Neuregulin 1 regulates pyramidal neuron activity via ErbB4 in parvalbumin-positive interneurons

Abstract

Neuregulin 1 (NRG1) is a trophic factor thought to play a role in neural development. Recent studies suggest that it may regulate neurotransmission, mechanisms of which remain elusive. Here we show that NRG1, via stimulating GABA release from interneurons, inhibits pyramidal neurons in the prefrontal cortex (PFC). Ablation of the NRG1 receptor ErbB4 in parvalbumin (PV)-positive interneurons prevented NRG1 from stimulating GABA release and from inhibiting pyramidal neurons. PV-ErbB4(-/-) mice exhibited schizophrenia-relevant phenotypes similar to those observed in NRG1 or ErbB4 null mutant mice, including hyperactivity, impaired working memory, and deficit in prepulse inhibition (PPI) that was ameliorated by diazepam, a GABA enhancer. These results indicate that NRG1 regulates the activity of pyramidal neurons by promoting GABA release from PV-positive interneurons, identifying a critical function of NRG1 in balancing brain activity. Because both NRG1 and ErbB4 are susceptibility genes of schizophrenia, our study provides insight into potential pathogenic mechanisms of schizophrenia and suggests that PV-ErbB4(-/-) mice may serve as a model in the study of this and relevant brain disorders.

Fig. 1.

NRG1 inhibition of spontaneous spikes of PFC pyramidal neurons. (A) NRG1 reduced whereas ecto-ErbB4 increased spontaneous spike activities of layers II–V pyramidal neurons. PFC slices were treated with vehicle (control), 5 nM NRG1, or 1 μg/mL ecto-ErbB4 in the absence (Upper) or presence (Lower) of 20 μM bicuculline. Pyramidal neuron firings were recorded under loose patch-clamp configuration. (B) Quantitative analysis of spontaneous firing rates. n = 7, *P < 0.05 in comparison with control; ^#P < 0.05 in comparison with NRG1. There was no significant difference in firing rates of the three groups: bicuculline alone, bicuculline/NRG1, and bicuculline/ecto-ErbB4 (P > 0.05).

Fig. 2.

NRG1 inhibition of evoked firing of PFC pyramidal neurons. (A) NRG1 reduced whereas ecto-ErbB4 enhanced action potential frequency of pyramidal neurons in a whole-cell patch-clamping configuration. Representative action potentials are shown of a single neuron before (baseline) and after bath application of vehicle (control), 5 nM NRG1, 5 nM denatured NRG1, or 1 μg/mL ecto-ErbB4, or 20 μM bicuculline and 5 nM NRG1, or 20 μM bicuculline and 1 μg/mL ecto-ErbB4. (B) Quantitative analysis of data in A. n = 9, *P < 0.05, compared with control; ^#P < 0.05, compared with NRG1. (C) Dose-dependent inhibition of NRG1 on evoked spikes of pyramidal neurons (n = 11).

Fig. 3.

Ablation of ErbB4 in PV-positive neurons prevented NRG1 from increasing GABAergic transmission. (A) Reduced levels of ErbB4 in PV-Cre;ErbB4^−/− PFC. PFC was isolated from PV-Cre;ErbB4^−/− and control littlemates (PV-Cre;ErbB4^+/+) and homogenized. Resulting homogenates (40 μg of protein) were subjected to Western blotting analysis with the indicated antibodies. Equal loading was shown by immunoblotting for β-actin. (B) Specific ablation of ErbB4 in PV-positive neurons. PFC slices of PV-Cre;ErbB4^−/− and PV-Cre;ErbB4^+/+ were stained with anti-ErbB4 and PV antibody. Immunoactivity was visualized by Alexa 488- and Alexa 594-conjugated secondary antibodies, respectively. Slices were also stained with DAPI to indicate nuclei. Arrows, PV-positive neurons; arrowheads, PV-negative neurons. (Scale bar, 10 μm.) (C) NRG1 increased eIPSCs in PFC slices of control, but not PV-Cre;ErbB4^−/−, mice. Concentrations were 5 nM for NRG1 and 40 μM for bicuculline. (D) Quantitative analysis of data in C (n = 8, *P < 0.01, compared with control). Control eIPSC amplitudes were 2,150 ± 128 and 1,650 ± 153 pA for PV-Cre;ErbB4^+/+ and PV-Cre;ErbB4^−/− mice, respectively (n = 8, P < 0.05). (E) No effect of NRG1 on eIPSCs at higher concentrations in PV-Cre;ErbB4^−/− PFC. Dose–response curves are shown for both control and mutant PFC (n = ∼5–8; *P < 0.05, compared with mutant PFC).

Fig. 4.

NRG1 was unable to inhibit pyramidal neuron firing in PV-Cre;ErbB4^−/− mice. (A and B) No effect of NRG1 or ecto-ErbB4 on pyramidal neuron firing in PV-Cre;ErbB4^−/− PFC. (A) Representative spontaneous firing patterns of pyramidal neurons of control or treated slices (5 nM NRG1 or 1 μg/mL ecto-ErbB4) of PV-Cre;ErbB4^+/+ and PV-Cre;ErbB4^−/− mice. (B) Quantitative analysis of data in A (n > 6 for both PV-Cre;ErbB4^+/+ and PV-Cre;ErbB4^−/− mice, *P < 0.01, compared with control; ^#P < 0.05, compared with PV-Cre;ErbB4^+/+ samples). (C and D) Inability of NRG1 to inhibit evoked action potential frequency of pyramidal neurons in mutant PFC. (C) Representative action potentials produced by a 200-pA current before (Upper) and after (Lower) bath application of 5 nM NRG1 in PFC slices from PV-Cre;ErbB4^+/+ and PV-Cre;ErbB4^−/− mice. (D) Quantitative analysis of evoked spike frequency of pyramidal neurons (n = 9; *P < 0.05, compared with control; ^#P < 0.05, compared with PV-Cre;ErbB4^+/+ samples).

Fig. 5.

Hyperactivity and impaired working memory in PV-Cre;ErbB4^−/− mice. (A–C) Mice were placed in a chamber and movements were monitored for 30 min in the open field test. (A and B) Enhanced ambulatory and stereotypic activity in PV-Cre;ErbB4^−/− mice (repeated measures for genotype, P = 0.045 for ambulatory activity and P = 0.043 for stereotypic activity). Ambulatory activity was measured as the total horizontal photobeam breaks, and stereotypy was quantified in terms of repetitive breaks of a given beam with intervals of <1 s. Activity was summated at 5-min intervals over a 30-min period. (C) Similar vertical activity between PV-Cre;ErbB4^−/− and PV-Cre;ErbB4^+/+ mice (repeated measures for genotype, P = 0.202). Vertical activity (rearing) was evaluated as the total number of vertical beam breaks at 5-min intervals over a 30-min period. n = 7 for PV-Cre;ErbB4^+/+ mice; n = 6 for PV-Cre;ErbB4^−/− mice. (D–F) Working memory of food-restricted mutant (n = 10) and control (n = 9) mice in four-arm or eight-arm radial maze tests. (D) Total number of errors (revisits and omission) were significantly higher in PV-Cre;ErbB4^−/− mice in both four-arm and eight-arm tests (repeated measures, P = 0.002 and P = 0.021, respectively). A significant trial effect was observed in the four-arm test (P < 0.001), but not in the eight-arm test (P = 0.290). (E) Time spent by mutant and control mice to retrieve all food pellets was similar (P = 0.096 for four-arm and P = 0.085 for eight-arm test). A significant trial effect was observed for both four-arm and eight-arm tests (P < 0.001). (F) Percentage of correct entries within the first four and eight entries was significantly lower in mutant mice in the four-arm test (P = 0.044) and the eight-arm test (P = 0.040), respectively. A significant trial effect was observed in both tests (P < 0.001 and P = 0.002, respectively).

Fig. 6.

PPI deficit in PV-Cre;ErbB4^−/− mice and its amelioration by diazepam. (A) Similar mean baseline startle response in PV-Cre;ErbB4^−/− mice and control littermates. Response to background white noise (no stimulus, 70 dB) and auditory-evoked startle stimulus (120 dB) was measured. n = 7 and 6 for PV-Cre;ErbB4^+/+ and PV-Cre;ErbB4^−/− mice, respectively; P > 0.05. AU, arbitrary units. (B) Reduced PPI in PV-Cre;ErbB4^−/− mice. PPI (%) was calculated according to the formula: [100 − (startle amplitude on prepulse-pulse trials/startle amplitude on pulse alone trials) × 100]. n = 7 and 6 for PV-Cre;ErbB4^+/+ and PV-Cre;ErbB4^−/− mice, respectively; repeated measures, P = 0.004. (C) Diazepam attenuated PPI deficit in PV-Cre;ErbB4^−/− mice. Diazepam (3 mg/kg) or vehicle was injected intraperitoneally 30 min before PPI test. Repeated measures, *P < 0.05, ^#P = 0.573. For vehicle treatment, n = 10 and 12 for PV-Cre;ErbB4^−/− mice and control littermates, respectively; for diazepam treatment, n = 9 and 11 for PV-Cre;ErbB4^−/− and controls, respectively.