Translational profiling of hypocretin neurons identifies candidate molecules for sleep regulation

Abstract

Hypocretin (orexin; Hcrt)-containing neurons of the hypothalamus are essential for the normal regulation of sleep and wake behaviors and have been implicated in feeding, anxiety, depression, and reward. The absence of these neurons causes narcolepsy in humans and model organisms. However, little is known about the molecular phenotype of these cells; previous attempts at comprehensive profiling had only limited sensitivity or were inaccurate. We generated a Hcrt translating ribosome affinity purification (bacTRAP) line for comprehensive translational profiling of all ribosome-bound transcripts in these neurons in vivo. From this profile, we identified >6000 transcripts detectably expressed above background and 188 transcripts that are highly enriched in these neurons, including all known markers of the cells. Blinded analysis of in situ hybridization databases suggests that ~60% of these are expressed in a Hcrt marker-like pattern. Fifteen of these were confirmed with double labeling and microscopy, including the transcription factor Lhx9. Ablation of this gene results in a >30% loss specifically of Hcrt neurons, without a general disruption of hypothalamic development. Polysomnography and activity monitoring revealed a profound hypersomnolence in these mice. These data provide an in-depth and accurate profile of Hcrt neuron gene expression and suggest that Lhx9 may be important for specification or survival of a subset of these cells.

Figure 1.

Characterization of a Hcrt bacTRAP line. (A) Immunohistochemistry for Hcrt1 (orexin A) in a wild-type (WT) mouse brain (left panel) shows the same pattern of expression as eGFP antibodies on Hcrt∷eGFP-RpL10a mice (middle panel). (Right panel) Both label scattered cells with neuronal morphology in the lateral hypothalamus. (B) Confocal immunofluorescence on the Hcrt∷eGFP-RpL10a mouse line reveals >85% of Hcrt-positive neurons (red; middle panel) are eGFP-RpL10a-positive (green; left panel). No Hcrt-negative, eGFP-RpL10a-positive cells were seen anywhere in brain. Bar, 50 μm.

Figure 2.

TRAP purification of Hcrt cell mRNA. (A) Scatter plot of mRNA levels from microarray data from replicate Hcrt neuron TRAP assays. Log10 expression values. (B) Scatter plot of mRNA levels from microarray data from Hcrt neuron TRAP assays (X-axis, average of four arrays), compared with whole hypothalamic RNA, shows robust enrichment (>16-fold) of known Hcrt neuron-expressed genes (blue) and depletion (<0.5-fold) of negative controls (red, markers of glia). Multiple probe sets per gene are labeled when available. (C) Scatter plot of Pabp arrays (downloaded from Gene Expression Omnibus [GEO]: GSE17617; average of n = 9 arrays per group) shows a larger number of probe sets enriched more than twofold, including some glial genes. Most of the marker probe sets showed no signal. (D) Scoring rubric for Allen Brain Atlas analysis. (E) Blinded scoring of ISH patterns reveals a significant enrichment within the TRAP gene list for transcripts with Hcrt marker-like expression (scores of 1–3, P < 3 × 10⁻¹⁰, χ² test). Each list normalized to the total number of scorable transcripts on the list (n = 32–67).

Figure 3.

TRAP accurately predicts gene expression in Hcrt neurons. (A) Confocal immunofluorescence for positive control anti-Nptx2 (green) and anti-Hcrt (red) antibodies shows Hcrt is expressed in a subset of Npt2x cells, consistent with their known expression patterns. Colocalization of nine novel Hcrt neuron gene products identified as enriched in Hcrt neurons (Table 1) shows substantial overlap (Table 2). Lhx9 protein was expressed in a subset of Hcrt neurons, and a subset of Lhx9 cells were positive for Hcrt. (B) In situ hybridization for potential novel markers of Hcrt neurons (scored as a 1 or 2 in Supplemental Table S1), identifies six genes highly enriched in Hcrt neurons. (C) Genes identified as expressed but not substantially enriched by TRAP (Supplemental Table S2) also show robust expression in Hcrt neurons. Note the molecular diversity of Hcrt neurons in their expression of calcium-binding proteins Calb1 and Calb2 (white arrows point to triple expression of Calb1, Calb2, and Hcrt; yellow arrows point to double expression of Calb1 or Calb2 and Hcrt; cyan arrows point to single expression of Calb1). All of the images are captured at 40× magnification.

Figure 4.

Genetic ablation of Lhx9 results in hypersomnolence. (A) Representative hypnograms for a single Lhx9 knockout (KO) and wild-type (WT) mouse over a 12-h active period show a lack of sleep-onset REM periods but an apparent increase in NREM sleep in the knockout mouse. (B, top panel) Quantification of minutes per hour spent in REM and NREM sleep and wakefulness for Lhx9 knockout and wild-type mice reveals a significant increase in NREM sleep at the expense of wakefulness (n = 5 per group, two-tailed t-test). (Bottom panel) There was no difference in bout duration of wakefulness and NREM sleep between Lhx9 knockout and wild-type mice (n = 5 per group, two-tailed t-test). (C) Both Lhx9 knockout and wild-type mice show normal circadian differences in activity between night and day (n = 5 per group, two-tailed t-test) with no significant difference between genotypes. (*) P < 0.05; (**) P < 0.01. (***) P < 0.001. Values represent mean ± SEM.

Figure 5.

Disturbances in behavior and Hcrt expression of Lhx9 knockout (KO) mice are not rescued by adult reinstatement of Lhx9 expression. During 48-h activity monitoring, Lhx9 knockout mice exhibited a 20% increase in time at rest, an estimate of sleep, compared with wild-type (WT) mice (P < 0.05, ANOVA) (A), while both groups demonstrated intact circadian differences in time at rest between the light and dark periods (P < 0.05, paired t-test) (B). (C) Lhx9 knockout mice also demonstrated a 57% decrease in ambulatory activity, an estimate of wakefulness, relative to wild-type mice (P < 0.01, ANOVA). (D) The ambulatory differences were maintained across the light and dark periods (P < 0.05, repeated measures ANOVA). (E) Confocal microscopy revealed double-immunofluorescent labeling of GFP with orexin within Hcrt neurons, confirming successful targeting of hypocretinergic regions. (F) Representative example of Lhx9 expression in the targeted region of the hypothalamus in Lhx9 knockout mice. The white asterisk represents a labeling artifact at injection sites. (G,H) No differences were observed in time at rest or ambulatory activity between pre- and post-injection activity monitoring for Lhx9-injected Lhx9 knockout mice. In addition, time at rest and ambulatory activity was comparable during post-injection activity monitoring for both the Lhx9-injected Lhx9 knockout mice and the GFP-injected Lhx9 knockout mice (repeated measures ANOVA). (I) No difference was noted in the number of Hcrt neurons between Lhx9-injected and GFP-injected animals. (*) P < 0.05; (**) P < 0.01. Values represent mean ± SEM.