Decoding Arc transcription: a live-cell study of stimulation patterns and transcriptional output

  1. Hye Yoon Park1,2,3
  1. 1Department of Physics and Astronomy, Seoul National University, Seoul 08826, Republic of Korea
  2. 2Institute of Applied Physics, Seoul National University, Seoul 08826, Republic of Korea
  3. 3Department of Electrical and Computer Engineering, University of Minnesota, Minneapolis, Minnesota 55455, USA
  1. Corresponding author: hyp{at}umn.edu
 
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Abstract

Activity-regulated cytoskeleton-associated protein (Arc) plays a crucial role in synaptic plasticity, a process integral to learning and memory. Arc transcription is induced within a few minutes of stimulation, making it a useful marker for neuronal activity. However, the specific neuronal activity patterns that initiate Arc transcription have remained elusive due to the inability to observe mRNA transcription in live cells in real time. Using a genetically encoded RNA indicator (GERI) mouse model that expresses endogenous Arc mRNA tagged with multiple GFPs, we investigated Arc transcriptional activity in response to various electrical field stimulation patterns. The GERI mouse model was generated by crossing the Arc-PBS knock-in mouse, engineered with binding sites in the 3′ untranslated region (UTR) of Arc mRNA, and the transgenic mouse expressing the cognate binding protein fused to GFP. In dissociated hippocampal neurons, we found that the pattern of stimulation significantly affects Arc transcription. Specifically, theta-burst stimulation consisting of high-frequency (100 Hz) bursts delivered at 10 Hz frequency induced the highest rate of Arc transcription. Concurrently, the amplitudes of nuclear calcium transients also reached their peak with 10 Hz burst stimulation, indicating a correlation between calcium concentration and transcription. However, our dual-color single-cell imaging revealed that there were no significant differences in calcium amplitudes between Arc-positive and Arc-negative neurons upon 10 Hz burst stimulation, suggesting the involvement of other factors in the induction of Arc transcription. Our live-cell RNA imaging provides a deeper insight into the complex regulation of transcription by activity patterns and calcium signaling pathways.

Learning and memory are complex cognitive functions that are critical for the survival and adaptation of an organism. A central biological mechanism underlying these functions is synaptic plasticity, the ability of synapses to change their strength and efficiency in response to varying levels of neuronal activity (Ho et al. 2011; Magee and Grienberger 2020). This dynamic process is modulated by a complex set of molecular mechanisms. Among these, activity-dependent gene expression plays an indispensable role in the regulation of synaptic plasticity (Kandel 2001; Flavell and Greenberg 2008). The induction of specific genes following neuronal stimulation leads to the production of proteins that can restructure synaptic connections. As neuronal activity levels fluctuate, this gene expression adapts accordingly, fostering the creation of dynamic neural networks that shape in accordance with an organism's experiences. Proteins produced as a result of this activity-dependent gene expression can augment synaptic strength, form new synaptic connections, or prune unnecessary ones, thereby sculpting the neural architecture that forms the basis of learning and memory.

The onset of activity-dependent gene expression is primarily characterized by the activation of immediate-early genes (IEGs) (Sheng and Greenberg 1990; Minatohara et al. 2016). These genes exhibit a rapid and transient response to a wide range of cellular stimuli, including neuronal activity. Most IEGs encode proteins that act as transcription factors, influencing the expression of other genes and modulating neuronal function and synaptic plasticity. A notable exception is the activity-regulated cytoskeleton-associated protein (Arc/Arg3.1), a well-characterized IEG. Unlike most IEGs, Arc is an effector protein that acts directly at synapses (Zhang and Bramham 2020). Arc protein is involved in AMPA receptor endocytosis, changes in synaptic morphology, and regulation of actin dynamics, which collectively contribute to synaptic plasticity (Chowdhury et al. 2006; Nair et al. 2017; Zhang and Bramham 2020). However, despite its well-established role, understanding how different neuronal activity patterns influence Arc transcriptional dynamics has been limited by the difficulty of observing Arc mRNA transcription in real time in living cells.

Previously, transcription has been studied either by isolating RNA after breaking up the cells, or by in situ hybridization, which can localize specific gene transcripts to individual cells (Vera et al. 2016). However, the former does not recapitulate the complex interactions and the spatial relationships between the cells, and the latter requires fixation of the sample, which loses the dynamic information of the process. Most live-cell imaging methods rely on the use of exogenous reporter genes, which typically do not contain all cis-regulatory elements, raising doubts about the physiological relevance of the results.

To decipher the neuronal activity pattern that triggers Arc transcription, we used a genetically encoded RNA indicator (GERI) mouse model (Lee et al. 2022). This mouse model expresses endogenous Arc mRNA tagged with multiple GFPs, enabling real-time monitoring of Arc transcriptional dynamics in live neurons. In this study, we specifically focused on the effects of different electrical stimulation patterns on Arc transcription in dissociated hippocampal neurons. We also examined the link between Arc transcription and calcium transient amplitude, a factor implicated in numerous signaling pathways in synaptic plasticity. These results shed new light on the relationship between neuronal activity and transcriptional output in live neurons.

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Results

Imaging Arc mRNA transcription in live neurons

To visualize the transcription of Arc mRNA in live neurons, we used the specific binding of PP7 capsid protein (PCP) to the stem–loop structure of PP7-binding site (PBS). We used the PCP × PBS GERI mouse model, which was generated by crossing the Arc-PBS knock-in (KI) mouse (Das et al. 2018) with the PCP-GFP transgenic mouse (Lee et al. 2022). In the Arc-PBS KI mouse, 24 repeats of the PBS are knocked in the 3′ untranslated region (3′ UTR) of Arc mRNA (Fig. 1A, left; Das et al. 2018). In the PCP-GFP transgenic mouse, tandem PCP fused with tandem GFP is expressed under the human synapsin promoter (Fig. 1A, right; Lee et al. 2022). We cultured hippocampal neurons from PCP × PBS mice carrying homozygous Arc-PBS and heterozygous PCP-GFP transgenes (Fig. 1A). In these neurons, all endogenous Arc mRNAs are labeled with up to 48 GFPs with a relatively low PCP-GFP background (Fig. 1B; Lee et al. 2022). We have not found any substantial effect on viability, fertility, or behavior of the PCP × PBS mice. Additionally, we previously verified that PBS tagging and the binding of the PCP-GFP to PBS-tagged mRNA did not affect the transcriptional output or stability of Arc mRNA (Das et al. 2018).

Figure 1.
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Figure 1.

Arc-PBS tagging and electrical stimulation system for investigating Arc transcription in live neurons. (A) Schematic of the Arc-PBS gene with 24×PBS cassette inserted in the 3′-UTR region. Yellow boxes, UTR; black lines, introns; NLS, nuclear localization sequence. (B) Arc mRNAs labeled with 24×PBS repeats are transcribed from the template DNA, and PCP-GFP fusion proteins bind to the 24×PBS repeats. Pol II, RNA polymerase II. (C) Schematic of electrical stimulation system. (D) Schematic of TBS. Each burst consists of four biphasic pulses with 2 msec duration and 10 msec intervals (top). Each train consists of 10 bursts with 200 msec intervals (middle). TBS consists of 10 trains with 20 sec intervals (bottom). (E) Schematic of LFS. LFS consists of 900 pulses with 1 sec interval. (F) Time-lapse images of Arc transcription induced by TBS. Red arrowheads indicate transcription sites of Arc mRNA. Scale bar, 10 μm.

To stimulate neurons, we constructed a three-dimensional (3D) printed frame that holds two parallel platinum wires in close proximity to the neurons on a glass-bottom dish and applied electric fields (Fig. 1C). First, we applied stimulation patterns known to induce long-term potentiation (LTP) and long-term depression (LTD). For the LTP-inducing stimulus, we used theta-burst stimulation (TBS) pattern, which consisted of four pulses at 100 Hz in each burst, with a total of 10 bursts per train separated by intervals of 200 msec (Fig. 1D). For the LTD-inducing stimulus, we used low-frequency stimulation (LFS), comprising 900 pulses at 1 Hz (Fig. 1E). We found that both TBS (Fig. 1F; Supplemental Movie S1) and LFS induced de novo Arc transcription in hippocampal neuron cultures. Although the induction of LTP or LTD was not directly confirmed in our experimental setup, it is intriguing that Arc expression was induced by both stimulation patterns associated with LTP and LTD. Our results suggest that the electrical field stimulation patterns applied to cultured neurons elicit transcriptional responses associated with synaptic plasticity.

Calcium activity and Arc transcription induced by TBS and LFS

Since electrical stimulation induces calcium activity and triggers transcription of immediate-early genes, we monitored calcium activity using a ratiometric calcium indicator, Fura Red. In this study, we focused on the effects of nuclear calcium transients on the induction of Arc transcription. Fluorescence signals representing nuclear calcium transients were recorded during stimulation. The region of interest (ROI) in each cell was selected based on the nuclear GFP signal due to the nuclear localization sequence (NLS) in the tandem PCP fused with tandem GFP construct. We obtained the fluorescence intensity ratio, R = F405nm/F488nm, by dividing the emission at 405 nm excitation by the emission at 488 nm excitation (Fig. 2A,B). Figure 2C shows the average ΔR/R profile of the neurons stimulated by TBS (n = 19 cells from three independent neuron cultures). Each train of TBS elicited abrupt surges in calcium concentration. On the other hand, LFS resulted in an initial calcium spike followed by a steady calcium level throughout the stimulation period (Fig. 2D; n = 13 cells from three independent neuron cultures).

Figure 2.
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Figure 2.

Calcium activity elicited by TBS and LFS. (A) Fluorescence intensities of Fura Red in a neuron during 10 Hz burst stimulation; F405nm at 405 nm and F488nm at 488 nm excitation. (B) Calcium intensity ratio calculated by F405nm/F488nm. (C) Average ΔR/R profile of the cells stimulated by TBS (n = 19 cells from three independent neuron cultures). (D) Average ΔR/R profile of the cells stimulated by LFS (n = 13 cells from three independent neuron cultures).

We then examined Arc mRNA transcription after TBS or LFS in live neurons. The fraction of neurons showing one or two Arc transcription sites was highest at 12–24 min after the onset of either stimulation (Fig. 3A). This peak timing of Arc transcription is consistent with previous findings using TTX withdrawal (Saha et al. 2011; Das et al. 2018) or bicuculline treatment (Das et al. 2018) in cultured neurons, although slightly longer than those observed in vivo (<7 min) (Vazdarjanova et al. 2002; Miyashita et al. 2009; Lee et al. 2022). While there exist transcribing cells in basal condition due to spontaneous activities (Das et al. 2018), we found that both TBS and LFS rapidly induced de novo Arc transcription within ∼20 min (Fig. 3B). Because Arc transcription gradually decreased thereafter (Fig. 3A), we focused on the transcriptional activity within 30 min of stimulation. The neurons that showed transcription sites during this period are hereafter referred to as Arc+ cells. The average fractions of Arc+ cells were 33 ± 3% after TBS and 27 ± 3% after LFS, which were significantly higher than 13 ± 2% without any stimulation (negative control, NC) (Fig. 3C). The average onset time, the time to observe the first transcription site within 30 min, was ∼13 min for all three conditions (Fig. 3D). The duration of Arc transcription on-time was also similar (16–18 min) for all three conditions (Fig. 3E). Therefore, both TBS and LFS increased the probability of transcription activation, but not the onset time nor the duration of on-time.

Figure 3.
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Figure 3.

Transcription of Arc mRNA induced by TBS and LFS. (A) Time course of Arc transcription. Each point represents the fraction of cells that were transcribing Arc mRNA. (B) Time course of the cumulative fraction of de novo Arc transcription alleles. (C) Fraction of cells showing Arc transcription within 30 min of stimulation (n = 622 cells from four independent neuron cultures for TBS, n = 684 cells from four independent neuron cultures for LFS, n = 353 cells from three independent neuron cultures for NC, P < 0.0001 for one-way ANOVA test, (**) P < 0.01, (****) P < 0.0001, post hoc Tukey's t-tests). Imaging and stimulation started simultaneously at t = 0. TBS (10 stimulations of 5-Hz bursts) was given for 200 sec, and LFS (900 pulses at 1 Hz) was given for 900 sec. (D) Mean onset time of Arc transcription (n = 622 cells for TBS, n = 684 cells for LFS, n = 353 cells for NC. P = 0.9456 for one-way ANOVA test). (E) Mean on-time duration of Arc transcription (n = 622 cells for TBS, n = 684 cells for LFS, n = 353 cells for NC. P = 0.3855 for one-way ANOVA test). Error bars indicate standard error of mean (SEM).

Effect of electrical burst frequency on Arc transcription

Next, we sought to determine whether a specific frequency of the electrical bursts could significantly affect Arc transcription. We varied the interval between each burst (Fig. 1D, middle) from 50 msec to 1 sec, while keeping the other parameters of the burst stimulation constant. The average ΔR/R profiles at 1, 5, 10, 15, and 20 Hz burst stimulation (Fig. 4A–E) indicate that 10 Hz burst induced significantly higher calcium amplitude than the other frequencies at each stimulation train (Fig. 4F).

Figure 4.
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Figure 4.

Calcium activity elicited by electrical burst stimulation with various burst frequencies. (A–E) Average ΔR/R profiles during burst stimulation at the frequency of 1 Hz (A), 5 Hz (B), 10 Hz (C), 15 Hz (D), and 20 Hz (E). (F) Average peak value of ΔR/R at each frequency (n = 7–19 cells, from three independent neuron cultures for each experiment, P = 0.0002 for one-way ANOVA test, (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, post hoc Tukey's t-tests). Error bars indicate SEM.

We then examined the fractions of Arc transcribing cells (Fig. 5A) and cumulative de novo Arc transcription alleles (Fig. 5B) after the onset of each stimulation pattern. We found that 10 Hz burst stimulation also resulted in the highest Arc transcription rate compared to the other frequencies (Fig. 5A,B). All tested burst frequencies (1–20 Hz) induced Arc transcription in a greater proportion of neurons than the NC (no stimulation). Notably, we found a significantly higher proportion of Arc+ neurons after 10 Hz burst stimulation than any other stimulation frequency (Fig. 5C). However, the average onset time of Arc transcription was 12–14 min for all six conditions (Fig. 5D). These data suggest that there exists an optimal frequency for inducing Arc transcription. The results also highlight a correlated response in calcium amplitude and Arc transcription rate, both modulated by stimulation patterns.

Figure 5.
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Figure 5.

Transcription of Arc mRNA induced by the electrical burst stimulation with various burst frequencies. (A) Time course of Arc transcription. Each point represents the fraction of cells that were transcribing Arc mRNA at each time. (B) Cumulative fraction of de novo Arc transcription alleles. (C) Fraction of cells that transcribed Arc mRNA within 30 min after the onset of stimulation (n = 436–1096 cells, from three to six independent neural cultures, P < 0.0001 for one-way ANOVA test, (*) P < 0.05, (***) P < 0.001, (****) P < 0.0001, post hoc Tukey's t-tests). (D) Mean onset time of Arc transcription (n = 436–1096 cells, P = 0.0086 for one-way ANOVA test, (*) P < 0.05, (**) P < 0.01, post hoc Tukey's t-tests). Error bars indicate SEM.

Effect of CREB phosphorylation on Arc transcription

Given the similar trends observed in calcium amplitude and Arc transcription at the population level, we sought to determine whether the amplitude of the calcium transient governs Arc transcription at the single-cell level. To do this, we imaged both Arc transcription and calcium activity in the same cells (Fig. 6A), whereas in the previous experiments, we imaged calcium and transcriptional activity separately. We began with recording baseline images of Arc mRNA in a neuron. Next, we imaged calcium signals of the neuron using Fura Red during 10 Hz burst stimulation. Lastly, we proceeded to image Arc mRNA in the neuron every 1 min after stimulation. Figure 6B,C shows time-lapse images of a representative Arc+ and Arc− cell, respectively. The average ΔR/R profiles of the Arc+ and Arc− neurons are shown in Figure 6D and E, respectively. The average peak ΔR/R values were similar for Arc+ and Arc− neurons (Fig. 6F). Therefore, we did not find a significant relationship between the calcium amplitude and Arc transcription at the single-cell level.

Figure 6.
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Figure 6.

Calcium activity in Arc+ and Arc− cells. (A) Experimental scheme to record calcium activity and Arc transcription. (B) Time-lapse images of an Arc+ cell. Red arrowheads indicate transcription sites of Arc mRNA. (C) Time-lapse images of an Arc− cell. (D) Average ΔR/R profile of Arc+ cells (n = 10 cells from three independent neuron cultures). (E) Average ΔR/R profile of Arc− cells (n = 17 cells from three independent neuron cultures). (F) Average peak ΔR/R values of the Arc+ and Arc− cells. Scale bars, 10 μm. Error bars indicate SEM.

Next, we examined another key component of the signaling pathway—the cAMP response element-binding protein (CREB). CREB is a well-characterized transcription factor that regulates activity-dependent gene expression. Neuronal activity triggers a signaling cascade that leads to rapid phosphorylation of CREB and its binding to the cAMP response element (CRE) present in the promoter of many activity-regulated genes. Once bound to CRE, phosphorylated CREB (pCREB) recruits other proteins to form a transcription complex, facilitating initiation of target gene transcription (Bito et al. 1996; Hardingham et al. 2001; West et al. 2001). To detect any differences in CREB expression levels between Arc+ and Arc− neurons, we performed immunofluorescence (IF) and RNA fluorescence in situ hybridization (FISH) on neurons that were fixed after 10 Hz burst stimulation. Figure 7A shows a representative image of Arc− (left) and Arc+ (right) neurons expressing similar levels of CREB (magenta). Neurons were sorted into Arc+ and Arc− cells by the presence of one or two bright spots labeled with FISH probes (green) targeting the Arc coding sequence (Table 1) in the nucleus (blue). There was no significant difference in the mean intensity of CREB signal between Arc+ cells with one or two Arc transcription sites and Arc− cells (Fig. 7B,C). There was no correlation between the CREB signal and the intensity of Arc transcription sites (Fig. 7D). However, as shown in the representative image in Figure 7E, Arc+ cells exhibited higher levels of pCREB than Arc− cells (Fig. 7F). Also, cells with two Arc transcription sites exhibited higher levels of pCREB than the cells with one Arc transcription site or Arc− cells (Fig. 7G). However, there was no correlation between pCREB intensity and Arc transcription site intensity (Fig. 7H). These results suggest that the on-rate of Arc transcription is correlated with the phosphorylation rate of CREB rather than the total CREB expression level.

Figure 7.
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Figure 7.

CREB and pCREB levels in Arc+ and Arc− cells. (A,E) Representative multichannel IF-smFISH image of CREB (A) or pCREB (E) protein (magenta), Arc mRNA (green), and DAPI (blue). Yellow arrowheads indicate transcription sites of Arc mRNA. (B) CREB intensity of Arc+ and Arc− cells (n = 346 for Arc+ cells, n = 279 for Arc− cells). (C) CREB intensity in neurons with 0, 1, and 2 Arc transcription sites (P = 0.1264 for one-way ANOVA test). (D) Scatter plot of CREB intensity and Arc transcription site intensity (n = 346 cells, r2 = 0.04469). (F) pCREB intensity of Arc+ and Arc− cells (n = 210 for Arc+ cells, n = 269 for Arc− cells, (****) P < 0.0001, two-tailed t-test). (G) pCREB intensity and the number of Arc transcription alleles (P < 0.0001 for one-way ANOVA test, (****) P < 0.0001, post hoc Tukey's t-tests). (H) Scatter plot of pCREB intensity and Arc transcription site intensity (n = 269 cells, r2 = 0.01987). Scale bars, 10 μm.

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Table 1.

Sequence of the probes used for FISH against Arc coding sequence. Probe sets were modified at both 5′ and 3′ ends for conjugation to Quasar 570 dyes

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Discussion

In this study, we investigated the transcriptional response of neurons following diverse patterns of electrical stimulation. Using dissociated hippocampal neurons cultured from the GERI mouse model (Lee et al. 2022), we monitored transcriptional activity of endogenous Arc gene in live neurons. This approach allowed us to analyze hundreds of individual cells, yielding highly quantitative and statistical information about Arc transcription.

The role of Arc in synaptic plasticity and long-term memory formation has been the subject of extensive research. Several studies have demonstrated the role of Arc in the LTP induction and maintenance, as evidenced by impaired LTP in an Arc knockout (KO) animal (Plath et al. 2006) or after infusion of Arc antisense oligodeoxynucleotides (Guzowski et al. 2000; Messaoudi et al. 2007). It has also been shown that Arc is selectively expressed in neurons that show increased correlated activity during chemically induced LTP (Jiang and VanDongen 2021). Interestingly, recent research has presented conflicting results, suggesting that LTP is unaffected in Arc KO animals (Kyrke-Smith et al. 2021). While uncertainty remains regarding the precise role of Arc in LTP, it has been established that Arc is crucial for LTD through the endocytosis of AMPAR receptors (Chowdhury et al. 2006; Park et al. 2008; Waung et al. 2008), implicating Arc in the regulation of Hebbian synaptic plasticity. Furthermore, evidence supports the idea that Arc plays a role in stabilizing synaptic responses, thereby influencing homeostatic plasticity (Shepherd et al. 2006; Gao et al. 2010; Peebles et al. 2010).

Since Arc is involved in multiple forms of synaptic plasticity, we first investigated whether the stimulation patterns known to induce either LTP or LTD could trigger Arc transcription in dissociated neurons. High-frequency stimulation (HFS) and TBS are known to induce LTP (Bliss and Lomo 1973; Larson et al. 1986; Capocchi et al. 1992), whereas LFS typically results in LTD (Dudek and Bear 1992). Past studies indicated that while HFS or TBS could trigger Arc transcription, LFS fails to do so in the dentate gyrus in vivo (Link et al. 1995; Lyford et al. 1995; Steward et al. 1998; Waltereit et al. 2001). In the CA1 region, it was shown that LTP- and LTD-inducing stimulations lead to opposite changes in Arc mRNA levels in vivo (Yilmaz-Rastoder et al. 2011). The authors applied paired-pulse stimulation at 0.5 Hz to dorsal CA3 commissural fibers and observed LTD induction in CA1 neurons. However, Arc mRNA levels initially decreased and then displayed a transient increase after 70 min relative to control levels.

Our study, focusing on dissociated hippocampal neurons, found an increase in Arc transcription rates in response to both TBS and LFS. While we did not directly confirm the induction of LTP or LTD, previous studies have reported supportive evidence of LTP or LTD induction following field stimulations in cultured hippocampal neurons (Deisseroth et al. 1996; Balkowiec and Katz 2002; Gärtner and Staiger 2002). We found a significant surge in the number of neurons showing Arc transcription sites within 30 min of either TBS or LFS stimulation, compared to the NC (Fig. 3). It is interesting to note that LFS induced immediate early transcription of Arc mRNA in neuron cultures, in contrast to previous studies conducted in vivo (Link et al. 1995; Lyford et al. 1995; Steward et al. 1998; Waltereit et al. 2001). It remains to be determined whether LFS elicits a differential effect on Arc expression between neuron cultures and in vivo conditions.

We then focused on high-frequency burst stimulation and investigated how Arc transcription and calcium amplitude are modulated by different bursting frequencies. Interestingly, we found that the proportion of Arc+ cells varied with different stimulation patterns, with the highest Arc transcription rate observed at 10 Hz burst stimulation (Fig. 5). This frequency falls within the theta oscillation range in rodents (6–10 Hz) (Buzsáki et al. 2013). This result is also in accordance with a previous study that found higher theta-burst activity in Arc+ cells in vivo (Lee et al. 2022). The highest Arc transcription and calcium amplitude at this specific burst frequency could be attributed to the cumulative effect of both excitatory and inhibitory influences from repeated burst stimulation (Larson and Munkacsy 2015).

Our observations suggesting a correlation between Arc transcription and nuclear calcium concentration led us to hypothesize a potential difference in calcium amplitude between Arc+ and Arc− cells. However, when stimulated at the optimal burst frequency of 10 Hz, our results showed no distinguishable difference in the amplitude of calcium transients between Arc+ and Arc− cells (Fig. 6F). This outcome suggests that factors beyond calcium transients also substantially contribute to inducing Arc transcription. One notable factor is the CREB protein, which is known for its role in activity-dependent gene expression (Bito et al. 1996; Mayr and Montminy 2001; Kornhauser et al. 2002; Lonze and Ginty 2002). CREB undergoes phosphorylation by various protein kinases, such as protein kinase A (PKA), extracellular signal-regulated kinase (ERK), and calcium/calmodulin-dependent protein kinase IV (CaMKIV) (Benito and Barco 2010). Upon phosphorylation, CREB binds to the synaptic activity-responsive element (SARE) in the enhancer region of the Arc gene, triggering Arc transcription (Kawashima et al. 2009). In our study, we found significantly higher levels of pCREB but not total CREB expression in Arc+ neurons compared to Arc− neurons (Fig. 7). Previous studies have demonstrated that activity-dependent Arc expression requires CREB phosphorylation, as evidenced by experiments using a selective inhibitor (Chen et al. 2017) or a dominant negative form of CREB (Pulimood et al. 2021). These findings, along with our results indicating correlations between pCREB levels and Arc transcription, suggest that the activation of CREB phosphorylation, possibly influenced by factors beyond calcium transients, further modulates Arc transcription. While some studies have shown that nuclear calcium transients regulate CREB phosphorylation and IEG transcription (Hardingham et al. 2001; Yu et al. 2017), other studies have suggested the importance of submembrane and cytosolic calcium transients to activate other intracellular pathways to initiate IEG transcription (Deisseroth et al. 1996; Nonaka et al. 2014; Colgan et al. 2023). In this study, we focused on the nuclear calcium signaling, but the effects of the calcium transients in other parts of the neurons could also be investigated in the future.

Since our experiments were performed in a cultured neuronal environment, it is worthwhile to compare our in vitro results with those of previous in vivo studies. Most in vivo studies have been performed using RNA FISH to examine Arc transcription immediately after animal behavioral experiments (Guzowski et al. 1999, 2005). Vazdarjanova et al. (2002) reported a significant increase in the percentage of neurons with Arc transcription foci at 0 and 8 min after novel environment exploration (5 and 13 min after the onset of stimulation). Therefore, animals were typically sacrificed ∼5 min after the start of the behavioral session, and RNA FISH was performed on brain slices (Miyashita et al. 2009). Our in vivo imaging results using GERI mice also showed that the percentage of neurons with Arc transcription sites peaked within 7 min after contextual fear conditioning experiments (Lee et al. 2022). However, in cultured neurons, we observed peak Arc transcription at 12–24 min after electrical stimulation, which was consistent with previous findings using TTX withdrawal or bicuculline treatment (Saha et al. 2011; Das et al. 2018). The observed discrepancy in the timing of peak Arc transcription between neuron culture experiments (12–30 min) and in vivo experiments (<7 min) could arise from several factors. For instance, cultured neurons lack the complex environment present in vivo, including interactions with other cell types and extracellular matrix components. These interactions can influence the timing and magnitude of Arc transcription in response to neuronal activity. In addition, cultured neurons tend to exhibit relatively high intrinsic firing, and the repeated periods of activation could reduce the ability to activate Arc transcription (Guzowski et al. 2006).

While the regulatory mechanisms governing Arc transcription may differ between cultured neurons and in vivo conditions, we used cultured neurons in this study for several reasons. First, cultured neurons provide a controlled experimental environment allowing systematic investigations into the effects of specific neuronal activity patterns. Second, culture neurons offer a simplified model system compared to intact tissue, allowing the identification of causal relationships between neuronal activity patterns and Arc expression. Finally, cultured neuron experiments enable high temporal resolution imaging, allowing for precise characterization of transcription in response to neuronal stimulation. These advantages of the in vitro neuron culture system allowed us to investigate Arc transcriptional dynamics upon various electrical stimulation patterns in this work.

The analysis on expression levels and spatial dynamics of Arc mRNA could be extended to various scales and regions using the Arc-PBS KI mouse model (Das et al. 2018). One of the recent studies has analyzed the transcriptional dynamics of single Arc mRNA in a time-resolved manner (Choi et al. 2022). By tracking Arc mRNA particles in the dendrites, other recent studies have revealed localization and transport dynamics of Arc mRNA (Das et al. 2018, 2023; Ahn et al. 2023). In addition, synaptic stimulation in acute brain slices showed detailed aspects of Arc transcription ex vivo (Lituma et al. 2022). Since GERI imaging offers sensitive detection of pre-mRNA synthesis in individual neurons both in culture and in vivo (Lee et al. 2022), future studies using GERI in conjunction with electrophysiology or voltage imaging could provide important insights into the relationship between Arc transcription and electrical activity at the single-cell level.

In conclusion, our findings have provided insights into the dynamic nature of Arc transcription in response to different neuronal stimulation patterns. We have established that Arc transcription is sensitive to the type of stimulation and the amplitude of calcium transients and is further regulated by other signaling pathways such as CREB phosphorylation. These results highlight the complexity of the mechanisms underlying activity-dependent gene expression and its potential implications for long-term synaptic plasticity. Although our study was carried out using dissociated neurons in a controlled environment, the principles revealed could provide a foundation for further studies investigating the relationship between neuronal activity and gene expression in vivo. Future research could benefit from exploring different mRNA species (Park et al. 2014; Lee et al. 2022) and brain regions in live mouse models, advancing our understanding of the intricate interplay between neuronal activity, gene expression, and synaptic plasticity.

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Materials and Methods

Hippocampal neuron preparation

All animal care and experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of Seoul National University. The day before neuronal culture, a glass-bottom dish (SPL 100350) was coated with poly-D-lysine (PDL) hydrobromide (Sigma-Aldrich P7886). The next day, hippocampi were dissected from the brains of 1-day-old Arc-PBS homozygous and PCP-GFP heterozygous mouse pups. The hippocampi were digested with 2.5% trypsin (Gibco 15090046), plated onto the PDL-coated glass-bottom dishes at a concentration of 420,000 cells/mL, and incubated at 37°C and 5% CO2 for ∼4 h until they adhered to the bottom of the dishes. Then, B27 media consisting of 1× B-27 supplement (Gibco 17504044), 1× GlutaMax supplement (Gibco 35050079), and 0.1 mg/mL Primocin (InvivoGen, ant-pm-1) in Neurobasal-A media (Gibco 10888022) was added to the dishes. Neurons were imaged between 16 and 18 days in vitro (DIV).

Loading cells with calcium indicator dye

Fura Red ratiometric calcium indicator (Invitrogen F3021) was used to monitor the intracellular calcium concentration of neurons. Before imaging, culture media were replaced with HEPES buffered saline (HBS) containing 119 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 30 mM D-glucose, and 20 mM HEPES (Gibco 15630-080) at pH 7.4. Neurons were incubated in HBS with 4 μM Fura Red and 0.04% Pluronic F-127 (Invitrogen P3000MP) for 30 min at 37°C. Neurons were washed twice with HBS and incubated for another 30 min at 37°C for de-esterification.

Electrical stimulation

Electrical stimulation was delivered using a 3D printed polylactic acid (PLA, Ultimaker) frame, which was designed to fit to the glass-bottom dish and hold two platinum wires close to the glass bottom. Each platinum wire was placed parallel to each other at a distance of 7.4 mm. Stimulation patterns were generated by an isolated pulse stimulator (A-M Systems 2100) using trigger signals from the Arduino Uno. A single pulse was a biphasic pulse of 2 msec duration and 3.7 V amplitude. LFS consisted of 900 pulses at a frequency of 1 Hz. Burst simulation consisted of 10 stimulations with an interval of 20 sec, and each stimulation consisted of 10 bursts of various frequencies. Each burst consisted of four pulses at a frequency of 100 Hz.

Live-cell imaging

To monitor Arc mRNA transcription, neurons were imaged by using an inverted microscope (Olympus IX83) equipped with an electron multiplying charge-coupled device (EMCCD) camera (Andor, iXon Ultra 888), 60×/1.3 NA silicon immersion objective (Olympus, UPLANSAPO60X), SOLA light-emitting diode (LED) Light Engine (Lumencor), motorized stage (Marzhauser), and Chamlide TC top stage incubator system (Live Cell Instruments). GFP signal was monitored with an ET-EGFP filter cube (Chroma 49002). Z-stack images were acquired at 0.5 μm intervals. Neurons were imaged at 3-min intervals for TBS/LFS experiments and at 90-sec intervals for frequency change experiments. For each experiment, neurons were imaged in the baseline condition before stimulation.

For real-time recording of intracellular calcium concentration, neurons were imaged using an inverted microscope (Olympus IX73) equipped with two EMCCD cameras (Andor, iXon Ultra 897), 60×/1.3 NA silicon immersion objective (Olympus, UPLANSAPO60X), MS-2000-500 XYZ automated stage (ASI), and Chamlide TC top stage incubator system (Live Cell Instruments). Calcium-bound Fura Red signals were obtained by excitation with a 405 nm diode laser (Cobolt), and calcium-free signals were obtained with a 488 nm diode laser (Cobolt). Each excitation laser was applied sequentially with an exposure time of 20 msec. Both emission signals were filtered with a 635/60 bandpass filter (Chroma). Calcium signals were recorded at 500 msec intervals. To compare the calcium signal between Arc+ and Arc− neurons, Arc mRNA signals were also observed immediately after calcium imaging. A 488 nm diode laser (Cobolt) was used to visualize Arc mRNA, and the fluorescence emission was filtered with a 525/50 bandpass filter (Chroma). Arc images were acquired at 60-sec intervals.

Arc transcription analysis

Imaging processing was performed using a similar approach described previously (Choi et al. 2022). Acquired z-stack images were projected to the maximum intensity profile at each time point. The mean intensity of a background region without cells was subtracted from the max-projected images. Transcription sites of Arc mRNA were tracked manually. When comparing Arc transcription rate between stimulation patterns, transcription sites activated before stimulation were excluded from the positive cell count. Cells showing transcription sites within 30 min of stimulation were considered positive cells. Time points at which transcription was initiated were also recorded, averaged across cells, and compared between stimulation patterns.

Calcium signal analysis

We corrected shading and background variations in the time-lapse images by using the BaSiC plugin (Peng et al. 2017) and performed exponential bleach correction and background subtraction using Fiji (Schindelin et al. 2012). We selected cells expressing nuclear GFP signals and segmented the nuclei of the cells as ROIs. The calcium intensity ratio R was calculated by F405nm/F488nm. The initial baseline of calcium intensity ratio R0 was calculated by averaging the bottom 50% of the first 50 frames, and the final baseline of the calcium intensity was calculated in the same way with the last 50 frames. For calcium intensity in Figure 6, the baseline was selected in the bottom 10% of the first or last 100 frames. R was adjusted by linearly fitting the final baseline to the initial baseline. ΔR/R was calculated as (R−R0)/R0. The calcium signal profile was obtained by averaging the ΔR/R of cells at each time point. The intensity of the peak signal elicited by each stimulation train was averaged per cell and compared between stimulation patterns.

IF-FISH

Neurons were stimulated with 10-Hz burst stimulation and fixed with 4% PFA (Electron Microscopy Sciences 15714) on ice 15 min after the onset of stimulation. After fixation, neurons were washed in phosphate buffered saline with 1 mM MgCl2 and 1 mM CaCl2 (PBS-MC) and quenched in PBS-MC with 50 mM glycine. Then neurons were permeabilized with 0.1% Triton X-100 on ice and washed three times with PBS-MC. Neurons were incubated with 10% formamide, 2× SSC, and 0.5% BSA (Sigma A7906) in RNase-free water for 30 min at room temperature for prehybridization and blocking. Neurons were hybridized with primary antibodies targeting CREB (Cell Signaling 9197S) and pCREB (Sigma 06-519), FISH probes targeting 20 sites in the open reading frame of the Arc gene (Stellaris; sequences in Table 1), 1 mg/mL tRNA (Sigma R1753), 10% dextran sulfate (Sigma D8906), 0.2 mg/mL BSA (Roche, 10711454001), 2× SSC, 2 mM vanadyl ribonucleoside complex (Sigma R3380), and 10 U/mL SUPERase-In (Invitrogen AM2694) in RNase-free water at 37°C for 3 h. After hybridization, neurons were washed twice with 10% formamide and 0.5% BSA in 2× SSC and incubated twice with goat anti-rabbit secondary antibody labeled with Alexa 647 (Invitrogen A21245), 10% formamide, and 0.5% BSA in 2× SSC at 37°C for 20 min each time. Neurons were washed four times with 2× SSC and stained with 0.5 µg/mL DAPI (Thermo Scientific 62248) in 2× SSC for 1 min. Neurons were washed with PBS-MC for 10 min and mounted with Prolong Gold (Invitrogen P10144).

Arc-CREB analysis

After IF-FISH, neurons were imaged using an inverted microscope (Olympus IX83) equipped with the same devices described above. Arc-CDS probe signal was monitored with an ET-Cy3/TRITC filter cube (Chroma 49004). The CREB/pCREB signal was monitored with an ET-Cy5 filter cube (Chroma 49006). DAPI signal was monitored using an ET-DAPI filter cube (Chroma 49000). The GFP signal was monitored using an ET-EGFP filter cube (Chroma 49002). Z-stack images were acquired at 0.5 μm intervals. After multichannel imaging, nuclear ROIs were selected based on DAPI and GFP signals. The number of Arc transcription sites in each cell was counted manually, and the mean CREB/pCREB intensity within the nuclear ROI was measured. Arc TXN intensity was calculated by dividing the intensity of the Arc transcription site by the average intensity of individual Arc mRNAs in the nucleus.

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Acknowledgments

This work was supported by the National Research Foundation (NRF) of Korea grant no. 2020R1A2C2007285 and a startup fund from the University of Minnesota.

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Footnotes

  • Received May 14, 2024.
  • Accepted August 5, 2024.

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first 12 months after the full-issue publication date (see http://learnmem.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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References

  1. ↵
    Ahn H, Durang X, Shim JY, Park G, Jeon JH, Park HY. 2023. Statistical modeling of mRNP transport in dendrites: a comparative analysis of β-actin and Arc mRNP dynamics. Traffic 24: 522–532. doi:10.1111/tra.12913
    CrossRefGoogle Scholar
  2. ↵
    Balkowiec A, Katz DM. 2002. Cellular mechanisms regulating activity-dependent release of native brain-derived neurotrophic factor from hippocampal neurons. J Neurosci 22: 10399–10407. doi:10.1523/JNEUROSCI.22-23-10399.2002
    Abstract/FREE Full Text
  3. ↵
    Benito E, Barco A. 2010. CREB's control of intrinsic and synaptic plasticity: implications for CREB-dependent memory models. Trends Neurosci 33: 230–240. doi:10.1016/j.tins.2010.02.001
    CrossRefMedlineWeb of ScienceGoogle Scholar
  4. ↵
    Bito H, Deisseroth K, Tsien RW. 1996. CREB phosphorylation and dephosphorylation: a Ca2+- and stimulus duration-dependent switch for hippocampal gene expression. Cell 87: 1203–1214. doi:10.1016/S0092-8674(00)81816-4
    CrossRefMedlineWeb of ScienceGoogle Scholar
  5. ↵
    Bliss TV, Lømo T. 1973. Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J Physiol 232: 331–356. doi:10.1113/jphysiol.1973.sp010273
    CrossRefMedlineWeb of ScienceGoogle Scholar
  6. ↵
    Buzsáki G, Logothetis N, Singer W. 2013. Scaling brain size, keeping timing: evolutionary preservation of brain rhythms. Neuron 80: 751–764. doi:10.1016/j.neuron.2013.10.002
    CrossRefMedlineWeb of ScienceGoogle Scholar
  7. ↵
    Capocchi G, Zampolini M, Larson J. 1992. Theta burst stimulation is optimal for induction of LTP at both apical and basal dendritic synapses on hippocampal CA1 neurons. Brain Res 591: 332–336. doi:10.1016/0006-8993(92)91715-Q
    CrossRefMedlineWeb of ScienceGoogle Scholar
  8. ↵
    Chen T, Zhu J, Yang LK, Feng Y, Lin W, Wang YH. 2017. Glutamate-induced rapid induction of Arc/Arg3.1 requires NMDA receptor-mediated phosphorylation of ERK and CREB. Neurosci Lett 661: 23–28. doi:10.1016/j.neulet.2017.09.024
    CrossRefMedlineGoogle Scholar
  9. ↵
    Choi H, Lee BH, Park HY. 2022. Time-resolved analysis of transcription kinetics in single live mammalian cells. Front Phys Lausanne 10: 977125. doi:10.3389/fphy.2022.977125
    CrossRefGoogle Scholar
  10. ↵
    Chowdhury S, Shepherd JD, Okuno H, Lyford G, Petralia RS, Plath N, Kuhl D, Huganir RL, Worley PF. 2006. Arc/Arg3.1 interacts with the endocytic machinery to regulate AMPA receptor trafficking. Neuron 52: 445–459. doi:10.1016/j.neuron.2006.08.033
    CrossRefMedlineWeb of ScienceGoogle Scholar
  11. ↵
    Colgan LA, Parra-Bueno P, Holman HL, Tu X, Jain A, Calubag MF, Misler JA, Gary C, Oz G, Suponitsky-Kroyter I, et al. 2023. Dual regulation of spine-specific and synapse-to-nucleus signaling by PKCδ during plasticity. J Neurosci 43: 5432–5447. doi:10.1523/JNEUROSCI.0208-22.2023
    Abstract/FREE Full Text
  12. ↵
    Das S, Moon HC, Singer RH, Park HY. 2018. A transgenic mouse for imaging activity-dependent dynamics of endogenous Arc mRNA in live neurons. Sci Adv 4: eaar3448. doi:10.1126/sciadv.aar3448
    FREE Full Text
  13. ↵
    Das S, Lituma PJ, Castillo PE, Singer RH. 2023. Maintenance of a short-lived protein required for long-term memory involves cycles of transcription and local translation. Neuron 111: 2051–2064.e6. doi:10.1016/j.neuron.2023.04.005
    CrossRefMedlineGoogle Scholar
  14. ↵
    Deisseroth K, Bito H, Tsien RW. 1996. Signaling from synapse to nucleus: postsynaptic CREB phosphorylation during multiple forms of hippocampal synaptic plasticity. Neuron 16: 89–101. doi:10.1016/S0896-6273(00)80026-4
    CrossRefMedlineWeb of ScienceGoogle Scholar
  15. ↵
    Dudek SM, Bear MF. 1992. Homosynaptic long-term depression in area CA1 of hippocampus and effects of N-methyl-D-aspartate receptor blockade. Proc Natl Acad Sci 89: 4363–4367. doi:10.1073/pnas.89.10.4363
    Abstract/FREE Full Text
  16. ↵
    Flavell SW, Greenberg ME. 2008. Signaling mechanisms linking neuronal activity to gene expression and plasticity of the nervous system. Annu Rev Neurosci 31: 563–590. doi:10.1146/annurev.neuro.31.060407.125631
    CrossRefMedlineWeb of ScienceGoogle Scholar
  17. ↵
    Gao M, Sossa K, Song L, Errington L, Cummings L, Hwang H, Kuhl D, Worley P, Lee HK. 2010. A specific requirement of Arc/Arg3.1 for visual experience-induced homeostatic synaptic plasticity in mouse primary visual cortex. J Neurosci 30: 7168–7178. doi:10.1523/JNEUROSCI.1067-10.2010
    Abstract/FREE Full Text
  18. ↵
    Gärtner A, Staiger V. 2002. Neurotrophin secretion from hippocampal neurons evoked by long-term-potentiation-inducing electrical stimulation patterns. Proc Natl Acad Sci 99: 6386–6391. doi:10.1073/pnas.092129699
    Abstract/FREE Full Text
  19. ↵
    Guzowski JF, McNaughton BL, Barnes CA, Worley PF. 1999. Environment-specific expression of the immediate-early gene Arc in hippocampal neuronal ensembles. Nat Neurosci 2: 1120–1124. doi:10.1038/16046
    CrossRefMedlineWeb of ScienceGoogle Scholar
  20. ↵
    Guzowski JF, Lyford GL, Stevenson GD, Houston FP, McGaugh JL, Worley PF, Barnes CA. 2000. Inhibition of activity-dependent arc protein expression in the rat hippocampus impairs the maintenance of long-term potentiation and the consolidation of long-term memory. J Neurosci 20: 3993–4001. doi:10.1523/JNEUROSCI.20-11-03993.2000
    Abstract/FREE Full Text
  21. ↵
    Guzowski JF, Timlin JA, Roysam B, McNaughton BL, Worley PF, Barnes CA. 2005. Mapping behaviorally relevant neural circuits with immediate-early gene expression. Curr Opin Neurobiol 15: 599–606. doi:10.1016/j.conb.2005.08.018
    CrossRefMedlineWeb of ScienceGoogle Scholar
  22. ↵
    Guzowski JF, Miyashita T, Chawla MK, Sanderson J, Maes LI, Houston FP, Lipa P, McNaughton BL, Worley PF, Barnes CA. 2006. Recent behavioral history modifies coupling between cell activity and Arc gene transcription in hippocampal CA1 neurons. Proc Natl Acad Sci 103: 1077–1082. doi:10.1073/pnas.0505519103
    Abstract/FREE Full Text
  23. ↵
    Hardingham GE, Arnold FJ, Bading H. 2001. Nuclear calcium signaling controls CREB-mediated gene expression triggered by synaptic activity. Nat Neurosci 4: 261–267. doi:10.1038/85109
    CrossRefMedlineWeb of ScienceGoogle Scholar
  24. ↵
    Ho VM, Lee JA, Martin KC. 2011. The cell biology of synaptic plasticity. Science 334: 623–628. doi:10.1126/science.1209236
    Abstract/FREE Full Text
  25. ↵
    Jiang Y, VanDongen AMJ. 2021. Selective increase of correlated activity in Arc-positive neurons after chemically induced long-term potentiation in cultured hippocampal neurons. eNeuro 8: ENEURO.0540-20.2021. doi:10.1523/ENEURO.0540-20.2021
    Abstract/FREE Full Text
  26. ↵
    Kandel ER. 2001. The molecular biology of memory storage: a dialogue between genes and synapses. Science 294: 1030–1038. doi:10.1126/science.1067020
    Abstract/FREE Full Text
  27. ↵
    Kawashima T, Okuno H, Nonaka M, Adachi-Morishima A, Kyo N, Okamura M, Takemoto-Kimura S, Worley PF, Bito H. 2009. Synaptic activity-responsive element in the Arc/Arg3.1 promoter essential for synapse-to-nucleus signaling in activated neurons. Proc Natl Acad Sci 106: 316–321. doi:10.1073/pnas.0806518106
    Abstract/FREE Full Text
  28. ↵
    Kornhauser JM, Cowan CW, Shaywitz AJ, Dolmetsch RE, Griffith EC, Hu LS, Haddad C, Xia Z, Greenberg ME. 2002. CREB transcriptional activity in neurons is regulated by multiple, calcium-specific phosphorylation events. Neuron 34: 221–233. doi:10.1016/S0896-6273(02)00655-4
    CrossRefMedlineWeb of ScienceGoogle Scholar
  29. ↵
    Kyrke-Smith M, Volk LJ, Cooke SF, Bear MF, Huganir RL, Shepherd JD. 2021. The immediate early gene Arc is not required for hippocampal long-term potentiation. J Neurosci 41: 4202–4211. doi:10.1523/JNEUROSCI.0008-20.2021
    Abstract/FREE Full Text
  30. ↵
    Larson J, Munkácsy E. 2015. Theta-burst LTP. Brain Res 1621: 38–50. doi:10.1016/j.brainres.2014.10.034
    CrossRefMedlineGoogle Scholar
  31. ↵
    Larson J, Wong D, Lynch G. 1986. Patterned stimulation at the theta frequency is optimal for the induction of hippocampal long-term potentiation. Brain Res 368: 347–350. doi:10.1016/0006-8993(86)90579-2
    CrossRefMedlineWeb of ScienceGoogle Scholar
  32. ↵
    Lee BH, Shim JY, Moon HC, Kim DW, Kim J, Yook JS, Kim J, Park HY. 2022. Real-time visualization of mRNA synthesis during memory formation in live mice. Proc Natl Acad Sci 119: e2117076119. doi:10.1073/pnas.2117076119
    CrossRefGoogle Scholar
  33. ↵
    Link W, Konietzko U, Kauselmann G, Krug M, Schwanke B, Frey U, Kuhl D. 1995. Somatodendritic expression of an immediate early gene is regulated by synaptic activity. Proc Natl Acad Sci 92: 5734–5738. doi:10.1073/pnas.92.12.5734
    Abstract/FREE Full Text
  34. ↵
    Lituma PJ, Singer RH, Das S, Castillo PE. 2022. Real-time imaging of Arc/Arg3.1 transcription ex vivo reveals input-specific immediate early gene dynamics. Proc Natl Acad Sci 119: e2123373119. doi:10.1073/pnas.2123373119
    CrossRefGoogle Scholar
  35. ↵
    Lonze BE, Ginty DD. 2002. Function and regulation of CREB family transcription factors in the nervous system. Neuron 35: 605–623. doi:10.1016/S0896-6273(02)00828-0
    CrossRefMedlineWeb of ScienceGoogle Scholar
  36. ↵
    Lyford GL, Yamagata K, Kaufmann WE, Barnes CA, Sanders LK, Copeland NG, Gilbert DJ, Jenkins NA, Lanahan AA, Worley PF. 1995. Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites. Neuron 14: 433–445. doi:10.1016/0896-6273(95)90299-6
    CrossRefMedlineWeb of ScienceGoogle Scholar
  37. ↵
    Magee JC, Grienberger C. 2020. Synaptic plasticity forms and functions. Annu Rev Neurosci 43: 95–117. doi:10.1146/annurev-neuro-090919-022842
    CrossRefMedlineGoogle Scholar
  38. ↵
    Mayr B, Montminy M. 2001. Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol 2: 599–609. doi:10.1038/35085068
    CrossRefMedlineWeb of ScienceGoogle Scholar
  39. ↵
    Messaoudi E, Kanhema T, Soule J, Tiron A, Dagyte G, da Silva B, Bramham CR. 2007. Sustained Arc/Arg3.1 synthesis controls long-term potentiation consolidation through regulation of local actin polymerization in the dentate gyrus in vivo. J Neurosci 27: 10445–10455.
    Abstract/FREE Full Text
  40. ↵
    Minatohara K, Akiyoshi M, Okuno H. 2016. Role of immediate-early genes in synaptic plasticity and neuronal ensembles underlying the memory trace. Front Mol Neurosci 8: 78. doi:10.3389/fnmol.2015.00078
    CrossRefMedlineGoogle Scholar
  41. ↵
    Miyashita T, Kubik S, Haghighi N, Steward O, Guzowski JF. 2009. Rapid activation of plasticity-associated gene transcription in hippocampal neurons provides a mechanism for encoding of one-trial experience. J Neurosci 29: 898–906. doi:10.1523/JNEUROSCI.4588-08.2009
    Abstract/FREE Full Text
  42. ↵
    Nair RR, Patil S, Tiron A, Kanhema T, Panja D, Schiro L, Parobczak K, Wilczynski G, Bramham CR. 2017. Dynamic Arc SUMOylation and selective interaction with F-actin-binding protein drebrin A in LTP consolidation in vivo. Front Synaptic Neurosci 9: 8. doi:10.3389/fnsyn.2017.00008
    CrossRefMedlineGoogle Scholar
  43. ↵
    Nonaka M, Kim R, Fukushima H, Sasaki K, Suzuki K, Okamura M, Ishii Y, Kawashima T, Kamijo S, Takemoto-Kimura S, et al. 2014. Region-specific activation of CRTC1-CREB signaling mediates long-term fear memory. Neuron 84: 92–106. doi:10.1016/j.neuron.2014.08.049
    CrossRefMedlineGoogle Scholar
  44. ↵
    Park S, Park JM, Kim S, Kim JA, Shepherd JD, Smith-Hicks CL, Chowdhury S, Kaufmann W, Kuhl D, Ryazanov AG, et al. 2008. Elongation factor 2 and fragile X mental retardation protein control the dynamic translation of Arc/Arg3.1 essential for mGluR-LTD. Neuron 59: 70–83. doi:10.1016/j.neuron.2008.05.023
    CrossRefMedlineWeb of ScienceGoogle Scholar
  45. ↵
    Park HY, Lim H, Yoon YJ, Follenzi A, Nwokafor C, Lopez-Jones M, Meng X, Singer RH. 2014. Visualization of dynamics of single endogenous mRNA labeled in live mouse. Science 343: 422–424. doi:10.1126/science.1239200
    Abstract/FREE Full Text
  46. ↵
    Peebles CL, Yoo J, Thwin MT, Palop JJ, Noebels JL, Finkbeiner S. 2010. Arc regulates spine morphology and maintains network stability in vivo. Proc Natl Acad Sci 107: 18173–18178. doi:10.1073/pnas.1006546107
    Abstract/FREE Full Text
  47. ↵
    Peng T, Thorn K, Schroeder T, Wang L, Theis FJ, Marr C, Navab N. 2017. A BaSiC tool for background and shading correction of optical microscopy images. Nat Commun 8: 14836. doi:10.1038/ncomms14836
    CrossRefMedlineGoogle Scholar
  48. ↵
    Plath N, Ohana O, Dammermann B, Errington ML, Schmitz D, Gross C, Mao XS, Engelsberg A, Mahlke C, Welzl H, et al. 2006. Arc/Arg3.1 is essential for the consolidation of synaptic plasticity and memories. Neuron 52: 437–444. doi:10.1016/j.neuron.2006.08.024
    CrossRefMedlineWeb of ScienceGoogle Scholar
  49. ↵
    Pulimood NS, Contreras M, Pruitt ME, Tarasiewicz A, Medina AE. 2021. Phosphorylation of CREB at serine 142 and 143 is essential for visual cortex plasticity. eNeuro 8: ENEURO.0217-21.2021. doi:10.1523/ENEURO.0217-21.2021
    Abstract/FREE Full Text
  50. ↵
    Saha RN, Wissink EM, Bailey ER, Zhao M, Fargo DC, Hwang JY, Daigle KR, Fenn JD, Adelman K, Dudek SM. 2011. Rapid activity-induced transcription of Arc and other IEGs relies on poised RNA polymerase II. Nat Neurosci 14: 848–856. doi:10.1038/nn.2839
    CrossRefMedlineGoogle Scholar
  51. ↵
    Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, et al. 2012. Fiji: an open-source platform for biological-image analysis. Nat Methods 9: 676–682. doi:10.1038/nmeth.2019
    CrossRefMedlineWeb of ScienceGoogle Scholar
  52. ↵
    Sheng M, Greenberg ME. 1990. The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron 4: 477–485. doi:10.1016/0896-6273(90)90106-P
    CrossRefMedlineWeb of ScienceGoogle Scholar
  53. ↵
    Shepherd JD, Rumbaugh G, Wu J, Chowdhury S, Plath N, Kuhl D, Huganir RL, Worley PF. 2006. Arc/Arg3.1 mediates homeostatic synaptic scaling of AMPA receptors. Neuron 52: 475–484. doi:10.1016/j.neuron.2006.08.034
    CrossRefMedlineWeb of ScienceGoogle Scholar
  54. ↵
    Steward O, Wallace CS, Lyford GL, Worley PF. 1998. Synaptic activation causes the mRNA for the IEG Arc to localize selectively near activated postsynaptic sites on dendrites. Neuron 21: 741–751. doi:10.1016/S0896-6273(00)80591-7
    CrossRefMedlineWeb of ScienceGoogle Scholar
  55. ↵
    Vazdarjanova A, McNaughton BL, Barnes CA, Worley PF, Guzowski JF. 2002. Experience-dependent coincident expression of the effector immediate-early genes Arc and Homer 1a in hippocampal and neocortical neuronal networks. J Neurosci 22: 10067–10071. doi:10.1523/JNEUROSCI.22-23-10067.2002
    Abstract/FREE Full Text
  56. ↵
    Vera M, Biswas J, Senecal A, Singer RH, Park HY. 2016. Single-cell and single-molecule analysis of gene expression regulation. Annu Rev Genet 50: 267–291. doi:10.1146/annurev-genet-120215-034854
    CrossRefMedlineGoogle Scholar
  57. ↵
    Waltereit R, Dammermann B, Wulff P, Scafidi J, Staubli U, Kauselmann G, Bundman M, Kuhl D. 2001. Arg3.1/Arc mRNA induction by Ca2+ and cAMP requires protein kinase A and mitogen-activated protein kinase/extracellular regulated kinase activation. J Neurosci 21: 5484–5493. doi:10.1523/JNEUROSCI.21-15-05484.2001
    Abstract/FREE Full Text
  58. ↵
    Waung MW, Pfeiffer BE, Nosyreva ED, Ronesi JA, Huber KM. 2008. Rapid translation of Arc/Arg3.1 selectively mediates mGluR-dependent LTD through persistent increases in AMPAR endocytosis rate. Neuron 59: 84–97. doi:10.1016/j.neuron.2008.05.014
    CrossRefMedlineWeb of ScienceGoogle Scholar
  59. ↵
    West AE, Chen WG, Dalva MB, Dolmetsch RE, Kornhauser JM, Shaywitz AJ, Takasu MA, Tao X, Greenberg ME. 2001. Calcium regulation of neuronal gene expression. Proc Natl Acad Sci 98: 11024–11031. doi:10.1073/pnas.191352298
    Abstract/FREE Full Text
  60. ↵
    Yilmaz-Rastoder E, Miyamae T, Braun AE, Thiels E. 2011. LTP- and LTD-inducing stimulations cause opposite changes in arc/arg3.1 mRNA level in hippocampal area CA1 in vivo. Hippocampus 21: 1290–1301. doi:10.1002/hipo.20838
    CrossRefMedlineWeb of ScienceGoogle Scholar
  61. ↵
    Yu Y, Oberlaender K, Bengtson CP, Bading H. 2017. One nuclear calcium transient induced by a single burst of action potentials represents the minimum signal strength in activity-dependent transcription in hippocampal neurons. Cell Calcium 65: 14–21. doi:10.1016/j.ceca.2017.03.003
    CrossRefGoogle Scholar
  62. ↵
    Zhang H, Bramham CR. 2020. Arc/Arg3.1 function in long-term synaptic plasticity: emerging mechanisms and unresolved issues. Eur J Neurosci 54: 6696–6712. doi:10.1111/ejn.14958
    CrossRefGoogle Scholar
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