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Santa Cruz Biotechnology rabbit polyclonal anti erbb4 antibody

(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) <t>ErbB4</t> 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.

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1) Product Images from "Parvalbumin interneuron ErbB4 controls ongoing network oscillations and olfactory behaviors in mice"

Article Title: Parvalbumin interneuron ErbB4 controls ongoing network oscillations and olfactory behaviors in mice

Journal: bioRxiv

doi: 10.1101/2024.07.25.604407

Figure Legend Snippet: (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.

Techniques Used: Sampling, Activity Assay, Control, Western Blot, Virus, Injection, Expressing, Concentration Assay

(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.

Figure Legend Snippet: (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.

Techniques Used: In Vitro, Imaging, Generated, Fluorescence, In Situ Hybridization, Expressing, Staining, Injection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

(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.

Figure Legend Snippet: (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.

Techniques Used: Concentration Assay

(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 ( χ 2 (2) =53.85, P < 0.0001, Chi-Square tests). ** P < 0.01, *** P < 0.001, **** P < 0.0001, n.s. = not significant.

Figure Legend Snippet: (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 ( χ 2 (2) =53.85, P < 0.0001, Chi-Square tests). ** P < 0.01, *** P < 0.001, **** P < 0.0001, n.s. = not significant.

Techniques Used: In Vivo, Activity Assay, Standard Deviation

(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.

Figure Legend Snippet: (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.

Techniques Used:

(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.

Figure Legend Snippet: (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.

Techniques Used: Injection

(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.

Figure Legend Snippet: (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.

Techniques Used:

(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.

Figure Legend Snippet: (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.

Techniques Used: Inhibition

(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.

Figure Legend Snippet: (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.

Techniques Used: Virus, Injection, Expressing, Control, Concentration Assay

Immunoprecipitation:

Article Title: Neuregulin Upregulates Microglial α7 Nicotinic Acetylcholine Receptor Expression in Immortalized Cell Lines: Implications for Regulating Neuroinflammation
Article Snippet: ErbB receptors were immunoprecipitated from protein samples by incubation with rabbit polyclonal anti-ErbB-2 (SC-284; Santa Cruz Biotechnologies, Santa Cruz, CA), 3 (SC-285; Santa Cruz), or 4 (SC-283; Santa Cruz) antibodies for 1 hour at 4 degrees Celsius with gentle rocking.

Incubation:

Image Search Results

Journal: bioRxiv

Article Title: Parvalbumin interneuron ErbB4 controls ongoing network oscillations and olfactory behaviors in mice

doi: 10.1101/2024.07.25.604407

Figure Lengend Snippet: (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.

Article Snippet: Rabbit polyclonal anti-ErbB4 antibody (sc-283, 1:2000, or 1:1000 for blotting, 1:100 for staining) was purchased from Santa Cruz, mouse monoclonal anti-PV antibody (P3088, 1:7000 for staining) was purchased from Sigma, anti-β-actin antibody (4970, 1:5000 for blotting) was purchased from Cell Signaling Technology, goat anti-rabbit IgG conjugated with Alexa Fluor 488 (A11089, 1:400 for staining) and goat anti-mouse IgG conjugated with Alexa Fluor 594 (A11037, 1:400 for staining) were purchased from Invitrogen.

Techniques: Sampling, Activity Assay, Control, Western Blot, Virus, Injection, Expressing, Concentration Assay

Journal: bioRxiv

Article Title: Parvalbumin interneuron ErbB4 controls ongoing network oscillations and olfactory behaviors in mice

doi: 10.1101/2024.07.25.604407

Figure Lengend Snippet: (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.

Techniques: In Vitro, Imaging, Generated, Fluorescence, In Situ Hybridization, Expressing, Staining, Injection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: bioRxiv

Article Title: Parvalbumin interneuron ErbB4 controls ongoing network oscillations and olfactory behaviors in mice

doi: 10.1101/2024.07.25.604407

Figure Lengend Snippet: (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.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Parvalbumin interneuron ErbB4 controls ongoing network oscillations and olfactory behaviors in mice

doi: 10.1101/2024.07.25.604407

Figure Lengend Snippet: (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 ( χ 2 (2) =53.85, P < 0.0001, Chi-Square tests). ** P < 0.01, *** P < 0.001, **** P < 0.0001, n.s. = not significant.

Techniques: In Vivo, Activity Assay, Standard Deviation

Journal: bioRxiv

Article Title: Parvalbumin interneuron ErbB4 controls ongoing network oscillations and olfactory behaviors in mice

doi: 10.1101/2024.07.25.604407

Figure Lengend Snippet: (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.

Techniques:

Journal: bioRxiv

Article Title: Parvalbumin interneuron ErbB4 controls ongoing network oscillations and olfactory behaviors in mice

doi: 10.1101/2024.07.25.604407

Figure Lengend Snippet: (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.

Techniques: Injection

Journal: bioRxiv

Article Title: Parvalbumin interneuron ErbB4 controls ongoing network oscillations and olfactory behaviors in mice

doi: 10.1101/2024.07.25.604407

Figure Lengend Snippet: (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.

Techniques:

Journal: bioRxiv

Article Title: Parvalbumin interneuron ErbB4 controls ongoing network oscillations and olfactory behaviors in mice

doi: 10.1101/2024.07.25.604407

Figure Lengend Snippet: (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.

Techniques: Inhibition

Journal: bioRxiv

Article Title: Parvalbumin interneuron ErbB4 controls ongoing network oscillations and olfactory behaviors in mice

doi: 10.1101/2024.07.25.604407

Figure Lengend Snippet: (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.

Techniques: Virus, Injection, Expressing, Control, Concentration Assay

( A ) HEK293T cells transiently transfected with human iRhom2-3xHA or UNC93B1-3xHA were stained with DAPI (blue) to label nuclei, anti-HA to label iRhom2-HA (red), and anti-calnexin to label the ER (green). Scale bar = 10 μm. ( B ) Lysates and anti-HA immunoprecipitation (HA-IP) from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells stably expressing iRhom2-3xHA (where indicated) were immunoblotted for HA and FRMD8. Nonspecific bands are marked with an asterisk.

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A ) HEK293T cells transiently transfected with human iRhom2-3xHA or UNC93B1-3xHA were stained with DAPI (blue) to label nuclei, anti-HA to label iRhom2-HA (red), and anti-calnexin to label the ER (green). Scale bar = 10 μm. ( B ) Lysates and anti-HA immunoprecipitation (HA-IP) from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells stably expressing iRhom2-3xHA (where indicated) were immunoblotted for HA and FRMD8. Nonspecific bands are marked with an asterisk.

Article Snippet: 0.5 × 10 6 HEK293T cells were washed with ice-cold FACS buffer (0.25% BSA, 0.1% sodium azide in PBS) and stained with rabbit polyclonal anti-HA antibody (Santa Cruz, sc-805; 0.5 μg diluted in FACS buffer), mouse monoclonal anti-ADAM10 (Biolegend, 352702; 4 μg diluted in FACS buffer) or mouse monoclonal anti-ADAM17 (A300E antibody , kindly provided by Dr Stefan Düsterhöft; 8 μg diluted in FACS buffer) on ice for 45 min. After two washes with FACS buffer, the cells were incubated with Alexa Fluor 488-coupled secondary antibody (Invitrogen, A21202 or A21206); 1:1000 dilution in FACS buffer) on ice for 30 min.

Techniques: Transfection, Staining, Immunoprecipitation, Knock-Out, Stable Transfection, Expressing

( A ) Volcano plot representing results from three iRhom2 co-immunoprecipitations. The fold change of label-free quantification values (in log2 ratio) was plotted against the p value (-log10 transformed). The grey dotted line indicates p-values <0.05 (analysed with a two-sample t-test). Benjamini-Hochberg correction was applied to adjust the p-value for multiple hypothesis testing (dark grey dotted line). ( B ) Lysates of HEK293T cells stably expressing human iRhom1-3xHA or iRhom2-3xHA transfected with human FRMD8-V5 (where indicated) were subjected to anti-HA and anti-V5 immunoprecipitation (HA-IP, V5–IP) and a western blot using anti-HA and anti-V5 antibodies was performed. Black arrowheads indicated the co-immunoprecipitated FRMD8-V5; white arrowheads indicated the co-immunoprecipitated iRhoms.

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A ) Volcano plot representing results from three iRhom2 co-immunoprecipitations. The fold change of label-free quantification values (in log2 ratio) was plotted against the p value (-log10 transformed). The grey dotted line indicates p-values <0.05 (analysed with a two-sample t-test). Benjamini-Hochberg correction was applied to adjust the p-value for multiple hypothesis testing (dark grey dotted line). ( B ) Lysates of HEK293T cells stably expressing human iRhom1-3xHA or iRhom2-3xHA transfected with human FRMD8-V5 (where indicated) were subjected to anti-HA and anti-V5 immunoprecipitation (HA-IP, V5–IP) and a western blot using anti-HA and anti-V5 antibodies was performed. Black arrowheads indicated the co-immunoprecipitated FRMD8-V5; white arrowheads indicated the co-immunoprecipitated iRhoms.

Techniques: Quantitative Proteomics, Transformation Assay, Stable Transfection, Expressing, Transfection, Immunoprecipitation, Western Blot

( A ) Schematic representation of truncated human iRhom2 constructs used in ( B–E ). ( B, C ) Lysates and anti-HA immunoprecipitation (HA-IP) from HEK293T cells transiently co-transfected with FRMD8-V5 and either empty vector (vect) or truncated human iRhom2-3xHA constructs were immunoblotted for V5 and HA. ( D ) iRhom1/2 double knockout HEK293T cells stably expressing empty vector (vect) or human iRhom2-3xHA constructs were transiently transfected with alkaline phosphatase (AP)-tagged AREG and then incubated with 200 nM PMA or with DMSO for 30 min. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed using a Mann-Whitney test. ****=p value<0.0001. ( E ) Lysates from iRhom1/2 double knockout HEK293T cells transiently transfected with empty vector (vect) or human iRhom2-3xHA constructs were immunoblotted for ADAM17 and HA.

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A ) Schematic representation of truncated human iRhom2 constructs used in ( B–E ). ( B, C ) Lysates and anti-HA immunoprecipitation (HA-IP) from HEK293T cells transiently co-transfected with FRMD8-V5 and either empty vector (vect) or truncated human iRhom2-3xHA constructs were immunoblotted for V5 and HA. ( D ) iRhom1/2 double knockout HEK293T cells stably expressing empty vector (vect) or human iRhom2-3xHA constructs were transiently transfected with alkaline phosphatase (AP)-tagged AREG and then incubated with 200 nM PMA or with DMSO for 30 min. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed using a Mann-Whitney test. ****=p value<0.0001. ( E ) Lysates from iRhom1/2 double knockout HEK293T cells transiently transfected with empty vector (vect) or human iRhom2-3xHA constructs were immunoblotted for ADAM17 and HA.

Techniques: Construct, Immunoprecipitation, Transfection, Plasmid Preparation, Double Knockout, Stable Transfection, Expressing, Incubation, Activity Assay, MANN-WHITNEY

The region required for FRMD8 binding is highlighted in red. Conserved phosphorylation sites that have been mutated to alanine in the iRhom2 pDEAD are marked in yellow. Grey residues indicate additional phosphorylation sites that have been reported on PhosphoSitePlus ( www.phosphosite.org ). An asterisk (*) indicates positions which have a fully conserved residue, a colon (:) indicates strongly similar properties of the amino acids, and a period (.) indicates weakly similar properties according to the Clustal Omega tool. ( B ) Lysates and anti-HA immunoprecipitation (HA-IP) from HEK293T cells transiently transfected with FRMD8-V5 and either empty vector (vect), mouse iRhom2 WT (WT) or Rhom2 cub (Δ268) were immunoblotted for V5 and HA.

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: The region required for FRMD8 binding is highlighted in red. Conserved phosphorylation sites that have been mutated to alanine in the iRhom2 pDEAD are marked in yellow. Grey residues indicate additional phosphorylation sites that have been reported on PhosphoSitePlus ( www.phosphosite.org ). An asterisk (*) indicates positions which have a fully conserved residue, a colon (:) indicates strongly similar properties of the amino acids, and a period (.) indicates weakly similar properties according to the Clustal Omega tool. ( B ) Lysates and anti-HA immunoprecipitation (HA-IP) from HEK293T cells transiently transfected with FRMD8-V5 and either empty vector (vect), mouse iRhom2 WT (WT) or Rhom2 cub (Δ268) were immunoblotted for V5 and HA.

Techniques: Binding Assay, Phospho-proteomics, Residue, Immunoprecipitation, Transfection, Plasmid Preparation

( A ) Lysates, anti-HA and anti-V5 immunoprecipitations (HA-IP, V5–IP) of HEK293T cells co-expressing human iRhom2-3xHA and human FRMD8-V5 were immunoblotted for ADAM17, HA and V5. ( B ) Lysates of wild-type (WT) and ADAM17 knockout (KO) HEK293T cells were transiently transfected with human iRhom2-3xHA and FRMD8-V5 (where indicated), anti-HA and anti-V5 immunoprecipitated (HA-IP; V5–IP) and immunoblotted for ADAM17, HA, and V5. ( C ) Lysates of WT and FRMD8 KO HEK293T cells stably expressing human iRhom2-3xHA were anti-HA immunoprecipitated (HA-IP) and stained for ADAM17 and HA. Nonspecific bands are indicated by an asterisk. ( D ) Lysates of WT and iRhom1/2 double knockout (DKO) HEK293T cells stably expressing human iRhom2 WT -3xHA or iRhom2 Δ201-300 -3xHA were anti-V5 immunoprecipitated (V5–IP) and immunoblotted for ADAM17, HA and V5.

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A ) Lysates, anti-HA and anti-V5 immunoprecipitations (HA-IP, V5–IP) of HEK293T cells co-expressing human iRhom2-3xHA and human FRMD8-V5 were immunoblotted for ADAM17, HA and V5. ( B ) Lysates of wild-type (WT) and ADAM17 knockout (KO) HEK293T cells were transiently transfected with human iRhom2-3xHA and FRMD8-V5 (where indicated), anti-HA and anti-V5 immunoprecipitated (HA-IP; V5–IP) and immunoblotted for ADAM17, HA, and V5. ( C ) Lysates of WT and FRMD8 KO HEK293T cells stably expressing human iRhom2-3xHA were anti-HA immunoprecipitated (HA-IP) and stained for ADAM17 and HA. Nonspecific bands are indicated by an asterisk. ( D ) Lysates of WT and iRhom1/2 double knockout (DKO) HEK293T cells stably expressing human iRhom2 WT -3xHA or iRhom2 Δ201-300 -3xHA were anti-V5 immunoprecipitated (V5–IP) and immunoblotted for ADAM17, HA and V5.

Techniques: Expressing, Knock-Out, Transfection, Immunoprecipitation, Stable Transfection, Staining, Double Knockout

( A ) Unpermeabilised WT (black) and FRMD8 KO HEK293T (cyan) cells stably expressing human iRhom2-3xHA were immunostained on ice for HA. Wild-type HEK293T cells immunostained for HA served as a negative control (grey). ( B ) Cells were permeabilised and stained at room temperature with an anti-HA antibody. Immunostaining with the Alexa Fluor 488-coupled secondary antibody served as a control (grey). The flow cytometry graphs shown are one representative experiment out of three experiments. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test; ns = p value>0.05; *=p value<0.05. ( C ) Lysates of HEK293T cells stably expressing human iRhom2-3xHA and transiently transfected with FRMD8-V5 (where indicated) were analysed by western blot for iRhom2 levels using anti-HA, anti-V5 and anti-actin immunostaining. Nonspecific bands are marked with an asterisk. ( D ) Lysates of WT and FRMD8 KO HEK293T cells stably expressing human iRhom2-3xHA (where indicated) were immunoblotted for HA, FRMD8 and actin. An asterisk marks nonspecific bands. ( E ) FRMD8 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used in ( D ).

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A ) Unpermeabilised WT (black) and FRMD8 KO HEK293T (cyan) cells stably expressing human iRhom2-3xHA were immunostained on ice for HA. Wild-type HEK293T cells immunostained for HA served as a negative control (grey). ( B ) Cells were permeabilised and stained at room temperature with an anti-HA antibody. Immunostaining with the Alexa Fluor 488-coupled secondary antibody served as a control (grey). The flow cytometry graphs shown are one representative experiment out of three experiments. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test; ns = p value>0.05; *=p value<0.05. ( C ) Lysates of HEK293T cells stably expressing human iRhom2-3xHA and transiently transfected with FRMD8-V5 (where indicated) were analysed by western blot for iRhom2 levels using anti-HA, anti-V5 and anti-actin immunostaining. Nonspecific bands are marked with an asterisk. ( D ) Lysates of WT and FRMD8 KO HEK293T cells stably expressing human iRhom2-3xHA (where indicated) were immunoblotted for HA, FRMD8 and actin. An asterisk marks nonspecific bands. ( E ) FRMD8 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used in ( D ).

Techniques: Stable Transfection, Expressing, Negative Control, Staining, Immunostaining, Control, Flow Cytometry, Fluorescence, Software, Transfection, Western Blot

( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.

Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet:

Techniques: Generated, Control, Transduction, CRISPR, Knock-In, Double Knockout, Clone Assay, Recombinant, Plasmid Preparation, Transfection, Construct, Sequencing, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Protease Inhibitor, Magnetic Beads, Knock-Out, Electron Microscopy, Software

Linear correlation analyses for Nrg1 with either pErbB4 (A and B) or pNeu (C and D) based on integrated fluorescence intensity of the human frontal cortex tissue array. Double immunofluorescence staining of Nrg1 with either pErbB4 (A) or pNeu (C) at each tissue point is shown. Linear correlation graph depicting the association between Nrg1 and pErbB4 (B) or pNeu (D). Each corresponding Pearson correlation coefficient ( r ) is shown. Nrg1, neuregulin 1; pErbB4, phosphorylated ErbB4; pNeu, phosphorylated Neu.

Journal: Molecular Medicine Reports

Article Title: Neuregulin-1 (Nrg1) signaling has a preventive role and is altered in the frontal cortex under the pathological conditions of Alzheimer's disease

doi: 10.3892/mmr.2016.5542

Figure Lengend Snippet: Linear correlation analyses for Nrg1 with either pErbB4 (A and B) or pNeu (C and D) based on integrated fluorescence intensity of the human frontal cortex tissue array. Double immunofluorescence staining of Nrg1 with either pErbB4 (A) or pNeu (C) at each tissue point is shown. Linear correlation graph depicting the association between Nrg1 and pErbB4 (B) or pNeu (D). Each corresponding Pearson correlation coefficient ( r ) is shown. Nrg1, neuregulin 1; pErbB4, phosphorylated ErbB4; pNeu, phosphorylated Neu.

Article Snippet: Following blocking in 10% PBS-buffered normal goat serum for 30 min, sections were incubated with primary antibodies, including mouse monoclonal anti-Nrg1 antibody (1:200, cat. no. MS-272-P1, Thermo Fisher Scientific, Inc.), rabbit polyclonal anti-pErbB4 antibody (1:200, cat. no. sc-33040, Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-pNeu antibody (1:200, cat. no. sc-12352-R, Santa Cruz Biotechnology, Inc.) and rabbit polyclonal anti-Aβ 1–42 antibody (1:1,000, cat. no. ab39377, Abcam) overnight at 4°C, followed by incubation with an Enhanced Polymer DAB Detection kit (cat. no. PV-900; ZSGB-Bio, Beijing, China) and an AEC kit (cat. no. ZLI-9036; ZSGB-Bio).

Techniques: Fluorescence, Double Immunofluorescence Staining

Changes in the Nrg1 signaling pathway molecules in the frontal lobe of a human AD brain. (A) Congo red staining and abnormal aggregation of Aβ 1–42 , indicating the formation of the amyloid plaques (indicated by the arrowheads) in the frontal cortical gray matter of a human AD brain. The scale bar represents 20 μ m. (B) Western blotting analysis of Nrg1, phosphorylation levels of ErbB4, Neu and Erk1/2 in the frontal lobe of a human AD patient brain. (C) Immunohistochemical detection of Nrg1, pErbB4 and pNeu (indicated by the arrowheads) in the frontal cortical gray matter from either the normal indivdual or the human AD patient. Double immunofluorescence staining images are shown for co-localization of Nrg1 with either (D) pErbB4 or (E) pNeu in the frontal cortical gray matter from either the normal individual or the human AD patient. The scale bar represents 20 μ m. AD, Alzheimer's disease; Aβ, β-amyloid; Nrg1, neuregulin 1; pErbB4, phosphorylated ErbB4; pNeu, phosphorylated Neu; Erk, extracellular-signal regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Journal: Molecular Medicine Reports

Article Title: Neuregulin-1 (Nrg1) signaling has a preventive role and is altered in the frontal cortex under the pathological conditions of Alzheimer's disease

doi: 10.3892/mmr.2016.5542

Figure Lengend Snippet: Changes in the Nrg1 signaling pathway molecules in the frontal lobe of a human AD brain. (A) Congo red staining and abnormal aggregation of Aβ 1–42 , indicating the formation of the amyloid plaques (indicated by the arrowheads) in the frontal cortical gray matter of a human AD brain. The scale bar represents 20 μ m. (B) Western blotting analysis of Nrg1, phosphorylation levels of ErbB4, Neu and Erk1/2 in the frontal lobe of a human AD patient brain. (C) Immunohistochemical detection of Nrg1, pErbB4 and pNeu (indicated by the arrowheads) in the frontal cortical gray matter from either the normal indivdual or the human AD patient. Double immunofluorescence staining images are shown for co-localization of Nrg1 with either (D) pErbB4 or (E) pNeu in the frontal cortical gray matter from either the normal individual or the human AD patient. The scale bar represents 20 μ m. AD, Alzheimer's disease; Aβ, β-amyloid; Nrg1, neuregulin 1; pErbB4, phosphorylated ErbB4; pNeu, phosphorylated Neu; Erk, extracellular-signal regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Techniques: Staining, Western Blot, Phospho-proteomics, Immunohistochemical staining, Double Immunofluorescence Staining

Western blot analysis of Nrg1, pNeu and pErbB4 in primary mouse cortical neurons in response to cell senescence. (A) Protein levels of Nrg1 isoforms and (B) the phosphorylation levels of Neu and ErbB4 in cortical neurons cultured for 0 to 10 days (n=5, one-way analysis of variance with Tukey's post-hoc test; data are expressed as the mean ± standard error of the mean). * P<0.05, ** P<0.01 and *** P<0.001 compared with the 0-day control. Nrg1, neuregulin 1; pErbB4, phosphorylated ErbB4; pNeu, phosphorylated Neu; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Journal: Molecular Medicine Reports

Article Title: Neuregulin-1 (Nrg1) signaling has a preventive role and is altered in the frontal cortex under the pathological conditions of Alzheimer's disease

doi: 10.3892/mmr.2016.5542

Figure Lengend Snippet: Western blot analysis of Nrg1, pNeu and pErbB4 in primary mouse cortical neurons in response to cell senescence. (A) Protein levels of Nrg1 isoforms and (B) the phosphorylation levels of Neu and ErbB4 in cortical neurons cultured for 0 to 10 days (n=5, one-way analysis of variance with Tukey's post-hoc test; data are expressed as the mean ± standard error of the mean). * P<0.05, ** P<0.01 and *** P<0.001 compared with the 0-day control. Nrg1, neuregulin 1; pErbB4, phosphorylated ErbB4; pNeu, phosphorylated Neu; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Techniques: Western Blot, Phospho-proteomics, Cell Culture, Control

Protective role of rNrg1β in primary mouse cortical neurons in response to oxidative stress and neuraxon damage. (A) Protein levels of Nrg1 isoforms and (B) phosphorylation levels of Neu and ErbB4 receptors in primary cortical neurons after a 24 h treatment with 0-20 μ M H 2 O 2 (n=6, one-way ANOVA with Tukey's post-hoc test; data are expressed as the mean ± SEM). * P<0.05, ** P<0.01 and *** P<0.001 compared with the control group. (C) The phosphorylation levels of Neu and ErbB4, and (D) the pAkt1 levels and (E) the pErk1/2 levels in primary cortical neurons pretreated with rNrg1β at a concentration of 5 or 10 nM for 2 h prior to a 24-h treatment with 2.5 μ M oligomeric H 2 O 2 (n=5, one-way ANOVA with Tukey's post-hoc test; data are expressed as the mean ± SEM). ## P<0.01 and ### P<0.001 vs. the vehicle control and * P<0.05, ** P<0.01 and *** P<0.001 vs. the H 2 O 2 -treated group. Double immunofluorescence staining of Nrg1 with either (F) pErbB4 or (G) pNeu in the cortical neurons pretreated with rNrg1β at a concentration of 5 or 10 nM for 2 h prior to a 24 h treatment with 2.5 μ M oligomeric H 2 O 2 . Scale bars=20 μ m. ANOVA, analysis of variance; SEM, standard error of the mean; H 2 O 2 , hydrogen peroxide; rNRG1β, recombinant neuregulin 1β, pErbB4, phosphorylated ErbB4; pNeu, phosphorylated Neu; pErk1/2, phosphorylated extracellular-regulated signal kinase 1/2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Journal: Molecular Medicine Reports

Article Title: Neuregulin-1 (Nrg1) signaling has a preventive role and is altered in the frontal cortex under the pathological conditions of Alzheimer's disease

doi: 10.3892/mmr.2016.5542

Figure Lengend Snippet: Protective role of rNrg1β in primary mouse cortical neurons in response to oxidative stress and neuraxon damage. (A) Protein levels of Nrg1 isoforms and (B) phosphorylation levels of Neu and ErbB4 receptors in primary cortical neurons after a 24 h treatment with 0-20 μ M H 2 O 2 (n=6, one-way ANOVA with Tukey's post-hoc test; data are expressed as the mean ± SEM). * P<0.05, ** P<0.01 and *** P<0.001 compared with the control group. (C) The phosphorylation levels of Neu and ErbB4, and (D) the pAkt1 levels and (E) the pErk1/2 levels in primary cortical neurons pretreated with rNrg1β at a concentration of 5 or 10 nM for 2 h prior to a 24-h treatment with 2.5 μ M oligomeric H 2 O 2 (n=5, one-way ANOVA with Tukey's post-hoc test; data are expressed as the mean ± SEM). ## P<0.01 and ### P<0.001 vs. the vehicle control and * P<0.05, ** P<0.01 and *** P<0.001 vs. the H 2 O 2 -treated group. Double immunofluorescence staining of Nrg1 with either (F) pErbB4 or (G) pNeu in the cortical neurons pretreated with rNrg1β at a concentration of 5 or 10 nM for 2 h prior to a 24 h treatment with 2.5 μ M oligomeric H 2 O 2 . Scale bars=20 μ m. ANOVA, analysis of variance; SEM, standard error of the mean; H 2 O 2 , hydrogen peroxide; rNRG1β, recombinant neuregulin 1β, pErbB4, phosphorylated ErbB4; pNeu, phosphorylated Neu; pErk1/2, phosphorylated extracellular-regulated signal kinase 1/2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Techniques: Phospho-proteomics, Control, Concentration Assay, Double Immunofluorescence Staining, Recombinant

Effects of rNrg1β pretreatment on the Nrg1 signaling in primary mouse cortical neurons exposed to Aβ 1–42 . The (A) relative levels of the Nrg1 isoforms, (B) phosphorylation levels of Neu and ErbB4, (C) pAkt1 levels and (D) pErk1/2 levels in primary cortical neurons pretreated with rNrg1β at a concentration of 5 or 10 nM for 2 h prior to a 24-h treatment with 10 μ M oligomeric Aβ 1–42 are shown (n=5, one-way analysis of variance with Tukey's post-hoc test; data are expressed as the mean ± standard error of the mean). Double immunofluorescence staining of β-III tubulin with either (E) pErbB4 or (F) pNeu in the cortical neurons pretreated with rNrg1β at a concentration of 5 or 10 nM for 2 h prior to a 24 h treatment with 10 μ M oligomeric Aβ 1–42 . Scale bars=20 μ m. rNRG1β, recombinant neuregulin 1β, pErbB4, phosphorylated ErbB4; pNeu, phosphorylated Neu; pErk1/2, phosphorylated extracellular-regulated signal kinase 1/2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Aβ, amyloid-beta; DAPI, 4′,6-diamidino-2-phenylindole. # P<0.05 and ## P<0.01 vs. vehicle control and * P<0.05, ** P<0.01 and *** P<0.001 compared with the control group.

Journal: Molecular Medicine Reports

Article Title: Neuregulin-1 (Nrg1) signaling has a preventive role and is altered in the frontal cortex under the pathological conditions of Alzheimer's disease

doi: 10.3892/mmr.2016.5542

Figure Lengend Snippet: Effects of rNrg1β pretreatment on the Nrg1 signaling in primary mouse cortical neurons exposed to Aβ 1–42 . The (A) relative levels of the Nrg1 isoforms, (B) phosphorylation levels of Neu and ErbB4, (C) pAkt1 levels and (D) pErk1/2 levels in primary cortical neurons pretreated with rNrg1β at a concentration of 5 or 10 nM for 2 h prior to a 24-h treatment with 10 μ M oligomeric Aβ 1–42 are shown (n=5, one-way analysis of variance with Tukey's post-hoc test; data are expressed as the mean ± standard error of the mean). Double immunofluorescence staining of β-III tubulin with either (E) pErbB4 or (F) pNeu in the cortical neurons pretreated with rNrg1β at a concentration of 5 or 10 nM for 2 h prior to a 24 h treatment with 10 μ M oligomeric Aβ 1–42 . Scale bars=20 μ m. rNRG1β, recombinant neuregulin 1β, pErbB4, phosphorylated ErbB4; pNeu, phosphorylated Neu; pErk1/2, phosphorylated extracellular-regulated signal kinase 1/2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Aβ, amyloid-beta; DAPI, 4′,6-diamidino-2-phenylindole. # P<0.05 and ## P<0.01 vs. vehicle control and * P<0.05, ** P<0.01 and *** P<0.001 compared with the control group.

Techniques: Phospho-proteomics, Concentration Assay, Double Immunofluorescence Staining, Recombinant, Control

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