Coordinated Activity of Transcriptional Networks Responding to the Pattern of Action Potential Firing in Neurons

Abstract

Transcriptional responses to the appropriate temporal pattern of action potential firing are essential for long-term adaption of neuronal properties to the functional activity of neural circuits and environmental experience. However, standard transcriptome analysis methods can be too limited in identifying critical aspects that coordinate temporal coding of action potential firing with transcriptome response. A Pearson correlation analysis was applied to determine how pairs of genes in the mouse dorsal root ganglion (DRG) neurons are coordinately expressed in response to stimulation producing the same number of action potentials by two different temporal patterns. Analysis of 4728 distinct gene-pairs related to calcium signaling, 435,711 pairs of transcription factors, 820 pairs of voltage-gated ion channels, and 86,862 pairs of calcium signaling genes with transcription factors indicated that genes become coordinately activated by distinct action potential firing patterns and this depends on the duration of stimulation. Moreover, a measure of expression variance revealed that the control of transcripts abundances is sensitive to the pattern of stimulation. Thus, action potentials impact intracellular signaling and the transcriptome in dynamic manner that not only alter gene expression levels significantly (as previously reported) but also affects the control of their expression fluctuations and profoundly remodel the transcriptional networks.

Keywords: DRG neurons; calcium signaling; calcium voltage-dependent channels; covariance analysis; gene expression variability; potassium voltage-gated channels; sodium voltage-gated channels; transcription factors.

Figure A1

Comparison of the expression coordination of the 93 adequately quantified calcium signaling genes among stimulation paradigms. A red/blue/yellow square indicates a statistically significant (p value < 0.05) positive/negative/independent coordination while a blank square indicates lack of statistical significance to assess the type of coordination. Numbers indicate the percentages of gene pairs exhibiting statistically significant positive (S), negative (A) and independent (I) coordination in the respective pattern of stimulation.

Figure A2

Part of the expression coordination of the all 93 quantified calcium signaling genes with 200 randomly selected (out of 934) transcription factors. A red/blue/ square indicates a statistically significant (p value < 0.05) positive/negative coordination, an yellow square indicates no correlation, while a blank square indicates lack of statistical significance to assess the type of correlation. Note the significantly higher degree of coordination between the transcription factors and the calcium signaling genes for the 5 h stimulation with the 18/1 pattern.

Figure 1

Average relative expression variability (REV) (A) and normalized median expression (B) of all quantified (ALL, 13,974 distinct genes), calcium signaling related genes (CAS, 93 genes) and transcription factors (TRF, 934 genes) after electrical stimulation at two different patterns for 2 and 5 h. The means of the REV distributions in stimulated conditions were significantly different from the control (unstimulated) ones as measured by the p-values of the heteroscedastic (two-sample unequal variance) t-test of the two means equality: 18/1 2 h (ALL < 10 ⁻³⁰⁸; CAS 6.28 × 10 ⁻⁶; TRF 3.78 × 10 ⁻⁵¹), 18/1 5 h (ALL 3.2 × 10 ⁻²¹⁶; CAS 2.76 × 10 ⁻¹¹; TRF 2.14 × 10 ⁻¹³²), 90/5 2 h (ALL <10 ⁻³⁰⁸; CAS 3.59 × 10 ⁻⁹; TRF 2.557 × 10 ⁻⁹⁷), 90/5 5 h (ALL <10 ⁻³⁰⁸; CAS 7.10 × 10 ⁻⁶; TRF 1.52 × 10 ⁻³⁷).

Figure 2

Pearson correlation of gene expressions. Percentage of (p < 0.05) significantly uncorrelated and positively or negatively correlated (A) calcium signaling (CAS–CAS) gene pairs, (B) transcription factor (TRF–TRF) gene pairs and (C) CAS–TRF gene pairs in all experimental conditions. Note that the percentage of positive coordination is substantially higher for the 18/1 stimulation pattern at 5 h and strong positive coordination of the calcium signaling genes with the transcription factors. The difference between 100% and sum of the represented percentages is composed by the gene-pairs whose coordination did not meet the statistical evidence to be categorized as significantly uncorrelated, or as significantly positively or negatively correlated.

Figure 3

Expression regulation and coordination of some voltage-gated channels in stimulated DRG neurons. (A) unstimulated neurons. (B) neurons stimulated for 2 h with 18/1 pattern. (C) neurons stimulated for 5 h with 18/1 pattern. (D) neurons stimulated for 2 h with 90/5 pattern. (E) neurons stimulated for 5 h with 90/5 pattern. Red/green background of the gene symbol indicates significant up-/down-regulation of that gene in that stimulation condition with respect to unstimulated neurons. Yellow background indicates that the expression change was not statistically significant. Red/blue line indicates that the expressions of the linked genes are significantly positively/negatively correlated. Note the substantial differences among the stimulation paradigms. Missing lines indicate that the expression coordination between the corresponding genes were not statistically significant.

Figure 4

Positive and negative expression coordination of the first 50 alphabetically ordered calcium signaling genes in each pattern of stimulation. (A) positive correlations in unstimulated neurons. (B) negative correlations in unstimulated neurons. (C) positive correlations in neurons stimulated for 2 h with 18/1 pattern. (D) negative correlations in neurons stimulated for 2 h with 18/1 pattern. (E) positive correlations in neurons stimulated for 5 h with 18/1 pattern. (F) negative correlations in neurons stimulated for 5 h with 18/1 pattern. (G) positive correlations in neurons stimulated for 2 h with 90/5 pattern. (H) negative correlations in neurons stimulated for 2 h with 90/5 pattern. (I) positive correlations in neurons stimulated for 5 h with 90/5 pattern. (J) negative correlations in neurons stimulated for 5 h with 90/5 pattern. A red/blue line indicates a statistically significant (p-value < 0.05) positive/negative coordination of the linked genes. Missing lines indicate that the expression coordination between the corresponding genes is not statistically significant.

Figure 5

Positive and negative expression coordination of 50 alphabetically ordered, randomly selected transcription factor genes (TRF) in all experimental conditions. (A) positive correlations in unstimulated neurons. (B) negative correlations in unstimulated neurons. (C) positive correlations in neurons stimulated for 2 h with 18/1 pattern. (D) negative correlations in neurons stimulated for 2 h with 18/1 pattern. (E) positive correlations in neurons stimulated for 5 h with 18/1 pattern. (F) negative correlations in neurons stimulated for 5 h with 18/1 pattern. (G) positive correlations in neurons stimulated for 2 h with 90/5 pattern. (H) negative correlations in neurons stimulated for 2 h with 90/5 pattern. (I) positive correlations in neurons stimulated for 5 h with 90/5 pattern. (J) negative correlations in neurons stimulated for 5 h with 90/5 pattern. A red/blue line indicates a statistically significant (p-value < 0.05) positive/negative coordination of the linked genes. Missing lines indicate that the expression coordination is not statistically significant. As in the case of CAS genes, the TRF network responds to the action potential firing pattern.

Figure 6

Pair-wise relevance (PWR) analysis of the interaction between 50 calcium signaling genes (CAS) and 50 transcription factors (TRF) in all patterns of stimulation. (A) unstimulated neurons. (B) neurons stimulated for 2 h with 18/1 pattern. (C) neurons stimulated for 5 h with 18/1 pattern. (D) neurons stimulated for 2 h with 90/5 pattern. (E) neurons stimulated for 5 h with 90/5 pattern. The medallions present the relevant TRF–CAS gene pairs (and their PWR scores) in each condition.