Biocytin-labelling and its impact on late 20th century studies of cortical circuitry

Abstract

In recognition of the impact that a powerful new anatomical tool, such as the Golgi method, can have, this essay highlights the enormous influence that biocytin-filling has had on modern neuroscience. This method has allowed neurones that have been recorded intracellularly, 'wholecel' or juxta-cellularly, to be identified anatomically, forming a vital link between functional and structural studies. It has been applied throughout the nervous system and has become a fundamental component of our technical armoury. A comprehensive survey of the applications to which the biocytin-filling approach has been put, would fill a large volume. This essay therefore focuses on one area, neocortical microcircuitry and the ways in which combining physiology and anatomy have revealed rules that help us the explain its previously indecipherable variability and complexity.

Statistical analysis and data mining of digital reconstructions of dendritic morphologies

“…For example, the separate clustering of different mouse S1 pyramidal cell datasets can be explained by the differences between intracellular biocytin injection (e.g., Yuste's archive) and bulk Golgi staining (e.g., Brumberg's archive). While the mechanisms underlying the different visualization by these techniques are not yet fully understood (Thomson and Armstrong, 2011 ), the histological labeling information is available as metadata in NeuroMorpho.Org, thus aiding interpretation.…”

Fast neuronal labeling in live tissue using a biocytin conjugated fluorescent probe

“…Long-range transport of biocytin has been used to trace neuronal tracts under in vivo or in vitro conditions (Kobbert et al, 2000;Thomson and Armstrong, 2011). Uptake can be facilitated by coinjection of NMDA or KCL (Chang et al, 2000;Jiang et al, 1993;Tarras-Wahlberg and Rekling, 2009;Zheng et al, 1998), but it is not entirely clear how extracellular deposited biocytin is taken up, nor have the mechanisms responsible for active transport in tracts been identified.…”

“…It might have specific affinity to molecules in the transport machinery, or piggyback on vesicles or other transported components. The main advantage of biocytin as a neuronal tracer lies in the comprehensive intracellular distribution of the molecule, which in some cases can fill neurons in their entirety, and the ease with which it can be detected in histochemical reactions based on the high-affinity binding to avidin (Thomson and Armstrong, 2011). Thus, iontophoretic, pressure, or pellet-based extracellular deposition of solutions containing 2-10% biocytin lead to antero-and retrograde labeling of nearby neurons under in vivo or in vitro conditions (Chang et al, 2000;Kenan-Vaknin et al, 1992;King et al, 1989;Sorensen et al, 1993).…”

“…This effective transport machinery has been utilized in numerous neuroanatomical studies where antero-and retrograde tracers such as horseradish peroxidase (HRP), choleratoxin subunit b (CTN), Fluoro-Gold (FG), Mini Ruby (MR), Phaseolus vulgaris leucoagglutinin (PHAL), fluorescently tagged or biotinylated dextran amines, and others have been used to label pathways and neurons (Kita and Armstrong, 1991;Kobbert et al, 2000;Lanciego and Wouterlood, 2011;Pinault, 1996),. Notably, biocytin, a low molecular weight conjugate of biotin and lysine, has found multiple uses as an antero-and retrograde neuroanatomical tracer, and in labeling individual neurons using intracellular, whole-cell patch or juxtacellular techniques (Kobbert et al, 2000;Thomson and Armstrong, 2011).…”

Minimizing shrinkage of acute brain slices using metal spacers during histological embedding

“…The morphology of neurons provides the structural framework for their functions, including the integration of synaptic inputs and the generation of action potentials (Kasper et al 1994 ; Norenberg et al 2010 ; DeFelipe et al 2013 ; de Sousa et al 2015 ; Gulledge and Bravo, 2016 ; Mihaljevic et al 2018 ). Indeed, from Cajal’s morphological studies of Golgi stained neurons (Ramón y Cajal 1909 , 1911) up to today’s high-resolution confocal images of genetically identified and biocytin stained neurons (Bartos et al 2002 ; Thomson and Armstrong 2011 ; Booker et al 2014 ), anatomical investigations offered important insights for the understanding of the physiological and circuit functions of neurons and also provided essential data for computational analysis (Traub et al 1994 ; Major et al 1994 ; Norenberg et al 2010 ; Gidon et al 2020 ). While identification, labeling and imaging of neurons have shown substantial advances in recent decades, a major technical problem of morphological analysis remains: the shrinkage of tissue during histological processing and embedding.…”