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. 2020 Sep 14;21(1):633.
doi: 10.1186/s12864-020-07050-7.

RNA-seq analysis of galaninergic neurons from ventrolateral preoptic nucleus identifies expression changes between sleep and wake

Xiaofeng Guo  1 , Xiaoling Gao  2 , Brendan T Keenan  1 , Jingxu Zhu  1 , Dimitra Sarantopoulou  3   4 , Jie Lian  1 , Raymond J Galante  1 , Gregory R Grant  3   5 , Allan I Pack  6
Affiliations

RNA-seq analysis of galaninergic neurons from ventrolateral preoptic nucleus identifies expression changes between sleep and wake

Xiaofeng Guo et al. BMC Genomics. .

Abstract

Background: Previous studies show that galanin neurons in ventrolateral preoptic nucleus (VLPO-Gal) are essential for sleep regulation. Here, we explored the transcriptional regulation of the VLPO-Gal neurons in sleep by comparing their transcriptional responses between sleeping mice and those kept awake, sacrificed at the same diurnal time.

Results: RNA-sequencing (RNA-seq) analysis was performed on eGFP(+) galanin neurons isolated using laser captured microdissection (LCM) from VLPO. Expression of Gal was assessed in our LCM eGFP(+) neurons via real time qPCR and showed marked enrichment when compared to LCM eGFP(-) cells and to bulk VLPO samples. Gene set enrichment analysis utilizing data from a recent single-cell RNA-seq study of the preoptic area demonstrated that our VLPO-Gal samples were highly enriched with galanin-expressing inhibitory neurons, but not galanin-expressing excitatory neurons. A total of 263 genes were differentially expressed between sleep and wake in VLPO-Gal neurons. When comparing differentially expressed genes in VLPO-Gal neurons to differentially expressed genes in a wake-active neuronal region (the medial prefrontal cortex), evidence indicates that both systemic and cell-specific mechanisms contribute to the transcriptional regulation in VLPO-Gal neurons. In both wake-active and sleep-active neurons, ER stress pathways are activated by wake and cold-inducible RNA-binding proteins are activated by sleep. In contrast, expression of DNA repair genes is increased in VLPO-Gal during wakefulness, but increased in wake-active cells during sleep.

Conclusion: Our study identified transcriptomic responses of the galanin neurons in the ventrolateral preoptic nucleus during sleep and sleep deprivation. Data indicate that VLPO contains mainly sleep-active inhibitory galaninergic neurons. The VLPO galanin neurons show responses to sleep and wake similar to wake-active regions, indicating these responses, such as ER stress and cold-inducible RNA-binding proteins, are systemic affecting all neuronal populations. Region-specific differences in sleep/wake responses were also identified, in particular DNA repair. Our study expands knowledge about the transcriptional response of a distinct group of neurons essential for sleep.

Keywords: Galaninergic neurons; Next-generation RNA-sequencing; Sleep deprivation; Spontaneous sleep; Ventrolateral preoptic nucleus.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Confirmation for the enrichment of galanin-expressing neurons in eGFP(+) samples collected using LCM. a eGFP(+) samples showed a median 116.9-fold increase in the expression of Gal when compared to the VLPO bulk tissues, indicating effective enrichment of galanin-expressing neurons in our eGFP(+) LCM samples. b Expression of the astrocyte gene Aldh1l1 in both the eGFP(+) and eGFP(−) samples was significantly lower when compared to the VLPO bulk tissues, indicating effective removal of contaminating astrocytes from the neuron samples collected by LCM. Kruskal-Wallis tests were made for comparisons among all three groups. Separate pairwise comparisons were then made between the VLPO bulk tissues and the LCM samples (eGFP+ and eGFP-) using one-tailed Wilcoxon exact test (see Methods). Y-axis shows fold difference in gene expression relative to VLPO bulk tissue on log10 scale
Fig. 2
Fig. 2
Plot of the multidimensional scaling (MDS) result. SS and SDep samples formed separate clusters separated primarily on the first dimension, demonstrating that behavioral state explains the largest proportion of gene expression variability. Mice collected at ZT0 were between the SS and SDep. Results are shown with confidence ellipse at 95% confidence intervals
Fig. 3
Fig. 3
Differentially expressed genes between spontaneous sleep (SS) and sleep deprivation (SDep). a 184 genes were identified to be significantly up-regulated by SDep (orange) and 79 genes were identified to be significantly up-regulated by sleep (green) with a cutoff of FDR < 0.05. b Selected Biological Processes GO terms enriched in the differentially expressed genes. Protein folding, response to unfolded protein, and regulation of gene expression are among the functions enriched from the genes up-regulated in SDep (red), whereas nucleosome assembly and regulation of translation are among the functions enriched from the genes up-regulated in sleep (green). X-axis shows the negative log10 of the P-values of the GO enrichment
Fig. 4
Fig. 4
Venn Diagrams comparing DEGs identified in VLPO-Gal neurons and mPFC. The Venn diagram on the left shows 127 genes are commonly up-regulated by SDep in both VLPO-Gal neurons and mPFC. The Venn diagram on the right shows 40 genes are commonly up-regulated by SS in both VLPO-Gal neurons and mPFC. Specifically in VLPO-Gal neurons, 54 genes are up-regulated by SDep (left) and 33 genes are up-regulated by SS (right)
Fig. 5
Fig. 5
Heat map of fold-changes between sleep deprivation and sleep at each time point in VLPO-Gal neurons and mPFC. Cell colors with red indicates up-regulation with SDep and blue indicates up-regulation with sleep. Row annotations indicate biological functions. a Genes involved in protein folding and transcription are commonly up-regulated with SDep in both VLPO-Gal and mPFC, whereas translation and cell differentiation are commonly up-regulated with sleep in both VLPO-Gal and mPFC. b Sixteen genes show opposite direction of change between VLPO-Gal and mPFC cells. Twelve genes were up-regulated with SDep in VLPO-Gal neurons but up-regulated with sleep in mPFC. Functions played by these genes include DNA repair and neuronal development. Four genes (Mnt, Cry2, Lrrc23, and Igsf11) were up-regulated with sleep in VLPO-Gal but up-regulated with SDep in mPFC
Fig. 6
Fig. 6
Expression of Fos across multiple time points during SS or SDep in VLPO-Gal neurons and mPFC. a Expression differences of Fos between SS and SDep. In VLPO-Gal, Fos was not significantly different between SS and SDep at earlier time points ZT3 and ZT6, but expressed at a higher level during SDep at ZT9 and ZT12 when compared using a two sample T-test. In mPFC, Fos was significantly elevated by SDep at all four time points. b Pearson’s correlation of Fos expression [log2(CPM + 0.5)] with duration of SS in VLPO-Gal (left) or mPFC (right). In VLPO-Gal, Fos showed a moderately large negative correlation with duration of SS (R = -0.40, p = 0.055), although not statistically significant. In mPFC, Fos was not significantly correlated with duration of SS but the direction of change was opposite from that of VLPO-Gal (R = 0.25, p = 0.23)
Fig. 7
Fig. 7
Pearson’s correlation of Fos with the amount (minutes) of sleep in the last 1 h before sacrifice in VLPO-Gal neurons and mPFC. Fos expression [log2(CPM + 0.5)] in VLPO-Gal had a moderate positive correlation (R = 0.33, p = 0.12) with the amount of sleep in the last hour before sacrifice, although not statistically significant. Fos expression in mPFC showed opposite direction of change as that of VLPO-Gal (R = -0.38, p = 0.063)
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