Merits and Limitations of Studying Neuronal Depolarization-Dependent Processes Using Elevated External Potassium

Abstract

Elevated extracellular potassium chloride is widely used to achieve membrane depolarization of cultured neurons. This technique has illuminated mechanisms of calcium influx through L-type voltage sensitive calcium channels, activity-regulated signaling, downstream transcriptional events, and many other intracellular responses to depolarization. However, there is enormous variability in these treatments, including durations from seconds to days and concentrations from 3mM to 150 mM KCl. Differential effects of these variable protocols on neuronal activity and transcriptional programs are underexplored. Furthermore, potassium chloride treatments in vitro are criticized for being poor representatives of in vivo phenomena and are questioned for their effects on cell viability. In this review, we discuss the intracellular consequences of elevated extracellular potassium chloride treatment in vitro, the variability of such treatments in the literature, the strengths and limitations of this tool, and relevance of these studies to brain functions and dysfunctions.

The Perils of Navigating Activity-Dependent Alternative Splicing of Neurexins

“…Treatment of cultured neurons with 8 mM KCl retains spontaneous neuronal activity, but shifts the pattern of that activity from burst to tonic spike firing (Golbs et al, 2011 ). Treatment of cultured neurons with KCl above 10 mM conversely diminishes spontaneous activity (Grubb and Burrone, 2010 ; Rienecker et al, 2020 ), suggesting that persistent neuronal depolarization does not necessarily induce neuronal stimulation. Furthermore, persistent neuronal depolarization produces sustained increases in intracellular calcium, a key second messenger for numerous cellular processes, including transcription and alternative splicing (Collins et al, 1991 ; Grubb and Burrone, 2010 ; Rienecker et al, 2020 ).…”

“…Treatment of cultured neurons with KCl above 10 mM conversely diminishes spontaneous activity (Grubb and Burrone, 2010 ; Rienecker et al, 2020 ), suggesting that persistent neuronal depolarization does not necessarily induce neuronal stimulation. Furthermore, persistent neuronal depolarization produces sustained increases in intracellular calcium, a key second messenger for numerous cellular processes, including transcription and alternative splicing (Collins et al, 1991 ; Grubb and Burrone, 2010 ; Rienecker et al, 2020 ). In addition, neurons die at concentrations of >50 mm KCl (Collins and Lile, 1989 ).…”

“…In the light of this relationship of KCl to neuronal survival, the utility of the persistent depolarization paradigm as an “activity-inducing system” to study alternative splicing may need re-evaluation. While there are clear merits of using elevated extracellular KCl (extensively reviewed in Rienecker et al, 2020 ), the inherent confounds and limitations of this approach in studying complex and heterogeneous splice isoforms could outweigh its benefits. The results of our KCl treatment experiments and RNA-seq analyses exemplify and highlight potential disadvantage of this approach.…”

“…However, the various papers report quite variable results and use diverse treatment conditions. Moreover, KCl and KA treatment are known to be neurotoxic (Sun et al, 1992 ; Cheng and Sun, 1994 ; Pollard et al, 1994 ; Kasof et al, 1995 ; Simonian et al, 1996 ; Cheung et al, 1998 ; Takahashi et al, 1999 ; Gluck et al, 2000 ; Milatovic et al, 2002 ; Le Duigou et al, 2005 ; Wang et al, 2005 ; Rienecker et al, 2020 ), thus raising the questions of validity. To address these issues, we here re-evaluated the activity-mediated regulation of Nrxn1 SS4 alternative splicing using simple in vitro and in vivo paradigms that replicate those employed by Iijima et al ( 2011 ) and Ding et al ( 2017 ), and we extended these experiments to Nrxn2 and Nrxn3 SS4 splice forms.…”

Transmission-selective muscle pathology induced by active propagation of mutant huntingtin across the human neuromuscular synapse

“…This impairs release of synaptic vesicles content from the presynaptic membrane. On the contrary, depolarization of neurons results in increased AP firing which triggers synaptic vesicle release 39 . To test whether an increase in neuronal activity effects Q72-mCherry NM transmission, we depolarized the Neu Q72-mCherry Clone#72 by exposing them for 10 minutes to an artificial cerebrospinal fluid (ACSF) solution with high KCl concentration (10mM) and kept them after this acute treatment for 2 hours in ACSF with 2.5mM KCl (Fig.…”