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.

Keywords

L-type voltage sensitive calcium channels immediate early genes (IEG)transcription intracellular calcium extracellular potassium

Introduction

Our understanding of the complex molecular mechanisms of brain development, physiology, and disorder are greatly aided by simple model systems, including dissociated neuronal cell culture. Here, immature neurons obtained from embryonic brains are cultured into mature neurons in vitro. Such dissociated cells are extensively used in neuroscience and are particularly useful in the study of neuronal excitation-transcription coupling. In vitro, elevated extracellular potassium is often employed to depolarize the neuronal membrane (Greenberg et al., 1985; Bartel et al., 1989; Gotoh et al., 1999; Michod et al., 2012) and induce Ca²⁺ influx through L-type voltage sensitive calcium channels (L-type VSCCs) (Ghosh et al., 1994; Xia et al., 1996; Tao et al., 1998; Martinowich et al., 2003; Greer and Greenberg, 2008; Malik et al., 2014; Qiu et al., 2016; Azad et al., 2018; Tyssowski et al., 2018), consequently initiating signaling cascades (Bading et al., 1993; Dolmetsch et al., 2001; Redmond et al., 2002; Kingsbury et al., 2007; Greer and Greenberg, 2008; Li et al., 2009; Lyons and West, 2011; Pruunsild et al., 2011; Evans et al., 2013; Cohen et al., 2018; Tyssowski et al., 2018) that result in activity responsive gene transcription (West et al., 2002; Martinowich et al., 2003; Greer and Greenberg, 2008; Kim et al., 2010; Pruunsild et al., 2011; Michod et al., 2012; Malik et al., 2014; Madabhushi et al., 2015; Azad et al., 2018; Tyssowski et al., 2018). In this review, we provide a general overview of these processes. We also discuss strengths and weaknesses of experimental approaches using elevated extracellular KCl (K⁺), their relevance to certain brain disorders, and offer suggestions regarding their possible use in other neurobiology subfields.

Mechanisms at Play

Elevated Extracellular KCl Induces Neuronal Depolarization, But Often, Not Activity

The neuronal membrane voltage is regulated by, among other factors, the concentration of potassium and sodium ions in the intracellular versus extracellular space. At rest, K⁺ is more concentrated inside the neuron, while Na⁺ concentration is higher outside the cell. Sodium-potassium pumps maintain these gradients, using ATP to pump Na⁺ out of the cell and K⁺ into the cell, against their gradients. Neurons at rest in vivo have a membrane voltage around -65 mV, whereas in vitro, they tend to be around -60 mV due to slightly higher KCl concentrations in commercial culture media formulations (5mM) in comparison to cerebrospinal fluid (3mM). Because the resting neuronal membrane is highly permeable to K⁺, the membrane potential is sensitive to changes in the extracellular potassium concentration – increasing extracellular potassium depolarizes neurons. As the extracellular concentration of potassium rises, the magnitude of the potassium gradient is reduced, and the potassium equilibrium potential becomes more positive. As external Na⁺ continues to leak into the soma, global depolarization occurs. As calculated by the Goldman equation, a tenfold change in extracellular K⁺ from 5 to 50mM will cause a 48mV membrane depolarization under physiological conditions. Experimentally, reminiscent of the Nernst relationship, elevation of external K⁺ to 20, 30, and 40mM concentration in one study depolarized the membrane to -37, -26, and -19mV respectively; the specific membrane potential achieved at 10 mM KCl was not reported in text, but was approximately -56mV in Figure 2B (Wheeler et al., 2008).

What happens to neuronal activity post membrane depolarization in response to elevated external potassium? When treated with 8mM KCl, spontaneous activity is retained in cortical neurons, but the pattern changes from burst firing to tonic spike firing (Golbs et al., 2011). However, application of 15mM KCl or higher, somewhat unintuitively, leads to a complete attenuation of spontaneous neuronal activity in dissociated hippocampal neurons (Grubb and Burrone, 2010). In our laboratory, we see similar activity arrest in dissociated cortical neurons, when treated with 10mM or higher concentrations of external K⁺ (Figure 1). Such obliteration of neuronal activity may be explained by depolarization-sensitive inactivation of voltage-gated channels. Alternatively, or in addition, mild depolarization could deplete pre-synaptic terminals of neurotransmitters, thereby preventing subsequent rounds of activity (Grubb and Burrone, 2010). Although 10mM or higher external K⁺-induced depolarization doesn’t produce neuronal activity, the sustained depolarization produces a chronic increase in an important second messenger, intracellular calcium (more in the next segment, 2.2) (Collins et al., 1991; Grubb and Burrone, 2010). Also, such sustained depolarization-coupled inactivation of neuronal activity may have notable functional repercussions in the brain (see segment 4.3.3).

Figure 1.

Neuronal Activity Under Elevated Extracellular KCl In Vitro. A: Spikes under KCl treatment as a % of baseline activity before treatment. Spiking is observed under 5mM KCl treatment, which matches [KCl] in standard media conditions, but disappears as [KCl]_o rises. Spike data were compared using ANOVA. *** P < 0.001. B: Example recordings during baseline measurements (top) and KCl treatments (bottom).

Calcium Influx

Both brief and prolonged stimulation with KCl elevate intracellular calcium in a sustained fashion for the duration of treatment (Dolmetsch et al., 2001; Kingsbury et al., 2007; Evans et al., 2013; Tyssowski et al., 2018). When the calcium chelator EGTA is included during KCl treatment, it blocks the increase in intracellular calcium, induction of BDNF mRNA, dendritic growth, and enhanced survival brought about by KCl (Zafra et al., 1990; Ghosh et al., 1994; Tao et al., 1998; Redmond et al., 2002; Tao-Cheng, 2018). In some cases, these changes are likewise prevented by reduction of the calcium concentration in the culture medium (Zafra et al., 1990). Together, these observations suggest that most of the biological effects of elevated external potassium are mediated by intracellular calcium signaling. Notably, in contrast to the localized depolarization that comes with synaptic stimulation, KCl administration induces global depolarization. Depolarization-induced calcium influx to the soma is possible through several different channels, including NMDA receptors, calcium permeable AMPA receptors, various voltage-sensitive calcium channels (VSCCs), and intracellular Ca²⁺ stores in the endoplasmic reticulum (Lyons and West, 2011). NMDA receptors are found at the synapse, but VSCCs are found globally across the plasma membrane–both at the synapse and at the soma. Studies with L-type Ca²⁺ channel antagonists like the dihydropyridines (DHPs) nimodipine and nifedipine show that L-type VSCCs mediate much of the effect of membrane depolarization by elevated extracellular KCl. Nimodipene is reported to attenuate KCl-induced Ca²⁺ influx by 15-20% (Dolmetsch et al., 2001) and nifedipine by 30-50% (Redmond et al., 2002). However, N- and P/Q-type channel blockers and NMDA receptor blockers can also reduce intracellular calcium concentrations induced by membrane depolarizations by 15-20% (Dolmetsch et al., 2001). Together, L-type VSCCs, N- and P/Q-type channels, and NMDARs each contribute to sustained intracellular calcium elevation in the cytoplasm and nucleus during potassium mediated depolarization (Dolmetsch et al., 2001; Lyons and West, 2011). It is notable that although Ca²⁺ influx is an absolute requirement for depolarization-induced downstream signaling, reduction of Ca²⁺ influx only marginally impairs signaling, suggesting the signaling strength is non-linearly coupled with intracellular calcium levels.

One of the key downstream mediators of calcium influx is the transcription factor CREB. CREB-dependent transcription signaled by Ca²⁺-CaMKII activation relies on the frequency of L-type channel openings, rather than bulk intracellular Ca²⁺ levels (Wheeler et al., 2008). Furthermore, N- and P/Q-type channel inhibitors (ω-Conotoxin GVIA and ω-Agatoxin IVA respectively), are unable to inhibit CREB activation and CREB-dependent transcription across various extracellular Ca²⁺ concentrations. In contrast, the L-type channel inhibitor nimodipine inhibits CREB Ser¹³³ phosphorylation at concentrations of extracellular Ca²⁺ up to 20 mM (Dolmetsch et al., 2001). For these reasons, Ca²⁺ influx through the L-type channels is thought to be primarily responsible for Ca²⁺-dependent signaling and CREB-dependent transcription induced by elevated extracellular potassium. The majority of this review will focus on signaling from L-type VSCCs.

L-Type VSCCs

Although multiple channels contribute to depolarization-coupled Ca²⁺ influx, L-type (long-lasting activation) channels provide specificity to the effects of elevated extracellular KCl in comparison to other stimulation paradigms (Ghosh et al., 1994; Lyons and West, 2011; Lyons et al., 2016). Induction of BDNF expression by KCl is completely blocked by L-type channel blockers nifedipine and nimodipine (Zafra et al., 1990; Ghosh et al., 1994; Lyons et al., 2016), but is not affected by the NMDAR blocker APV (D-2-amino-5-phosphonovaleric acid) or by CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), an AMPAR/kainate (aka, non-NMDAR) blocker (Ghosh et al., 1994). In contrast, BDNF induced by glutamate is sensitive to APV, but not nifedipine or CNQX (Ghosh et al., 1994). Differential regulation and expression profiles of BDNF induced by signaling through NMDARs and VSCCs may explain the observation that calcium influx through NMDARs is more excitotoxic than calcium influx through L-type VSCCs (Ghosh et al., 1994). VSCCs sustain BDNF expression longer than NMDAR signaling. While calcium influx through either channel may be potentially excitotoxic, robust BDNF induction after VSCC activation may be an effective neuroprotectant, in contrast to the transient and weaker BNDF induction by NMDAR activation (Ghosh et al., 1994). More information about L-type VSCCs may be found in Lipscombe et al. (2004), Berger and Bartsch (2014), and Kobrinsky (2015).

Signaling to the Nucleus

Once Ca²⁺ influx occurs through L-type VSCCs, signaling cascades transmit this information to the nucleus. On certain occasions, information is postulated to be directly relayed by C-terminus fragments of L-type VSCCs (Gomez-Ospina et al., 2006). However, in many instances, the L-type channel activity is locally computed by Ca²⁺/CaM-dependent protein kinase II (CaMKII; see “Calmodulin Kinase (CaMKII/CaMKIV)” section), which then conveys the signal to downstream pathways. The principal cascades include the calmodulin kinase (CaMK) (Bading et al., 1993; Cohen et al., 2018), mitogen activated protein kinase (MAPK) (Ghosh and Greenberg, 1995; Tyssowski et al., 2018), and calcineurin pathways (Figure 2) (Rajadhyaksha and Kosofsky, 2005; Greer and Greenberg, 2008; Li et al., 2009), which converge on CREB-mediated transcription of activity-regulated genes. While all three pathways may be activated by calcium influx, high intracellular Ca²⁺ concentrations [Ca²⁺]_i preferentially promote kinase activation (CaMK and MAPK), while modest or low [Ca²⁺]_I preferentially activates the phosphatases (CaN) (Rajadhyaksha and Kosofsky, 2005). Additional recommended readings about signaling originating at L-type VSCCs include: Hagenston and Bading (2011), Greer and Greenberg (2008), and West et al. (2001).

Figure 2.

CaMK, MAPK/ERK, and CaN Signaling From L-VSCCs to Transcription. Signaling pathways initiating from Ca²⁺ influx at L-type VSCCs leading to transcription in the nucleus. Solid arrows represent intra-somatic interactions. Dashed arrows represent translocation into the nucleus. Black arrows represent the CaMK pathway, orange arrows represent the MAPK/ERK pathway, and blue arrows represent the CaN pathway. Dephosphorylated CRTC1 (in dark blue) translocates into the nucleus, and phosphorylated CRTC1 (in light blue) moves out of the nucleus.

Calmodulin Kinase (CaMKII/CaMKIV)

Calmodulin (CaM) binds the L-type VSCCs at the “IQ” motif in the carboxyl terminus of the channel (Dolmetsch et al., 2001). When extracellular KCl induces calcium influx through these channels, Ca²⁺ binds CaM (Ca²⁺/CaM), inducing a conformational switch that allows calmodulin to activate calmodulin kinase signaling. Ca²⁺/CaM activates cytosolic CaMKK, which in turn activates cytosolic CaMKI and CaMKII, or nuclear CaMKIV (Ghosh and Greenberg, 1995; Greer and Greenberg, 2008; Hagenston and Baing, 2011). CaMKII shuttles Ca²⁺/CaM to the nucleus and phosphorylates activity-dependent transcription factors including CREB, NeuroD, NF-kB, and MECP2 (Greer and Greenberg, 2008; Cohen et al., 2018). CaMKIV, activated by CaMKK and increases in nuclear [Ca²⁺], also activates CREB by phosphorylation at Ser 133 (Redmond et al., 2002). To successfully induce CREB-mediated transcription, CaMKIV must also activate the CREB co-activator CBP (CREB binding protein), a histone acetyltransferase (Hagenston and Baing, 2011). Kinase-dead CaMKIV, elimination of CaMKII, and dominant-negative CREB each block KCl-induced IEG transcription and dendritic growth (Redmond et al., 2002; Cohen et al., 2018). Both L-type VSCCs and NMDARs evoke CaMK signaling, which is critical for propagating the L-type VSCC signaling to the nucleus. However, distinctions between signaling from NMDARs and L-type VSCCs activate transcription at different regulatory elements of IEGs, indicating the mode of Ca²⁺ entry to the cell results in transcriptional specificity in the nucleus (Bading et al., 1993).

Ras/MAPK/ERK

CaM bound to the L-type VSCCs also activates Ras/MAPK/ERK signaling (Dolmetsch et al., 2001; Hagenston and Baing, 2011). Ca²⁺/CaM activates the GTP-binding protein Ras, which binds and activates Raf (mitogen activated protein kinase kinase), which phosphorylates and activates MEK. MEK phosphorylates and activates MAPK/ERK, which translocates to the nucleus to phosphorylate TCF-family transcription factors and stimulate TCF/SRF-dependent transcription (Hagenston and Baing, 2011). Ras/MAPK signaling also activates nuclear Rsk kinases, including Rsk2, which phosphorylates CREB at Ser-133 (West et al., 2001). Ca²⁺/CaM activates Ras/MAPK and CaMK pathways in concert, and both may lead to CREB-dependent transcription, but these signaling pathways may be responsive to different timing and magnitudes of neuronal activation (West et al., 2001; Hagenston and Baing, 2011). For instance, MAPK/ERK inhibition blunts and delays the first wave of IEG induction, whereas later waves of IEG transcription are less impacted, if at all (Tyssowski et al., 2018). Also, crosstalk exists between the CaMK and the MAPK pathways. Several groups, including ours, have shown that CaMKI regulates depolarization-induced MEK-ERK activity and their physiological functions (Schmitt et al., 2004, 2005; Wayman et al., 2006; Poston et al., 2020). We have recently identified that certain ortho-hydroxylated brominated ethers, which are persistent environmental toxins widely detectable in organisms including humans, bind to and inhibit CaMKI, which subsequently impairs MEK-ERK-dependent neuronal functions (Poston et al., 2020). Such inhibition by environmental toxins may have significant neurodevelopmental implications.

Calcineurin (CaN)

Calcineurin (CaN) is a phosphatase associated to L-type channels by the AKAP family of proteins. CaN is activated when it binds Ca²⁺/CaM on its catalytic subunit, and Ca²⁺ on its B subunit (Groth et al., 2003). Its affinity for Ca²⁺/CaM is much higher than the CaM-dependent protein kinases, and consequently initial elevations of intracellular Ca²⁺ preferentially induce CaN-mediated phosphatase activity over CaM kinase signaling (Groth et al., 2003). Inhibition of CaN by FK506 or cyclosporine A blocks CREB-dependent gene expression induced by depolarization and does not inhibit depolarization-induced Ca²⁺ influx or CREB Ser-133 phosphorylation (Kingsbury et al., 2007; España et al., 2010). Calcium and cAMP signaling converge on CREB-dependent gene transcription through CRTC1/TORC, a CREB transcriptional coactivator (Kovács et al., 2007). Active CaN phosphorylates CRTC1, causing it to translocate from the cytosol to the nucleus, where it accumulates and initiates expression of CREB-target genes, including Sik1 (España et al., 2010,Li et al., 2009). SIK1 phosphorylates CRTC1, reversing translocation and depleting it from the nucleus. This negative feedback mechanism prevents persistent CREB/CRTC1-dependent transcription and dendritic growth in the face of long lasting neuronal activity (Li et al., 2009).

Chromatin Architecture at IEGs

In addition to activating CREB and CREB co-activators like CRTC1, CBP, and p300, extracellular KCl induces changes in the chromatin architecture at IEGs (West et al., 2002; Kim et al., 2010; Pruunsild et al., 2011). Neuronal depolarization by KCl induces changes in histone H1 and H3.3, concomitant with IEG transcription (Michod et al., 2012; Azad et al., 2018). KCl also induces activity-dependent enhancers, cis-element activity, and DNA breaks regulating transcription (Pruunsild et al., 2011; Malik et al., 2014; Madabhushi et al., 2015). Promoters and enhancers of rapid IEGs –a subset of IEGs which are transcribed almost immediately after activity– are marked by paused RNA polymerase II and an accessible chromatin state (Saha et al., 2011; Tyssowski et al., 2018). Delayed IEGs –a subset of IEGs that occur post-activity later in the hour– do not have similar chromatin features at their promoters and/or enhancers. Sustained activity-dependent transcription of delayed IEGs and other secondary response genes (Tyssowski et al., 2018) may be epigenetically regulated by chromatin remodeling (Martinowich et al., 2003) and negative feedback by histone deacetylases (Kyrke-Smith and Williams, 2018). The specifics of these transcriptional regulatory mechanisms may be highly dependent on cell identity, transcriptional history, and gene or even transcript identity.

Effects of Duration and Doses of Elevated External Potassium

Different types and durations of neuronal stimulation can induce different gene expression programs (Tyssowski et al., 2018). Moreover, the mode of Ca²⁺ entry into the neuron plays a key role in determining which signaling pathways and gene expression programs are activated (Dolmetsch et al., 2001; Martinowich et al., 2003; Kingsbury et al., 2007). While extracellular KCl concentration and membrane voltage are directly proportional (Leslie et al., 2001; Somjen, 2002), signaling strength may be non-linearly correlated with Ca²⁺ influx levels (Wheeler et al., 2008). Here, we will discuss variations in the types of extracellular KCl protocols published for in vitro work, including pre-treatments, variations in KCl concentration ([KCl]), and variations in time of treatment.

Silencing Activity With Pre-Treatments

Experimentation with KCl and in vitro neuronal cultures began in the 1980s with treatments at 20-85 mM and treatment times between 10 minutes and 48 hours, with no pre-treatments to silence basal activity (Greenberg et al., 1985; Morgan and Curran, 1986; Bartel et al., 1989; Collins et al., 1989). The addition of Ca²⁺ channel inhibitors like APV and CNQX used in conjunction with KCl treatments distinguished the roles of NMDARs, non-NMDARs (AMPARs), and voltage-sensitive calcium channels (VSCCs) in c-fos and Bdnf induction (Zafra et al., 1990; Bading et al., 1993; Ghosh et al., 1994). Many authors have subsequently employed inhibitors in the same manner, including APV and D-AP5 (NMDARs), CNQX and NBQX (AMPARs), nimodipine and nifedipine (L-type VSCCs), KN93 (CaMK), and PD (MEK) (Dolmetsch et al., 2001; Redmond et al., 2002; Kingsbury et al., 2007; Evans et al., 2013; Cohen et al., 2018). These inhibitors are usually added to neuronal cultures several minutes (15-30 min) prior to KCl-depolarization to allay concerns about effectivity. For the experiments cited above, these inhibitors were used as experimental variants, and were compared to KCl-treatment without inhibition.

Historically, as KCl became a common means of depolarization, inhibitors became a means of standardizing activity levels and/or conferring specificity to the channels available for Ca²⁺ influx. Pre-silencing all conditions with TTX and APV excludes NMDARs from the response to depolarization. Some cultures are treated overnight in 1µM TTX to reduce endogenous activity (Xia et al., 1996; Tao et al., 1998, 2002; Lyons et al., 2012; Michod et al., 2012; Malik et al., 2014; Lyons et al., 2016). 100 µM APV is added 30 minutes prior to potassium depolarization to block the NMDARs (Tao et al., 1998, 2002). Some experiments vary concentration and time of pre-treatment, or add other inhibitors, like CNQX (Xia et al., 1996; Grubb and Burrone, 2010; Malik et al., 2014; Cohen et al., 2018; Tyssowski et al., 2018). Experimenters must decide whether pre-treatment to silence synaptic activity is appropriate for their KCl depolarization experiments. For those attempting to isolate the activity of the L-type Ca²⁺ channels, TTX + APV + CNQX pre-treatments may be useful. However, some treatments have their own constraints and effects; for instance, prolonged neuronal silencing with TTX triggers homeostatic changes (Turrigiano and Nelson, 2004), which may add artifacts to observations. Also, washout of TTX after prolonged application induces elevated activity (Saha et al., 2011; Lyons et al., 2016). Pre-silencing with TTX may be unnecessary for many experimental paradigms, especially considering the activity silencing effects of KCl concentrations above 10mM (see Figure 1).

Variation in Stimulation by Concentration and Time

While 50-55 mM KCl has become the standard treatment concentration in the excitation-transcription coupling field, the range historically spans from low doses of 3 mM to high doses of 120 mM. According to the Goldman equation, 50 mM is sufficient to induce a +48 mV depolarization in cultures maintained in media with 5 mM KCl. Experimentally, 50mM KCl has been shown to depolarize the membrane to about -30mV (Furness, 1970; Somjen, 2002). This does not bring the resting membrane potential to zero, but it is sufficient to induce robust gene expression. We have found that lower concentrations of KCl (mild depolarization) are also potent inducer of neuronal IEG transcription (Rienecker et al., 2020). Treatment times also vary widely, from 10 seconds to 48 hours. Contrary to a common presumption, prolonged treatment with reasonable concentrations of elevated external potassium does not induce apoptosis; instead, it facilitates cell survival (see “Cell Viability Under KCl Treatment” section). Furthermore, different gene programs are triggered by different duration of KCl-induced (or, any) depolarization (Tyssowski et al., 2018). However, to our knowledge, there has not been a methodical investigation into whether varied concentrations and treatment times interact to produce different gene expression profiles. We might expect variation, as different concentrations of intracellular Ca²⁺ and modes of Ca²⁺ entry differentially induce different signaling pathways, resulting in distinct transcriptional profiles (Rajadhyaksha and Kosofsky, 2005; Tyssowski et al., 2018; Rienecker et al., 2020). In Table 1, we have compiled and sorted selected papers by KCl concentration and time of treatment.

Table 1.

KCl Treatments by Time and Concentration.

	0–5'	6'–30'	31'–60'	1.1–2 hr	2.1–4 hr	4.1–6 hr	6.1–8 hr	8.1–12 hr	2.1–23 hr	24 hr	48 hr
3–8 mM								(Golbs et al., 2011)			(Leslie et al., 2001)
10 mM		(Collins et al., 1989; Evans et al., 2013; Madabhushi et al., 2015)			(Evans et al., 2013)						(Leslie et al., 2001; Evans et al., 2013; 2017)
12.5 mM		(Azad et al., 2018)	(Azad et al., 2018)				(Azad et al., 2018)				(Leslie et al., 2001)
15 mM	(Kato et al., 1998)				(Evans et al., 2017)						(Grubb and Burrone, 2010)
20–25 mM		(Collins et al., 1989; Pruunsild et al., 2011)	(Pruunsild et al., 2011)	(Collins et al., 1989; Pruunsild et al., 2011)	(Pruunsild et al., 2011)	(Pruunsild et al., 2011)	(Pruunsild et al., 2011)	(Pruunsild et al., 2011)	(Kingsbury et al., 2007)	(Collins et al., 1989; D’Mello et al., 1993)	(D’Mello et al., 1993; Bok et al., 2007)
30–35mM	(Kato et al., 1998)	(Collins et al., 1989)
40 mM	(Collins et al., 1991)	(Collins et al., 1989; 1991)		(Cohen et al., 2018)						(Collins et al., 1989; 1991)	(Collins et al., 1989)
45 mM	(Kato et al., 1998)	(Greenberg et al., 1985; Collins et al., 1989)
50 mM	(Kingsbury et al., 2007; Casalbore et al., 2010; Berger et al., 2018)	(Kruijer et al., 1985; Morgan and Curran, 1986; Zafra et al., 1990; Ghosh et al., 1994; Tao et al., 1998; Kingsbury et al., 2007; Li et al., 2009; Casalbore et al., 2010; Berger et al., 2018)	(Kruijer et al., 1985; Zafra et al., 1990; Ghosh et al., 1994; Tao et al., 1998; 2002; Li et al., 2009; Casalbore et al., 2010; Berger et al., 2018; Pastuzyn et al., 2018)	(Zafra et al., 1990; Tao et al., 1998; 2002; Li et al., 2009; Casalbore et al., 2010)	(Kruijer et al., 1985; Zafra et al., 1990; Ghosh et al., 1994; Tao et al., 1998; 2002; Kingsbury et al., 2007; Michod et al., 2012; Qiu et al., 2016)	(Tao et al., 1998; 2002; Kingsbury et al., 2007; Li et al., 2009)	(Zafra et al., 1990; Ghosh et al., 1994)	(Tao et al., 1998; 2002)	(Kruijer et al., 1985; Zafra et al., 1990; Redmond et al., 2002)	(Redmond et al., 2002; Martinowich et al., 2003; Li et al., 2009)	(Crowder and Freeman, 1999; Redmond et al., 2002)
55 mM	(Tyssowski et al., 2018)	(Xia et al., 1996; Madabhushi et al., 2015; Tyssowski et al., 2018)	(Xia et al., 1996; Malik et al., 2014)	(Malik et al., 2014; Lyons et al., 2016)	(Lyons et al., 2012; Malik et al., 2014)	(Lyons et al., 2012; Malik et al., 2014; Lyons et al., 2016; Tyssowski et al., 2018)		(Tyssowski et al., 2018)
60 mM	(Kato et al., 1998; Gotoh et al., 1999; Dolmetsch et al., 2001)	(Bartel et al., 1989; Gotoh et al., 1999; Dolmetsch et al., 2001)	(Bartel et al., 1989; Dolmetsch et al., 2001)	(Bartel et al., 1989)	(Bartel et al., 1989)	(Dolmetsch et al., 2001)
85 mM		(Collins et al., 1989)
90 mM	(Tao-Cheng, 2018; 2018)	(Rothman, 1985)
120 mM+	(Kato et al., 1998)	(Rothman, 1985)

A categorization of literature using extracellular potassium treatments by duration of treatment and concentration of potassium.

Strengths and Limitations of Extracellular KCl Treatment

General Assessment of Extracellular KCl as an Experimental Paradigm

Extracellular KCl treatment is a relatively easy method of inducing global depolarization in vitro to study channel physiology, signaling messengers and cascades, and gene transcription. Methods involve media replacement–partially or completely– to induce depolarization, and wash out to remove the stimulus. This method is scalable, enabling experiments across a variety of KCl concentrations applied for anywhere from seconds to days and allows studies of mild graded potential changes without sophisticated equipment. Because this method induces global depolarization, it is unsuitable for distinguishing differences amongst inter-compartmental signaling events in a neuron that may exist in the brain. For example, unlike approaches such as localized glutamate uncaging, this method cannot distinguish activity-induced local events in the synapse from their dendritic, somatic, or axonal counterparts. Sustained global depolarization also cannot replicate variable stimuli on physiologically relevant timescales. Extracellular potassium is unsuitable for exploring how temporal features of action potentials are coded into specific intracellular signaling and transcriptional programs (Fields et al., 1997; Tyssowski et al., 2018). Another consideration is that experimental readouts are often population averages where cell-specific responses, which might differ based on history of activity and cell type, remain obscure. However, in nearly uniform cultures this simultaneously strengthens detection of small populational effects. As global depolarization induces calcium influx through both synapse- and soma-associated channels, multiple signaling pathways may be recruited by simple KCl treatments. Channel-specific inhibitors, gene constructs, and other tools can be used to impose specificity on KCl-induced Ca²⁺ influx and signaling (Dolmetsch et al., 2001; Redmond et al., 2002; Kingsbury et al., 2007; Evans et al., 2013; Cohen et al., 2018).

Cell Viability Under KCl Treatment

Elevated extracellular potassium promotes cell survival in a range of neuronal cell types and species. Reports of enhanced survival include peripheral neurons from chicken dorsal root, sympathetic, and parasympathetic ganglia (Bennett and White, 1979; Wakade and Thoenen, 1984; Koike et al., 1989; Collins et al., 1991; Crowder and Freeman, 1999), the mollusk giant neuron from ganglia tissue (Kostenko et al., 1982), murine cerebellar neurons (Didier et al., 1989), and neonatal rat cortical and cerebellar neurons (Lasher and Zagon, 1972; Golbs et al., 2011). Optimal potassium concentrations that promote survival lie between 25-40 mM, with survival falling off above 50 mM, and dropping to 20-50% of maximal survival at 85 mM (Collins et al., 1989). These cell survival effects are dependent on elevated potassium and Ca²⁺ influx through DHP sensitive L-type channels. Both must be sustained to promote cell survival; calcium chelators, replacement of elevated potassium with normal media, or L-type channel inhibition blocks the protective effects (Collins et al., 1989; Koike et al., 1989; D’Mello et al., 1993). Likewise, the L-type channel agonist BAYK 8644 enhances the protective effect, achieving high survival rates at lower potassium concentrations. BAYK 8644 allows L-type channels to open at significantly lower levels of depolarization, increasing Ca²⁺ influx (Collins et al., 1989). While there is a strong quantitative correlation between mean sustained intracellular calcium levels and the percentage of surviving neurons (Collins et al., 1991), simple flux in intracellular calcium concentration is not the mechanism of neuroprotection. Concentrations of 90-200 mM KCl are reported to be lethal to glutamate sensitive neurons (Rothman, 1985; Choi, 1987; Mattson et al., 1988; Collins et al., 1989). Moreover, levels of intracellular calcium which are protective when induced by potassium-mediated depolarization, are toxic when induced by glutamate signaling, particularly in the presence of NMDARs including GluN2 (Rothman et al., 1987; Collins et al., 1989; Martel et al., 2012). This is likely because potassium-mediated depolarization relies on L-type VSCCs, whereas glutamate signals through NMDARs (Kudo and Ogura, 1986; Murphy et al., 1987; Choi, 1988; Collins et al., 1989). Ca²⁺ influx through non-NMDARs is neither sufficient nor necessary for glutamate-induced neurotoxic injury, suggesting the mode of Ca²⁺ entry determines toxicity or neuroprotection (Choi, 1988; Collins et al., 1989). Likewise, potassium-mediated and neurotrophic neuroprotection mechanisms are distinct; DHP inhibition of L-type channels does not block neuronal survival mediated by neurotrophins (Collins et al., 1989). The neuroprotective effects of elevated extracellular potassium are rather mediated by calcium-dependent intracellular signaling, although the relative importance of these different pathways may vary with neuronal identity. In neonatal rat spiral ganglion neurons treated with 25mM K⁺ for 96 hrs, nuclear CaMKIV promotes survival by activating CREB (see Figure 2), while CaMKII functionally inactivates the proapoptotic regulator Bad (Bok et al., 2007). At this sustained depolarization, CaN signaling is inactivated (Bito et al., 1997; Groth et al., 2003). The mechanism of elevated potassium-mediated neuroprotection may also partially depend on the treatment’s effect on neuronal activity. A small elevation in extracellular potassium to 8 mM reportedly shift the frequency distribution of neuronal activity toward high-frequency bursts associated with reduced caspase-3 dependent apoptosis and increased neuronal survival (Golbs et al., 2011). In cortical neurons, this neuroprotective effect of high-frequency activity is mediated by a PI3K dependent pathway (Golbs et al., 2011). The PI3K/Akt pathway is involved in both depolarization and neurotrophin promoted survival in sympathetic neurons, but may not be a universal neuroprotective mechanism in all cell types (Crowder and Freeman, 1999). At extremely high concentrations (exceeding 85mM), elevated extracellular potassium becomes antagonistic to neuronal survival due to the osmotic effects of excess sodium and chloride influx. Removal of chloride from the external medium prevents toxicity of 90mM to 140mM K⁺ (Rothman, 1985). In summary, while the optimal extracellular potassium concentration for cell survival may vary between differing neuronal types, KCl is generally neuroprotective at concentrations between 25-40 mM and at lower concentrations when supplemented with BAYK 8644. Mechanistically, this neuroprotection is mediated by Ca²⁺ dependent intracellular signaling including calmodulin kinases (CaMKII and CaMKIV) and the PI3K/Akt pathway, but the relative importance of different pathways to neuroprotection may also depend on the cell type and strength of the depolarization.

Implications of In Vitro KCl Research for In Vivo Functions

Extracellular KCl is useful for investigating cellular mechanisms of neurons in vitro. However, concerns are frequently raised about its relevance to events in vivo. KCl treatments are indeed different from physiological stimuli in both dose and duration of application. Many protocols apply 50-55mM KCl for hours or days at a time, periods that do not reflect the duration of sensory stimuli or seizures. Nevertheless, many mechanisms mediating responses to elevated extracellular KCl also prove necessary for similar in vivo functions. Seizures, sensory stimuli, and learning events induce IEGs likewise induced by extracellular KCl (Morgan et al., 1987; Hunt et al., 1988; Guzowski et al., 2001; Lyons and West, 2011; West and Greenberg, 2011; Mukherjee et al., 2018; Heinz and Bloodgood, 2020). Furthermore, while sustained global depolarization by elevated KCl is not a feature of healthy neuronal signaling, it is a proposed mechanism of Leão’s spreading depression of neuronal activity (Leao, 1944; Grafstein, 1956; Somjen, 2001; de Curtis et al., 2018). Below we discuss the in vivo phenomena that highlight the relevance of in vitro findings from research using extracellular KCl.

IEG Transcription Is Important for Learning and Memory, and Is Induced in Physiological Conditions

IEG induction is a prominent consequence of elevated extracellular KCl treatment (Zafra et al., 1990; West et al., 2002; Tyssowski et al., 2018). While there are key differences between KCl and physiological stimuli, induction mechanisms discovered in vitro have held true in vivo (Greer and Greenberg, 2008). Transcriptional responses to depolarizing KCl in vitro have downstream effects on homeostatic plasticity, dendritic growth, and the remodeling of synaptic structures (Leslie et al., 2001; Redmond et al., 2002; Kovács et al., 2007; Grubb and Burrone, 2010; Tao-Cheng, 2018). Likewise, similar programs of IEG transcription in vivo are crucial for homeostatic plasticity, neurite outgrowth, synapse development and strength, neural adaptations to drugs of abuse, and learning and memory (Ploski et al., 2008; Lyons and West, 2011). These IEG programs are so reliable, expression of genes such as Bdnf, Arc, and Fos are often used in immunohistochemistry as proxies of neural activity induced by learning paradigms and sensory stimuli (West and Greenberg, 2011; Lyons et al., 2016).

L-Type Channels and Ca2+ Signaling Are Involved in IEG Transcription, Learning and Memory, and Homeostatic Plasticity

Both in culture and under physiological conditions, IEG transcription is regulated both by synaptic (NMDAR, AMPAR signaling) and extra-synaptic signaling, including somatically (L-type VSCCs) distributed ion channels. In vitro, extracellular KCl generates Ca²⁺ influx through L-type VSCCs and stimulates signaling to IEG transcription in the nucleus. Such signaling is highly relevant in vivo where L-type VSCCs induce IEG transcription and are involved in learning and memory, as well as in pathological brain functions, autism, aging, and pain (Shinnick-Gallagher et al., 2003; Jerome et al., 2012; Berger and Bartsch, 2014; Ghosh et al., 2017; Navakkode et al., 2018; Roca-Lapirot et al., 2018). Models of aging and neurodegenerative diseases like Parkinson’s disease suggest alterations in Ca²⁺ homeostasis through L-type VSCCs increase vulnerability to cognitive decline and synaptic dysfunction (Berger and Bartsch, 2014; Navakkode et al., 2018). Genome wide association studies (GWAS) and natural channel variants in humans link L-type VSCCs to working memory performance (Berger and Bartsch, 2014). Olfactory and fear memory in rats also depends on L-type VSCCs. L-type VSCCs induce transcription of the plasticity-related genes necessary for protein-synthesis dependent long term olfactory memory (Jerome et al., 2012; Ghosh et al., 2017). In brain slices from fear-conditioned rats, nimodipine reduces EPSC amplitude and increases paired pulse facilitation. When administered in vivo by intraperitoneal-injection, nimodipine also blocks fear-potentiated startle in a dose dependent manner (Shinnick-Gallagher et al., 2003). L-type VSCCs play a selective role in long-term, but not short term, fear conditioning memory by activating Ca²⁺/calmodulin and MAPK/ERK signaling to induce CREB-mediated transcription (Schafe et al., 2000; Bauer et al., 2002).

Extracellular KCl in vitro induces Ca²⁺ dependent signaling cascades involving calmodulin kinases, MAPK/ERK, and calcineurin (Ghosh and Greenberg, 1995; Rajadhyaksha and Kosofsky, 2005; Greer and Greenberg, 2008; Hagenston and Baing, 2011; Tyssowski et al., 2018). In vivo, elimination of CaMKII prevents activity-dependent expression of IEGs like Bdnf, Fos, and Arc, inhibits persistent synaptic strengthening, and impairs spatial memory (Cohen et al., 2018). Blockade of MAPK/ERK activation in the lateral amygdala impairs fear memory consolidation and synaptic plasticity in vivo (Schafe et al., 2000). CRTC1, aka TORC1, is downstream of CaN signaling and is crucial for contextual fear and spatial memory (España et al., 2010; Nonaka et al., 2014; Uchida et al., 2017). Knockdown of CRTC1 in CA1 and CA3 reduces long-term but not short-term contextual fear memory, implying its effect on activity-induced gene transcription, which is necessary for long-term memory. In response to neural activity and learning, CRTC1 localizes to the nucleus in a CaN dependent manner and induces Fgf1b. (Uchida et al., 2017). Furthermore, gene transcription mediated by CRTC1, including fos, Bdnf, and Nr4a2, is impaired in Alzheimer’s disease transgenic mice expressing human beta-amyloid precursor protein. These transcriptional deficits coincide with long-term spatial memory deficits in the transgenic mice (España et al., 2010).

Homeostatic alterations and remodeling of cellular structures are also consequences of global depolarization by KCl (Leslie et al., 2001; Grubb and Burrone, 2010; Evans et al., 2013; Tao-Cheng, 2018, 2018). In hippocampal cultures, chronic depolarization with high potassium moves the axon initial segment (AIS) away from the soma, dependent on the activation of L-type VSCCs (Grubb and Burrone, 2010). Changes in AIS position and activity-dependent plasticity are thought to fine tune neuronal excitability according to ongoing electrical activity (Grubb and Burrone, 2010). In Aplysia, subthreshold depolarizing changes in the presynaptic holding potential of the sensory neuron B21 increase the rate at which homosynaptic facilitation occurs. L-type VSCC-dependent calcium elevation is partially responsible for these membrane potential-induced alterations in facilitation (Ludwar et al., 2020).

Potassium Hypotheses of Brain Disorders

Brain disorders such as migraine aura, epilepsy, haemorrhagic and ischaemic stroke, subarachnoid hemmorahage, and traumatic brain injury are associated with a robust phenomenon called spreading depression (SD), first described by Leão in 1944 and subsequently linked to increases in extracellular K⁺ in vivo (Leao, 1944; Somjen, 2001; Gupta, 2006; Fabricius et al., 2008; Richter and Lehmenkühler, 2008; Charles and Brennan, 2009; Cui et al., 2014; de Curtis et al., 2018). SD is a transient (60-120s), self-propagating wave of neuronal and glial depolarization accompanied by a negative shift of the current potential of about 20-35 mV and followed by prolonged quiescence (Richter and Lehmenkühler, 2008; Zhang et al., 2011; Cui et al., 2014). This propagation occurs in all directions at ∼3 mm/min, a rate similar to the spread of aura in classical migraine (Richter and Lehmenkühler, 2008; Cui et al., 2014). SD is accompanied by changes in blood flow, vascular caliber, and energy metabolism. While SD can occur in all neural tissues (Charles and Brennan, 2009; Dong et al., 2020), it is most often studied in the cortex, where it is referred to as cortical spreading depression (CSD). The phenomenon occurs in rat, mouse, rabbit, pigeon, and human models, among others (Leao, 1944; Fabricius et al., 2008; Richter and Lehmenkühler, 2008; Charles and Brennan, 2009).

The mechanistic hypothesis of SD postulates increases in extracellular [K⁺] trigger massive neuronal depolarization that leads to network hyperactivity and further K⁺ accumulation, which eventually depresses activity (Grafstein, 1956; Somjen, 2001; de Curtis et al., 2018). When the brain tissue is unable to clear the excessive K⁺ in the extracellular space, prolonged accumulation of K⁺ and sustained depolarization compromises synaptic transmission (de Curtis et al., 2018). Mild depolarization moves the membrane potential closer to the firing threshold and increases excitability, but stronger depolarization (as extracellular [K⁺] continues to rise) inactivates some voltage sensitive ion channels, raises the threshold, and eventually–reminiscent of in vitro findings mentioned in “Elevated Extracellular KCl Induces Neuronal Depolarization, But Often, Not Activity” section–suppresses excitability (Kager et al., 2002; Somjen, 2002; Dreier et al., 2012). During SD, increases in extracellular [K⁺] above the baseline 3 mM have been reported, ranging from 7.3 mM (Enger et al., 2015) up to 35 mM (Kraio and Nicholson, 1978), and are accompanied by a sharp drop in extracellular Na⁺ and Ca²⁺. This suggests K⁺ leaving the cells is exchanged against an influx of Na⁺ and Ca²⁺ (Brinley et al., 1960; Nicholson, 1984; Silver and Erecńska, 1990; Somjen, 2001). Membrane potentials consequently depolarize significantly, briefly approaching zero (Somjen, 2001). In K⁺ induced models of CSD in vivo, K⁺ rise precedes CSD and intracellular increases in Ca²⁺. These neuronal Ca²⁺ transients lag behind the negative direct current potential shift but last twice as long. Interestingly, astrocytic Ca²⁺ increases lagged further behind negative potential shifts, and were shorter than neuronal Ca²⁺ transients (Enger et al., 2015). Astrocytes can prevent the occurrence of SD through buffering of extracellular K⁺ (Cui et al., 2014). However, glial cells also experience the depolarization induced by SD, and extracellular [K⁺] may exceed their capacity to maintain physiologically tolerable limits. As neuronal dendrites and glia uptake K⁺ and swell, they restrict the interstitial space and further concentrate extracellular [K⁺] (Somjen, 2001).

Both elevations in extracellular [K⁺] and glutamate contribute to SD, but synaptic signaling is not required (Brinley et al., 1960; Charles and Brennan, 2009; Enger et al., 2015). CSD becomes less frequent and harder to trigger if P/Q calcium channels are genetically modified or specifically blocked. Likewise, blocking NMDARs by MK-801 stops the spread of CSD (Richter and Lehmenkühler, 2008). TTX, however, does not block the propagation of CSD, even though it suppresses action potential firing. Most likely, increases in extracellular [K⁺] and neuronal voltage-activated currents are key to the ignition and evolution of SD, as opposed to interstitial glutamate (Enger et al., 2015). However, extracellular [K⁺] is reported not to increase ahead of changes in voltage, as it should if it were the agent of propagation of SD (Somjen, 2001).

The difference between in vitro KCl treatments and in vivo SD is that SD events accompany other activity. In epilepsy for instance, SD interacts with synchronous ictal epileptic events and paroxysmal depolarization shifts (Dreier et al., 2012; Kubista et al., 2019; Zakharov et al., 2019). Thus, it is ambiguous whether the in vivo cellular consequences of SD associated with epilepsy relate to increased synchronous activity, or to the sustained depolarization that follows. Furthermore, as SD propagates across neural tissue at ∼3 mm/min, extracellular [K⁺] rises more slowly in physiological conditions than KCl treatments applied in vitro. The rate of depolarization by extracellular [K⁺] may also impact membrane voltage and cellular responses. Nevertheless, extracellular KCl can be applied to induce SD in experimental models in vivo (Marshall, 1959; Jing et al., 1991; Herdegen et al., 1993; Herreras and Somjen, 1993; Busch et al., 1996; Fujikawa et al., 1996; Somjen, 2001; Cui et al., 2014). While comparisons between in vitro KCl treatments and physiological events like SD must be cautious, they could be reciprocally informative.

Potential Use of Extracellular KCl in Other Fields

Fields broader than that of activity-regulated transcription may benefit from using potassium-mediated depolarization in vitro. For example, in environmental toxicology, toxins like polybrominated diphenyl ethers (PBDEs) and methylmercury (MeHg) act via pathways involving neuronal excitability, intracellular calcium concentrations, and MAPK, PKA/CREB, and PI3K/Akt signaling. PBDEs are a bioaccumulative class of brominated flame retardants with neurodevelopmental toxicity. PBDE metabolites like 6-OH-BDE-47 impair MAPK signaling (Poston et al., 2020) and disturb intracellular [Ca²⁺] by causing an influx of extracellular Ca²⁺ and release of Ca²⁺ from intracellular stores in the ER and mitochondria (Dingemans et al., 2010). Disruption of normal MAPK signaling to IEGs and of excitability could play a role in the neurodevelopmental toxicity of PBDEs. Likewise, methylmercury increases intracellular Ca²⁺, disrupts MAPK and ERK1/2 signaling, and upregulates IEGs like fos, c-jun, and Bdnf (Bulleit and Cui, 1998; Dao et al., 2017; Fujimura and Usuki, 2017; Heimfarth et al., 2018, 2018). MeHg induced MAPK and PKA/CREB pathways trigger site specific neural hyperactivity and degeneration (Fujimura and Usuki, 2017, 2018; Heimfarth et al., 2018). These similarities with mechanisms of potassium-mediated depolarization may prove informative.

Wrap Up

Elevated extracellular potassium is a useful in vitro treatment for elucidating the signaling, transcriptional, and plasticity-related events associated with global neuronal depolarization. Because it is applied by media replacement, it is scalable by dose and time. Many in vitro depolarization-induced mechanisms, including Ca²⁺ influx through L-type VSCCs, Ca²⁺ dependent signaling, IEG transcription, homeostasis, and long term potentiation, are also recruited for sensory stimuli, learning, memory, disease, and seizures in vivo. Indeed, KCl treatments have strong similarities to the potassium hypothesis of SD and epilepsy. However, researchers should justify the parameters of KCl treatments cautiously; prolonged periods, rapid change in concentration, and high doses of depolarizing KCl are not comparable to in vivo stimuli. Given the wide range of potassium stimulation protocols, more investigation is required into whether different stimulation parameters have differing results. For example, different neuronal activity patterns (short and prolonged) induce different IEG expression profiles (Greer and Greenberg, 2008; Tyssowski et al., 2018). Consequently, treatment length, cell type, and activity history of the neurons may heavily impact findings. The selected stimulation paradigm also has consequences for the final data type. Electrophysiological recordings allow internal measurements of the depolarization of single cells and can stimulate single cells to depolarize at graded stages. However, the modulation of only one or a few cells at a time is incompatible with certain molecular biology techniques that require higher cell loads, like chromatin immunoprecipitation. KCl treatment, which can be applied homogenously to a population of cells in vitro, is more appropriate for these measures. While global depolarization induced by potassium recruits multiple ion channels and several signaling cascades, experimenters can still achieve some degree of specificity. Inhibitors and genetic engineering can limit the effects of global depolarization to the mechanisms of interest. Inhibitors may be used as acute treatments or prolonged pre-treatments, depending on the intended comparisons. The body of work using elevated extracellular potassium in vitro has significantly contributed to our understanding of depolarization-transcription coupling mechanisms in neurons, and these findings are robustly confirmed by in vivo work. Consolidating an understanding of the boundaries of this stimulation protocol will ensure it remains a productive research tool.

Summary Statement

Elevated extracellular potassium chloride is widely used to achieve membrane depolarization, but there is great variability between protocols. We summarize the mechanisms of potassium-mediated depolarization, their relevance to brain functions and dysfunctions, and strengths and drawbacks of this technique.

Footnotes

Acknowledgements

We thank all Saha Lab members for their support and constructive criticism during manuscript preparation, including Ayna Rejepova, who contributed to the literature search.

Author Contributions

KDAR (lead) and RNS wrote the manuscript with feedback from RP. RP contributed MEA data for KCL depolarizations in .

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The study was conducted under the auspices of (NIH) a National Institute of Environmental Health Sciences (NIEHS), NIH grant to RNS (R01ES028738). The contents of this paper is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

ORCID iD

Ramendra N. Saha

References

Azad

G. K.

Ito

Sailaja

B. S.

Biran

Nissim-Rafinia

Yamada

Brown

D. T.

Takizawa

Meshorer

(2018). PARP1-dependent eviction of the linker histone H1 mediates immediate early gene expression during neuronal activation. J Cell Biol. Advance online publication. https://doi.org/10.1083/jcb.201703141

Bading

Ginty

D. D.

Greenberg

M. E.

(1993). Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways. Science. Advance online publication. https://doi.org/10.1126/science.8097060

Bartel

D. P.

Sheng

Lau

L. F.

Greenberg

M. E.

(1989). Growth factors and membrane depolarization activate distinct programs of early response gene expression: Dissociation of fos and jun induction. Genes Dev. Advance online publication. https://doi.org/10.1101/gad.3.3.304

Bauer

E. P.

Schafe

G. E.

LeDoux

J. E.

(2002). NMDA receptors and L-type voltage-gated calcium channels contribute to long-term potentiation and different components of fear memory formation in the lateral amygdala. J Neurosci. Advance online publication. https://doi.org/10.1523/jneurosci.22-12-05239.2002

Bennett

M. R.

White

(1979). The survival and development of cholinergic neurons in potassium-enriched media. Brain Res. Advance online publication. https://doi.org/10.1016/0006-8993(79)90250-6

Berger

S. L.

Leo-Macias

Yuen

Khatri

Pfennig

Zhang

Agullo-Pascual

Caillol

Zhu

M. S.

Rothenberg

Melendez-Vasquez

C. V.

Delmar

Leterrier

Salzer

J. L.

(2018). Localized myosin II activity regulates assembly and plasticity of the axon initial segment. Neuron. Advance online publication. https://doi.org/10.1016/j.neuron.2017.12.039

Berger

S. M.

Bartsch

(2014). The role of L-type voltage-gated calcium channels Cav1.2 and Cav1.3 in normal and pathological brain function. Cell Tissue Res. Advance online publication. https://doi.org/10.1007/s00441-014-1936-3

Bito

Deisseroth

Tsien

R. W.

(1997). Ca2+-dependent regulation in neuronal gene expression. Curr Opin Neurobiol. Advance online publication. https://doi.org/10.1016/S0959-4388(97)80072-4

Bok

Wang

Huang

Green

S. H.

(2007). CaMKII and CaMKIV mediate distinct prosurvival signaling pathways in response to depolarization in neurons. Mol Cell Neurosci. Advance online publication. https://doi.org/10.1016/j.mcn.2007.05.008

10.

Brinley

F. J.

Kandel

E. R.

Marshall

W. H.

(1960). Potassium outflux from rabbit cortex during spreading depression. J Neurophysiol. Advance online publication. https://doi.org/10.1152/jn.1960.23.3.246

11.

Bulleit

R. F.

Cui

(1998). Methylmercury antagonizes the survival-promoting activity of insulin-like growth factor on developing cerebellar granule neurons. Toxicol Appl Pharmacol. Advance online publication. https://doi.org/10.1006/taap.1998.8561

12.

Busch

Gyngell

M. L.

Eis

Hoehn-Berlage

Hossmann

K. A.

(1996). Potassium-induced cortical spreading depressions during focal cerebral ischemia in rats: Contribution to lesion growth assessed by diffusion- weighted NMR and biochemical imaging. J Cerebr Blood Flow Metabol. Advance online publication. https://doi.org/10.1097/00004647-199611000-00002

13.

Casalbore

Barone

Felsani

D’Agnano

Michetti

Maira

Cenciarelli

(2010). Neural stem cells modified to express BDNF antagonize trimethyltin-induced neurotoxicity through PI3K/Akt and MAP kinase pathways. J Cell Physiol, 224, 710–721. https://doi.org/10.1002/jcp.22170

14.

Charles

Brennan

K. C.

(2009). Cortical spreading depression – New insights and persistent questions. Cephalalgia. Advance online publication. https://doi.org/10.1111/j.1468-2982.2009.01983.x

15.

Choi

D. W.

(1987). Ionic dependence of glutamate neurotoxicity. J Neurosci. Advance online publication. https://doi.org/10.1523/jneurosci.07-02-00369.1987

16.

Choi

D. W.

(1988). Calcium-mediated neurotoxicity: Relationship to specific channel types and role in ischemic damage. Trends Neurosci. Advance online publication. https://doi.org/10.1016/0166-2236(88)90200-7

17.

Cohen

S. M.

Suutari

Wang

Sanchez

Tirko

N. N.

Mandelberg

N. J.

Mullins

Zhou

Wang

Kats

Salah

Tsien

R. W.

(2018). Calmodulin shuttling mediates cytonuclear signaling to trigger experience-dependent transcription and memory. Nature Commun. Advance online publication. https://doi.org/10.1038/s41467-018-04705-8

18.

Collins

Schmidt

M. F.

Guthrie

P. B.

Kater

S. B.

(1991). Sustained increase in intracellular calcium promotes neuronal survival. J Neurosci. Advance online publication. https://doi.org/10.1523/jneurosci.11-08-02582.1991

19.

Collins F. Lile J . D. (1989). The role of dihydropyridine-sensitive voltage-gated calcium channels in potassium-mediated neuronal survival. Brain Res. Advance online publication. https://doi.org/10.1016/0006-8993(89)90465-4

20.

Crowder

R. J.

Freeman

R. S.

(1999). The survival of sympathetic neurons promoted by potassium depolarization, but not by cyclic AMP, requires phosphatidylinositol 3- kinase and Akt. J Neurochem. Advance online publication. https://doi.org/10.1046/j.1471-4159.1999.0730466.x

21.

Cui

Kataoka

Watanabe

(2014). Role of cortical spreading depression in the pathophysiology of migraine. Neurosci Bull. Advance online publication. https://doi.org/10.1007/s12264-014-1471-y

22.

Dao

C. V.

Shiraishi

Miyamoto

(2017). The MARCKS protein amount is differently regulated by calpain during toxic effects of methylmercury between SH-SY5Y and EA.hy926 cells. J Veterinary Med Sci 79(12), 1931–1938. https://doi.org/0.1292/jvms.17-0473

23.

de Curtis

Uva

Gnatkovsky

Librizzi

(2018). Potassium dynamics and seizures: Why is potassium ictogenic? Epilepsy Res. Advance online publication. https://doi.org/10.1016/j.eplepsyres.2018.04.005

24.

Didier

Roux

Piechaczyk

Verrier

Bockaert

J. P.

(1989). Cerebellar granule cell survival and maturation induced by K+ and NMDA correlate with c-fos proto-oncogene expression. Neurosci Lett. Advance online publication. https://doi.org/10.1016/0304-3940(89)90790-8

25.

Dingemans

M. M. L.

Heusinkveld

H. J.

Bergman

Å.

van den Berg

Westerink

R. H. S.

(2010). Bromination pattern of hydroxylated metabolites of BDE-47 affects their potency to release calcium from intracellular stores in PC12 cells. Environ Health Perspect. Advance online publication. https://doi.org/10.1289/ehp.0901339

26.

D’Mello

S. R.

Galli

Ciotti

Calissano

(1993). Induction of apoptosis in cerebellar granule neurons by low potassium: Inhibition of death by insulin-like growth factor I and cAMP. Proc Natl Acad Sci U S A. Advance online publication. https://doi.org/10.1073/pnas.90.23.10989

27.

Dolmetsch

R. E.

Pajvani

Fife

Spotts

J. M.

Greenberg

M. E.

(2001). Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway. Science. Advance online publication. https://doi.org/10.1126/science.1063395

28.

Dong

Zhang

Wang

Liu

Wei

Chen

Yuan

Zhang

Hou

Jiao

Guo

Wei

Shen

(2020). Simultaneous calcium recordings of hippocampal CA1 and primary motor cortex M1 and their relations to behavioral activities in freely moving epileptic mice. Exp Brain Res. Advance online publication. https://doi.org/10.1007/s00221-020-05815-w

29.

Dreier

J. P.

Major

Pannek

H. W.

Woitzik

Scheel

Wiesenthal

Martus

Winkler

M. K. L.

Hartings

J. A.

Fabricius

Speckmann

E. J.

Gorji

(2012). Spreading convulsions, spreading depolarization and epileptogenesis in human cerebral cortex. Brain. Advance online publication. https://doi.org/10.1093/brain/awr303

30.

Enger

Tang

Vindedal

G. F.

Jensen

Helm

P. J.

Sprengel

Looger

L. L.

Nagelhus

E. A.

(2015). Dynamics of ionic shifts in cortical spreading depression. Cerebral Cortex. Advance online publication. https://doi.org/10.1093/cercor/bhv054

31.

España

Valero

Miñano-Molina

A. J.

Masgrau

Martín

Guardia-Laguarta

Lleó

Giménez-Llort

Rodríguez-Alvarez

Saura

C. A.

(2010). β-amyloid disrupts activity-dependent gene transcription required for memory through the CREB coactivator CRTC1. J Neurosci. Advance online publication. https://doi.org/10.1523/JNEUROSCI.2154-10.2010

32.

Evans

M. D.

Sammons

R. P.

Lebron

Dumitrescu

A. S.

Watkins

T. B. K.

Uebele

V. N.

Renger

J. J.

Grubb

M. S.

(2013). Calcineurin signaling mediates activity-dependent relocation of the Axon Initial segment. J Neurosci. Advance online publication. https://doi.org/10.1523/JNEUROSCI.0277-13.2013

33.

Evans

M. D.

Tufo

Dumitrescu

A. S.

Grubb

M. S.

(2017). Myosin II activity is required for structural plasticity at the axon initial segment. Eur J Neurosci. Advance online publication. https://doi.org/10.1111/ejn.13597

34.

Fabricius

Fuhr

Willumsen

Dreier

J. P.

Bhatia

Boutelle

M. G.

Hartings

J. A.

Bullock

Strong

A. J.

Lauritzen

(2008). Association of seizures with cortical spreading depression and peri-infarct depolarisations in the acutely injured human brain. Clin Neurophysiol. Advance online publication. https://doi.org/10.1016/j.clinph.2008.05.025

35.

Fields

R. D.

Eshete

Stevens

Itoh

(1997). Action potential-dependent regulation of gene expression: Temporal specificity in Ca2+, cAMP-responsive element binding proteins, and mitogen- activated protein kinase signaling. J Neurosci. Advance online publication. https://doi.org/10.1523/jneurosci.17-19-07252.1997

36.

Fujikawa

D. G.

Kim

J. S.

Daniels

A. H.

Alcaraz

A. F.

Sohn

T. B.

(1996). In vivo elevation of extracellular potassium in the rat amygdala increases extracellular glutamate and aspartate and damages neurons. Neuroscience. Advance online publication. https://doi.org/10.1016/0306-4522(96)00171-6

37.

Fujimura

Usuki

(2017). Site-specific neural hyperactivity via the activation of MAPK and PKA/CREB pathways triggers neuronal degeneration in methylmercury-intoxicated mice. Toxicol Lett, 271, 66–73. https://doi.org/10.1016/j.toxlet.2017.03.001

38.

Fujimura

Usuki

(2018). Methylmercury induces oxidative stress and subsequent neural hyperactivity leading to cell death through the p38 MAPK-CREB pathway in differentiated SH-SY5Y cells. Neurotoxicology, 67, 226–233. https://doi.org/10.1016/j.neuro.2018.06.008

39.

Furness

J. B.

(1970). The effect of external potassium ion concentration on autonomic neuro-muscular transmission. Pflügers Archiv Eur J Physiol. Advance online publication. https://doi.org/10.1007/BF00586580

40.

Ghosh

Carew

S. J.

Chen

Yuan

(2017). The role of L-type calcium channels in olfactory learning and its modulation by norepinephrine. Front Cell Neurosci. Advance online publication. https://doi.org/10.3389/fncel.2017.00394

41.

Ghosh

Carnahan

Greenberg

M. E.

(1994). Requirement for BDNF in activity-dependent survival of cortical neurons. Science. Advance online publication. https://doi.org/10.1126/science.7907431

42.

Ghosh

Greenberg

M. E.

(1995). Calcium signaling in neurons: Molecular mechanisms and cellular consequences. Science. Advance online publication. https://doi.org/10.1126/science.7716515

43.

Golbs

Nimmervoll

Sun

J. J.

Sava

I. E.

Luhmann

H. J.

(2011). Control of programmed cell death by distinct electrical activity patterns. Cereb Cortex. Advance online publication. https://doi.org/10.1093/cercor/bhq200

44.

Gomez-Ospina

Tsuruta

Barreto-Chang

Dolmetsch

(2006). The C terminus of the L-type voltage-gated calcium channel CaV1.2 encodes a transcription factor. Cell. Advance online publication. https://doi.org/10.1016/j.cell.2006.10.017

45.

Gotoh

Kajikawa

Kato

Suto

(1999). Intracellular Mg2+ surge follows Ca2+ increase during depolarization in cultured neurons. Brain Res. Advance online publication. https://doi.org/10.1016/S0006-8993(99)01298-6

46.

Grafstein

(1956). Mechanism of spreading cortical depression. J Neurophysiol. Advance online publication. https://doi.org/10.1152/jn.1956.19.2.154

47.

Greenberg

M. E.

Greene

L. A.

Ziff

E. B.

(1985). Nerve growth factor and epidermal growth factor induce rapid transient changes in proto-oncogene transcription in PC12 cells. J Biol Chem, 260(26), 14101–10.

48.

Greer

P. L.

Greenberg

M. E.

(2008). From synapse to nucleus: Calcium-dependent gene transcription in the control of synapse development and function. Neuron. Advance online publication. https://doi.org/10.1016/j.neuron.2008.09.002

49.

Groth

R. D.

Dunbar

R. L.

Mermelstein

P. G.

(2003). Calcineurin regulation of neuronal plasticity. Biochem Biophys Res Commun. Advance online publication. https://doi.org/10.1016/j.bbrc.2003.09.002

50.

Grubb

M. S.

Burrone

(2010). Activity-dependent relocation of the axon initial segment fine-tunes neuronal excitability. Nature. Advance online publication. https://doi.org/10.1038/nature09160

51.

Gupta

V. K.

(2006). Migrainous scintillating scotoma and headache is ocular in origin: A new hypothesis. Med Hypotheses. Advance online publication. https://doi.org/10.1016/j.mehy.2005.11.010

52.

Guzowski

J. F.

Setlow

Wagner

E. K.

McGaugh

J. L.

(2001). Experience-dependent gene expression in the rat hippocampus after spatial learning: A comparison of the immediate-early genes Arc, c-fos, and zif268. J Neurosci. Advance online publication. https://doi.org/10.1523/jneurosci.21-14-05089.2001

53.

Hagenston

A. M.

Baing

(2011). Calcium signaling in synapse-to-nucleus communicationI. Cold Spring Harbor Perspect Biol. Advance online publication. https://doi.org/10.1101/cshperspect.a004564

54.

Heimfarth

Delgado

Mignori

M. R.

Gelain

D. P.

Moreira

J. C. F.

Pessoa-Pureur

(2018). Developmental neurotoxicity of the hippocampus following in utero exposure to methylmercury: impairment in cell signaling. Archiv Toxicol, 92(1), 513–527. https://doi.org/10.1007/s00204-017-2042-6

55.

Heimfarth

Delgado

Mingori

M. R.

Moresco

K. S.

Pureur

R. P.

Gelain

D. P.

Moreira

J. C. F.

(2018). Delayed neurochemical effects of prenatal exposure to MeHg in the cerebellum of developing rats. Toxicol Lett, 284, 161–169. https://doi.org/10.1016/j.toxlet.2017.12.006

56.

Heinz

D. A.

Bloodgood

B. L.

(2020). Mechanisms that communicate features of neuronal activity to the genome. Curr Opin Neurobiol. Advance online publication. https://doi.org/10.1016/j.conb.2020.03.002

57.

Herdegen

Sandkühler

Gass

Kiessling

Bravo

Zimmermann

(1993). JUN, FOS, KROX, and CREB transcription factor proteins in the rat cortex: Basal expression and induction by spreading depression and epileptic seizures. J Compar Neurol. Advance online publication. https://doi.org/10.1002/cne.903330212

58.

Herreras

Somjen

G. G.

(1993). Effects of prolonged elevation of potassium on hippocampus of anesthetized rats. Brain Res. Advance online publication. https://doi.org/10.1016/0006-8993(93)91086-8

59.

Hunt

S. P.

Pini

Evan

(1988). Induction of c-fos-like protein in spinal cord neurons following sensory stimulation. Nature. Advance online publication. https://doi.org/10.1038/328632a0

60.

Jerome

Hou

Yuan

(2012). Interaction of NMDA receptors and L-type calcium channels during early odor preference learning in rats. Eur J Neurosci. Advance online publication. https://doi.org/10.1111/j.1460-9568.2012.08210.x

61.

Jing

Aitken

P. G.

Somjen

G. G.

(1991). Lasting neuron depression induced by high potassium and its prevention by low calcium and NMDA receptor blockade. Brain Res. Advance online publication. https://doi.org/10.1016/0006-8993(91)90132-F

62.

Kager

Wadman

W. J.

Somjen

G. G.

(2002). Conditions for the triggering of spreading depression studied with computer simulations. J Neurophysiol. Advance online publication. https://doi.org/10.1152/jn.00237.2002

63.

Kato

Gotoh

Kajikawa

Suto

(1998). Depolarization triggers intracellular magnesium surge in cultured dorsal root ganglion neurons. Brain Res. Advance online publication. https://doi.org/10.1016/S0006-8993(97)01232-8

64.

Kim

T. K.

Hemberg

Gray

J. M.

Costa

A. M.

Bear

D. M.

Harmin

D. A.

Laptewicz

Barbara-Haley

Kuersten

Markenscoff-Papadimitriou

Kuhl

Bito

Worley

P. F.

Kreiman

Greenberg

M. E.

(2010). Widespread transcription at neuronal activity-regulated enhancers. Nature. Advance online publication. https://doi.org/10.1038/nature09033

65.

Kingsbury

T. J.

Bambrick

L. L.

Roby

C. D.

Krueger

B. K.

(2007). Calcineurin activity is required for depolarization-induced, CREB-dependent gene transcription in cortical neurons. J Neurochem. Advance online publication. https://doi.org/10.1111/j.1471-4159.2007.04801.x

66.

Kobrinsky

(2015). Heterogeneity of calcium channel/cAMP-dependent transcriptional activation. Curr Mol Pharmacol. Advance online publication. https://doi.org/10.2174/1874467208666150507093601

67.

Koike

Martin

D. P.

Johnson

E. M.

(1989). Role of Ca2+ channels in the ability of membrane depolarization to prevent neuronal death induced by trophic-factor deprivation: Evidence that levels of internal Ca2+ determine nerve growth factor dependence of sympathetic ganglion cells. Proc Natl Acad Sci U S A. Advance online publication. https://doi.org/10.1073/pnas.86.16.6421

68.

Kostenko

M. A.

Tretjak

N. N.

Musienko

V. S.

(1982). The effect of elevated potassium on the adult mollusc giant neurone survival and neurite formation in culture. Brain Res. Advance online publication. https://doi.org/10.1016/0006-8993(82)90044-0

69.

Kovács

K. A.

Steullet

Steinmann

K. Q.

Magistretti

P. J.

Halfon

Cardinaux

J. R.

(2007). TORC1 is a calcium- and cAMP-sensitive coincidence detector involved in hippocampal long-term synaptic plasticity. Proc Natl Acad Sci U S A. Advance online publication. https://doi.org/10.1073/pnas.0607524104

70.

Kraio

R. P.

Nicholson

(1978). Extracellular ionic variations during spreading depression. Neuroscience. Advance online publication. https://doi.org/10.1016/0306-4522(78)90122-7

71.

Kruijer

Schubert

Verma

I. M.

(1985). Induction of the proto-oncogene fos by nerve growth factor. Proc Natl Acad Sci U S A. Advance online publication. https://doi.org/10.1073/pnas.82.21.7330

72.

Kubista

Boehm

Hotka

(2019). The paroxysmal depolarization shift: Reconsidering its role in epilepsy, epileptogenesis and beyond. Int J Mol Sci. Advance online publication. https://doi.org/10.3390/ijms20030577

73.

Kudo

Ogura

(1986). Glutamate‐induced increase in intracellular Ca2+ concentration in isolated hippocampal neurones. Br J Pharmacol. Advance online publication. https://doi.org/10.1111/j.1476-5381.1986.tb11135.x

74.

Kyrke-Smith

Williams

J. M.

(2018). Bridging synaptic and epigenetic maintenance mechanisms of the engram. Front Mol Neurosci. Advance online publication. https://doi.org/10.3389/fnmol.2018.00369

75.

Lasher

R. S.

Zagon

I. S.

(1972). The effect of potassium on neuronal differentiation in cultures of dissociated newborn rat cerebellum. Brain Res. Advance online publication. https://doi.org/10.1016/0006-8993(72)90521-5

76.

Leao

A. A. P.

(1944). Spreading depression of activity in the cerebral cortex. J Neurophysiol. Advance online publication. https://doi.org/10.1152/jn.1944.7.6.359

77.

Leslie

K. R.

Nelson

S. B.

Turrigiano

G. G.

(2001). Postsynaptic depolarization scales quantal amplitude in cortical pyramidal neurons. J Neurosci. Advance online publication. https://doi.org/10.1523/jneurosci.21-19-j0005.2001

78.

Zhang

Takemori

Zhou

Xiong

Z. Q.

(2009). TORC1 regulates activity-dependent CREB-target gene transcription and dendritic growth of developing cortical neurons. J Neurosci. Advance online publication. https://doi.org/10.1523/JNEUROSCI.2296-08.2009

79.

Lipscombe

Helton

T. D.

(2004). L-type calcium channels: The low down. J Neurophysiol. Advance online publication. https://doi.org/10.1152/jn.00486.2004

80.

Ludwar

B. C.

Weiss

K. R.

Cropper

E. C.

(2020). Background calcium induced by subthreshold depolarization modifies homosynaptic facilitation at a synapse in aplysia. Sci Rep. Advance online publication. https://doi.org/10.1038/s41598-019-57362-2

81.

Lyons

M. R.

Chen

L. F.

Deng

J. V.

Finn

Pfenning

A. R.

Sabhlok

Wilson

K. M.

West

A. E.

(2016). The transcription factor calcium-response factor limits NMDA receptor-dependent transcription in the developing brain. J Neurochem. Advance online publication. https://doi.org/10.1111/jnc.13556

82.

Lyons

M. R.

Schwarz

C. M.

West

A. E.

(2012). Members of the myocyte enhancer factor 2 transcription factor family differentially regulate Bdnf transcription in response to neuronal depolarization. J Neurosci. Advance online publication. https://doi.org/10.1523/JNEUROSCI.0534-12.2012

83.

Lyons

M. R.

West

A. E.

(2011). Mechanisms of specificity in neuronal activity-regulated gene transcription. Progr Neurobiol. Advance online publication. https://doi.org/10.1016/j.pneurobio.2011.05.003

84.

Madabhushi

Gao

Pfenning

A. R.

Pan

Yamakawa

Seo

Rueda

Phan

T. X.

Yamakawa

Pao

P. C.

Stott

R. T.

Gjoneska

Nott

Cho

Kellis

Tsai

L. H.

(2015). Activity-induced DNA breaks govern the expression of neuronal early-response genes. Cell. Advance online publication. https://doi.org/10.1016/j.cell.2015.05.032

85.

Malik

A. N.

Vierbuchen

Hemberg

Rubin

A. A.

Ling

Couch

C. H.

Stroud

Spiegel

Farh

K. K. H.

Harmin

D. A.

Greenberg

M. E.

(2014). Genome-wide identification and characterization of functional neuronal activity-dependent enhancers. Nature Neurosci. Advance online publication. https://doi.org/10.1038/nn.3808

86.

Marshall

W. H.

(1959). Spreading cortical depression of Leao. Physiol Rev. Advance online publication. https://doi.org/10.1152/physrev.1959.39.2.239

87.

Martel

M. A.

Ryan

T. J.

Bell

K. F. S.

Fowler

J. H.

McMahon

Al-Mubarak

Komiyama

N. H.

Horsburgh

Kind

P. C.

Grant

S. G. N.

Wyllie

D. J. A.

Hardingham

G. E.

(2012). The subtype of GluN2 C-terminal domain determines the response to excitotoxic insults. Neuron. Advance online publication. https://doi.org/10.1016/j.neuron.2012.03.021

88.

Martinowich

Hattori

Fouse

Fan

Sun

Y. E.

(2003). DNA methylation-related chromatin remodeling in activity-dependent Bdnf gene regulation. Science. Advance online publication. https://doi.org/10.1126/science.1090842

89.

Mattson

M. P.

Dou

Kater

S. B.

(1988). Outgrowth-regulating actions of glutamate in isolated hippocampal pyramidal neurons. J Neurosci. Advance online publication. https://doi.org/10.1523/jneurosci.08-06-02087.1988

90.

Michod

Bartesaghi

Khelifi

Bellodi

Berliocchi

Nicotera

Salomoni

(2012). Calcium-dependent dephosphorylation of the histone chaperone DAXX regulates H3.3 loading and transcription upon neuronal activation. Neuron. Advance online publication. https://doi.org/10.1016/j.neuron.2012.02.021

91.

Morgan

J. I.

Cohen

D. R.

Hempstead

J. L.

Curran

(1987). Mapping patterns of c-fos expression in the central nervous system after seizure. Science. Advance online publication. https://doi.org/10.1126/science.3037702

92.

Morgan

J. I.

Curran

(1986). Role of ion flux in the control of c-fos expression. Nature. Advance online publication. https://doi.org/10.1038/322552a0

93.

Mukherjee

Ignatowska-Jankowska

B. M.

Itskovits

Gonzales

B. J.

Turm

Izakson

Haritan

Bleistein

Cohen

Amit

Shay

Grueter

Zaslaver

Citri

(2018). Salient experiences are represented by unique transcriptional signatures in the mouse brain. ELife. Advance online publication. https://doi.org/10.7554/eLife.31220

94.

Murphy

S. N.

Thayer

S. A.

Miller

R. J.

(1987). The effects of excitatory amino acids on intracellular calcium in single mouse striatal neurons in vitro. J Neurosci. Advance online publication. https://doi.org/10.1523/jneurosci.07-12-04145.1987

95.

Navakkode

Liu

Soong

T. W.

(2018). Altered function of neuronal L-type calcium channels in ageing and neuroinflammation: Implications in age-related synaptic dysfunction and cognitive decline. Ageing Res Rev. Advance online publication. https://doi.org/10.1016/j.arr.2018.01.001

96.

Nicholson

(1984). Comparative neurophysiology of spreading depression in the cerebellum. An Acad Bras Cienc.

97.

Nonaka

Kim

Fukushima

Sasaki

Suzuki

Okamura

Ishii

Kawashima

Kamijo

Takemoto-Kimura

Okuno

Kida

Bito

(2014). Region-specific activation of CRTC1-CREB signaling mediates long-term fear memory. Neuron. Advance online publication. https://doi.org/10.1016/j.neuron.2014.08.049

98.

Pastuzyn

E. D.

Day

C. E.

Kearns

R. B.

Kyrke-Smith

Taibi

A. V.

McCormick

Yoder

Belnap

D. M.

Erlendsson

Morado

D. R.

Briggs

J. A. G.

Feschotte

Shepherd

J. D.

(2018). The neuronal gene arc encodes a repurposed retrotransposon Gag protein that mediates intercellular RNA transfer. Cell. Advance online publication. https://doi.org/10.1016/j.cell.2017.12.024

99.

Ploski

J. E.

Pierre

V. J.

Smucny

Park

Monsey

M. S.

Overeem

K. A.

Schafe

G. E.

(2008). The activity-regulated cytoskeletal-associated protein (Arc/Arg3.1) is required for memory consolidation of pavlovian fear conditioning in the lateral amygdala. J Neurosci. Advance online publication. https://doi.org/10.1523/JNEUROSCI.1662-08.2008

100.

Poston

R. G.

Murphy

Rejepova

Ghaninejad-Esfahani

Segales

Mulligan

Saha

R. N.

(2020). Certain ortho-hydroxylated brominated ethers are promiscuous kinase inhibitors that impair neuronal signaling and neurodevelopmental processes. J Biol Chem. Advance online publication. https://doi.org/10.1074/jbc.RA119.011138

101.

Pruunsild

Sepp

Orav

Koppel

Timmusk

(2011). Identification of cis-elements and transcription factors regulating neuronal activity-dependent transcription of human BDNF gene. J Neurosci. Advance online publication. https://doi.org/10.1523/JNEUROSCI.4540-10.2011

102.

Qiu

, et al. (2016). Evidence for evolutionary divergence of activity-dependent gene expression in developing neurons. ELife. Advance online publication. https://doi.org/10.7554/eLife.20337

103.

Rajadhyaksha

A. M.

Kosofsky

B. E.

(2005). Psychostimulants, L-type calcium channels, kinases, and phosphatases. Neuroscientist. Advance online publication. https://doi.org/10.1177/1073858405278236

104.

Redmond

Kashani

A. H.

Ghosh

(2002). Calcium regulation of dendritic growth via CaM kinase IV and CREB-mediated transcription. Neuron. Advance online publication. https://doi.org/10.1016/s0896-6273(02)00737-7

105.

Richter

Lehmenkühler

(2008). Cortical spreading depression (CSD): a neurophysiological correlate of migraine aura. Schmerz, 22(5): 544–546, 548–550. https://doi.org/10.1007/s00482-008-0653-9

106.

Rienecker

K. D. A.

Poston

R. G.

Segales

J. S.

Saha

R. N.

(2020). Mild membrane depolarization in neurons induces immediate early gene transcription and acutely subdues responses to successive stimulus. BioRxiv 2020. Advance online publication. https://doi.org/10.1101/2020.07.23.217844

107.

Roca-Lapirot

Radwani

Aby

Nagy

Landry

Fossat

(2018). Calcium signalling through L-type calcium channels: role in pathophysiology of spinal nociceptive transmission. Br J Pharmacol. Advance online publication. https://doi.org/10.1111/bph.13747

108.

Rothman

S. M.

(1985). The neurotoxicity of excitatory amino acids is produced by passive chloride influx. J Neurosci. Advance online publication. https://doi.org/10.1523/jneurosci.05-06-01483.1985

109.

Rothman

S. M.

Thurston

J. H.

Hauhart

R. E.

(1987). Delayed neurotoxicity of excitatory amino acids in vitro. Neuroscience. Advance online publication. https://doi.org/10.1016/0306-4522(87)90347-2

110.

Saha

R. N.

Wissink

E. M.

Bailey

E. R.

Zhao

Fargo

D. C.

Hwang

J. Y.

Daigle

K. R.

Fenn

J. D.

Adelman

Dudek

S. M.

(2011). Rapid activity-induced transcription of Arc and other IEGs relies on poised RNA polymerase II. Nature Neurosci. Advance online publication. https://doi.org/10.1038/nn.2839

111.

Schafe

G. E.

Atkins

C. M.

Swank

M. W.

Bauer

E. P.

Sweatt

J. D.

Ledoux

J. E.

(2000). Activation of ERK/MAP kinase in the amygdala is required for memory consolidation of Pavlovian fear conditioning. J Neurosci. Advance online publication. https://doi.org/10.1523/jneurosci.20-21-08177.2000

112.

Schmitt

J. M.

Guire

E. S.

Saneyoshi

Soderling

T. R.

(2005). Calmodulin-dependent kinase kinase/calmodulin kinase I activity gates extracellular-regulated kinase-dependent long-term potentiation. J Neurosci. Advance online publication. https://doi.org/10.1523/JNEUROSCI.4086-04.2005

113.

Schmitt

J. M.

Wayman

G. A.

Nozaki

Soderling

T. R.

(2004). Calcium activation of ERK mediated by calmodulin kinase I. J Biol Chem. Advance online publication. https://doi.org/10.1074/jbc.M401501200

114.

Shinnick-Gallagher

McKernan

M. G.

Xie

Zinebi

(2003). L-type voltage-gated calcium channels are involved in the in vivo and in vitro expression of fear conditioning. Ann N Y Acad Sci, 985(1), 135–149. https://doi.org/10.1111/j.1749-6632.2003.tb07078.x

115.

Silver

I. A.

Erecńska

(1990). Intracellular and extracellular changes of [Ca2+] in hypoxia and ischemia in rat brain in vivo. J Gen Physiol. Advance online publication. https://doi.org/10.1085/jgp.95.5.837

116.

Somjen

G. G.

(2001). Mechanisms of spreading depression and hypoxic spreading depression-like depolarization. Physiol Rev. Advance online publication. https://doi.org/10.1152/physrev.2001.81.3.1065

117.

Somjen

G. G.

(2002). Ion regulation in the brain: Implications for pathophysiology. Neuroscientist. Advance online publication. https://doi.org/10.1177/1073858402008003011

118.

Tao

Finkbeiner

Arnold

D. B.

Shaywitz

A. J.

Greenberg

M. E.

(1998). Ca2+ influx regulates BDNF transcription by a CREB family transcription factor-dependent mechanism. Neuron. Advance online publication. https://doi.org/10.1016/S0896-6273(00)81010-7

119.

Tao

West

A. E.

Chen

W. G.

Corfas

Greenberg

M. E.

(2002). A calcium-responsive transcription factor, CaRF, that regulates neuronal activity-dependent expression of BDNF. Neuron. Advance online publication. https://doi.org/10.1016/S0896-6273(01)00561-X

120.

Tao-Cheng

J. H.

(2018). Activity-dependent decrease in contact areas between subsurface cisterns and plasma membrane of hippocampal neurons. Mol Brain. Advance online publication. https://doi.org/10.1186/s13041-018-0366-7

121.

Tao-Cheng

J. H.

(2018). Stimulation-induced structural changes at the nucleus, endoplasmic reticulum and mitochondria of hippocampal neurons. Mol Brain. Advance online publication. https://doi.org/10.1186/s13041-018-0387-2

122.

Turrigiano

G. G.

Nelson

S. B.

(2004). Homeostatic plasticity in the developing nervous system. Nature Rev Neurosci. Advance online publication. https://doi.org/10.1038/nrn1327

123.

Tyssowski

K. M.

DeStefino

N. R.

Cho

J. H.

Dunn

C. J.

Poston

R. G.

Carty

C. E.

Jones

R. D.

Chang

S. M.

Romeo

Wurzelmann

M. K.

Ward

J. M.

Andermann

M. L.

Saha

R. N.

Dudek

S. M.

Gray

J. M.

(2018). Different neuronal activity patterns induce different gene expression programs. Neuron. Advance online publication. https://doi.org/10.1016/j.neuron.2018.04.001

124.

Uchida

Teubner

B. J. W.

Hevi

Hara

Kobayashi

Dave

R. M.

Shintaku

Jaikhan

Yamagata

Suzuki

Watanabe

Zakharenko

S. S.

Shumyatsky

G. P.

(2017). CRTC1 nuclear translocation following learning modulates memory strength via exchange of chromatin remodeling complexes on the Fgf1 gene. Cell Rep. Advance online publication. https://doi.org/10.1016/j.celrep.2016.12.052

125.

Wakade

A. R.

Thoenen

(1984). Interchangeability of nerve growth factor and high potassium in the long-term survival of chick sympathetic neurons in serum-free culture medium. Neurosci Lett. Advance online publication. https://doi.org/10.1016/0304-3940(84)90331-8

126.

Wayman

G. A.

Impey

Marks

Saneyoshi

Grant

W. F.

Derkach

Soderling

T. R.

(2006). Activity-dependent dendritic arborization mediated by CaM-kinase I activation and enhanced CREB-dependent transcription of Wnt-2. Neuron. Advance online publication. https://doi.org/10.1016/j.neuron.2006.05.008

127.

West

A. E.

Chen

W. G.

Dalva

M. B.

Dolmetsch

R. E.

Kornhauser

J. M.

Shaywitz

A. J.

Takasu

M. A.

Tao

Greenberg

M. E.

(2001). Calcium regulation of neuronal gene expression. Proc Natl Acad Sci U S A. Advance online publication. https://doi.org/10.1073/pnas.191352298

128.

West

A. E.

Greenberg

M. E.

(2011). Neuronal activity-regulated gene transcription in synapse development and cognitive function. Cold Spring Harbor Perspect Biol. Advance online publication. https://doi.org/10.1101/cshperspect.a005744

129.

West

A. E.

Griffith

E. C.

Greenberg

M. E.

(2002). Regulation of transcription factors by neuronal activity. Nature Rev Neurosci. Advance online publication. https://doi.org/10.1038/nrn987

130.

Wheeler

D. G.

Barrett

C. F.

Groth

R. D.

Safa

Tsien

R. W.

(2008). CaMKII locally encodes L-type channel activity to signal to nuclear CREB in excitation - transcription coupling. J Cell Biol. Advance online publication. https://doi.org/10.1083/jcb.200805048

131.

Xia

Dudek

Miranti

C. K.

Greenberg

M. E.

(1996). Calcium influx via the NMDA receptor induces immediate early gene transcription by a MAP kinase/ERK-dependent mechanism. J Neurosci. Advance online publication. https://doi.org/10.1523/jneurosci.16-17-05425.1996

132.

Zafra

Hengerer

Leibrock

Thoenen

Lindholm

(1990). Activity dependent regulation of BDNF and NGF mRNAs in the rat hippocampus is mediated by non-NMDA glutamate receptors. EMBO J. Advance online publication. https://doi.org/10.1002/j.1460-2075.1990.tb07564.x

133.

Zakharov

Chernova

Burkhanova

Holmes

G. L.

Khazipov

(2019). Segregation of seizures and spreading depolarization across cortical layers. Epilepsia. Advance online publication. https://doi.org/10.1111/epi.16390

134.

Zhang

Levy

Kainz

Noseda

Jakubowski

Burstein

(2011). Activation of central trigeminovascular neurons by cortical spreading depression. Ann Neurol. Advance online publication. https://doi.org/10.1002/ana.22329