Neuroligin 3/NLGN3 Antibody NBP1-90080 from Novus Biologicals, a Bio-Techne Brand

Published Figures
Supplier Images

Register or Sign In to View Figures.

iScience
August 19 2022

Fig 1: Nlgn1 and Nlgn3 are present at cochlear ribbon synapses in a modiolar-pillar position dependent pattern(A) Representative immunofluorescent images from Nlgn1 KO cochlear whole mounts demonstrating the absence of Nlgn1 at ribbon synapse (CtBP2 = red, GluA2 = green).(B) Representative images from apical (8–12 kHz), middle (12–20 kHz), and basal (20–28 kHz) regions of the cochlea immunostained with antibodies for Nlgn1 (blue) and IHC ribbon synapse markers CtBP2 (red) and GluA2 (green).(C) Quantification of (B) reveals that on average Nlgn1 is present at greater than 65% of all IHC ribbon synapses within the cochlea.(D) Representative immunofluorescent images from Nlgn3 KO cochlear whole mounts demonstrating the absence of Nlgn3 at ribbon synapse (CtBP2 = red, GluA2 = green).(E) Representative images from apical (8–12 kHz), middle (12–20 kHz), and basal (20–28 kHz) regions of the cochlea immunostained with antibodies for Nlgn3 (blue), CtBP2 (red), and GluA2 (green).(F) Quantification of (E) reveals that on average Nlgn3 is present at greater than 78% of inner hair cell ribbon synapses within the cochlea.(G) Representative images of ribbon synapses immunostained with antibodies for Nlgn1 (magenta), Nlgn3 (blue), CtBP2 (red), and GluA2 (green); subpanels one to four are enlargements of four individual synapses. Data from 16 to 20 kHz region of the cochlea.(H) Quantification of (G) revealed that 40 ± 6.7% of all ribbon synapses contain both Nlgn1 and 3, 17 ± 9.5% were positive for only Nlgn1, 31 ± 4.2% were positive for only Nlgn3, and 11 ± 8.1% of ribbon synapses had undetectable levels of either Nlgn1 or 3 (n = 1306 synapses).(I) Nlgn1 and Nlgn3 positive synapse coordinates plotted according to the IHC pillar-to-modiolar axis from the 16–20 kHz region of the cochlea. Dotted lines represent the basal pole and the line demarcating the pillar to modiolar boarder used for analysis.(J) Quantification of (I) reveals that on the modiolar side a larger proportion of ribbon synapses contained exclusively Nlgn1 (42 ± 5.4%) compared to exclusively Nlgn3 (29 ± 4.8).(K) Quantification of (I) revealed that on the pillar face the majority of synapses were populated with Nlgn3 (Nlgn1 exclusive: 18 ± 5.4%, Nlgn3 exclusive: 49 ± 1.3%, and Nlgn1 and Nlgn3: 33 ± 1.0%).Data are represented as mean ± SD. * = p value <0.05, ** = p value <0.01, *** = p value <0.001 by one-way ANOVA with Tukey post hoc correction for pairwise comparison. Scale bar = 10 µm (A, B, D, and E), 2 µm (G). N = 5 mice per genotype aged P60-65.
Register or Sign In to View Figures.

iScience
August 19 2022

Fig 2: Nlgn1 and three are required for cochlear ribbon synapse maturation(A–D) Representative images from WT, Nlgn1 KO, Nlgn3 KO and Nlgn1/3 dKO cochlear wholemounts from P60-65 mice immunostained with the indicated antibodies for ribbon synapses. Instances of orphan ribbons are marketed by arrows.(E) Synaptic density was significantly reduced at all measured regions: 8 kHz: WT 15.5 ± 0.28, Nlgn1 KO 14.4 ± 0.72, Nlgn3 KO 15 ± 0.22, Nlgn1/3 dKO 11.7 ± 0.45; 12 kHz: WT 16.2 ± 0.32, Nlgn1 KO 15.1 ± 0.14, Nlgn3 KO 14.6 ± 0.33, Nlgn1/3 dKO 11.4 ± 1.0; 16 kHz: WT 17.0 ± 0.49, Nlgn1 KO 15.3 ± 0.37, Nlgn3 KO 15 ± 0.13, Nlgn1/3 dKO 13.3 ± 0.93; 20 kHz: WT 17.3 ± 0.19, Nlgn1 KO 16 ± 0.31, Nlgn3 KO 15.8 ± 0.17, Nlgn1/3 dKO 13.5 ± 0.79; 24 kHz: WT 16.6 ± 0.26, Nlgn1 KO 15.3 ± 0.12, Nlgn3 KO 15.8 ± 0.32, Nlgn1/3 dKO 11.6 ± 0.79; 28 kHz: WT 16.5 ± 0.64, Nlgn1 KO 15.1 ± 0.66, Nlgn3 KO 15.3 ± 0.5, Nlgn1/3 dKO 12.4 ± 0.98.(F) The number of orphan ribbons per IHC was elevated in both Nlgn1 KO and dKO mice compared to WT in the apical (WT: 0.23 ± 0.04; Nlgn1 KO: 1.2 ± 0.61; Nlgn1/3 dKO: 4.9 ± 0.85), middle (WT: 0.41 ± 0.16; Nlgn1 KO: 1.7 ± 0.69; Nlgn1/3 dKO: 4.8 ± 2.0) and basal (WT: 0.31 ± 0.09; Nlgn1 KO: 1.5 ± 0.54; Nlgn1/3 dKO: 7.2 ± 1.6) regions of the cochlea. Box and whisker plot highlighting the 25th-75th quartile distribution middle line is the median of the distribution.(G) Single gene KO cochlea had significantly reduced afferent synapse volume, on average, compared to WT (Nlgn1 KO: 0.21 ± 0.05 µm3, Nlgn3 KO: 0.19 ± 0.04 µm3, WT: 0.40 ± 0.19 µm3). Analysis of the Nlgn1/3 dKO cochlea reveal an exacerbated phenotype (0.07 ± 0.01 µm3).(H) Schematic representation of how distances between pre- and postsynaptic elements were measured to identify nearest neighbors. Distances were measured from the center of each CtBP2 punctum to its nearest neighboring GluA2 punctum to generate a list of nearest neighbors from CtBP2, and vice versa. Lists were then compared to keep only the closest pairs common to both lists, thus eliminating mismatched lone puncta.(I) The average distance between presynaptic CtBP2 and postsynaptic GluA2 is enhanced in Nlgn1 KO cochlea (0.26 ± 0.05 µm) and Nlgn3 KO cochlea (0.26 ± 0.06 µm) compared to WT (0.18 ± 0.02 µm) and Nlgn1/3 dKO (0.21 ± 0.03 µm) cochlea.Data are represented as mean ± SD. * = p value <0.05, ** = p value <0.01, *** = p value <0.001 by one-way ANOVA with Tukey post hoc correction. Scale bar = 10 µm (A–D), N = 4 (A–E), 8 (F), 5 (G–I) P60-65 mice per genotype.
Register or Sign In to View Figures.

iScience
August 19 2022

Fig 3: Nlgn3 KOs have increased sensitivity to noise(A) In WT mice ABR thresholds fully recovered to before noise (BN) threshold levels by 14 DAN at 8 kHz (BN = 30 ± 5.8 dB SPL, one DAN = 33 ± 9.5 dB SPL, seven DAN = 36 ± 11 dB SPL, 14 DAN = 34 ± 9.8 dB SPL), 16 kHz (BN = 24 ± 5.3 dB SPL, one DAN = 52 ± 7.5 dB SPL, seven DAN = 34 ± 7.9 dB SPL, 14 DAN = 27 ± 4.8 dB SPL), and 24 kHz (BN = 31 ± 6.8 dB SPL, one DAN = 60 ± 17.3 dB SPL, seven DAN = 52 ± 18.9 dB SPL, 14 DAN = 38 ± 10.7 dB SPL) after 30 min exposure to 94 dB SPL.(B) In Nlgn1 KO mice ABR thresholds fully recovered to BN threshold levels by 14 DAN at 8 kHz (BN = 26 ± 8.16 dB SPL, one DAN = 55 ± 15.2 dB SPL, seven DAN = 43 ± 10.3 dB SPL, 14 DAN = 32 ± 15.0 dB SPL), 16 kHz (BN = 25 ± 5.47 dB SPL, one DAN = 53 ± 10.3 dB SPL, seven DAN = 47 ± 10.3 dB SPL, 14 DAN = 32 ± 7.5 dB SPL), and 24 kHz (BN = 25 ± 5.47 dB SPL, one DAN = 50 ± 15.5 dB SPL, seven DAN = 42 ± 14.7 dB SPL, 14 DAN = 31 ± 7.5 dB SPL).(C) In Nlgn3 KO mice ABR thresholds fully recovered to BN threshold levels by 14 DAN at 8 kHz (BN = 27 ± 4.9 dB SPL, one DAN = 56 ± 16 dB SPL, seven DAN = 34 ± 5.3 dB SPL, 14 DAN = 60 ± 11 dB SPL) and 16 kHz (BN = 27 ± 4.9 dB SPL, one DAN = 70 ± 10.0 dB SPL, seven DAN = 66 ± 14 dB SPL, 14 DAN = 35 ± 5.3 dB SPL) but failed to recover to BN thresholds at 24 kHz (BN = 31 ± 6.9 dB SPL, one DAN = N/A, seven DAN = 77 ± 4.9 dB SPL, 14 DAN = 60 ± 14 dB SPL).(D) In Nlgn1/3 dKO mice ABR thresholds fully recovered to BN threshold levels by 14 DAN at 8 kHz (BN = 35 ± 5.7 dB SPL, one DAN = 56 ± 16 dB SPL, seven DAN = 34 ± 5.3 dB SPL, 14 DAN = 60 ± 11 dB SPL) but failed to recover to BN thresholds at 16 kHz (BN = 32 ± 5.1 dB SPL, one DAN = N/A, seven DAN = N/A, 14 DAN = 76.6 ± 5.8 dB SPL) and moreover failed to generate any ABR activity above 24 kHz following noise.(E–H) Wave I amplitudes in response to 80 dB SPL stimulation fully recovered to BN amplitudes across WT mice 14 DAN (8 kHz = 97 ± 3.4%, 16 kHz = 94 ± 8%, 24 kHz = 88 ± 5.3%). Conversely, Nlgn1 KOs remained dramatically reduced and never recovered to baseline levels 14 DAN at 8 kHz (77 ± 11%) while Nlgn3 KOs failed to recover at 16 and 24 kHz (16 kHz = 76 ± 7.1, 24 kHz = 26 ± 20%). Nlgn1/3 dKOs never recovered Wave I amplitudes at any frequency compared to BN (8 kHz = 62 ± 13, 16 kHz = 19 ± 14, 24 kHz = N/A).(I) Representative images from WT, Nlgn1 KO, Nlgn3 KO and Nlgn1/3 dKO cochlear wholemounts from P60-65 mice 14 DAN, immunostained with antibodies for ribbon synapses. Scale bar = 10 µm(J) Quantification of (I) revealed Nlgn3 KOs and Nlgn1/3 dKO had significantly disorganized pre- and postsynaptic elements with considerable reductions in synapse density 14 DAN (Nlgn3 KO = 4.23 ± 14, Nlgn1/3 dKO = 5.15 ± 1.1). Synaptic density in WT and Nlgn1 KO cochlea had no significant difference 14 DAN.Data are represented as mean ± SD * = p value <0.05, ** = p value <0.01, *** = p value <0.001 by one-way ANOVA with Tukey post hoc correction. N = 7 mice for (A–F), five mice for (G and H) at P60-65 per genotype.
Register or Sign In to View Figures.

iScience
August 19 2022

Fig 4: Nlgn1 and Nlgn3 are predominantly expressed by spiral ganglion neurons(A) Representative fluorescent images from RNAScope in situ hybridization analysis from Organ of Corti sections. Top: Nlgn1 (green); Bottom: Nlgn3 (green), expression was undetectable in cells positive for Myo7a (red) and Tubb3 (blue).(B) Representative fluorescent images from RNAScope in situ hybridization analysis from 12 μm-thick sections of spiral ganglion in WT, Nlgn1 KO, or Nlgn3 KO cochlea. Nlgn1 (green) and Nlgn3 (red) expression was detectable in cells positive for Tubb3 (blue). Probe specificity was validated in KO tissues which demonstrate little to no reactivity for Nlgn1 or Nlgn3 probes.Scale bar = 10 μm (A and B). Mice were aged P60-65 per genotype.
Register or Sign In to View Figures.

iScience
August 19 2022

Fig 5: Nlgn1 and three are required for cochlear function(A) Representative ABR waveforms at 8 kHz and 80 dB SPL by genotype. Wave-I amplitude was measured peak to trough; latency was measured from sound onset to P1; duration was measured as time from P1 to N1.(B) Quantification reveals ABR thresholds were largely unaffected in either Nlgn1 or Nlgn3 KOs compared to WT. Nlgn3 KOs had elevated threshold levels only at 28 kHz (WT: 25 ± 6.0 dB SPL, Nlgn3 KO: 33 ± 6.6 dB SPL). Nlgn1/3 dKO mice had significantly elevated thresholds at all tested frequencies.(C–E) Growth response curves for 8, 16, and 24 kHz from 20 to 80 dB SPL show significant reduction in ABR responses predominantly between Nlgn3 KO and Nlgn1/3 dKOs compared to WT. Wave I amplitude at 8 kHz 80 dB SPL: WT 3.6 ± 0.31 μV, Nlgn1 KO 2.9 ± 0.66 μV, Nlgn3 KO 2.2 ± 0.45 μV, Nlgn1/3 dKO 2.71 ± 0.73 μV. Wave I amplitude at 16 kHz 80 dB SPL: WT 6.0 ± 0.58 μV, Nlgn1 KO 5.4 ± 1.01 μV, Nlgn3 KO 3.9 ± 0.25 μV, Nlgn1/3 dKO 3.8 ± 0.53 μV. Wave I amplitude at 24 kHz 80 dB SPL: WT 3.7 ± 0.38 μV, Nlgn1 KO 3.5 ± 0.55 μV, Nlgn3 KO 2.1 ± 0.33 μV, Nlgn1/3 dKO 2.6 ± 0.43 μV.(F–H) Wave-I latency was significantly increased in dKO mice compared to Nlgn1 KO, Nlgn3 KO, or WT mice (8 kHz: Nlgn1/3 dKO: 1.6 ± 0.18 ms, WT: 1.3 ± 0.04 ms, Nlgn1 KO: 1.3 ± 0.13 ms, Nlgn3 KO: 1.3 ± 0.13 ms; 16 kHz: Nlgn1/3 dKO: 1.5 ± 0.15 ms, WT: 1.2 ± 0.07 ms, Nlgn1 KO: 1.2 ± 0.03 ms, Nlgn3 KO: 1.2 ± 0.06 ms; 24 kHz: Nlgn1/3 dKO: 1.5 ± 0.05 ms, WT: 1.2 ± 0.10 ms, Nlgn1 KO: 1.2 ± 0.05 ms, Nlgn3 KO: 1.2 ± 0.03 ms). The duration of Wave I was significantly increased in Nlgn1/3 dKO mice compared to WT at higher intensities at 8 kHz: (40 dB: WT 0.4 ± 0.03 ms, Nlgn1 KO 0.43 ± 0.07 ms, Nlgn3 KO 0.41 ± 0.03 ms, Nlgn1/3 dKO 0.50 ± 0.14 ms; 80 dB: WT 0.37 ± 0.01 ms, Nlgn1 KO 0.42 ± 0.04 ms, Nlgn3 KO 0.47 ± 0.05 ms, Nlgn1/3 dKO 0.53 ± 0.14 ms), 16 kHz: (40 dB: WT 0.44 ± 0.02 ms, Nlgn1 KO 0.44 ± 0.04 ms, Nlgn3 KO 0.43 ± 0.05 ms, Nlgn1/3 dKO 0.54 ± 0.04 ms; 80 dB: WT 0.45 ± 0.02 ms, Nlgn1 KO 0.49 ± 0.04 ms, Nlgn3 KO 0.45 ± 0.05 ms, Nlgn1/3 dKO 0.52 ± 0.06 ms) and 24 kHz: (40 dB: WT 0.38 ± 0.05 ms, Nlgn1 KO 0.44 ± 0.02 ms, Nlgn3 KO 0.42 ± 0.04 ms, Nlgn1/3 dKO 0.51 ± 0.11 ms; 80 dB: WT 0.41 ± 0.04 ms, Nlgn1 KO 0.53 ± 0.03 ms, Nlgn3 KO 0.53 ± 0.06 ms, Nlgn1/3 dKO 0.56 ± 0.03 ms).Data are represented as mean ± SD. ∗ = p value <0.05, ∗∗ = p value <0.01, ∗∗∗ = p value <0.001 by one-way ANOVA with Tukey post hoc correction. N = 8 mice for (A–C), five mice for (D–H) at P60-65 per genotype