Abstract
The potassium chloride cotransporter 2 (KCC2) is the main Cl− extruder in neurons. Any alteration in KCC2 levels leads to changes in Cl− homeostasis and, consequently, in the polarity and amplitude of inhibitory synaptic potentials mediated by GABA or glycine. Axotomy downregulates KCC2 in many different motoneurons and it is suspected that interruption of muscle-derived factors maintaining motoneuron KCC2 expression is in part responsible. In here, we demonstrate that KCC2 is expressed in all oculomotor nuclei of cat and rat, but while trochlear and oculomotor motoneurons downregulate KCC2 after axotomy, expression is unaltered in abducens motoneurons. Exogenous application of vascular endothelial growth factor (VEGF), a neurotrophic factor expressed in muscle, upregulated KCC2 in axotomized abducens motoneurons above control levels. In parallel, a physiological study using cats chronically implanted with electrodes for recording abducens motoneurons in awake animals, demonstrated that inhibitory inputs related to off-fixations and off-directed saccades in VEGF-treated axotomized abducens motoneurons were significantly higher than in control, but eye-related excitatory signals in the on direction were unchanged. This is the first report of lack of KCC2 regulation in a motoneuron type after injury, proposing a role for VEGF in KCC2 regulation and demonstrating the link between KCC2 and synaptic inhibition in awake, behaving animals.
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Funding
This work was funded by NIH (NINDS) R01 NS111969 and R21 NS114839 to F.J.A. This publication is also part of the I + D + i project P20_00529 Consejería de Transformación Económica Industria y Conocimiento, Junta de Andalucía-FEDER. Research materials were also supported by project PGC2018-094654-B-100 and PID2021-124300NB-I00 both funded by MCIN/AEI/FEDER “A way of making Europe” to A.M.P and RRC. P.M.C. was a scholar of Ministerio de Educación y Ciencia (BES-2016–077912) in Spain and now a “Margarita Salas” postdoctoral fellow from Spain at Emory University Atlanta, USA.
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The experiments were designed by FJA and AMP. Immunocytochemistry and image analysis were carried out by PMC. Electrophysiological experiments and analysis were carried out by PMC, AMP and RRC. The manuscript was written by FJA, RRC and AMP. All authors have revised and accept the final version of the manuscript.
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All animal procedures were performed at the University of Seville (Spain) and in accordance with the guidelines of the European Union (2010/63/EU) and Spanish legislation (R.D. 53/2013, BOE 34/11370-421) for the use and care of laboratory animals. They were approved by the local ethics committee (Protocol #04/11/15/349). Animal procedures also followed NIH guidelines and legislation in the US. No animal experimentation was performed in the US for this project. Work at the US (Emory University) consisted in immunocytochemical and morphological analyses of tissues collected at University of Seville in Spain. All efforts were made to reduce the number of animals used and their suffering during the present experiments and, in fact, some of the material derives from the tissue bank of previously published studies (Calvo et al. 2018, 2020).
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Calvo, P.M., de la Cruz, R.R., Pastor, A.M. et al. Preservation of KCC2 expression in axotomized abducens motoneurons and its enhancement by VEGF. Brain Struct Funct 228, 967–984 (2023). https://doi.org/10.1007/s00429-023-02635-w
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DOI: https://doi.org/10.1007/s00429-023-02635-w