MicroRNA-124a (miR-124a) is the most abundant microRNA expressed in the vertebrate CNS. Despite past investigations into the role of miR-124a, inconsistent results have left the in vivo function of miR-124a unclear. We examined the in vivo function of miR-124a by targeted disruption of Rncr3 (retinal non-coding RNA 3), the dominant source of miR-124a. Rncr3(-/-) mice exhibited abnormalities in the CNS, including small brain size, axonal mis-sprouting of dentate gyrus granule cells and retinal cone cell death. We found that Lhx2 is an in vivo target mRNA of miR-124a. We also observed that LHX2 downregulation by miR-124a is required for the prevention of apoptosis in the developing retina and proper axonal development of hippocampal neurons. These results suggest that miR-124a is essential for the maturation and survival of dentate gyrus neurons and retinal cones, as it represses Lhx2 translation.

Nucleus accumbens (NAc) is involved in behaviors that depend on heightened wakefulness, but its impact on arousal remains unclear. Here, we demonstrate that NAc dopamine D1 receptor (D1R)-expressing neurons are essential for behavioral arousal. Using in vivo fiber photometry in mice, we find arousal-dependent increases in population activity of NAc D1R neurons. Optogenetic activation of NAc D1R neurons induces immediate transitions from non-rapid eye movement sleep to wakefulness, and chemogenetic stimulation prolongs arousal, with decreased food intake. Patch-clamp, tracing, immunohistochemistry, and electron microscopy reveal that NAc D1R neurons project to the midbrain and lateral hypothalamus, and might disinhibit midbrain dopamine neurons and lateral hypothalamus orexin neurons. Photoactivation of terminals in the midbrain and lateral hypothalamus is sufficient to induce wakefulness. Silencing of NAc D1R neurons suppresses arousal, with increased nest-building behaviors. Collectively, our data indicate that NAc D1R neuron circuits are essential for the induction and maintenance of wakefulness.

Caffeine, the most widely used psychoactive compound, is an adenosine receptor antagonist. It promotes wakefulness by blocking adenosine A2A receptors (A2ARs) in the brain, but the specific neurons on which caffeine acts to produce arousal have not been identified. Using selective gene deletion strategies based on the Cre/loxP technology in mice and focal RNA interference to silence the expression of A2ARs in rats by local infection with adeno-associated virus carrying short-hairpin RNA, we report that the A2ARs in the shell region of the nucleus accumbens (NAc) are responsible for the effect of caffeine on wakefulness. Caffeine-induced arousal was not affected in rats when A2ARs were focally removed from the NAc core or other A2AR-positive areas of the basal ganglia. Our observations suggest that caffeine promotes arousal by activating pathways that traditionally have been associated with motivational and motor responses in the brain.

To insure an adequate supply of nutrients, omnivores choose among available food sources. This process is exemplified by the well-characterized innate aversion of omnivores to otherwise nutritious foods of imbalanced amino acid content. We report that brain-specific inactivation of GCN2, a ubiquitously expressed protein kinase that phosphorylates translation initiation factor 2 alpha (eIF2alpha) in response to intracellular amino acid deficiency, impairs this aversive response. GCN2 inactivation also diminishes phosphorylated eIF2alpha levels in the mouse anterior piriform cortex following consumption of an imbalanced meal. An ancient intracellular signal transduction pathway responsive to amino acid deficiency thus affects feeding behavior by activating a neuronal circuit that biases consumption against imbalanced food sources.