Array tomography of physiologically-characterized CNS synapses

Abstract

Background The ability to correlate plastic changes in synaptic physiology with changes in synaptic anatomy has been very limited in the central nervous system because of shortcomings in existing methods for recording the activity of specific CNS synapses and then identifying and studying the same individual synapses on an anatomical level. New method We introduce here a novel approach that combines two existing methods: paired neuron electrophysiological recording and array tomography, allowing for the detailed molecular and anatomical study of synapses with known physiological properties. Results The complete mapping of a neuronal pair allows determining the exact number of synapses in the pair and their location. We have found that the majority of close appositions between the presynaptic axon and the postsynaptic dendrite in the pair contain synaptic specializations. The average release probability of the synapses between the two neurons in the pair is low, below 0.2, consistent with previous studies of these connections. Other questions, such as receptor distribution within synapses, can be addressed more efficiently by identifying only a subset of synapses using targeted partial reconstructions. In addition, time sensitive events can be captured with fast chemical fixation. Comparison with existing methods Compared to existing methods, the present approach is the only one that can provide detailed molecular and anatomical information of electrophysiologically-characterized individual synapses. Conclusions This method will allow for addressing specific questions about the properties of identified CNS synapses, even when they are buried within a cloud of millions of other brain circuit elements.

A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses

“…Embedding into LR White is routinely performed in tissue blocks, and it is not used for so-called flat embedding (i.e., embedding a 60-to 100-mm-thick section between a glass slide and a coverslip), which is essential for LM identification of functionally characterized synapses and their consequent EM or LM molecular analysis. Although Valenzuela et al (2016) identified synapses in LR-White-embedded large tissue blocks, it requires the resectioning of the whole block and 3D reconstructing and inspecting the entire tissue block from Biocytin is shown with blue, and pre-and postsynaptic molecules are shown with green and red, respectively. vGluT1 immunosignal occupies most of the presynaptic bouton area, whereas the other 3 presynaptic molecules are more restricted toward the apposition of the pre-and postsynaptic structures.…”

“…They also demonstrated that the Abs can be eluted after a labeling round and that the sections can be relabeled, allowing the visualization of dozens of molecules. Probably the major shortcoming of AT was its limited sensitivity, and its application to functionally characterized synapses was challenging ( Valenzuela et al., 2016 ). Here, we aim to overcome these limitations by optimizing fixation, resin, embedding, etching, retrieval, and elution conditions.…”

Design and implementation of multi-signal and time-varying neural reconstructions

“…For instance, super resolution imaging has revealed the arbor-wide distribution of proteins associated with post-synaptic receptors 35 . Combining paired recording with array tomography enabled researchers to study the interrelation between functional plasticity and molecular composition of synapses 36 . The positions of synapses have been detected with high spatial accuracy using mGRASP 37 .…”

Array tomography: 15 years of synaptic analysis

“…Some groups have combined the study of form and function by performing AT on physiologically characterised synapses [ 80–82 ]. They achieved this by filling neurons with a dye via their recording electrode, and then embedding the tissue in resin before performing AT.…”