A Transgenic Mouse Model to Selectively Identify α3 Na,K-ATPase Expressing Cells in the Nervous System

Abstract

The α Na,K-ATPase (αNKA) is one of four known α isoforms of the mammalian transporter. A deficiency in αNKA is linked to severe movement control disorders. Understanding the pathogenesis of these disorders is limited by an incomplete knowledge of αNKA expression in the brain as well as the challenges associated with identifying living cells that express the isoform for subsequent electrophysiological studies. To address this problem, transgenic mice were generated on the C57BL/6 genetic background, which utilize the mouse α subunit gene (Atp1a3) promoter to drive the expression of ZsGreen1 fluorescent protein. Consistent with published results on αNKA distribution, a ZsGreen1 signal was detected in the brain, but not in the liver, with Atp1a3-ZsGreen1 transgenic mice. The intensity of ZsGreen1 fluorescence in neuronal cell bodies varied considerably in the brain, being highest in the brainstem, deep cerebellar and select thalamic nuclei, and relatively weak in cortical regions. Fluorescence was not detected in astrocytes or white matter areas. ZsGreen1-positive neurons were readily observed in fresh (unfixed) brain sections, which were amenable to patch-clamp recordings. Thus, the αNKA-ZsGreen1 mouse model provides a powerful tool for studying the distribution and functional properties of αNKA-expressing neurons in the brain.

Clock Scan Protocol for Image Analysis: ImageJ Plugins

“…The images that are used here for illustration purpose, are taken from databases created during our previous cell and tissue biological studies 5,6,7 and from the Allen Mouse Brain Atlas 8…”

“…(D)Average clock-scan profile for selected ROIs (panel A) with SD bars (option "plot with standard deviation" selected). (E) RGB image of cultured mouse preBI-lymphocytes, labeled with 4,6-Diamidino-2-phenylindole (DAPI, nuclear stain, blue) and with fluorescently-labeled antibodies for β1-integrin (green) and F-actin (red; see Dobretsov et al7 for the cell culture technique and Yuryev et al11 for staining details). Eleven cells (see number labels) were outlined using the ImageJ polygon selection tool.…”

Alternating Hemiplegia of Childhood: gastrointestinal manifestations and correlation with neurological impairments

“…On the other hand, they may be, at least in part, related to the specific underlying AHC pathophysiology. Recent investigations have revealed high concentrations of the α3 Na, K-ATPase subunit in all hypothalamic nuclei [ 8 , 9 ]. The hypothalamus projects to the lateral medulla and regulates sympathetic and parasympathetic pathway functions [ 19 ].…”

“…Interestingly, however, we have commonly observed gastrointestinal problems in AHC patients being followed in our Duke Multidisciplinary AHC Clinic. In addition, ATP1A3 is highly expressed in brain areas that control the autonomic nervous system and consequently gastrointestinal motility, such as the hypothalamic and vagus nerve nuclei [ 8 , 9 ]. This gene is also expressed in motor brain stem nuclei that control swallowing and in GABAergic interneurons that can modulate motility [ 10 ].…”

Comparative description of themRNAexpression profile of Na+/K+‐ATPaseisoforms in adult mouse nervous system

“…Four α subunit isoforms are expressed in mammalian cells in a spatiotemporally regulated manner (Blanco & Mercer, 1998; Clausen et al, 2017; Lingrel et al, 1990). Within the nervous system, the α1 isoform is ubiquitously expressed, while α2 and α3 isoforms are predominantly observed in glia and neurons, respectively (Bottger et al, 2011; Cameron et al, 1994; Dobretsov et al, 2019; Edwards et al, 2013; Murata et al, 2020; Richards et al, 2007; Sweadner, 1991). The α4 isoform expresses exclusively in testis and is essential for sperm motility and fertility (Jimenez, McDermott, Sanchez, & Blanco, 2011a; Jimenez, Sanchez, et al, 2011; Shamraj & Lingrel, 1994).…”