Leptin/LepRb in the Ventral Tegmental Area Mediates Anxiety-Related Behaviors

Abstract

Background: Leptin, an adipose-derived hormone, has been implicated in emotional regulation. We have previously shown that systemic administration of leptin produces anxiolytic-like effects and deletion of the leptin receptor, LepRb, in midbrain dopamine neurons leads to an anxiogenic phenotype. This study investigated whether activation or deletion of LepRb in the ventral tegmental area of adult mice is capable of inducing anxiolytic and anxiogenic effects, respectively.

Methods: Mice were cannulated in the ventral tegmental area and received bilateral intra-ventral tegmental area infusions of leptin or the JAK2/STAT3 inhibitor AG490. Anxiety-like behaviors were assessed using the elevated plus-maze, light-dark box, and novelty suppressed feeding tests. Deletion of LepRb in the ventral tegmental area was achieved by bilateral injection of AAV-Cre into the ventral tegmental area of adult Lepr(flox/flox) mice. Anxiety-related behaviors were evaluated 3 weeks after viral injection.

Results: Intra-ventral tegmental area infusions of leptin reduced anxiety-like behaviors, as indicated by increased percent open-arm time and open-arm entries in the elevated plus-maze test, increased time spent in the light side and decreased latency to enter the light side of the light-dark box, and decreased latency to feed in the novelty suppressed feeding test. Blockade of JAK2/STAT3 signaling in the ventral tegmental area by AG490 attenuated the anxiolytic effect produced by systemic administration of leptin. Lepr(flox/flox) mice injected with AAV-Cre into the ventral tegmental area showed decreased leptin-induced STAT3 phosphorylation and enhanced anxiety-like behaviors in the elevated plus-maze test and the novelty suppressed feeding test.

Conclusions: These findings suggest that leptin-LepRb signaling in the ventral tegmental area plays an important role in the regulation of anxiety-related behaviors.

Keywords: JAK2/STAT3; Leptin receptor; elevated plus-maze test; light-dark box; novelty suppessed feeding.

Figure 1.

Intra-ventral tegmental area (VTA) infusion of leptin dose-dependently induces anxiolytic effects in the elevated plus-maze test. Mice received an intra-VTA infusion of leptin (0.05, 0.5 μg/side) or vehicle (artificial cerebrospinal fluid [aCSF]) 30 minutes before the test. (A) Percent open arm entries. (B) Percent open arm time. (C) The number of total entries made into open and closed arms. n = 12 to 14 each group. *P < .05, compared with the aCSF-injected control group.

Figure 2.

Effects of intra-ventral tegmental area (VTA) infusion of leptin on anxiety behavior in the light-dark box test. Mice received an intra-VTA infusion of leptin (0.5 μg/side) or vehicle (artificial cerebrospinal fluid [aCSF]) 30 minutes before the test. The latency to enter the light side (A), total time spent in the light side (B), and number of transition between light and dark compartments (C) were measured during the 5-minute test. n = 8 each group. *P < .05, compared with the aCSF-injected control group.

Figure 3.

Effects of intra-ventral tegmental area (VTA) infusion of leptin on anxiety behavior in the novelty suppressed feeding test. Mice fasted for 24 hours received an intra-VTA infusion of leptin (0.5 μg/side) or vehicle (artificial cerebrospinal fluid [aCSF]) 30 minutes before the test. (A) The latency to feed. (B) Home-cage food consumption within 5 minutes immediately after the test. n = 6–7 each group. * P < .05 compared with the aCSF-injected control group.

Figure 4.

Effects of intra-ventral tegmental area (VTA) infusion of leptin on locomotor activity. Mice received an intra-VTA infusion of leptin (0.5 μg/side) or vehicle (artificial cerebrospinal fluid [aCSF]) 30 minutes before the locomotion test. (A) The distance traveled in 5-minute bins. (B) Total distance traveled within the 15 minutes. n = 6 each group.

Figure 5.

Histological verification of intra-ventral tegmental area (VTA) injection sites. (A) Representative images showing the deposition sites within the VTA. Arrows indicated the rostral-caudal sequence of the coronal sections. (B) Schematic illustration of bilateral injection sites in the VTA. The drawings of coronal sections were derived from the atlas of Paxinos and Franklin (2001).

Figure 6.

Effects of intra-ventral tegmental area (VTA) infusion of AG490 on leptin’s anxiolytic effects in the elevated plus-maze test. Mice received an intra-VTA infusion of AG490 (0.15 nmol/side) or vehicle 60 minutes before an i.p. injection of leptin (1mg/kg) or saline. The elevated plus-maze test was performed 30 minutes after leptin injection. (A) Percent open arm entries. (B) Percent open arm time. (C) The number of total entries made into open and closed arms. n = 10 to 14 each group. ***P < .001, compared with the Vehicle+Saline control group; ^## P < .01, compared with the Vehicle+Leptin group. (D) Schematic illustration of bilateral injection sites in the VTA.

Figure 7.

Adeno-associated virus (AAV)-Cre–mediated deletion of leptin receptor (LepRb) in the intra-ventral tegmental area (VTA) of adult Lepr^flox/flox mice. (A) Schematic diagram depicting the floxed Lepr allele and the Lepr floxed allele after Cre recombination. (B) Left, schematic illustration of AAV injection (Paxinos and Franklin, 2001). Right, Cre-GFP expression in the VTA. (C) Immunohistochemical staining showing leptin-induced STAT3 phosphorylation in the VTA in AAV-GFP (left) and AAV-Cre-GFP (right) injected mice. IP, interpeduncular nucleus; ML, medial lemniscus; SN, substantia nigra.

Figure 8.

Adeno-associated virus (AAV)-Cre–mediated leptin receptor (LepRb) deletion in the ventral tegmental area (VTA) results in anxiogenic-like behaviors. (A) Timeline of experimental procedures and behavioral tests. (B) Elevated plus-maze test. Left, percent open arm entries; middle, percent open arm time; right, number of total entries. n = 13 to 15 each group. (C) Light-dark box test. Left, latency to enter the light side; middle, time spent in the light compartment; right, number of transitions between light and dark compartments. n = 12 to 14 each group. (D) Novelty suppressed feeding test. Left, latency to feed; right, home-cage food consumption within 5 minutes immediately after the test. n = 12 to 15 each group. (E) Body weight before or after AAV injection. n = 10 to 13 each group. *P < .05; **P < .01 compared with the AAV-GFP control group.