Neuregulin 1 Type I Overexpression Is Associated with Reduced NMDA Receptor-Mediated Synaptic Signaling in Hippocampal Interneurons Expressing PV or CCK

Abstract

Hypofunction of N-methyl-d-aspartate receptors (NMDARs) in inhibitory GABAergic interneurons is implicated in the pathophysiology of schizophrenia (SZ), a heritable disorder with many susceptibility genes. However, it is still unclear how SZ risk genes interfere with NMDAR-mediated synaptic transmission in diverse inhibitory interneuron populations. One putative risk gene is neuregulin 1 (NRG1), which signals via the receptor tyrosine kinase ErbB4, itself a schizophrenia risk gene. The type I isoform of NRG1 shows increased expression in the brain of SZ patients, and ErbB4 is enriched in GABAergic interneurons expressing parvalbumin (PV) or cholecystokinin (CCK). Here, we investigated ErbB4 expression and synaptic transmission in interneuronal populations of the hippocampus of transgenic mice overexpressing NRG1 type I (NRG1^tg-type-I mice). Immunohistochemical analyses confirmed that ErbB4 was coexpressed with either PV or CCK in hippocampal interneurons, but we observed a reduced number of ErbB4-immunopositive interneurons in the NRG1^tg-type-I mice. NMDAR-mediated currents in interneurons expressing PV (including PV⁺ basket cells) or CCK were reduced in NRG1^tg-type-I mice compared to their littermate controls. We found no difference in AMPA receptor-mediated currents. Optogenetic activation (5 pulses at 20 Hz) of local glutamatergic fibers revealed a decreased NMDAR-mediated contribution to disynaptic GABAergic inhibition of pyramidal cells in the NRG1^tg-type-I mice. GABAergic synaptic transmission from either PV⁺ or CCK⁺ interneurons, and glutamatergic transmission onto pyramidal cells, did not significantly differ between genotypes. The results indicate that synaptic NMDAR-mediated signaling in hippocampal interneurons is sensitive to chronically elevated NGR1 type I levels. This may contribute to the pathophysiological consequences of increased NRG1 expression in SZ.

Keywords: Axo-axonic cell; ErbB4 receptor; NMDA receptors; basket cell; cholecystokinin; parvalbumin; schizophrenia.

Figure 1.

ErbB4 expression in PV⁺ and PV^– interneurons and the ErbB4 expression levels in hippocampus of WT and the NRG1^tg-type-I mice. A, Immunostaining for ErbB4, the NRG1 receptor, in the ventral hippocampus CA3 area neurons using highly specific rabbit anti-ErbB4 (polyclonal anti-antiserum 5941; Neddens and Buonanno, 2010). A1, Double immunolabeling for PV (Cy3) and ErbB4 (Alexa488). Merged image shows double-labeled neurons (arrowhead) and ErbB4⁺ interneurons immunonegative for PV (arrows). s.r, stratum radiatum. A2, In mice with genetic fluorescence marker (tdTomato) in CCK cells (tdTom-CCK), ErbB4 immunostaining with Alexa Fluor 488 shows the expression in many CCK⁺ neurons in s.r. and stratum pyramidale (s.p.). Cre-dependent tdTomato signal is strong in putative CA3 interneurons (soma in s.r.) and weaker in s.p., where the majority of pyramidal cell somata are located (contrast adjustment in the image). In merged image, arrowheads point at interneuron somata with both fluorescent signals. Scale bars, 50 µm. Confocal microscope images. B–D, Cell density analysis of hippocampal interneurons immunopositive for ErbB4 in the WT and NRG1 type I–overexpressing mice (NRG1^tg-type-I mice). B1, ErbB4 immunoreaction (20-µm stack image) in sample hippocampal sections of WT (left) and NRG1^tg-type-I mice (right). Scale bar, 100 µm. B2, Box plots show ErbB4⁺ cell soma density (measured up to 20-µm depth from the section surface) in WT (blue, n = 9 sections in 3 mice) and NRG1^tg-type-I (red, 12 sections in 3 mice) mice hippocampi. The plot shows median and interquartile range. Fewer ErbB4⁺ somata were detected in the NRG1^tg-type-I mice compared to the WT mice in all hippocampal areas. From the left: whole hippocampus including areas CA1, CA2, and CA3; area CA1–2 restricted to alveus, stratum oriens, and stratum pyramidale; area CA1–2 restricted to stratum radiatum and lacunosum-moleculare; area CA3 containing alveus with strata oriens and pyramidale; and area CA3 with strata lucidum and radiatum and lacunosum-moleculare. p-values compare data between genotypes (Mann–Whitney U test). C1, Immunoreaction for PV in the same sections as in B1. C2, Cell density analyses show no difference in the observed PV⁺ cell somata between the two genotypes as indicated by p values (Mann–Whitney U test). Box plots as in B2. D1, Merged ErbB4 and PV immunolabeling in the sample sections above. D2, Box plots show proportion of the double-labeled cells (co-immunoreactive for ErbB4 and PV) in the PV⁺ cell population in WT and NRG1^tg-type-I mice. The analyses show unaltered proportion in the whole hippocampus and in most subregions compared separately. The significant p value is bolded. E, Immunoblot analysis of ErbB4 expression levels in WT and NRG1^tg-type-I mice using hippocampal extracts. E1, The antibody against ErbB4 detects a band of the predicted protein size (∼150 kDa) in hippocampal protein extracts. Left lane, no nonspecific bands were detected in the secondary-only antibody control (right lane). E2, Hippocampal extracts from 6 WT and 6 NRG1^tg-type-I mice of both genders (3 males and 3 females in each genotype in scrambled order) tested for ErbB4 expression. GAPDH was used as a loading control. E3, Box plot shows (mean and interquartile range) densitometry analysis comparison of the ErbB4 levels normalized by the GAPDH in the 12 hippocampal extracts (6 in both genotypes including 3 males and 3 females). The results indicate a general trend to lower ErbB4 levels in NRG1^tg-type-I mice, but with no significant difference between the genotypes (Mann–Whitney U test).

Figure 2.

Reduced synaptic NMDAR-mediated currents in hippocampal interneurons expressing PV or CCK in the NRG1^tg-type-I mice. A, Interneurons expressing PV or CCK in the CA3 area. A1, Sample image of a recorded PV interneuron identified by PV expression-dependent fluorescent genetic marker tdTomato (tdTom-PV). Recorded cells were also visualized with filled neurobiotin (nb, Alexa Fluor 488). A2, Recorded cells not showing tdTomato signal were identified as CCK⁺ interneurons post hoc with positive somatic immunoreaction for pro-CCK (left; Cy5, arrowhead) or in the absence of recovered soma and dendrites (right) by positive reaction for axonal cannabinoid receptor type 1 (CB1R, Cy3). Scale bars from left: 10, 20, and 10 µm, respectively. B, Reduced NMDAR- versus AMPAR-mediated EPSCs ratio (N/A ratio) in glutamatergic synaptic input to interneurons expressing PV. Electrical stimulation was applied in CA3 stratum oriens aiming to activate associative/commissural pathways. AMPAR-mediated EPSCs were recorded at –60 mV (in PiTX, 100 µm) and blocked by NBQX (25 µm) to record NMDAR-mediated EPSCs (at 40 mV from their reversal potential). B1, Averaged EPSCs (10 traces) in sample PV⁺ interneurons in WT and NRG1^tg-type-I mouse (black, AMPAR EPSCs; green, NMDAR EPSCs in the presence of NBQX; gray, following application of NMDAR blocker DL-AP5). Scale bars, 100 pA, 25 ms. B2, Cumulative histograms of the N/A amplitude ratios in all studied PV⁺ interneurons (WT, blue line; NRG1^tg-type-I, red line). p indicates difference between the genotypes (Mann–Whitney U test). C, Reduced N/A ratio in glutamatergic synaptic input to the CCK⁺ interneurons. C1, Averaged EPSCs (10) in sample cells in the WT and in the NRG1^tg-type-I mouse with scaling as above. C2, Cumulative histogram quantifying the N/A ratios in CCK⁺ interneurons with p indicating significant difference between the genotypes (Mann–Whitney U test). D, The N/A ratio is unaltered between the genotypes in the CA3 pyramidal cells. D1, Averaged EPSCs (10 traces) in sample pyramidal cells with scaling as above. D2, Cumulative histograms of the N/A ratios.

Figure 3.

Reduced synaptic NMDAR-mediated currents in identified PV basket cells in the NRG1^tg-type-I mice. Identified PV basket cells (PVBCs) in the recorded interneuron population (see Fig. 2) show reduced N/A ratio in the NRG1^tg-type-I mice. A, Illustration of a sample PVBC (70-μm-thick section) in WT (axon, blue; soma and dendrites, red; s.r., stratum radiatum; s.luc., lucidum; s.p., pyramidale; s.o., oriens). Scale, 100 μm. B, The N/A EPSC amplitude ratio in identified basket cells. B1, Averaged (10) EPSCs in a PVBC from WT and NRG1^tg-type-I mouse. Black, AMPAR EPSCs at –60 mV; green, EPSCs (at 40 mV from their reversal potential in the presence of NBQX, 25 μm); gray, EPSCs following the application of DL-AP5 (100 μm). PiTX (100 µm) was present in all experiments. Scale bars, 50 pA, 25 ms. B2, Plot shows N/A ratio of every identified PVBC in WT (blue circles) and NRG1^tg-type-I mice (red circles), and their mean ± SEM (n = 10 and 10 cells). p value indicates highly significant difference (t test).

Figure 4.

Quantal current analysis in parvalbumin basket cells shows unaltered AMPAR mEPSCs in the NRG1^tg-type-I mice. A, Recording of miniature AMPAR- and NMDAR-mediated EPSCs (mEPSCs) in identified CA3 area PVBCs (in the presence of TTX 1 μm and PiTX 100 μm). A1, Illustration of a recorded and partially reconstructed PVBC (70-μm-thick section) in WT mouse. Scale, 100 μm. A2, AMPAR mEPSCs in a PVBC in the WT mouse (at –65 mV). Left, the mEPSCs shown in 45-s time window. Right, six events superimposed in 15-ms time window. A3, AMPAR mEPSCs in a PVBC in the NRG1^tg-type-I mouse. A4, NMDAR mEPSCs in the same WT mouse PVBC as in A2 after blockade of AMPARs by NBQX (25 µm, recorded at 40 mV). Left, the mEPSCs shown in 45-s time window. Middle, the mEPSCs blockade with DL-AP5 (100 µm, application indicated by horizontal bar). Right, six superimposed mEPSCs in 80-ms time window. A5, NMDAR mEPSCs blocked by DL-AP5 in NRG1^tg-type-I mouse PVBC shown in A3. A6, Box plot (median, interquartile range) summarizes AMPAR and NMDAR mEPSC frequency (measured at least 3 min) in PVBC in WT (blue) and NRG1^tg-type-I (red) mice. A7, Box plot summarizes mEPSC amplitude. Note moderately but significantly smaller NMDAR mEPSC in the NRG1^tg-type-I mice PVBCs (Mann–Whitney U test). The significant p value is bolded. B, Unaltered NMDAR- and AMPAR-mediated mEPSCs in the CA3 area pyramidal cells in NRG1^tg-type-I mice. B1, B2, Sample traces showing AMPAR mEPSCs in pyramidal cells of both genotypes. B3, B4, Respectively, NMDAR EPSCs in the same cells. B5, Box plot (median, interquartile range) summarizing the AMPAR- and NMDAR-mediated mEPSC frequency (WT, blue, NRG1^tg-type-I, red). B6, Summary of he AMPAR and NMDAR mEPSC amplitudes in the two genotypes (Mann–Whitney U test).

Figure 5.

GABAergic synaptic transmission from either PV⁺ or CCK⁺ cells is not significantly altered in NRG1^tg-type-I mice. A, Experimental design showing optogenetic stimulation (at 473-nm laser spot, 20-μm diameter) of GABAergic fibers in the CA3 stratum pyramidale in slices from ChR2-eYFP–transfected mice expressing Cre-protein in either PV⁺ cells (A1) or CCK⁺ cells (A2). Left, schematic illustration of the experiment with whole-cell recording in CA3 PCs and optogenetic stimulation focused on stratum pyramidale (s.p.). Right, confocal microscope images from sample slices (visualized post hoc) showing eYFP fluorescence (green) in the PV- (above) or CCK-Cre mice. Postsynaptic neurobiotin-filled pyramidal cells are shown red with an inset of a spiny pyramidal cell apical dendrite. Scale, 50 μm. B, Sample experiment showing optogenetically evoked GABAergic IPSCs in a postsynaptic pyramidal cell using minimal stimulation. Monosynaptic IPSCs (black circles) were evoked by smallest stimulation power eliciting IPSCs in the PC. Open circles, failures; red circles, additional IPSCs elicited by increased stimulation power. Timing of laser pulses with representative IPSCs in the experiment is shown above. C, The optogenetically evoked GABAergic IPSCs from PV cell fibers do not differ significantly between WT mice (blue) and NRG1^tg-type-I mice (red; Mann–Whitney U test). C1, A sample trace. Box plots (median, interquartile range) show data from all the PCs studied. C2, The evoked IPSC amplitudes. C3, The IPSC half decay. C4, The IPSC rise time. C5, The IPSC paired-pulse (50 ms) ratio (2nd/1st IPSC amplitude). p values with Mann-Whitney test. D, The IPSCs from CCK-fibers do not show significant difference between the genotypes. D1, Sample trace. D2–D5, The IPSC amplitude, IPSC half decay, rise time, and paired-pulse ratio, respectively (Mann–Whitney test).

Figure 6.

Reduced NMDAR-driven recurrent hippocampal inhibition in NRG1^tg-type-I mice. A, Schematic summarizes the experimental design. Optogenetic stimulation of CA1 area pyramidal cell fibers expressing ChR2 (green, CAMKII-Cre mice transfected with AAV2-ChR2-eYFP). Recurrent inhibitory IPSCs are generated by laser spot (473-nm, 3-ms) stimulation focused in stratum pyramidale (s.p.) and oriens (s.o.). B, Sample experiments showing averaged (5) recurrent IPSCs in the CA1 pyramidal cells, evoked by the optogenetic stimulation (5 pulses at 20 Hz) in WT (B1) and NRG1^tg-type-I (B2) mice. Black traces show IPSCs in baseline; green is in the presence of NMDAR blocker DL-AP5 (100 μm, at 5–8 min after DL-AP5 application). The IPSCs were recorded at the reversal potential of EPSCs. The IPSCs were fully blocked with NBQX (25 µm, gray traces). C, Plots show the recurrent IPSC charge in sample experiments in the WT (C1) and NRG1^tg-type-I (C2) mouse. Wash-in of DL-AP5 and NBQX is indicated by green and gray horizontal bars, respectively. D, The hippocampal recurrent IPSCs in the NRG1^tg-type-I mice show reduced sensitivity to the NMDAR antagonist. Box plot (median, interquartile range) summarizes the effect of DL-AP5 (100 µm) on the recurrent IPSC charge in WT (blue) and NRG1^tg-type-I (red) mice. The IPSC charge in the presence of DL-AP5 (and in the presence of NBQX) is normalized with the baseline for each experiment. p value with Mann–Whitney U test.