Calcitonin gene-related peptide pre-administration acts as a novel antidepressant in stressed mice

Abstract

Calcitonin gene-related peptide (CGRP) is a neuropeptide that has potent vasodilator properties and is involved in various behavioral disorders. The relationship between CGRP and depression-like behavior is unclear. In this study, we used chronically stressed mice to investigate whether CGRP is involved in depression-like behavior. Each mouse was exposed to restraint and water immersion stress for 15 days. After stress exposure, mice were assessed using behavioral tests: open field test, forced swim test and sucrose preference test. Serum corticosterone levels, hippocampal proliferation and mRNA expression of neurotrophins were measured. After stress exposure, mice exhibited depression-like behavior and decreased CGRP mRNA levels in the hippocampus. Although intracerebroventricular CGRP administration (0.5 nmol) did not alter depression-like behavior after 15-day stress exposure, a single CGRP administration into the brain, before the beginning of the 15-day stress exposure, normalized the behavioral dysfunctions and increased nerve growth factor (Ngf) mRNA levels in stressed mice. Furthermore, in the mouse E14 hippocampal cell line, CGRP treatment induced increased expression of Ngf mRNA. The NGF receptor inhibitor K252a inhibited CGRP's antidepressant-like effects in stressed mice. These results suggest that CGRP expression in the mouse hippocampus is associated with depression-like behavior and changes in Ngf mRNA levels.

Figure 1. Stressed mice display depression-like behavior and changes in hippocampal proliferation.

(A) Open field test analysis showing locomotor activity, rearing activity and time spent in the center area following 15-day stress exposure in mice (control n = 5, stress n = 5). (B) Immobility times in the forced swim test (control n = 5, stress n = 5). (C) Sucrose preferences and total fluid intake in the sucrose preference test (control n = 7, stress n = 7). (D) Photomicrographs of the dentate gyrus showing representative BrdU-positive cells. Scale bar indicates 100 μm. Arrows show immunopositive cells. (E) The number of BrdU-positive cells (control n = 4, stress n = 3). (F) Serum corticosterone level (control n = 7, stress n = 7). Each bar indicates the mean ± S.E.M. *P < 0.05 vs. control.

Figure 2. Effects of 15-day stress exposure on mRNA level of Cgrp, c-fos or neurotrophic factors in the mouse hippocampus.

Quantitative real-time RT-PCR analysis showing the expression levels of Cgrp, Ngf, Bdnf, c-fos, Cntf, Gdnf, Ntf3 and Ntf5 (control n = 8, stress n = 7). Each bar indicates the mean ± S.E.M. *P < 0.05 vs. control.

Figure 3. Effects of CGRP i.c.v. administration in mouse depression-like behavior in the forced swim test.

Mice were administered saline or CGRP (0.5 nmol). (A) On the day following the 15-day stress exposure (day 16), mice were administered saline or CGRP and 24 h later, assessed in the forced swim test (saline n = 5, CGRP n = 6). (B) Mice were administered saline or CGRP (0.5 nmol) i.c.v. on the day following the 15-day stress exposure (day 16) and the forced swim test was assessed on day 30 (saline n = 6, CGRP n = 7).

Figure 4. Exogenous CGRP administration rescues depression-like behavior.

Mice were administered saline, CGRP (0.5 nmol), or CGRP in combination with the CGRP antagonist CGRP_8-37 (0.5 nmol) i.c.v. before beginning the 15-day stress exposure. (A) Immobility times in the forced swim test (non-stress (saline n = 11, CGRP n = 10), stress (saline n = 9, CGRP n = 11)). (B) Immobility times in the forced swim test (saline n = 8, CGRP n = 8, CGRP + CGRP_8-37 n = 8). (C) Sucrose preferences and total fluid intake in the sucrose preference test (saline n = 5, CGRP n = 5). No treatment significantly affected general behavior, locomotor activity, rearing activity, or time spent in the center area in the open-field test (D) (saline n = 8, CGRP n = 8). (E) Photomicrographs of the dentate gyrus showing representative BrdU-positive cells. Scale bar indicates 100 μm. Arrows show immunopositive cells. (F) The number of BrdU-positive cells (saline n = 4, CGRP n = 4). (G) Serum corticosterone level (saline n = 5, CGRP n = 5). Each bar indicates the mean ± S.E.M. *P < 0.05 vs. saline.

Figure 5. Effects of CGRP administration on mRNA level of neurotrophic factors or c-fos in the mouse hippocampus.

Mice were administered saline or CGRP (0.5 nmol) i.c.v. before beginning the 15-day stress exposure. Quantitative real-time RT-PCR analysis showing the expression levels of Ngf, Bdnf, c-fos, Cntf, Gdnf, Ntf3 and Ntf5 (non-stress (saline n = 9, CGRP n = 5), stress (saline n = 10, CGRP n = 9)). Each bar indicates the mean ± S.E.M. *P < 0.05 vs. saline.

Figure 6. Ngf mRNA levels in mouse embryonic E14 hippocampal cell line.

CGRP (100 nM) alone or CGRP with a CGRP antagonist, CGRP_8-37 (100 nM) incubated for 60 min. CGRP_8-37 was added 30 min before the addition of CGRP (saline n = 4, CGRP n = 4, CGRP + CGRP_8-37 n = 4). Each bar indicates the mean ± S.E.M.

Figure 7. The NGF receptor inhibitor K252a suppresses CGRP-induced antidepressant-like effects.

(A) No treatment significantly affected general behavior, locomotor activity, rearing activity, or time spent in the center area in the open field test (saline n = 8, K252a n = 8). (B) Immobility times in the forced swim test (saline n = 8, K252a n = 8). (C) Sucrose preference and total fluid intake in the sucrose preference test (saline n = 8, K252a n = 8). (D) Photomicrographs of the dentate gyrus showing representative BrdU-positive cells. Scale bar indicates 100 μm. Arrows show immunopositive cells. (E) The number of BrdU-positive cells (saline n = 4, K252a n = 4). K252a (25 μg/kg) was administered i.p. four times during stress exposure (day 1, 5, 10, 15). CGRP (0.5 nmol) was administered i.c.v. before beginning the 15-day stress exposure. Each bar indicates the mean ± S.E.M. *P < 0.05 vs. saline.

Figure 8. Experimental timelines.

Protocol 1: to observe spontaneous behavior and depression-related behavior, open field test (OF) and forced swim test (FST) performances were assessed on the day following the 15-day stress exposure (day 16). Protocol 2: to observe depression-related behavior, the sucrose preference test was assessed days 1–3 after the 15-day stress exposure (day 16–18). Protocol 3: to observe serum corticosterone and mRNA levels of neurotrophic factors, blood and brain samples were collected at the end of the 15-day stress exposure from mice that had not undergone behavioral testing. Protocol 4: to study cell proliferation in the hippocampus, a single injection of 5-bromo-2-deoxyuridine (BrdU; 50 mg/kg, i.p.), was administered 3 times on day 15 of the 15-day stress exposure.