Dissection Of The Projection Pattern Of Parvalbumin Neurons In The Basal Forebrain At The Synaptic Level

The basal forebrain is composed of various types of neurons,including cholinergic,glutamatergic,and γ-aminobutyric acid neurons.Parvalbumin(PV)neurons play important roles in brain activities such as attention,learning memory,and sleep,and these functions were degraded in clinical patients and animal models with Alzheimer’s disease(AD).Although the circuit structure of PV neurons had been explored,its long-range projection patterns are still unclear at the synaptic level.The main contents are as follows:(1)Establishing an embedding method for acquiring information on neuronal projection patterns in the whole brain at the synaptic level.To obtain weak signals of the axons and terminals during long-term imaging of the whole brain,the HM20-T low-temperature embedding method was developed,achieving sample polymerization at low temperatures.By introducing N,N-Dimethyl-p-toluidine as a polymerization accelerator and adjusting the parameters of the accelerator and initiator,stable polymerization of the whole brain was achieved at-20℃.Compared with the previous method,HM20-T was compatible with a variety of fluorescent proteins and fluorescein,while the fluorescence preservation ratio of presynaptic terminals was doubled.The fluorescent signals of multicolor-labeled precise structures were preserved and used to acquire the projection patterns of neurons in the whole brain at the synaptic level.(2)Constructing projection atlas of PV neurons in the basal forebrain at the synaptic level.The axons and presynaptic terminals of PV neurons in the basal forebrain were simultaneously labeled by adeno-associated virus.By using fluorescence micro-optical sectioning tomography system(f MOST),the projection dataset of PV neurons at a resolution of 0.23 μm × 0.23 μm × 1 μm was acquired in the whole brain.The axons innervated multiple cortical regions and subcutaneous nuclei.Presynaptic terminals were densely distributed in limbic systems such as the hippocampus,thalamus,cingulate cortex,and mamillary body and preferentially concentrated in the anterolateral hippocampus,layer5 of the retrosplenial area,and dorsal-medial medial mamillary nucleus(MM).To certify the synaptic types between PV neurons and downstream neurons,the virus was modified for double-transgenic mice to label the pre-and post-synaptic components.About 50% of the presynaptic terminals formed connections with inhibitory neurons,of which about 30%were connected to the somas,indicating that PV neurons might regulate the inhibitory circuits in the hippocampus through axosomatic connection.Combining f MOST with customized sparsely labeling virus for presynaptic terminals,the morphology of single PV neurons and the distribution of their presynaptic terminals were reconstructed and analyzed.The morphology of PV neurons was correlated to the spatial position of their somas,and these neurons were divided into four types according to their projection patterns.These four types of neurons projected to the hippocampal region,retrohippocampal region,subcortical nuclei and others,respectively.The presynaptic terminals of type I neurons were distributed in multiple regions of the bilateral hippocampus,and the presynaptic terminals of some type II neurons were concentrated in the MM.These data indicated that the PV neurons in the basal forebrain formed synaptic connections with memory-related regions.(3)Exploring the degenerative changes of projection patterns of PV neurons in the basal forebrain at the synaptic level.Comparing four,six,and eight-month-old AD mice,I found presynaptic terminals degenerated in brain regions such as the hippocampus,MM,and thalamus.These data indicated that the degeneration of terminals tended to occur in Papez memory-related circuits.The number of terminals was first reduced in the MM and hippocampus.As AD progressed,the number of terminals in the MM and hippocampus further were reduced,and the number of terminals in the thalamus began to decrease.Then,I explored the cause of presynaptic terminals degeneration and found that presynaptic degeneration preceded the changes in the neurons in the basal forebrain and downstream brain regions.The extent of degeneration of presynaptic terminals was inconsistent with the density of amyloid plaques.These results suggested that we should investigate neuronrelated circuits to study the mechanisms of degeneration of neural terminals.In summary,I established the resin embedding method of neuronal projection patterns at the synaptic level,analyzed the projection patterns atlas of PV neurons in the basal forebrain and synaptic degeneration in AD at the synaptic level,and constructed projection patterns of single PV neurons.These results provided new clues into the study of the regulation of the functions of multiple brain regions and treatment of degenerative diseases.