Fig. 3. Repeated treatment with a low dose of exendin-4 reduces aggressive behaviors in male mice.

The outline of the resident intruder experiment with a low dose of exendin-4 (Ex4) is illustrated in Panel A. At day 1, the mice were trained in the resident intruder paradigm and the time to first attack was scored. This allows stratification (#; with similar attack score between future treatment groups) into Ex4 (1.2 µg/kg, IP, n = 8) or vehicle (Veh, IP, n = 7) injections throughout training (day 2–5) and at the test day (day 6). During training the latency to attack was recorded (Panel B), and aggressive behaviors (attack, threat, Panel C) and non-aggressive behaviors (social and non-social, Panel D) were scored for 10 min at the test day. Directly after the test, the mice were euthanized, and the brains from each mouse were collected for ex-vivo analyses (Panel E, F). A In these mice the baseline latency to attack was similar between future treatment groups (t(13) = 0.20, P = 0.8476). A A low dose of Ex4 did not alter the latency to attack (treatment F(1,13) = 0.31, P = 0.5850, time F(3,39) = 1.64, P = 0.1959, interaction F(3,39) = 0.96, P = 0.4221; n = 8, Ex4, n = 7, veh), possibly implying that higher doses of Ex4 are required to influence attack latency during the training. B This is further evident as the area under the curve was similar between vehicle and Ex4 treated mice (t(13) = 0.30, P = 0.7704). At test day, a low dose of Ex4 affected some aggressive (Panel C), but not any non-aggressive (Panel D) behaviors. Ex4 (C) did not alter attack duration (t(13) = 0.75, P = 0.4646), or (D) attack frequency (t(13) = 1.48, P = 0.1619), but (E) tended to enhance the or attack latency (t(13) = 2.02, P = 0.0650). In addition, Ex4 (F) reduced threat duration (t(13) = 3.15, P = 0.0077), (G) suppressed threat frequency (t(13) = 3.94, P = 0.0017) and (H) enhanced threat latency (t(13) = 4.24, P = 0.0010). The low dose of Ex4 did not alter (I) social behavior duration (t(13) = 0.002, P = 0.9982), (J) social behavior frequency (t(13) = 0.65, P = 0.5281), (K) social behavior latency (t(13) = 0.59, P = 0.5639) or (L) non-social behavior duration (t(13) = 1.21, P = 0.2471). Western blot (Panel E) show (M) the expression of tryptophan hydroxylase (TPH2) of raphe from mice of the above resident intruder test, (N) and the TPH2 levels are higher after a low dose of Ex4 (n = 3) compared to vehicle (n = 3). Western blots show the expression of dopamine-β-hydroxylase (DBH), noradrenalin reuptake transporter (NET), serotonin reuptake transporter (SERT), glucagon-like peptide-1 receptor (GLP1R), serotonin-1B-receptor (5HT1B) of (O) nucleus accumbens (NAc) core, (P) where no differences are evident between treatments, or (Q) NAc shell. R Compared to vehicle, a low dose of Ex4 reduces the protein levels of DBH, NET, SERT, without alerting the levels of GLP1R, or 5HT1B in NAc shell. β-actin was always used as a loading control. The relative density of the target protein band was normalized to the density of the β-actin band to represent the relative expression of the target protein. FOS immunoreactivity (Panel F) of one representative picture of (S) dorsal raphe and (U) NAc core and shell, where the FOS positive cells were quantified. Compared to vehicle (n = 4, and 2–4 slices from each animal) the low dose of Ex4 (n = 3, and 2–4 slices from each animal) did not alter the number of cells positive for FOS immunoreactivity in (T) dorsal raphe (V) NAc core (W) NAc shell. Lateral ventricle (LV), anterior commissure (AC). Data are presented as mean ± SEM; significant data are illustrated by *P < 0.05, **P < 0.01, ***P < 0.001.