Schizophrenia susceptibility pathway neuregulin 1-ErbB4 suppresses Src upregulation of NMDA receptors

Abstract

Hypofunction of the N-methyl D-aspartate subtype of glutamate receptor (NMDAR) is hypothesized to be a mechanism underlying cognitive dysfunction in individuals with schizophrenia. For the schizophrenia-linked genes NRG1 and ERBB4, NMDAR hypofunction is thus considered a key detrimental consequence of the excessive NRG1-ErbB4 signaling found in people with schizophrenia. However, we show here that neuregulin 1β-ErbB4 (NRG1β-ErbB4) signaling does not cause general hypofunction of NMDARs. Rather, we find that, in the hippocampus and prefrontal cortex, NRG1β-ErbB4 signaling suppresses the enhancement of synaptic NMDAR currents by the nonreceptor tyrosine kinase Src. NRG1β-ErbB4 signaling prevented induction of long-term potentiation at hippocampal Schaffer collateral-CA1 synapses and suppressed Src-dependent enhancement of NMDAR responses during theta-burst stimulation. Moreover, NRG1β-ErbB4 signaling prevented theta burst-induced phosphorylation of GluN2B by inhibiting Src kinase activity. We propose that NRG1-ErbB4 signaling participates in cognitive dysfunction in schizophrenia by aberrantly suppressing Src-mediated enhancement of synaptic NMDAR function.

Figure 1

NRG1β-ErbB4 signaling prevents endogenous Src activation–induced potentiation of NMDAR-mediated synaptic responses in the hippocampal CA1. (a) Scatter plot of NMDAR EPSC peak amplitude over time (min) from three rat CA1 neurons recorded with control intracellular solution (ICS), ICS containing EPQ(pY)EEIPIA or ICS containing EPQ(pY)EEIPIA and NRG1β (2 nM) bath-applied beginning 20 min before whole-cell recording (black bar). Top, average NMDAR EPSC traces from the three neurons (scale bars, 45 ms; 50 pA) at the times indicated (1 and 2). (b) Summary scatter plot of NMDAR EPSC peak amplitude with control ICS (n = 20), EPQ(pY)EEIPIA (n = 13) or EPQ(pY)EEIPIA with NRG1β in ACSF (n = 7; *P < 0.05 or **P < 0.01 versus EPQ(pY)EEIPIA). (c) Current-voltage (I-V) relationship for pharmacologically isolated NMDARs with control solution, EPQ(pY)EEIPIA or EPQ(pY)EEIPIA with NRG1β in ACSF. Right, superimposed NMDAR EPSC traces at membrane potentials from −80 to +60 mV. Scale bars, 150 ms; 250 pA. (d) Summary scatter plot of normalized peak NMDAR EPSC amplitude from CA1 neurons from WT mice during intracellular administration of ICS containing EPQ(pY)EEIPIA with NRG1β (2 nM) in ACSF (n = 18), from neurons from Erbb4^−/−HER4^heart mice during intracellular administration of EPQ(pY)EEIPIA with NRG1β (2 nM) (n = 7) or from neurons from WT mice during intracellular administration of EPQ(pY)EEIPIA with bath-applied NRG1β (2 nM) and AG1478 (n = 16). Top, representative average NMDAR EPSCs at the indicated times (1 and 2) and after D-APV (scale bars, 50 ms; 50 pA). (e) Summary histogram of EPQ(pY)EEIPIA-induced increase in NMDAR EPSC amplitude at 30 min of recording from WT neurons with ICS containing EPQ(pY)EEIPIA (n = 13), ICS containing EPQ(pY)EEIPIA with NRG1β present in ACSF beginning 20 min before whole-cell recording (n = 18), from CA1 pyramidal neurons from Erbb4^−/−HER4^heart mice with ICS containing EPQ(pY)EEIPIA with NRG1β in ACSF (n = 7) or from WT CA1 pyramidal neurons with ICS containing EPQ(pY)EEIPIA with NRG1β and AG1478 in ACSF (n = 16). ***P < 0.001 versus wild-type. In all figures, group data are mean ± s.e.m. In voltage-clamp experiments the holding potential was −60 mV, except where otherwise indicated.

Figure 2

NRG1β has no effect on basal NMDAR-mediated synaptic responses in hippocampal CA1 or in prefrontal cortex but prevents endogenous Src activation-induced potentiation of NMDAR EPSCs at prefrontal cortex synapses. (a) Summary scatter plot of peak NMDAR EPSC amplitude over time from mouse CA1 neurons recorded with control ICS (n = 10) and NRG1β (2 nM) present in ACSF beginning at 10 min (black bar). Top, average NMDAR EPSCs from a representative neuron (scale bars, 50 ms; 50 pA) at the times indicated (1 and 2) and after D-APV. (b) Summary histogram of NMDAR EPSC decay from the CA1 neurons recorded in a at the 10-min time point immediately before NRG1β administration and at the 40-min time point during NRG1β perfusion. Results are percentage of NMDAR EPSC decay with mean decay during first 2 min of recording before NRG1β administration normalized to 100% (dotted line). (c) Summary histogram of pharmacologically isolated NMDAR fEPSP responses (n = 14) before or during PD158780 application. Results are percentage of NMDAR fEPSP slope at the beginning of the recording normalized to 100% (dotted line). (d) Scatter plot of NMDAR EPSC peak amplitude from four prefrontal cortex neurons: with control ICS, with control ICS with NRG1β (6 nM) in ACSF, with ICS containing EPQ(pY)EEIPIA or with ICS containing EPQ(pY)EEIPIA with NRG1β in ACSF (black bar). Top, average NMDAR EPSC traces from the four neurons (scale bars, 100 ms; 40 pA) at the times indicated (1 and 2). (e) Summary histogram of normalized NMDAR EPSC amplitude at 30 min of recording from prefrontal cortex neurons with control ICS (n = 11), ICS containing EPQ(pY)EEIPIA (n = 9), control ICS with NRG1β present in ACSF (n = 7) or ICS containing EPQ(pY)EEIPIA with NRG1β in ACSF (n = 5). ***P < 0.001.

Figure 3

NRG1β prevents but does not reverse endogenous Src-induced synaptic potentiation. (a) Scatter plot of EPSP slope over time from three rat CA1 neurons recorded with control ICS, ICS containing EPQ(pY)EEIPIA or ICS containing EPQ(pY)EEIPIA with NRG1β (2 nM) in the ACSF beginning 20 min before whole-cell recording (black bar). Right, average EPSP traces from the three neurons (scale bars, 50 ms; 10 mV). Bottom, scatter plots of simultaneously recorded fEPSPs from CA1 stratum radiatum. Right, average fEPSPs (scale bars, 30 ms; 0.5 mV) at the times indicated (1 and 2). (b) Summary scatter plot of EPSP slope with control ICS (n = 16), ICS containing EPQ(pY)EEIPIA (n = 9) or ICS containing EPQ(pY)EEIPIA with NRG1β in the ACSF (n = 6; *P < 0.05, **P < 0.01 or ***P < 0.001 versus EPQ(pY)EEIPIA). Bottom, averaged slope of fEPSPs recorded simultaneously during experiments when whole-cell recordings were carried out. (c) Scatter plot of EPSP slope over time from two WT mouse CA1 neurons recorded with ICS containing EPQ(pY)EEIPIA with NRG1β (2 nM) or vehicle administered to the ACSF during EPQ(pY)EEIPIA-induced potentiation of EPSP responses (black bar). Right, average EPSPs from the two neurons (scale bars, 100 ms; 15 mV). Bottom, scatter plots of simultaneously recorded fEPSPs from CA1. Right, average fEPSPs (scale bars, 10 ms; 0.5 mV) at the times indicated (1, 2 and 3). (d) Summary histogram of the EPQ(pY)EEIPIA-induced increase in EPSP slope at 30 min of recording from neurons with ICS containing EPQ(pY)EEIPIA (n = 19), ICS containing EPQ(pY)EEIPIA with NRG1β present in ACSF beginning 20 min before whole-cell recording at 0.2 nM (n = 16; **P < 0.01 versus vehicle) or 2 nM (n = 6; ***P < 0.001 versus vehicle) or ICS containing EPQ(pY)EEIPIA with NRG1β (2 nM) treatment during EPQ(pY)EEIPIA-induced potentiation of EPSPs (n = 12).

Figure 4

NRG1β prevents but does not reverse TBS-induced LTP in CA1 hippocampus and has no effect in Src^−/− mice. (a) Scatter plot of normalized fEPSP slope over time for a rat hippocampal slice treated with NRG1β (2 nM) or another slice treated with vehicle (black bar). Top, average fEPSP traces (scale bars, 10 ms; 0.5 mV) at the times indicated (1 and 2) from the two individual experiments. Bottom, summary scatter plot of grouped normalized fEPSP slope from control slices (n = 23) or NRG1β-treated slices (n = 14). (b) Scatter plot of normalized fEPSP slope over time for a rat hippocampal slice treated with NRG1β (2 nM) present in ACSF beginning 30 min after TBS (black bar). Top, average fEPSP traces (scale bars, 15 ms; 0.5 mV) at the times indicated (1, 2, 3 and 4) from the experiment plotted. Bottom, summary scatter plot of grouped normalized fEPSP slope (n = 19). (c) Summary scatter plot of grouped normalized fEPSP slope in slices from Src^+/+ mice treated with vehicle (VEH, n = 13) or NRG1β (NRG, 2 nM; n = 14; P < 0.001 versus VEH) (black bar). Inset, average fEPSP traces at the times indicated (1 and 2) (scale bars, 10 ms; 0.5 mV) from two representative experiments. Bottom, summary scatter plot of normalized fEPSP slope in slices from Src^−/− mice treated with NRG1β (2 nM, n = 11) or vehicle (n = 14) (black bar). Inset, average fEPSP traces recorded at the times indicated (1 and 2) (scale bars, 10 ms; 0.5 mV) from two representative experiments. (d) Summary histogram of TBS-induced increase in fEPSP slope 60 min after TBS in slices from Src^+/+ and Src^−/− mice with NRG1β (+) or vehicle (−) treatment. Data are percentage of tbLTP with vehicle normalized to 100%. ***P < 0.001 versus Src^+/+ with vehicle. (e) Immunoblot analysis of ErbB4, GluN1 and GluN2, in CA1 from a Src^+/+ mouse or a Src^−/− littermate. GAPDH was loading control.

Figure 5

NRG1β reduces depolarization of CA1 neurons during the period of TBS. (a) The first four pulse-induced burst EPSP of TBS for control mouse (Src^+/+) slices (n = 28), slices (from Src^+/+ mice) pretreated with D-APV (n = 17), slices from Src^−/− mice (n = 12) or slices from Src^+/+ mice pretreated with NRG1β (2 nM; n = 19). Traces are the mean membrane potential from all recordings in response to the first four stimuli. Gray region, ± s.e.m. Vertical arrows, start of stimulation for each of four pulses delivered at 100 Hz. x axis, time (200 ms). Bottom, average pre-TBS baseline single stimulus–evoked EPSPs (scale bars, 100 ms; 10 mV). (b) Burst EPSPs for the entire duration of TBS from control slices (n = 28) or slices pretreated with NRG1β (n = 19). (c) Summary scatter plot of peak amplitude of burst EPSPs in response to each of the four stimuli-elicited bursts in control slices (n = 28) or slices pretreated with NRG1β (n = 19; *P < 0.05 or **P < 0.01 versus control). (d) Summary scatter plot of grouped normalized EPSP slope over time. (e) First four pulse-induced burst EPSP of TBS for neurons from control slices (from Src^+/+mice), slices (from Src^+/+ mice) pretreated with NRG1β (2 nM) or slices (from Src^+/+ mice) pretreated with NRG1β (2 nM) and AG1478. x axis, time (200 ms). Inset, average representative pre-TBS baseline single stimulus–evoked EPSPs from a control slice and a slice pretreated with NRG1β and AG1478 (scale bars, 100 ms; 8 mV). (f) Summary scatter plot of peak amplitude of burst EPSPs in response to each of the four stimuli-elicited bursts in slices pretreated with NRG1β (n = 19) or with NRG1β and AG1478 (n = 25; *P < 0.05, **P < 0.01 or ‡P < 0.001 versus NRG1β).

Figure 6

NRG1β does not alter Src association with the NMDAR but reduces Src tyrosine kinase activity and prevents TBS-induced GluN2B phosphorylation in hippocampal CA1. (a) Immunoprecipitation (IP) of GluN2 subunits carried out from hippocampal proteins prepared from control (−) or NRG1β (2 nM)-treated (+) slices. Proteins that immunoprecipitated with antibody to GluN2B were probed with antibodies to GluN2B, PSD-95 or Src (left). In ‘input’ lanes, 60 μg of proteins without immunoprecipitation were loaded. (b) Histogram of Src kinase activity in CA1 lysates without (n = 4) or with (n = 4) NRG1β (2 nM) pretreatment. Src kinase activity normalized to activity of Src in control lysates (**P < 0.01, t-test versus control). (c) Summary histogram of TBS-induced pYGluN2B from CA1 region collected from Src^+/+ (n = 6) or Src^−/− (n = 6; **P < 0.01, t-test versus Src^+/+) mice. (d) Top image, representative immunoblots of lysates from rat CA1 region with TBS or without (baseline, BL) probed with antibody to phosphorylated tyrosine (pY). The same membrane was stripped and reprobed with GluN2B antibody. Top graph, four-point standard curve derived from serial dilutions of rat brain proteins. Bottom image, representative immunoblot of lysates from rat CA1 region with TBS or without (BL) and treated with NRG1β (2 nM) probed sequentially with antibodies to pY and GluN2B. Bottom graph, four-point standard curve derived from serial dilutions of rat brain lysate. Right, summary histogram of immunoblot analysis. Densiometric quantification was carried out for pY GluN2B from six immunoblot experiments for each condition. Band intensity was quantified as mean gray value and the ratio of pY GluN2B to total GluN2B was calculated. Bars correspond to mean ratios normalized to ratios obtained for baseline (BL; *P < 0.05, t-test versus BL). (e) Top, four-point standard curve derived from serial dilutions of Src^+/+ mouse brain lysate. Bottom, summary histogram of TBS-induced pY GluN2B from hippocampal CA1 region without (n = 4) or with (n = 4) NRG1β (2 nM) treatment or with NRG1β (2 nM) and AG1478 treatment (n = 4). **P < 0.01, t-test versus control or versus NRG1β and AG1478.