Isolation of Functional Presynaptic Complexes from CNS Neurons: A Cell-Free Preparation for the Study of Presynaptic Compartments In Vitro

Abstract

The difficulty in developing successful treatments to facilitate nerve regeneration has prompted a number of new in vitro experimental methods. We have recently shown that functional presynaptic boutons can be formed when neuronal cells are cocultured with surface-modified artificial substrates including poly(d-lysine)-coated beads and supported lipid bilayer-coated beads (Lucido(2009) J. Neurosci.29, 12449-12466; Gopalakrishnan(2010) ACS Chem. Neurosci.1, 86-94). We demonstrate here, using confocal microscopy combined with immunocytochemistry, that it is possible to isolate such in vitro presynaptic endings in an exclusive fashion onto glass substrates through a simple "sandwich/lift-off" technique (Perez(2006) Adv. Funct. Mater.16, 306-312). Isolated presynaptic complexes are capable of releasing and recycling neurotransmitter in response to an external chemical trigger. These bead-presynaptic complexes are facile to prepare and are readily dispersible in solution. They are thus compatible with many experimental methods whose focus is the study of the neuronal presynaptic compartment.

Robust Type-specific Hemisynapses Induced by Artificial Dendrites

“…One of the most striking features in synaptogenesis is that neurons can form ‘hemisynapses’ with a non-biological partner whose surface is covered with functional synaptic CAMs 5 6 7 . Such hemisynapses garner much attention because they can secure the connection between the two heterologous systems with defined geometry 8 9 10 . However, there has been lack of information about the dependency of neuronal morphology on synaptic specificity, the durability of excitatory and inhibitory hemisynapses, and the methodology of synaptic CAM engineering for the generation of robust artificial dendrites that enable the formation of type-specific hemisynapses.…”

Label-Free Visualization of Ultrastructural Features of Artificial Synapses via Cryo-EM

“…This is especially true when functionality studies were performed on artificial presynaptic boutons in culture and in isolated forms. 7,13 Importantly, spherical substrates also provide the advantage of accessing different radii of curvature simply by using different size beads. 14 The design of substrates for artificial synapse formation is of great interest both in applied and in fundamental aspects of in vitro studies involving synaptogenesis.…”

Lipid Microdomains in Synapse Formation

“…These probes are chosen on the basis of their differential ability to partition into different phases . Artificial synapse formation was explored via immunofluorescence methods for visualization of several synaptic proteins in hippocampal neurons. − These cells were grown for at least 14 days in vitro (DIV) and then cocultured with SS-BLMs of various lipid mixtures for 24 h. Quantification of the fluorescence intensity serves as an indicator of the level of accumulation of synaptic proteins in the vicinity of the SS-BLMs. This allows for exploration of the various chemical and physical parameters of lipid bilayers that trigger a functional synaptic response from the neurons.…”

“…We have previously demonstrated the in vitro formation of presynapses on micrometer-sized spherical substrates coated with either a synthetic polypeptide (i.e., poly- d -lysine; PDL) , or lipid bilayer membranes. , These reports have shown that the spherical geometry of these substrates is considerably favored for the observation of a functional synaptic response using microscopy techniques. In addition, preliminary evidence suggested that coexisting membrane domains direct lipid membrane-induced synapse formation .…”

“…In this proof-of-concept study, we demonstrate how extracellular membrane heterogeneity presented on SS-BLMs influences the dynamics of cellular microenvironments. This physicochemical model is (i) robust and can withstand cell culture conditions and characterization/imaging procedures, − , (ii) interactive with cells at any point in time during the period of cell culture, − , (iii) compositionally versatile, displaying structural properties of membranes with minimal components, , and (iv) of great potential for incorporating functional molecules, such as purified membrane raft components, into the bilayer structure of SS-BLMs. , …”