Figure 1.
The EGF domain of NRG3 permeates the neonatal mouse BBB in a dose dependent manner, activating ErbB4-Akt signaling.
Activation levels of pErbB4 (A), and pAktSer473 (B) in hemi-brain lysates of PND2 mice 3 hours following s.c. injection of vehicle (PBS/0.1% BSA), 1–3 mg/kg NRG3-EGF or 1 mg/kg NRG1-EGF. Representative blots of tErbB4, pErbB4, tAKT and pAKT ser473 protein levels are depicted (upper panels). N = 4–5/treatment group, data represents mean± s.e.m ratio of the respective phosphorylated/total protein expression relative to vehicle treatment group. *p<0.05, **p<0.01 compared to vehicle treated group.
Table 1.
Effects of neonatal peripheral overexposure to NRG1 and NRG3 on developmental milestones and general health in adulthood.
Figure 2.
Overexposure to NRG3 during the neonatal period induces an anxiety-like phenotype in adulthood, but has no impact on locomotor activity or temporal order recency discrimination.
(A) Total distance travelled in 5 minute intervals over a 1 hr exposure to the open field arena did not differ between NRG3-EGF or Vehicle treated mice (n = 12–14/treatment group). (B)Time spent in the center of the open field over a 1 hr period was significantly reduced in mice treated with NRG3-EGF as neonates compared to control treated mice (n = 12–14/treatment group). (C) Representative track plots of exploration in the open field of vehicle (left panel) and NRG3-EGF (right panel) overexposed mice. Inner area designated by white dashed line designates the center arena. (D) Adult mice treated with NRG3-EGF or vehicle during PND 2–10 performed successfully in the temporal order object recognition task of recency discrimination as adults (Discrimination ratio >0, n = 11–14/treatment group). (E) After a 10 minute period of habituation to the open field (indicated by dashed vertical line), mice neonatally overexposed to NRG3-EGF or vehicle were given a single i.p. injection of saline or 3 mg/kg amphetamine. No effect of treatment (NRG3-EGF) was observed on total distance travelled in 5 minute intervals of the subsequent 75 minutes in the context of amphetamine at a dose of 3 mg/kg (n = 6–7/group). Data represents mean ± s.e.m., *p<0.05 compared to vehicle treated mice.
Figure 3.
Neonatal overexposure to NRG1 but not NRG3 impairs sensorimotor gating in adulthood.
The effect of daily treatment from PND 2–10 with 3 mg/kg NRG3-EGF (A, B) or 1 mg/kg NRG1-EGF (C, D) on startle responses to the presentation of no stimulus or a 120 dB stimulus (A, C) and % prepulse inhibition of the startle response to the presentation of prepulse stimuli at amplitudes of 74, 78, 82, 86 and 90 dB (B, D) in adulthood. Data represents mean ± s.e.m., n = 8–14/treatment group. **p<0.01, main effect of treatment.
Figure 4.
Neonatal overexposure to NRG3 impairs social behaviors in adulthood.
(A) Vehicle and NRG3-EGF treated mice spent more time in chamber with the novel mouse 1 than a novel object in the 3-chamber test of sociability. (B) Both treatment groups showed preference for the novel mouse 1 in time spent sniffing; however, the preference was blunted in mice overexposed to NRG3-EGF compared to vehicle treated mice. (C) NRG3-EGF treated mice showed absence of preference for social novelty, spending equal time in the chamber with familiar mouse 1 and novel mouse 2. (D) Sniff time at familiar mouse 1 and novel mouse 2 was comparable in NRG3-EGF overexposed mice, whereas vehicle treated mice showed a significant preference for the more unfamiliar novel mouse 2. (E) During the initial 10-minute habituation period to the apparatus the number of entries was equal between right and left chambers. (F) Schematic showing the setup of the trials in the three-chambered social test apparatus. n = 11–12/treatment group. Data represents mean ± s.e.m. **p<0.01, ***p<0.001 within group comparison (novel mouse 1 vs. novel object or familiar mouse 1 vs. novel mouse 2), #p<0.05,##p<0.01 between group comparison (Vehicle vs. NRG3-EGF treated mice).