The importance of the NRG-1/ErbB4 pathway for synaptic plasticity and behaviors associated with psychiatric disorders

Abstract

Neuregulin 1 (NRG-1) and its receptor ErbB4 have emerged as biologically plausible schizophrenia risk factors, modulators of GABAergic and dopaminergic neurotransmission, and as potent regulators of glutamatergic synaptic plasticity. NRG-1 acutely depotentiates LTP in hippocampal slices, and blocking ErbB kinase activity inhibits LTP reversal by theta-pulse stimuli (TPS), an activity-dependent reversal paradigm. NRG-1/ErbB4 signaling in parvalbumin (PV) interneurons has been implicated in inhibitory transmission onto pyramidal neurons. However, the role of ErbB4, in particular in PV interneurons, for LTP reversal has not been investigated. Here we show that ErbB4-null (ErbB4(-/-)) and PV interneuron-restricted mutant (PV-Cre;ErbB4) mice, as well as NRG-1 hypomorphic mice, exhibit increased hippocampal LTP. Moreover, both ErbB4(-/-) and PV-Cre;ErbB4 mice lack TPS-mediated LTP reversal. A comparative behavioral analysis of full and conditional ErbB4 mutant mice revealed that both exhibit hyperactivity in a novel environment and deficits in prepulse inhibition of the startle response. Strikingly, however, only ErbB4(-/-) mice exhibit reduced anxiety-like behaviors in the elevated plus maze task and deficits in cued and contextual fear conditioning. These results suggest that aberrant NRG-1/ErbB4 signaling in PV interneurons accounts for some but not all behavioral abnormalities observed in ErbB4(-/-) mice. Consistent with the observation that PV-Cre;ErbB4 mice exhibit normal fear conditioning, we find that ErbB4 is broadly expressed in the amygdala, largely by cells negative for PV. These findings are important to better understand ErbB4's role in complex behaviors and warrant further analysis of ErbB4 mutant mice lacking the receptor in distinct neuron types.

Figure 1.

Effects of acute ErbB receptor activation on LTP induction at SC→CA1 synapses in WT mice. Synaptic responses were measured in whole-cell voltage-clamp mode beginning 10 min before and continuing for 60 min following LTP induction by pairing (arrow). EPSC amplitudes are relative to baseline (set as 100%; dotted line). Perfusion with 1 nm NRG-1β for 10 min before LTP induction (−15 min to −5 min relative to pairing; black bar) effectively inhibited LTP induction (blue squares). Following pretreatment with 10 μm PD158780, NRG-1β no longer inhibited LTP induction (black squares). Rather, EPSC amplitudes exhibited a small but significant increase compared with normal controls (filled circles). N = 5 (normal control), 6 (NRG-1β), 5 (NRG-1β + PD158780). **p < 0.01.

Figure 2.

LTP in ErbB4 and NRG-1 mutant mice. EPSCs were measured as described in Figure 1. Compared with normal controls (A, B, D, E; black circles), hippocampal slices from ErbB4^−/− (A; solid blue circles) and ErbB4^+/− mice (B; open blue circles) exhibited enhanced LTP throughout the entire recording period following pairing. N = 6 (WT), 8 (ErbB4^−/−), 4 (ErbB4^−/+). C, Top, Western blot analysis of ErbB4 protein levels in whole tissue lysates from the frontal cortex (FC) and hippocampus (HC) of control ErbB4^f/f (−cre) and PV-Cre;ErbB4 (+cre) mice. Representative results from two animals are shown for each genotype. Clathrin heavy chain (CHC) is included as a loading control. Bottom, Coimmunofluorescence of ErbB4 (red) and PV (green) in the frontal cortex of PV-Cre;ErbB4 mice. In ErbB4^f/f controls, numerous PV(+) cells coexpress ErbB4 (arrows). In contrast, ErbB4 is not detected in PV(+) cells from PV-Cre;ErbB4 mice. Arrowheads mark ErbB4(+) cells that are negative for PV. Scale bars, 100 μm. D, Selective ablation of ErbB4 in PV interneurons (green) results in higher LTP, mimicking the effects of complete loss of ErbB4. N = 8 (ErbB4^f/f controls) and 6 (PV-Cre;ErbB4; green). E, LTP was also increased in slices from NRG-1^+/− mice (gray), although it runs down somewhat 25 min postinduction. N = 5 (WT), 8 (NRG-1^+/−). ***p < 0.001.

Figure 3.

LTP reversal in full and conditional ErbB4 knock-outs and hypomorphic NRG-1 mice. A, No LTP reversal by NRG-1β in SC→CA1 synapses from ErbB4^−/− mice. EPSC amplitudes were recorded as described in Figure 1. Addition of 1 nm NRG-1β (black bar) triggers the rapid reversal of LTP in slices from wild-type control mice (black circles) but not from ErbB4 mutant mice (blue circles). N = 8 (WT controls and ErbB4^−/−). B–D, Analysis of LTP reversal by theta-pulse stimuli. TPS (arrowheads), delivered 5 min after LTP induction (arrows), rapidly reverse LTP in hippocampal slices from normal controls (black circles) but not from ErbB4^−/− (B, blue circles) or PV-Cre;ErbB4 (C, green) mice. In slices from NRG-1^+/− mice (D, gray), TPS mostly, but not completely, reverse LTP. N = 5 (ErbB4^−/− and WT controls), 7 (PV-Cre;ErbB4), 6 (ErbB4^f/f controls), 6 (NRG-1^+/− and WT controls). ***p < 0.002.

Figure 4.

ErbB4^−/− and PV-Cre;ErbB4 mice are hyperactive. A, B, Time course of spontaneous locomotor activity during the first 30 min following placement in the novel environment for ErbB4^−/− (gray triangles) and its littermate control group (Con, black diamonds) (A) and for PV-Cre;ErbB4 mice (gray squares) and its respective littermate controls (Con, black diamonds) (B). Data points represent 5 min bins of locomotor activity. N = 17 for ErbB4^−/− and its control group; N = 8 for PV-Cre;ErbB4 and 11 for its control group.

Figure 5.

ErbB4^−/− and PV-Cre;ErbB4 mice show deficits in PPI of the acoustic startle response. A, B, Percentage inhibition of the startle response as a function of increasing prepulse sound levels (68, 71, 77, 82 dB) for ErbB4^−/− and their controls (Con) (N = 8 for both groups; A) and for PV-Cre;ErbB4 and their controls (N = 12 for both groups; B). A 65 dB background sound was presented throughout the sessions. *p < 0.05; **p < 0.01.

Figure 6.

Reduced anxiety in ErbB4^−/− but not PV-Cre;ErbB4 mice. A, B, Time spent in open and closed arms of the elevated plus maze during 5 min trials for ErbB4^−/− (N = 7 for both groups; A) and for PV-Cre;ErbB4. N = 11 for controls (Con) and 8 for PV-Cre;ErbB4; B]. *p < 0.05.

Figure 7.

ErbB4^−/− mice, but not PV-Cre;ErbB4 mice, exhibit deficits in fear conditioning. A–D, Freezing behavior, plotted as the fraction of time spent motionless, was recorded for 3 min following presentation of the CS to assess cued fear conditioning (A, C) and for 5 min following return to the training chamber to measure contextual fear conditioning (B, D). Generalized fear (no cue) was assessed for 2 min before presentation of the CS. ErbB4^−/− mice exhibit reduced cued and contextual fear responses (A, B; N = 8 for both groups), whereas responses in PV-Cre;ErbB4 mice were not different from controls (Con) (C, D; N = 11 for controls and 8 for PV-Cre;ErbB4). *p < 0.05, **p < 0.01.

Figure 8.

ErbB4 is abundantly expressed in the amygdala and shows only modest colocalization with PV. A, B, In situ hybridization of PV and ErbB4 in coronal sections of P56 mice (Allen Brain Atlas, 2009). Boxed areas correspond to the region shown in C and include the amygdala (ventrally), caudate/putamen, and the lateral global pallidus (dorsally). PV mRNA signals are dense in the lateral globus pallidus (LGP), sparse in the BLA, and not detectable in the BMA. By contrast, ErbB4 mRNA signals are very dense in the BMA and more sparsely distributed in most other areas. C, Co(immuno)fluorescence micrograph image of tdTomato (tdTom; red), reporting ErbB4 expression in adult Ai14 × ErbB4-2A-CreERT2 mice, and endogenous PV (green). The boxed area is magnified in D–F. While few tdTomato-labeled cells in the BLA coexpress PV (arrows), virtually none of the large number of the ErbB4-expressing cells in the BMA and other intercalated areas coexpress PV. CPu, Caudate–putamen; Ctx, cerebral cortex; LGP, lateral globus pallidus. Scale bar, 100 μm.