Locus ceruleus control of state-dependent gene expression

Abstract

Wakefulness and sleep are accompanied by changes in behavior and neural activity, as well as by the upregulation of different functional categories of genes. However, the mechanisms responsible for such state-dependent changes in gene expression are unknown. Here we investigate to what extent state-dependent changes in gene expression depend on the central noradrenergic (NA) system, which is active in wakefulness and reduces its firing during sleep. We measured the levels of approximately 5000 transcripts expressed in the cerebral cortex of control rats and in rats pretreated with DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine], a neurotoxin that removes the noradrenergic innervation of the cortex. We found that NA depletion reduces the expression of approximately 20% of known wakefulness-related transcripts. Most of these transcripts are involved in synaptic plasticity and in the cellular response to stress. In contrast, NA depletion increased the expression of the sleep-related gene encoding the translation elongation factor 2. These results indicate that the activity of the central NA system during wakefulness modulates neuronal transcription to favor synaptic potentiation and counteract cellular stress, whereas its inactivity during sleep may play a permissive role to enhance brain protein synthesis.

Figure 1.

Levels of NA, DA, and 5-HT were measured in the left cerebral cortex of saline-treated controls (c, white boxes) and DSP-4 treated rats (4, gray boxes) using HPLC with electrochemical detection. Values (mean ± SEM) in DSP-4-treated rats are expressed as percentage relative to saline-treated controls (equivalent to 100%). NA, 14.2 ± 1.3; DA, 105.5 ± 10.3; 5-HT, 70.3 ± 5.7. Boxes equal mean ± SEM; error bars indicate mean ± SD; open circles, outliers; plus signs, extremes. Values refer to all experimental animals, including 17 controls (6 for mRNA differential display, 7 for microarrays analysis, and 4 for confirmation experiments) and 17 DSP-4-treated rats (6 for mRNA differential display, 7 for microarrays analysis, and 4 for confirmation experiments). Mann-Whitney U test: NA, ^*p = 0.0017; DA, p = 0.277; 5-HT, ^*p = 0.0017. Levels of metabolites DOPAC, HVA, and 5-HIAA were also measured and were as follows (data not shown): DOPAC, 84.4 ± 9.0 (p = 0.337); HVA, 84.6 ± 9.2 (p = 0.11); HIAA, 87.6 ± 4.6 (p = 0.14).

Figure 2.

Wakefulness (W), NREM, and REM sleep in saline-treated (controls; n = 17) and DSP-4 treated (n = 17) rats. Values refer to the 24 hr period preceding the final experimental day and are expressed as percentage of total recording time (mean ± SEM in parenthesis; Mann-Whitney U test: W, p = 0.72; NREM, p = 0.67; REM, p = 0.08).

Figure 3.

Examples of mRNA differential display analysis of cerebral cortex RNA in saline-treated (controls, lanes 1-6) and DSP-4 treated (lanes 7-12) rats. Each lane corresponds to a single rat. The figure shows a small section of three different gels. The band indicated by an asterisk in the two upper gels corresponds to NGFI-A (each gel used a different sets of primers), and the band indicated by an asterisk on the lower gel corresponds to minoxidil(aryl)sulfotransferase.

Figure 4.

A, Cortical transcripts (GeneChip RGU34A) regulated by sleep and wakefulness, by DSP-4, and their overlap. Numbers only include known genes and not ESTs. ↑ in wakefulness, Genes whose mRNA levels increased in both spontaneously awake and sleep deprived animals relative to sleeping animals according to Cirelli et al. (2004). ↑ in sleep, Genes whose mRNA levels increased in sleeping animals relative to both spontaneously awake and sleep deprived animals according to Cirelli et al. (2004). ↓ with DSP-4, Genes whose mRNA levels were decreased in DSP-4 animals (i.e., after cortical NA depletion). ↑ with DSP-4, Genes whose mRNA levels were increased in DSP-4 animals (i.e., after cortical NA depletion). The 16 overlapping genes up in wakefulness and with NA include c-fos and BDNF, which were identified using mRNA differential display. The only overlapping gene up in sleep and down with NA is EF2.B, Biological functions associated with transcripts with higher expression in wakefulness (light gray box) and sleep (dark gray box), as by Cirelli et al. (2004). Functional categories listed in bold are those that include one or more transcripts whose expression is also modulated by NA. The tree on the left (dots and connecting paths) represents biological process annotations according to the gene ontology hierarchy.