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.

A peptidergic amygdala microcircuit modulates sexually dimorphic contextual fear

“…However, it is possible that NMDA receptors play a role. NMDA receptors mediate calcium entry in response to synaptic stimulation and have been previously shown to modulate mICC function [17,32,33]. In a sub-population of mICCs neurons, we observed an EPSC followed by a longer latency IPSC, which could be due to collateral projections from nearby mICC neurons.…”

Punishment-Predictive Cues Guide Avoidance through Potentiation of Hypothalamus-to-Habenula Synapses

“…To corroborate these results and visualize the locus of expression for avoidance-driven AMPAR potentiation, we employed freeze-fracture replica immunolabeling (Schö nherr et al, 2016). Combined with infusion of rAAV2-hSyn-CoChR-EGFP, this approach allows the quantification of membrane AMPARs specifically at synapses formed by LH axons to LHb postsynaptic neurons (Figure 3C).…”

Structural and Functional Remodeling of Amygdala GABAergic Synapses in Associative Fear Learning

“…Freeze-fracture replica immunolabeling Only 5 animals/group/experiment were processed for FRIL, and were selected among those with the best behavioral response. FRIL was performed according to previously published procedures (Kasugai et al, 2010;Schö nherr et al, 2016). Mice were transcardially perfused with PB (0.1 M, pH 7.4) followed by a fixative containing 1% formaldehyde and 15% of a saturated solution of picric acid for 12 min.…”