The Optimization of Golgi-Cox Staining in Parallel with Immunofluorescence to Colocalize the Adhesion Protein, N-Cadherin, to Developing Synapses

Abstract

Numerous neurodevelopmental disorders, including Autism Spectrum Disorders (ASD), exhibit altered synapse formation. Post-mortem brain samples from idiopathic cases of ASD exhibit increased numbers of dendritic spines, which are the primary site of excitatory synapse formation. Excitatory synapses facilitate action potential formation, underlying cognitive functions such as learning and memory formation. Excitatory synapses develop in stages: synapse formation begins when post-synaptic spine precursors adhere to a pre-synaptic axon partner. As synapses mature, dendritic spines undergo a morphological transition from filopodia-like spine precursors to mushroom-shaped dendritic spines. These morphological changes increase the surface area adjacent to the axon partner, as well as the number of neurotransmitter receptors, thereby increasing the likelihood of action potential propagation. We are interested in the mechanisms of synapse formation, maturation, and maintenance. While there is considerable research on experience-dependent synapse maturation after birth, we lack understanding of the molecular mechanisms that initiate synapse formation and how these mechanisms are altered by ASD-associated genetic mutations and/or environmental factors. To address how synapse adherence occurs, we developed techniques to visualize association of the known synaptic adhesion molecule, N-Cadherin, with developing synapses. In this study we will be presenting the process of optimizing Golgi-Cox staining in embryonic mouse brain tissue to visualize post-synaptic dendritic spine morphology and using immunofluorescence in mouse brains to study N-Cadherin’s association with developing synapses. Our hypothesis stated that as synapse development progresses there will be an increase in N-Cadherin localized to synapses. Using these techniques, we saw trends which suggest that as the brain develops there will be an increase in the density of N-Cadherin positive synapses over time. Limitations of this study though prevented the acceptation or rejection of our hypothesis, instead it was inconclusive. The association we observed for the increase of N-Cadherin positive synapses over time, suggests a role for N-Cadherin in synapse stabilization/maturation. Further, significant differences were observed in that N-Cadherin positive synapses were larger than the negative N-Cadherin synapses on average. This supports the postulation that N-Cadherin promotes excitatory synapse maturation. Future research will use knockdown approaches to examine the role of N-Cadherin at distinct stages of synapse formation in neuronal cell cultures.