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Post hoc immunostaining of GABAergic neuronal subtypes following in vivo two-photon calcium imaging in mouse neocortex


Langer, D; Helmchen, F (2012). Post hoc immunostaining of GABAergic neuronal subtypes following in vivo two-photon calcium imaging in mouse neocortex. Pflügers Archiv: European Journal of Physiology (Pflugers Archiv), 463(2):339-354.

Abstract

GABAergic neurons in the neocortex are diverse with regard to morphology, physiology, and axonal targeting pattern, indicating functional specializations within the cortical microcircuitry. Little information is available, however, about functional properties of distinct subtypes of GABAergic neurons in the intact brain. Here, we combined in vivo two-photon calcium imaging in supragranular layers of the mouse neocortex with post hoc immunohistochemistry against the three calcium-binding proteins parvalbumin, calretinin, and calbindin in order to assign subtype marker profiles to neuronal activity. Following coronal sectioning of fixed brains, we matched cells in corresponding volumes of image stacks acquired in vivo and in fixed brain slices. In GAD67-GFP mice, more than 95% of the GABAergic cells could be unambiguously matched, even in large volumes comprising more than a thousand interneurons. Triple immunostaining revealed a depth-dependent distribution of interneuron subtypes with increasing abundance of PV-positive neurons with depth. Most importantly, the triple-labeling approach was compatible with previous in vivo calcium imaging following bulk loading of Oregon Green 488 BAPTA-1, which allowed us to classify spontaneous calcium transients recorded in vivo according to the neurochemically defined GABAergic subtypes. Moreover, we demonstrate that post hoc immunostaining can also be applied to wild-type mice expressing the genetically encoded calcium indicator Yellow Cameleon 3.60 in cortical neurons. Our approach is a general and flexible method to distinguish GABAergic subtypes in cell populations previously imaged in the living animal. It should thus facilitate dissecting the functional roles of these subtypes in neural circuitry.

Abstract

GABAergic neurons in the neocortex are diverse with regard to morphology, physiology, and axonal targeting pattern, indicating functional specializations within the cortical microcircuitry. Little information is available, however, about functional properties of distinct subtypes of GABAergic neurons in the intact brain. Here, we combined in vivo two-photon calcium imaging in supragranular layers of the mouse neocortex with post hoc immunohistochemistry against the three calcium-binding proteins parvalbumin, calretinin, and calbindin in order to assign subtype marker profiles to neuronal activity. Following coronal sectioning of fixed brains, we matched cells in corresponding volumes of image stacks acquired in vivo and in fixed brain slices. In GAD67-GFP mice, more than 95% of the GABAergic cells could be unambiguously matched, even in large volumes comprising more than a thousand interneurons. Triple immunostaining revealed a depth-dependent distribution of interneuron subtypes with increasing abundance of PV-positive neurons with depth. Most importantly, the triple-labeling approach was compatible with previous in vivo calcium imaging following bulk loading of Oregon Green 488 BAPTA-1, which allowed us to classify spontaneous calcium transients recorded in vivo according to the neurochemically defined GABAergic subtypes. Moreover, we demonstrate that post hoc immunostaining can also be applied to wild-type mice expressing the genetically encoded calcium indicator Yellow Cameleon 3.60 in cortical neurons. Our approach is a general and flexible method to distinguish GABAergic subtypes in cell populations previously imaged in the living animal. It should thus facilitate dissecting the functional roles of these subtypes in neural circuitry.

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Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Brain Research Institute
Special Collections > SystemsX.ch
Special Collections > SystemsX.ch > Research, Technology and Development Projects > Neurochoice
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2012
Deposited On:17 Jan 2012 15:58
Last Modified:10 Aug 2017 12:59
Publisher:Springer
ISSN:0031-6768
Funders:Roche Research Foundation, Swiss National Science Foundation (SNSF; grant 310030–127091), German-Swiss Research Group “Barrel Cortex Function” (FOR1341, SNSF 310030–130826), Swiss SystemsX.ch initiative (project 2008/2011-Neurochoice)
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1007/s00424-011-1048-9
PubMed ID:22134770

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