Genetic recovery of ErbB4 in adulthood partially restores brain functions in null mice

Abstract

Neurotrophic factor NRG1 and its receptor ErbB4 play a role in GABAergic circuit assembly during development. ErbB4 null mice possess fewer interneurons, have decreased GABA release, and show impaired behavior in various paradigms. In addition, NRG1 and ErbB4 have also been implicated in regulating GABAergic transmission and plasticity in matured brains. However, current ErbB4 mutant strains are unable to determine whether phenotypes in adult mutant mice result from abnormal neural development. This important question, a glaring gap in understanding NRG1-ErbB4 function, was addressed by using two strains of mice with temporal control of ErbB4 deletion and expression, respectively. We found that ErbB4 deletion in adult mice impaired behavior and GABA release but had no effect on neuron numbers and morphology. On the other hand, some deficits due to the ErbB4 null mutation during development were alleviated by restoring ErbB4 expression at the adult stage. Together, our results indicate a critical role of NRG1-ErbB4 signaling in GABAergic transmission and behavior in adulthood and suggest that restoring NRG1-ErbB4 signaling at the postdevelopmental stage might benefit relevant brain disorders.

Keywords: ErbB4; GABA; adulthood; schizophrenia; treatment.

Fig. 1.

Behavioral deficits in Tam-treated iKO mice. (A) Breeding diagram of iKO mice. (B) Times of Tam injection and analysis. Tam was injected once every other day for 20 d. (C and D) Diminished ErbB4 expression in iKO+Tam mice and ErbB4 null mice. From left to right, WT, ErbB4 null, ErbB4^f/f, iKO+Veh, and iKO+Tam; n = 3 mice in each group. CT, cortex; HC, hippocampus. (E) Representative travel traces of mice in an open-field test. (F) Increased travel distance (per 5 min) by iKO+Tam mice. (G) Increased total travel distance (within 30 min) by iKO+Tam mice. (H) Diagram of the PPI test. PPI (%) = 100 × (a − b)/a. (I) Impaired PPI of iKO+Tam mice. (J) Decreased preference for the social chamber by iKO+Tam mice. (K) Decreased preference for social novelty by iKO+Tam mice. (L) Diagram of contextual fear memory test. (M) Unaltered freezing response to foot shock in training session and reduced freezing time in test session by iKO+Tam mice. n = 12 per group for behavior tests; *P < 0.05, **P < 0.01, compared with ErbB4^f/f mice; ^#P < 0.05, ^##P < 0.01, compared with ErbB4 null mice; ^$P < 0.05, ^$$P < 0.01, compared with iKO+Veh mice.

Fig. 2.

Decreased inhibitory transmission in Tam-treated iKO mice. (A) Recording diagram. (B) Decreased eIPSC amplitude in iKO+Tam mice. n = 7/8 neurons from three mice. (C) Representative traces of mIPSCs. (Scale bar: 20 pA/s.) (D) No change in mIPSC amplitude. (E) Decreased mIPSC frequency. n = 8/9 neurons from three mice. (F) Representative images of PV+ inhibitory synapses onto NeuN+ postsynaptic somata. (Scale bar: Upper, 5 µm; Lower, 2 µm.) (G) Quantification data of F. n = 40/42/42 somata of six mice. (H) Representative IPSC traces induced by paired-pulse stimulation with a 100-ms interval. (Scale bar: 250 pA/50 ms.) (I) Increased paired-pulse ratios in iKO+Tam mice. n = 10 neurons from four mice. (J) Diagram of inhibitory synapses onto axon initial segments. (K) Representative images of GAD67+ inhibitory synapses onto AnkG+ axon initial segments. AnkG, Ankyrin G. (Scale bar: 5 µm.) (L) Quantification data of K. n = 42/40/43 neurons of six mice. *P < 0.05, **P < 0.01, compared with ErbB4^f/f mice; ^$P < 0.05, ^$$P < 0.01, compared with iKO+Veh mice.

Fig. 3.

Generation and characterization of rKO mice. (A) ErbB4 alleles of WT, Stop-ErbB4, and rKO+Tam mice. (B) Breeding diagram of rKO mice. (C) Expression of ErbB4 in Stop-ErbB4;α-MHC::ErbB4 mice. (D and E) Restored ErbB4 expression in the cortex and hippocampus of rKO+Tam mice. CT, cortex; HC, hippocampus; from left to right: control, ErbB4 null, iKO+Veh double, iKO+Tam. n = 4 mice; **P < 0.01, compared with control, ^$$P < 0.01, compared with rKO+Veh mice.

Fig. 4.

Diminished GABA transmission deficits in Tam-treated rKO mice. (A) Recording diagram. (B) Diminished reduction of eIPSC amplitude in rKO+Tam mice. n = 7/8 neurons from three mice. (C) Representative traces of mIPSCs. (Scale bar: 20 pA/s.) (D) Diminished reduction of mIPSC frequency in rKO+Tam mice. (E) No difference in mIPSC amplitude among groups. n = 8/9 neurons from three mice. (F) Representative images of PV+ inhibitory synapses onto NeuN+ postsynaptic somata. (Scale bar: Upper, 5 µm; Lower, 2 µm.) (G) No effect of Tam treatment on decreased number of inhibitory synapses. n = 45/45/47 somata of seven mice. (H) Representative IPSC traces induced by paired-pulse stimulation with a 100-ms interval. (Scale bar: 250 pA/50 ms.) (I) Recovered paired-pulse ratios in Tam-treated rKO mice. n = 9/10 neurons of four mice. (J) Diagram of inhibitory synapses onto axon initial segments. (K) Representative images of GAD67+ inhibitory synapses onto AnkG+ postsynaptic axon initial segments. AnkG, ankyrin G. (Scale bar: 5 µm.) (L) No effect of Tam treatment on decreased number of inhibitory synapses. n = 45/44/47 neurons of seven mice. *P < 0.05, **P < 0.01, compared with control, ^$P < 0.05, ^$$P < 0.01, compared with rKO+Veh mice.

Fig. 5.

Mitigated behavioral deficits in Tam-treated rKO mice. (A) Representative travel traces of mice in an open-field test. (B) Mitigation of increased travel distance (per 5 min) in rKO+Tam mice. (C) Mitigation of increased total travel distance (within 30 min) in rKO+Tam mice. (D) Diagram of the PPI test. PPI (%) = 100 × (a − b)/a. (E) Mitigation of impaired PPI in rKO+Tam mice. (F) Diagram of contextual fear memory test. (G) Mitigation of reduced freezing time in rKO+Tam mice. n = 12 mice; *P < 0.05, **P < 0.01, compared with control; ^$P < 0.05, ^$$P < 0.01, compared with rKO+Veh mice.