Figures and data
ErbB4 in the OB is critical for odor discrimination and dishabituation
(A) Behavioral paradigm for olfactory associative learning without odor discrimination (go/go task). Mice inserted their snout into the sampling port to trigger odors. The schematic describes the timeline of a single trial. Mice learned to lick the metal tube to receive water in response to either of the odors in the pair (reward, hit).
(B) Timeline for a single trial in the go/no-go odor discrimination task. Mice learned to avoid licking the metal tube for the unrewarded odor (correct rejection, CR). Licking when presented with the unrewarded odor (false alarm, FA) led to no water reward and a timeout of up to 10 s. (C and D) ErbB4 activity in the OB was elevated after training on the reinforced go/no-go odor discrimination task. Relative p-ErbB4 and ErbB4 levels were normalized to their respective β-actin control groups in the western blot analysis (n = 3 mice per group, t(2) = 4.34, P = 0.0492, paired t test). (E) Schema indicating virus injection sites. To specifically delete ErbB4 protein in the OB, AAV-Cre-GFP was injected into bilateral OB of neonatal loxP-flanked ErbB4 mice. (F) Reduced ErbB4 expression in the OB of a mouse injected with AAV-Cre-GFP. (G) Odor discrimination performance under the reinforced go/no-go task. The accuracy for simple odor pairs was similar for the control and experimental groups (n = 8 mice per group, F(1, 14) = 2.83, P = 0.1148, two-way ANOVA). However, the accuracy for difficult odor mixtures (6/4 V 4/6) was reduced in AAV-Cre-GFP mice (F(1, 14) = 16.14, P = 0.0013, two-way ANOVA). (H) Odor performance under a spontaneous habituation/dishabituation task. Both animal groups showed a decline in investigation time to isoamyl acetate over the habituation period (n = 9 AAV-GFP mice, P < 0.0001; n = 10 AAV-Cre-GFP mice, P < 0.0001, F(3, 51) = 21.83, two-way ANOVA). However, AAV-GFP (F(1, 17) = 3.52, P = 0.0065), but not AAV-Cre-GFP mice (P = 0.6296, two-way ANOVA), showed an increase in investigation time toward limonene in the dishabituation period. (I) Odor detection threshold to isoamyl acetate. For AAV-GFP mice, the sniffing time toward isoamyl acetate was significantly higher than that for mineral oil at concentrations of 10-5 and 10-4, but not 10-6. (n = 12 mice, F(1, 21) = 0.19, 4.18 and 14.88, P = 0.5689, 0.0111, and 0.0090, two-way ANOVA). These results show that AAV-GFP mice were able to detect isoamyl acetate at a concentration of 10−5. For AAV-Cre-GFP mice, the sniffing time toward isoamyl acetate was significantly higher than that for mineral oil at a concentration of 10-4, but not at concentrations of 10-5 and 10-6 (n = 11 mice, P = 0.9672, 0.8713, and 0.0172, two-way ANOVA). These results show that AAV-Cre-GFP mice only detect isoamyl acetate at a concentration of 10−4. (J) Similarly, AAV-GFP mice were able to detect limonene at a concentration of 10−5 (n = 12 mice, F(1, 21) = 0.03, 4.77 and 12.96, P = 0.9603, 0.0011, and 0.0069, two-way ANOVA), whereas AAV-Cre-GFP mice only detected limonene at a concentration of 10−4 (n = 11 mice, P = 0.7671, 0.5490, and 0.0463, two-way ANOVA). Data are presented as means ± s.e.m. * P < 0.05, ** P < 0.01, **** P < 0.0001, n.s. = not significant. EPL, external plexiform layer; GCL, granule cell layer; GL, glomerular layer; IPL, internal plexiform layer; MCL, mitral cell layer.
ErbB4 proteins are largely expressed in PV interneurons in the OB
(A) In vitro imaging of ErbB4 mRNA in sections from PV-Erbb4+/+ mice (generated by crossing PV-Cre mice with loxP-flanked Erbb4 mice). Double single-molecule fluorescence in situ hybridization of ErbB4 (green) and PV (red) in the OB. Scale bar, 200 μm. (B) Magnified view of the EPL box from A. Scale bar, 50 μm. (C) Magnified view of the IPL box from A. Scale bar, 50 μm. (D) Summarized data showing the proportion of ErbB4-expressing neurons in different layers (n = 24, 24, 24, 22, and 24 fields from 4 mice). (E) Summarized data showing the proportion of the ErbB4/PV double-positive neurons relative to the total number of PV interneurons in the EPL and IPL (n = 18 and 5 fields from 4 mice). DAPI staining was used to determine the total number of cells. (F) In vitro electrophysiology experiments performed in slices from PV-Erbb4+/+ or PV-Erbb4-/-mice (generated by crossing PV-Cre mice with loxP-flanked Erbb4 mice). Representative examples of action potentials (APs) elicited by positive current injection (500 ms, 300 pA), recorded from an MC (left) and a fast-spiking PV interneuron (right). (G) Corresponding single-cell RT-PCR analyses showing that ErbB4 mRNA is detected only in PV interneurons (PVN) from PV-Erbb4+/+ OB. Dl1000 was used as the size reference (M, 300, 200 and 100 base-pair fragments are indicated). (H) Specific deletion of ErbB4 in EPL PV interneurons of the OB. OB sections from PV-Erbb4+/+ and PV-Erbb4−/− mice (P28) were stained with DAPI, anti-PV and ErbB4 antibody. Scale bars represent 50 and 20 μm respectively. (I and J) Western blots showing that ErbB4 in PV-Erbb4−/−OB was largely reduced from P7 onward, whereas ErbB4 in the PFC and hippocampus began to decrease only at P21. Relative levels were normalized to their respective P7 groups of control littermates (n = 3 mice per group, OB: F(1, 8) = 245.70, P < 0.0001, P = 0.0002, 0.0006, and 0.0010; PFC: F(1, 8) = 61.20, P = 0.0532, 0.1791, 0.0075, and 0.0021; Hi: F(1, 8) = 38.25, P = 0.2585, 0.1005, 0.0139 and 0.0382, two-way ANOVA). Data are presented as means ±s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001, n.s. = not significant. EPL, external plexiform layer; GCL, granule cell layer; GL, glomerular layer; Hi, hippocampus; MCL, mitral cell layer; ONL, olfactory nerve layer; PFC, prefrontal cortex.
ErbB4 in PV interneurons is critical for olfactory behaviors
(A) Odor discrimination under the reinforced go/no-go task in PV-Erbb4-/- mice. The accuracy for simple odor pairs was indistinguishable (n = 5 and 6 mice, F(1, 9) = 0.70, P = 0.4260, two-way ANOVA). However, the accuracy for difficult odor pairs was significantly lower in PV-Erbb4−/−mice (F(1, 9) = 9.12, P = 0.0144, two-way ANOVA). (B) Odor performance under the spontaneous habituation/dishabituation task. Both animal groups habituated to isoamyl acetate (n = 12 mice per group, F(3, 66) = 6.68, P = 0.0349 and 0.0164, two-way ANOVA). However, PV-Erbb4+/+ mice (F(1, 22) = 8.93, P = 0.0025), but not PV-Erbb4−/−mice (P = 0.8451, two-way ANOVA), dishabituated to limonene. (C) Odor performance under the reversed habituation/dishabituation task. Both animal groups habituated to limonene (F(3, 54) = 16.33, P < 0.0001 and 0.0003, two-way ANOVA). However, PV-Erbb4+/+mice (F(1, 18) = 5.22, P = 0.0023), but not PV-Erbb4−/−mice (P = 0.7890, two-way ANOVA), dishabituated to isoamyl acetate. (D) PV-Erbb4+/+ mice were able to detect isoamyl acetate at a concentration of 10−5 (n = 10 mice, F(1, 18) = 0.60, 3.74 and 16.69, P = 0.6069, 0.0498, and 0.0096 for 10-6, 10-5, and 10-4, two-way ANOVA). PV-Erbb4−/− mice only detected isoamyl acetate at a concentration of 10−4 (n = 10 mice, P = 0.5764, 0.5353, and 0.0100 for 10-6, 10-5, and 10-4, two-way ANOVA). (E) PV-Erbb4+/+ mice could detect limonen at a concentration of 10−5 (n = 10 mice, F(1, 18) = 1.75, 7.95 and 32.51, P = 0.4540, 0.0106, and 0.0064 for 10-6, 10-5, and 10-4, two-way ANOVA). PV-Erbb4−/−mice only detected limonene at a concentration of 10−4 (n = 10 mice, P = 0.2842, 0.2709, and < 0.0001 for 10-6, 10-5, and 10-4, two-way ANOVA). (F) The latency for mice to locate the buried and visible food pellets did not differ between the groups (n = 14 and 11 mice, for the buried food pellet, F(1, 23) = 0.21, P = 0.9786, 0.9279, and 0.8873; for the visible food pellet, P > 0.9999, two-way ANOVA). Data are presented as means ±s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n.s. = not significant.
Odor-evoked responses in M/TCs are decreased in PV-Erbb4−/− mice
(A) Schematic of in vivo odor-evoked electrophysiological recordings in awake, head-fixed mice with ErbB4 knocked out in PV interneurons. (B) Representative raw traces of spike activity before (spontaneous), during (odor-evoked), and after 2-s odor stimulation in PV-Erbb4+/+ and PV-Erbb4−/−mice. (C) Examples of raster plots (top) and peristimulus time histograms (PSTHs) of the firing rate (bottom) for odor-evoked excitatory (left) and inhibitory (right) responses in PV-Erbb4+/+and PV-Erbb4−/− mice. PSTHs were smoothed with a Gaussian filter with a standard deviation of 1500 ms. (D) Heat maps of the mean firing rate (MFR) across all unit–odor pairs in PV-Erbb4+/+ mice (n = 240 unit–odor pairs from 4 mice) and PV-Erbb4−/− mice (n = 232 unit–odor pairs from 4 mice). (E) Quantitative analysis of the spontaneous firing rate (t(470) = 0.2665, P = 0.7899), odor-evoked MFR (t(470) = 3.304, P = 0.0010), absolute value of odor-evoked changes (t(470) = 5.046, P < 0.0001), and normalized signal-to-noise ratio (SNR) (t(470) = 5.152, P < 0.0001, unpaired t test) across all unit–odor pairs. (F) Odor-evoked excitatory changes in MFR (ΔMFR) for M/TCs recorded from PV-Erbb4+/+mice (n = 66 unit–odor pairs from 4 mice) and PV-Erbb4−/−mice (n = 16 unit–odor pairs from 4 mice). (G) Quantitative analysis of the spontaneous firing rate (t(80) = 7.666, P < 0.0001), odor-evoked MFR (t(80) = 1.426, P = 0.1578), odor-evoked ΔMFR (t(80) = 3.099, P = 0.0027), SNR (t(80) = 5.909, P < 0.0001), and normalized SNR (t(80) = 5.909, P < 0.0001, unpaired t test) across excitatory unit–odor pairs. (H) Odor-evoked inhibitory ΔMFR for M/TCs recorded from PV-Erbb4+/+ mice (n = 81 unit–odor pairs from 4 mice) and PV-Erbb4−/− mice (n = 148 unit–odor pairs from 4 mice). (I) Quantitative analysis of spontaneous firing rate (t(227) = 7.521, P < 0.0001), odor-evoked MFR (t(227) = 7.228, P < 0.0001), odor-evoked ΔMFR (t(227) = 2.724, P = 0.0070), SNR (t(227) = 6.905, P < 0.0001), and normalized SNR (t(227) = 6.905, P < 0.0001, unpaired t test) across inhibitory unit–odor pairs. (J) ΔMFR for units with no response to odor in PV-Erbb4+/+ mice (n = 93 unit–odor pairs from 4 mice) and PV-Erbb4−/− mice (n = 68 unit–odor pairs from 4 mice). (K) Quantitative analysis of spontaneous firing rate (t(159) = 3.129, P = 0.0021), odor-evoked MFR (t(159) = 3.378, P = 0.0009), absolute value of ΔMFR (t(159) = 1.627, P = 0.1057), and normalized SNR (t(159) = 1.713, P = 0.0886, unpaired t test) across “no response” unit–odor pairs. (L) Distribution of excitatory, inhibitory, and “no response” units in PV-Erbb4+/+ and PV-Erbb4−/− mice (χ2(2) =53.85, P < 0.0001, Chi-Square tests). ** P < 0.01, *** P < 0.001, **** P < 0.0001, n.s. = not significant.
The ongoing LFP in the OB is increased in PV-Erbb4−/− mice
(A) Examples of ongoing LFP signals recorded in the OB from PV-Erbb4+/+and PV-Erbb4−/− mice. The five rows show the raw traces and the filtered theta, beta, low-gamma, and high-gamma signals. (B-E) Quantitative analysis of the averaged power spectra in the theta, beta, low-gamma, and high-gamma bands for the two groups. (F-I) Comparisons of power in the theta (n = 5 mice per group, t(8) = 2.80, P = 0.0233, unpaired t test), beta (t(8) = 2.80, P = 0.0232, unpaired t test), low-gamma (t(8) = 3.80, P = 0.0053, unpaired t test), and high-gamma (t(8) = 3.73, P = 0.0058, unpaired t test) oscillations in the OB in the two groups. * P < 0.05, ** P < 0.01.
PV-Erbb4−/− mice have impaired information output from the OB
(A and B) Increased frequency of MC spontaneous action potentials (sAPs) and olfactory nerve-evoked APs (eAPs) in PV-Erbb4−/− mice, but decreased ratio of eAPs to sAPs (n = 9 from 3 mice per group, sAPs: t(16) = 4.173, P = 0.0007, eAPs: t(16) = 2.24, P = 0.0395; ratio: t(16) = 3.68, P = 0.0020, unpaired t test). (C and D) The eAP-to-sAP ratio was reduced in PV-Erbb4−/− mice as the intensity of the stimulus increased (n = 8 from 3 mice per group, F(1, 70) = 32.39, P < 0.0001, two-way ANOVA). (E and F) AP frequency elicited by injection of positive currents was higher in PV-Erbb4−/− mice than PV-Erbb4+/+ mice (n = 9 from 4 and 3 mice per group, F(1, 80) = 78.9, P < 0.0001, two-way ANOVA). Data are presented as means ±s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
Decreased GABAergic transmission mediates the OB output impairments in PV-Erbb4−/− mice
(A and B) The increased sAP frequency and decreased ratio of eAPs to sAPs in MCs could not be further enhanced by bicuculline in PV-Erbb4−/− mice (n = 7 from 3 PV-Erbb4+/+ mice, n = 9 from 3 PV-Erbb4−/− mice; for sAP, F(1, 14) = 17.08, P = 0.0008 and 0.5531; for ratio of eAPs to sAPs, F(1, 14) = 13.99, P = 0.0007 and 0.9360, two-way ANOVA). (C and D) The frequency but not the amplitude of MC mIPSCs was lower in PV-Erbb4−/− mice (n = 9 from 7 PV-Erbb4+/+and 6 PV-Erbb4−/−mice; for frequency, t(16) = 2.45, P = 0.0263; for amplitude, t(16) = 0.53, P = 0.6038, unpaired t test). (E and F) Neither frequency nor amplitude of MC mEPSCs was different in PV-Erbb4−/− mice versus PV-Erbb4+/+ mice (n = 10 from 5 PV-Erbb4+/+ mice, n = 9 from 7 PV-Erbb4−/−mice; for frequency, t(17) = 0.14, P = 0.8899; for amplitude, t(17) = 0.89, P = 0.3863, unpaired t test). Data are presented as means ±s.e.m. * P < 0.05, *** P < 0.001, **** P < 0.0001, n.s. = not significant.
Reduced recurrent inhibition and EPL lateral inhibition of MCs in PV-Erbb4−/− mice
(A and B) Recurrent inhibition did not increase alongside MC hyperactivity in PV-Erbb4−/− mice (n = 9 from 5 and 6 mice; for peak amplitude, t(16) = 1.57, P = 0.1351; for decay time constant, t(16) = 0.28, P = 0.7821, unpaired t test). (C and D) Recurrent IPSPs elicited by the same number of MC APs were smaller in PV-Erbb4−/− mice. In PV-Erbb4−/− mice, an 80 pA current induced ten APs, whereas in PV-Erbb4+/+ mice, a 100 pA current was needed to induce ten APs. Both the peak amplitude (n = 10 from 6 and 5 mice, t(18) = 2.46, P = 0.0242, unpaired t test) and the decay time constant (t(18) = 2.56, P = 0.0198, unpaired t test) of IPSPs evoked by the same number of APs were reduced in PV-Erbb4−/−mice. (E) Schematic of the EPL lateral inhibition experimental configuration. Whole-cell recording of an MC (right side of the schematic) upon stimulus of an adjacent glomerulus (left side of the schematic). A cut was made through the GL and GCL between the sites of conditioning stimulation and target glomerus to isolate EPL lateral inhibition. (F and G) EPL lateral inhibition was observed in the OB of PV-Erbb4+/+ mice but not PV-Erbb4−/− mice (n = 10 from 7 PV-Erbb4+/+ mice, F(1, 18) = 26.48, P < 0.0001; n = 10 from 6 PV-Erbb4−/− mice, P = 0.3426, two-way ANOVA). (H-J) Cutting through the EPL as well abolished the EPL lateral inhibition in PV-Erbb4+/+ mice. (H) Schematic of the experimental configuration. (I and J) Representative and quantitative analysis of spike frequency in PV-Erbb4+/+mice (n = 9 from 5 mice, t(8) = 0.80, P = 0.4468, paired t test). EPL, external plexiform layer; GL, glomerular layer; GCL, granule cell layer; MCL, mitral cell layer; ONL, olfactory nerve layer; PVN, PV interneuron. Data are presented as means ±s.e.m. * P < 0.05, **** P < 0.0001, n.s. = not significant.
ErbB4 in PV interneurons of the OB is critical for odor discrimination and sensitivity
(A) Schema indicating virus-injection sites. To specifically delete ErbB4 protein in the PV interneurons of the OB, AAV-PV-Cre-GFP was injected into the bilateral OB of neonatal loxP-flanked ErbB4 mice. (B) Reduced ErbB4 expression in AAV-PV-Cre-GFP mouse OB (n = 4 mice per group, t(3) = 3.93, P = 0.0293, paired t test). Relative levels were normalized to their respective control groups. (C) The accuracy in discriminating simple odor pairs was similar for the two groups (n = 6 and 7 mice, F(1, 11) = 0.06, P = 0.8147). The accuracy in discriminating difficult odor pairs was significantly lower in AAV-PV-Cre-GFP mice (F(1, 11) = 5.74, P = 0.0355, two-way ANOVA). (D) Both animal groups habituated to isoamyl acetate (n = 10 mice per group, F(3, 54) = 20.94, P < 0.0001 and = 0.0010, two-way ANOVA). However, the AAV-PV-GFP (F(1, 18) = 3.95, P = 0.0045), but not the AAV-PV-Cre-GFP mice (P = 0.7107, two-way ANOVA), dishabituated to limonene. (E) Both animal groups habituated to carvone+ (n = 10 mice per group, F(3, 54) = 9.92, P = 0.0019 and 0.0035, two-way ANOVA). However, AAV-PV-GFP (F(1, 18) = 6.72, P = 0.0025), but not AAV-PV-Cre-GFP mice (P = 0.9845, two-way ANOVA), dishabituated to carvone-. (F) AAV-PV-GFP mice were able to detect isoamyl acetate at a concentration of 10−5 (n = 10 mice, F(1, 18) = 0.04, 2.63 and 12.42, P = 0.8309, 0.0258, and 0.0164 for 10-6, 10-5, and 10-4, two-way ANOVA), whereas AAV-PV-Cre-GFP mice only detected isoamyl acetate at a concentration of 10−4 (n = 10 mice, P = 0.9484, 0.8930, and 0.0312, two-way ANOVA). (G) AAV-PV-GFP mice detected limonene at 10−5 (n = 10 mice, F(1, 18) = 0.28, 3.81 and 11.79, P = 0.7894, 0.0154, and 0.0131 for 10-6, 10-5, and 10-4, two-way ANOVA) but AAV-PV-Cre-GFP mice only detected limonene at a higher concentration of 10−4 (n = 10 mice, P = 0.6408, 0.9349, and 0.0496 for 10-6, 10-5, and 10-4, two-way ANOVA). Data are presented as means ±s.e.m. * P < 0.05, ** P < 0.01, **** P < 0.0001, n.s. = not significant. EPL, external plexiform layer; GCL, granule cell layer; GL, glomerular layer; IPL, internal plexiform layer; MCL, mitral cell layer.
