The Establishment Of An AFM-based Single Molecule Force Spectroscopy Technique For The Investigation Of The Recognition Of The Receptor On Hippocampal Neuronal Surface

Hippocampus is an important structure of the brain,which is involved in short-term memory,learning,cognition,emotion and other physiological functions.Hippocampal neurons are the main components of the hippocampus,and various important functional receptors are distributed on the neuronal surface.The interactions between the receptors and corresponding ligands determine a lot of signal pathways,which can affect many important neural activities.Currently,understanding the recognition process of the receptors by corresponding ligands is attracting enormous attention.To fully understand the recognition process,however it is necessary to study the interactions at the single molecule level because the traditional techniques which only provide averaged information on the interactions of bulk molecules cannot provide the underly mechanisms.Thanks to the emergence of atomic force microscopy-based single molecule force spectroscopy technique(AFM-SMFS),the scientists now are able to study the receptors on cell surface at the single molecule level.However,so far this kind of technique is mainly limited to detect the receptors on ordinary cell surface and rarely is used to study the receptors on neuronal cell surface.Therefore,it is an urgent work to establish an AFM-SMFS technique to explore the underlying mechanisms for the recognition of neuronal surface receptors by corresponding ligands.There are many receptors distributed on the neuronal cell surface of the hippocampus,such as tyrosine kinase B(Trk B),N-methyl-D-aspartate-type glutamate(NMDA)receptors,gamma-aminobutyric acid B(GABA B)receptors,etc.Among them,TrkB receptor is one of the most important receptors.Brain-derived neurotrophic factor(BDNF)is a ligand that can specificity interact with Trk B,and their interaction is vital for the function of neurons.In this project,by choosing TrkB and BDNF as the model molecules,we aimed to establish an AFM-SMFS technique which can be used to interrogate the recognition between neuronal receptors and ligands with high accuracy.First,we established the hippocampal neuron culture system.To be specifically,newborn suckling mice were selected as the experimental animals and the brain of the mouse was dissected to separate the hippocampus.Afterwards the hippocampus was digested and the hippocampus neurons were collected which were further cultured.The status of the growth of the neurons was monitored by an inverted optical microscopy.The mature neurons were examined by AFM high-resolution imaging in culture medium and the morphological information of neurons were obtained.Second,we linked one end of the α,ω-dicarboxy polyethylene glycol molecule(HOOC-PEG 3500-COOH)to the AFM probe via the carboxyl group by chemical modifications.Then we allowed another end the of α,ω-dicarboxy polyethylene glycol molecule to interact with the amino group grafted on a gold surface using the AFM-SMFS technique.By data fitting and analysis,we found that the force for α,ω-dicarboxy polyethylene glycol to peel off the gold substrate was 102.90 ± 7.03 pN,and that the contour length for the α,ω-dicarboxy polyethylene glycol molecule was 17.96 ± 0.93 nm(FJC model)and 19.40 ± 1.68 nm(WLC model).The obtained results are highly consistent with their theoretical values of length.The above results confirm that the α,ω-dicarboxy polyethylene glycol molecule has been successfully linked to the AFM probe,which lay a solid foundation for further linking BDNF to the AFM probe via –COOH of the α,ω-dicarboxy polyethylene glycol molecule.Finally,based on the above work,we further interrogated the recognition process between the BDNF and Trk B receptor at the single molecule level.We linked BDNF protein to the AFM probe via one end of the α,ω-dicarboxy polyethylene glycol molecule.Then we allowed the linked BDNF protein to recognize the TrkB receptors on live neuronal cell surface using the AFM-SMFS technique.By data fitting and analysis,we found that the pull-off between a BDNF and Trk B receptor was 206.81 ± 8.13 pN(in the force range for the receptor-ligand interaction),and that the contour length for the complex formed by the α,ω-dicarboxy polyethylene glycol molecule and BDNF protein was 25.34 ± 1.21 nm(FJC model)and 26.56 ± 1.52 nm(WLC model)which are consistent with the theoretical length of the complex.The above results confirm that we have linked BDNF to the AFM probe via the α,ω-dicarboxy polyethylene glycol molecule,and that BDNF can recognize the Trk B receptor on the neuronal cell specifically at single molecule level.In conclusion,we firstly established the hippocampal neuron culture system;then linked BDNF to the AFM probe by the linker molecule,α,ω-dicarboxy polyethylene glycol,through chemical modifications;afterwards investigated the interactions between the BDNF and TrkB receptors on neuronal cell surface.Our results suggest that we have successfully established the AFM-SFMS technique for interrogating the interactions of ligand-receptor systems for neurons.This work has laid a solid basis for further work aiming to understand the underlying in-depth biophysical mechanisms regarding the interactions between neuronal surface receptors and ligands at the single molecule level.