ABSTRACT
Sialic acid (SA) is a special monosaccharide widely distributed at the termini of sugar chains on the cell surface, and its expression level is closely connected with various biological and pathological processes. Therefore, accurate quantitative detection of SA on cancer cell surface is of great significance for clinical diagnosis and therapy. Here, we developed a whole-surface accessible method of accurate SERS quantification of SA level on a single cell, in which silver nanoparticles functionalized with 4-mercaptophenylboric acid and 4-mercaptobenzenitrile was used as the background-free SERS probe. The cyano group on the nanoprobe showed a unique Raman shift at 2232 cm-1, where most of the biological samples have no Raman response. Meanwhile, the boronic acid group had high specificity to SA molecules at physiological pH. The expression level of SA can be accurately quantitated on the basis of the CN Raman signal. The average number of expressed SA molecules on the surface of a single HeLa cell was 4.6 × 107. And SERS imaging of a single cell was achieved at 2232 cm-1 without biological interference. We evaluated SA expression level on the surface of different cancer cells and dynamically monitored SA expression under the influence of drugs. The proposed approach is accurate as well as sensitive for background-free quantification of SA on cell surface, which is promising for revealing the relationship between tumors and cell surface glycosylation.
Subject(s)
Metal Nanoparticles/chemistry , Sialic Acids/analysis , Silver/chemistry , Spectrum Analysis, Raman/methods , Sulfhydryl Compounds/chemistry , Boric Acids/chemistry , HEK293 Cells , HeLa Cells , Hep G2 Cells , Humans , Nitriles/chemistry , Single-Cell Analysis/methodsABSTRACT
Electrical coupling between excitatory neurons in the neocortex is developmentally regulated. It is initially prominent but eliminated at later developmental stages when chemical synapses emerge. However, it remains largely unclear whether early electrical coupling networks broadly contribute to neocortical circuit formation and animal behavior. Here, we report that neonatal electrical coupling between neocortical excitatory neurons is critical for proper neuronal development, synapse formation, and animal behavior. Conditional deletion of Connexin 26 (CX26) in the superficial layer excitatory neurons of the mouse neocortex around birth significantly reduces spontaneous firing activity and the frequency and size of spontaneous network oscillations at postnatal day 5-6. Moreover, CX26-conditional knockout (CX26-cKO) neurons tend to have simpler dendritic trees and lower spine density compared with wild-type neurons. Importantly, early, but not late, postnatal deletion of CX26, decreases the frequency of miniature excitatory postsynaptic currents (mEPSCs) in both young and adult mice, whereas miniature inhibitory postsynaptic currents (mIPSCs) were unaffected. Furthermore, CX26-cKO mice exhibit increased anxiety-related behavior. These results suggest that electrical coupling between excitatory neurons at early postnatal stages is a critical step for neocortical development and function.
Subject(s)
Anxiety/etiology , Anxiety/metabolism , Connexin 26/genetics , Connexin 26/metabolism , Neocortex/metabolism , Neocortex/physiopathology , Action Potentials/genetics , Animals , Animals, Newborn , Anxiety/psychology , Behavior, Animal , Dendrites/metabolism , Dendritic Spines/metabolism , Disease Models, Animal , Excitatory Postsynaptic Potentials/genetics , Female , Gene Deletion , Mice , Mice, Knockout , Mice, Transgenic , Neurons/metabolism , PregnancyABSTRACT
The coexistence of electrical and chemical synapses among interneurons is essential for interneuron function in the neocortex. However, it remains largely unclear whether electrical coupling between interneurons influences chemical synapse formation and microcircuit assembly during development. Here, we show that electrical and GABAergic chemical connections robustly develop between interneurons in neocortical layer 1 over a similar time course. Electrical coupling promotes action potential generation and synchronous firing between layer 1 interneurons. Furthermore, electrically coupled interneurons exhibit strong GABA-A receptor-mediated synchronous synaptic activity. Disruption of electrical coupling leads to a loss of bidirectional, but not unidirectional, GABAergic connections. Moreover, a reduction in electrical coupling induces an increase in excitatory synaptic inputs to layer 1 interneurons. Together, these findings strongly suggest that electrical coupling between neocortical interneurons plays a critical role in regulating chemical synapse development and precise formation of circuits.
