cholinergic neurons were divided into three types,which base on the difference in co-projection to higher and lower order cortices.Finally,we found that the cholinergic neurons from three types showed a preference of axonal distribution in neocortex and long-range projection route by quantitative analysis.At the same time,the cholinergic neurons with semblable cell bodies location have significant differences of the axonal projection to medial-lateral cortical areas.However,the dendritic morphology is irrelevant to the axonal projection targets.In summary,base on the single neuron reconstruction,this study provides a systematic result on the co-projection pattern of basal forebrain cholinergic neurons in the neocortex.These results reveal a novel organization of the cortical cholinergic system,which provide important reference value for further understanding the mechanism of the cholinergic system for regulating the plasticity of cortical network. |
