In situ labeling of non-accommodating interneurons based on metabolic rates

G C Gotti; M Kikhia; V Wuntke; L A Hasam-Henderson; B Wu; J R P Geiger; R Kovacs

doi:10.1016/j.redox.2020.101798

In situ labeling of non-accommodating interneurons based on metabolic rates

Redox Biol. 2021 Jan:38:101798. doi: 10.1016/j.redox.2020.101798. Epub 2020 Nov 28.

Authors

G C Gotti¹, M Kikhia¹, V Wuntke¹, L A Hasam-Henderson¹, B Wu², J R P Geiger¹, R Kovacs³

Affiliations

¹ Institut für Neurophysiologie, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, NeuroCure Cluster of Excellence, Berlin, Charité Platz 1, 10117, Berlin, Germany.
² Institut für Neurophysiologie, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, NeuroCure Cluster of Excellence, Berlin, Charité Platz 1, 10117, Berlin, Germany; Institute of Neuroinformatics, University of Zurich - Irchel, Winterthurerstrasse 190, 8057, Zürich, Switzerland.
³ Institut für Neurophysiologie, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, NeuroCure Cluster of Excellence, Berlin, Charité Platz 1, 10117, Berlin, Germany. Electronic address: richard.kovacs@charite.de.

Abstract

Maintaining high frequency firing of narrow action potentials puts a large metabolic load on fast spiking (FS), perisomatic-inhibitory interneurons compared to their slow-spiking, dendrite targeting counterparts. Although the relationship of action potential (AP) firing and metabolism is firmly established, there is no single method to differentiate interneurons in situ based on their firing properties. In this study, we explore a novel strategy to easily identify the metabolically active FS cells among different classes of interneurons. We found that the oxidation of the fluorescent free radical marker 2,7-dichlorodihydrofluorescein (H₂DCF) preferentially occurs in interneurons both in slice cultures and acute brain slices. Despite their morphological heterogeneity, almost all DCF-positive (DCF+) neurons belonged to the cluster of non-accommodating FS interneurons. Furthermore, all FS interneurons expressing parvalbumin (PV) both in slice cultures and in acute slices from tdTomato-PVCre transgenic mice were also DCF+. However, only half of the recorded DCF + cells were also PV+, indicating that H₂DCF-oxidation occurs in different interneuron classes characterized by non-accomodating AP-firing. Comprehensively enhancing spontaneous neuronal activity led to mitochondrial oxidation of DCF in pyramidal cells as well as interneurons, suggesting that the apparent selectivity towards interneurons represents differences in the underlying metabolic load. While radical-scavenging, inhibition of APs or NO-synthesis, and iron chelation had no effect on the staining pattern, exposure to the complex-I inhibitor, rotenone, prevented interneuronal DCF accumulation. We conclude that H₂DCF oxidation is independent of free radicals but correlates with the intensive oxidative energy metabolism and high mitochondrial mass in interneurons sharing the non-accommodating FS phenotype.

Keywords: 2',7'-Dichlorodihydrofluorescein; Energy metabolism; Fast spiking interneuron; Neurometabolic coupling; Seizure; VGAT-YFP.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Action Potentials
Animals
Interneurons*
Mice
Mice, Transgenic
Parvalbumins*
Pyramidal Cells

Substances

Parvalbumins