Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Abstract

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre-and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning. Video LinkThe video component of this article can be found at https://www.jove.com/video/53853/ . This original method had, however, several limitations, i.e., severe fragmentation of replicas, which hampered meaningful correlations of labeled molecules with individually mapped cells in complex tissues such as the brain. Approximately 10 years ago, Shigemoto and Fukazawa progressively improved the technique 2 . This was paralleled by efforts from another group of scientists at the Boulder laboratories of the Colorado State University, who also significantly improved the technique, in particular for the study of gap junctions 3 .