Perifornical Urocortin-3 mediates the link between stress-induced anxiety and energy homeostasis

Abstract

In response to physiological or psychological challenges, the brain activates behavioral and neuroendocrine systems linked to both metabolic and emotional outputs designed to adapt to the demand. However, dysregulation of integration of these physiological responses to challenge can have severe psychological and physiological consequences, and inappropriate regulation, disproportional intensity, or chronic or irreversible activation of the stress response is linked to the etiology and pathophysiology of mood and metabolic disorders. Using a transgenic mouse model and lentiviral approach, we demonstrate the involvement of the hypothalamic neuropeptide Urocortin-3, a specific ligand for the type-2 corticotropin-releasing factor receptor, in modulating septal and hypothalamic nuclei responsible for anxiety-like behaviors and metabolic functions, respectively. These results position Urocortin-3 as a neuromodulator linking stress-induced anxiety and energy homeostasis and pave the way toward better understanding of the mechanisms that mediate the reciprocal relationships between stress, mood and metabolic disorders.

Fig. 1.

Establishment of site-specific and inducible Ucn3-overexpressing transgenic mouse model. (A) Schematic representation of the tetracycline-responsive regulatory system for transcriptional transactivation in a combined transgenic mouse and lentiviral systems. (B and C) Injection of the transactivator (‘effector virus’) into a specific site of the transgenic mouse brain will result in incorporation of the reverse transactivator (rtTA)-expressing sequence at the infected neuron, ensuring constant and stable expression. In the presence of the inducer doxycycline (Dox), the rtTA protein binds TRE and activates transcription of Ucn3 at the injection site. The rtTA lentiviruses were injected to the rPFA, which endogenously expresses Ucn3 and projects to the LS and the VMH. In the presence of Dox, rPFA-Ucn3 is overexpressed, and its release at the LS and VMH nuclei is intensified. (D–F) Immunostaining of lateral septal Ucn3 terminal fields. In situ hybridization demonstrating lateral septal CRFR2 mRNA expression, indicated by arrowheads (D). Immunostaining for lateral septal Ucn3 terminal fields (fibers immunostaining, indicated by arrowheads) obtained from mouse that was not treated (E) or was treated (F) with Dox. RRE, Rev-responsive element; cPPT, central polypurine tract; WPRE, Woodchuck hepatitis posttranscriptional regulatory element; CMV, cytomegalovirus; IRES, internal ribosome entry site; pA, polyadenylation site.

Fig. 2.

Verification of rtTA-expressing lentiviruses, rPFA-Ucn3 injection site, and Ucn3 mRNA levels. (A) Schematic representation of the luciferase reporter construct designed and used for in vitro verification studies in HEK293T cells. Luciferase expression is controlled by TRE and activated in the presence of both rtTA and Dox. (B) HEK293T cells transfected with both TRE-luciferase and rtTA-expressing constructs demonstrated an inducible activation of the luciferase gene following exposure to Dox. (C) Brain coronal section adapted from the Paxinos & Franklin mouse brain atlas (49), showing the PFA location (Left), a specific Ucn3 radioactive in situ hybridization indicating Ucn3-expressing cells in the rPFA (Middle), and the site of lentiviral injection as verified by immunostaining using anti-GFP antibody (Right). (D) Ucn3 mRNA levels in mouse brain with or without exposure to Dox. f, fornix; pA, polyadenylation site. *, P < 0.05.

Fig. 3.

Ucn3 overexpression at the rPFA increases anxiety-like behavior. (A–D) Anxiety-like behavior of rPFA-Ucn3 overexpressing animals as measured by the open-field test. Time spent in the center of the arena (A), latency entering the center (B), visits to the center (C), and distance traveled during the test (D) are indicative of increase in anxiety-like behavior. (E–G) Anxiety-like behavior as measured by the dark–light transfer test. The latency entering the light compartment (E), percentage of time spent in the light compartment (F), and number of transitions into the light compartment (G) are indicative of anxiogenic behavior. (H) Home-cage locomotor activity was similar in the two experimental conditions. *, P < 0.05; **, P < 0.01 vs. without Dox (paired Student's t test). Values are expressed as the mean ± SEM (n = 10 mice per group).

Fig. 4.

Metabolic alterations in the conditional rPFA-Ucn3–overexpressing transgenic mice. (A) Food intake, (B) RER profile; (C) RER averaged values, (D) oxygen consumption (VO₂), (E) carbon dioxide production (VCO₂), (F) heat production, (G) locomotor activity, and (H) rearing events were measured continuously and simultaneously. Significant differences in RER, VO_2, VCO_2, and in heat production following rPFA-Ucn3 overexpression were observed (n = 8–10 mice per group). (I and J) All-body glucose homeostasis determined using glucose-tolerance test (I) and insulin-tolerance test (J) in mice treated with or without Dox demonstrates normal glucose tolerance but reduce insulin sensitivity. Mice (n = 6–11 per group) were injected i.p. with 2 g glucose/kg body weight (BW) (I) or with 0.75 U insulin/kg BW (J), and plasma glucose was measured at the indicated time points. (K) Insulin plasma level. Values are expressed as the mean ± SEM *, P < 0.05 **, P < 0.01 vs. without Dox (repeated measurements two-way ANOVA).