A Versatile Method for Viral Transfection of Calcium Indicators in the Neonatal Mouse Brain

Abstract

The first three postnatal weeks in rodents are a time when sensory experience drives the maturation of brain circuits, an important process that is not yet well understood. Alterations in this critical period of experience-dependent circuit assembly and plasticity contribute to several neurodevelopmental disorders, such as autism, epilepsy, and schizophrenia. Therefore, techniques for recording network activity and tracing neuronal connectivity over this time period are necessary for delineating circuit refinement in typical development and how it deviates in disease. Calcium imaging with GCaMP6 and other genetically encoded indicators is rapidly becoming the preferred method for recording network activity at the single-synapse and single-cell level in vivo, especially in genetically identified neuronal populations. We describe a protocol for intracortical injection of recombinant adeno-associated viruses in P1 neonatal mice and demonstrate its use for longitudinal imaging of GCaMP6s in the same neurons over several weeks to characterize the developmental desynchronization of cortical network activity. Our approach is ideally suited for chronic in vivo two-photon calcium imaging of neuronal activity from synapses to entire networks during the early postnatal period.

EZcalcium: Open-Source Toolbox for Analysis of Calcium Imaging Data

“…scientific community. Aspects of certain modules of the EZcalcium toolbox have already been successfully implemented in several published studies (He et al, 2017(He et al, , 2018Goel et al, 2018), and the toolbox is freely available on GitHub 2 .…”

“…In particular, calcium imaging with two-photon microscopy (Svoboda and Yasuda, 2006;Mostany et al, 2015) and microendoscopy with miniscopes or fiber photometry (Ghosh et al, 2011;Liberti et al, 2017;Jacob et al, 2018;Aharoni et al, 2019;Zhang et al, 2019) have provided key insights, such as how developing cortical networks undergo drastic transitions (Golshani et al, 2009;Rochefort et al, 2009) or how large neuronal ensembles encode spatial navigation (Dombeck et al, 2010). Calcium imaging offers several distinct advantages over traditional electrode recording techniques (Grewe and Helmchen, 2009;Grienberger and Konnerth, 2012): (1) it can be combined with genetic approaches (e.g., Cre-Lox) to probe neuronal activity in specific sub-populations of neurons (Goel et al, 2018;Yaeger et al, 2019) and glia (Srinivasan et al, 2016), either in specific subcellular compartments (e.g., axon boutons, dendritic spines, glial microdomains; Cichon and Gan, 2015;Broussard et al, 2018) or in specific brain regions or cortical layers (Lacefield et al, 2019); (2) recordings can be carried out over periods of days or even weeks (Chen et al, 2013;He et al, 2018); (3) recordings can be made simultaneously in a large population of hundreds or thousands of neurons in multiple brain regions (e.g., an entire sub-network; Sofroniew et al, 2016); (4) calcium imaging can also be combined with optogenetic manipulations, which makes it possible to perform all-optical probing of circuit function (Packer et al, 2015); (5) recordings can be performed in freely moving animals, providing a key link between circuit activity and behavior (Lin and Schnitzer, 2016); and (6) calcium imaging is less invasive than traditional electrode recordings (e.g., tetrodes, silicon probes). Another advantage the technique offers is the ability to precisely map the relative spatial location of groups of neurons.…”

EZcalcium: Open Source Toolbox for Analysis of Calcium Imaging Data

“…The goal of this project was to develop an open source toolbox for analysis of calcium imaging data that could be freely disseminated across the broader scientific community. Aspects of certain modules of the EZcalcium toolbox have already been successfully implemented in several published studies He et al, 2018;He et al, 2017) and the toolbox is freely available on GitHub (https://github.com/porteralab/EZcalcium).…”

“…In particular, calcium imaging with 2-photon microscopy (Mostany et al, 2015;Svoboda and Yasuda, 2006) and micro-endoscopy with miniscopes or fiber photometry (Aharoni et al, 2019;Ghosh et al, 2011;Jacob et al, 2018;Liberti et al, 2017;Zhang et al, 2019) have provided key insights, such as how developing cortical networks undergo drastic transitions (Golshani et al, 2009;Rochefort et al, 2009) or how large neuronal ensembles encode spatial navigation (Dombeck et al, 2010). Calcium imaging offers several distinct advantages over traditional electrode recording techniques (Grewe and Helmchen, 2009;Grienberger and Konnerth, 2012): 1) it can be combined with genetic approaches (e.g., Cre-Lox) to probe neuronal activity in specific sub-populations of neurons Yaeger et al, 2019) and glia (Srinivasan et al, 2016), either in specific subcellular compartments (e.g., axon boutons, dendritic spines, glial microdomains) (Broussard et al, 2018;Cichon and Gan, 2015) or in specific brain regions or cortical layers (Lacefield et al, 2019); 2) recordings can be carried out over periods of days or even weeks He et al, 2018); 3) recordings can be made simultaneously in a large population of hundreds or thousands of neurons in multiple brain regions (e.g., an entire sub-network) (Sofroniew et al, 2016); 4) calcium imaging can also be combined with optogenetic manipulations, which makes it possible to perform all-optical probing of circuit function (Packer et al, 2015); 5) recordings can be performed in freely moving animals, providing a key link between circuit activity and behavior (Lin and Schnitzer, 2016); and 6) calcium imaging is less invasive than traditional electrode recordings (e.g., tetrodes, silicon probes). Another advantage the technique offers is the ability to precisely map the relative spatial location of groups of neurons.…”

Alternative Anesthesia of Neonatal Mice for Global rAAV Delivery in the Brain With Non-detectable Behavioral Interference in Adults

“…Classical virus-mediated gene transfer approaches into pre-selected brain regions are mostly limited to adult animals via stereotactic injections (Cetin et al, 2007 ). For a global expression of transduced genes in the mouse brain, a neonatal rAAV delivery method was introduced and used successfully (Passini and Wolfe, 2001 ; Pilpel et al, 2009 ; Chakrabarty et al, 2013 ; Kim et al, 2013 , 2014 ; McLean et al, 2014 ; Ayers et al, 2015 ; He et al, 2018 ). As alternatives to this global, neonatal rAAV gene transduction, in utero electroporation (Saito, 2006 ; Huang and Carcagno, 2018 ), or systemic injection of rAAV into the tail vein of adult or adolescent mice (Foust et al, 2009 ; Stoica et al, 2013 ; Körbelin et al, 2016 ; Thomsen et al, 2017 ) achieved similar expression of transfected or transduced genes in the mouse brain.…”