Journal: Communications Biology

Article Title: Enhanced expression of spinal PTP1B contributes to peripheral inflammation- induced glycinergic disinhibition and mechanical pain

doi: 10.1038/s42003-026-10318-5

Figure Lengend Snippet: a Co-immunoprecipitation (Co-IP) with a specific antibody against GlyR α1 from the lysates of the spinal dorsal horn, and the immunoprecipitates were immunoblotted. The experiments were replicated three times, and each obtained a similar result. IP: immunoprecipitation. b Immunostaining of PTP1B (green) in spinal SOM + neurons (red) from SOM-tdtomato mice. Arrows indicated the colocalization. The graph summarizes the percentage of tdTomato + neurons expressing PTP1B (PTP1B/tdTomato) or the percentage of PTP1B + neurons co-localizing with tdTomato (tdTomato/PTP1B). n = 8 sections from three mice. Scale bar, 20 μm. c Immunofluorescence for PTP1B (green) and gephyrin (red) in SOM + neurons (blue) at days 21 after intraspinal injection of AAV2/9- EF1α-DIO-EGFP in SOM-Cre mice. The boxed area (left) was shown with higher magnification (middle). The arrow indicated the colocalization. The graph summarizes the percentage of PTP1B signals expressing gephyrin (gephyrin/PTP1B) or the percentage of gephyrin signals co-localizing with PTP1B (PTP1B/gephyrin). n = 8 sections from three mice. Scale bars, 10 μm (left) and 2 μm (middle). d PLA detection of PTP1B-GlyR α1 complex in EGFP-labeled SOM + interneurons. Scale bar, 10 µm. e GST-fused intracellular large loop of GlyR α1 (GST-α1-ILL) pulled down Myc-PTP1B(1-321/D181A) from transfected HEK293T cells. The experiments were replicated three times, and each obtained a similar result. TCL total cell lysates. All data were shown as mean ± SEM.

Article Snippet: After the antigen retrieval, the sections were permeabilized with PBS containing 0.25% Triton X-100 overnight, blocked for 1 h at 37 °C with the Duolink blocking solution, and incubated at 4 °C overnight with a mixture of rabbit anti-GPR39 (1:500; #NLS142; Novus Biologicals) and mouse anti-GlyR α1 antibody (1:200; #146111; Synaptic System), a mixture of rabbit anti-GlyR α1 (1:200; #146003; Synaptic System) and mouse phosphotyrosine (PY) antibody (1:200; #P4110; Sigma-Aldrich) or a mixture of mouse anti-PTP1B antibody (1:200; #PH01; Calbiochem) and rabbit anti-GlyR α1 (1:200; #146003; Synaptic System).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Immunostaining, Expressing, Immunofluorescence, Injection, Labeling, Transfection

Journal: Communications Biology

Article Title: Enhanced expression of spinal PTP1B contributes to peripheral inflammation- induced glycinergic disinhibition and mechanical pain

doi: 10.1038/s42003-026-10318-5

Figure Lengend Snippet: a GlyR α1 immunoprecipitates from HEK293T cells co-expressing Flag-tagged PTP1B (Flag-PTP1B) were immunoblotted for PY20 and GlyR α1. TCL total cell lysates, IP immunoprecipitation. ** P = 0.004 (Mann–Whitney U -test), n = 6 experiments. b GlyR α1 immunoprecipitates from spinal dorsal horn of PTP1B −/− and wild-type (WT) mice were immunoblotted for PY20 and GlyR α1. ** P = 0.004 (Mann–Whitney U -test), n = 6 experiments. c PLA assay of GlyR α1 tyrosine phosphorylation with a pair of PY20 and GlyR α1 antibody in spinal SOM + interneurons transfected with EGFP or shRNA-PTP1B. *** P < 0.001 (Mann–Whitney U -test). n = 30 neurons from three mice per group. Scale bar, 10 μm. d Spinal sections showing PTP1B (blue) and EGFP at days 21 after intraspinal injection of AAV2/9-EF1α-DIO-EGFP (EGFP) or AAV2/9-EF1α-DIO-shRNA-PTP1B-EGFP (shRNA-PTP1B) from SOM-Cre mice. Arrows indicated the transfected SOM + neurons. Scale bar: 10 µm. e Effects of extracellular perfusion of PTP1B inhibitor XXII (horizontal bar) on glycinergic currents mediated by GlyR α1 and GlyR α1(Y339F) in HEK293T cells. The original traces were taken at the time points indicated by the numbers 1–3. F (49,490) = 3.323, *** P < 0.001 (Repeated measurement). n = 6 cells per group. f GlyR-mIPSCs were recorded on SOM + neurons expressing EGFP or shRNA-PTP1B in SOM-tdTomato mice. * P = 0.023, ns P = 0.853 (Mann–Whitney U -test), n = 10 neurons from six mice per group. All data were shown as mean ± SEM.

Article Snippet: After the antigen retrieval, the sections were permeabilized with PBS containing 0.25% Triton X-100 overnight, blocked for 1 h at 37 °C with the Duolink blocking solution, and incubated at 4 °C overnight with a mixture of rabbit anti-GPR39 (1:500; #NLS142; Novus Biologicals) and mouse anti-GlyR α1 antibody (1:200; #146111; Synaptic System), a mixture of rabbit anti-GlyR α1 (1:200; #146003; Synaptic System) and mouse phosphotyrosine (PY) antibody (1:200; #P4110; Sigma-Aldrich) or a mixture of mouse anti-PTP1B antibody (1:200; #PH01; Calbiochem) and rabbit anti-GlyR α1 (1:200; #146003; Synaptic System).

Techniques: Expressing, Immunoprecipitation, MANN-WHITNEY, Phospho-proteomics, Transfection, shRNA, Injection

Journal: Communications Biology

Article Title: Enhanced expression of spinal PTP1B contributes to peripheral inflammation- induced glycinergic disinhibition and mechanical pain

doi: 10.1038/s42003-026-10318-5

Figure Lengend Snippet: a von Frey thresholds before (baseline) and after Spared Nerve Injury (SNI). *** P < 0.001 (one-way ANOVA and post hoc Bonferroni test). n = 10 mice/group. b Temporal changes of spinal PTP1B protein levels after CFA injection. * P = 0.042, ** P = 0.006 (One-way ANOVA and post hoc Bonferroni test), n = 6 experiments. c Immunohistochemistry for PTP1B (red) in spinal SOM + neurons (green) at 24 h post CFA injection. ***P = 0.001 (Mann–Whitney U -test). n = 30 neurons from three mice per group. Scale bar, 10 μm. d PLA assay of GlyR α1 tyrosine phosphorylation in SOM + interneurons expressing EGFP or shRNA-PTP1B at 24 h after saline or CFA injection. ** P = 0.005, *** P < 0.001 (one-way ANOVA and post hoc Bonferroni test). n = 30 neurons from three mice per group. Scale bar, 10 μm. e GlyR-mIPSCs record e d on SOM + neurons expressing EGFP or shRNA-PTP1B at 24 h after saline or CFA injection in SOM-Cre mice. *** P < 0.001, ## P = 0.004, ** P = 0.001, * P = 0.041, ns P = 1.00 (one-way ANOVA and post hoc Bonferroni test), n = 10 neurons per group from 3–4 mice. f Current-clamp recordings of action potential (AP) firings elicited by depolarizing current injections in SOM + interneurons expressing EGFP or shRNA-PTP1B at 24 h after saline or CFA injection (left). The firing frequencies at all current injection levels were quantified (right). F (60,560) = 2.453, *** P < 0.001 (repeated measurement); ** P = 0.001, ## P = 0.006, *** P < 0.001 (post hoc Bonferroni test). n = 8 neurons from 3 mice per group. ( g , h ) Rheobase ( g , * P = 0.01, # P = 0.025, & P = 0.022) and resting membrane potential ( h; * P = 0.031, ** P = 0.001, ## P = 0.004) of SOM + interneurons expressing EGFP or shRNA-PTP1B at 24 hours after saline or CFA injection. One-way ANOVA and post hoc Bonferroni test, n = 8–9 neurons from three mice per group. All data were shown as mean ± SEM.

Article Snippet: After the antigen retrieval, the sections were permeabilized with PBS containing 0.25% Triton X-100 overnight, blocked for 1 h at 37 °C with the Duolink blocking solution, and incubated at 4 °C overnight with a mixture of rabbit anti-GPR39 (1:500; #NLS142; Novus Biologicals) and mouse anti-GlyR α1 antibody (1:200; #146111; Synaptic System), a mixture of rabbit anti-GlyR α1 (1:200; #146003; Synaptic System) and mouse phosphotyrosine (PY) antibody (1:200; #P4110; Sigma-Aldrich) or a mixture of mouse anti-PTP1B antibody (1:200; #PH01; Calbiochem) and rabbit anti-GlyR α1 (1:200; #146003; Synaptic System).

Techniques: Injection, Immunohistochemistry, MANN-WHITNEY, Phospho-proteomics, Expressing, shRNA, Saline, Membrane

Journal: Communications Biology

Article Title: Enhanced expression of spinal PTP1B contributes to peripheral inflammation- induced glycinergic disinhibition and mechanical pain

doi: 10.1038/s42003-026-10318-5

Figure Lengend Snippet: a Schematic diagram of GPR39 and PTP1B that competitively bound to GlyR α1 and determined Tyr 339 phosphorylation. b Effects of Myc-tagged PTP1B(1-321/D181A) (Myc-PTP1B) on Flag-GPR39 interaction with GlyR α1 (left). Graphs summarized the ratio of precipitated Flag-GPR39 to GlyR α1 (middle; ** P = 0.002, Mann–Whitney U -test) or the ratio of precipitated Myc-PTP1B to GlyR α1 (right; ** P = 0.002, Mann–Whitney U -test). n = 6 experiments. c Effects of HA-GPR39ct on PTP1B/Myc-α1 interaction in HEK293T cells. ** P = 0.002 (Mann–Whitney U -test). n = 6 experiments. d GlyR α1 immunoprecipitates from spinal dorsal horn of WT and GPR39 −/− mice were immunoblotted for PTP1B and GlyR α1. * P = 0.015 (Mann–Whitney U - test), n = 6 experiments. e PLA detection of PTP1B-GlyR α1 complex in spinal SOM + neurons expressing EGFP or shRNA-GPR39. *** P < 0.001 (Mann–Whitney U -test). n = 30 neurons from three mice per group. Scale bar, 5 μm. f PLA detection of GPR39-GlyR α1 complex in spinal SOM + neurons expressing EGFP or shRNA-PTP1B. *** P < 0.001 (Mann–Whitney U -test). n = 29 neurons (EGFP) and 31 neurons (shRNA-PTP1B) from 3 mice per group. Scale bar, 5 μm. g PLA assay of GlyR α1 tyrosine phosphorylation in SOM + interneurons expressing EGFP or shRNA-GPR39. PTP1B inhibitor (10 nmol) was spinally injected for 4 h before PLA assay. * P = 0.0228, ** P = 0.0015 (one-way ANOVA and post hoc Bonferroni test). n = 30 neurons from three mice per group. Scale bar, 5 μm. h , i Responses of WT and GPR39 −/− mice to paintbrush ( h ; *** P < 0.001, ns P = 1.00) and von Frey stimulation ( i ; ** P = 0.003, * P = 0.015, ns P = 1.00) before (baseline) and 4 h after intrathecal PTP1B inhibitor XXII (10 nmol) injection. One-way ANOVA and post hoc Bonferroni test, n = 8 mice/group. j Effects of extracellular perfusion of PTP1B inhibitor XXII (1 μM) on GlyR-mIPSCs on SOM + neurons expressing EGFP or shRNA - GPR39 in SOM-tdTomato mice. ** P = 0.017, *** P < 0.001 (one-way ANOVA and post hoc Bonferroni test), n = 9 neurons from three mice per group. All data were shown as mean ± SEM.

Article Snippet: After the antigen retrieval, the sections were permeabilized with PBS containing 0.25% Triton X-100 overnight, blocked for 1 h at 37 °C with the Duolink blocking solution, and incubated at 4 °C overnight with a mixture of rabbit anti-GPR39 (1:500; #NLS142; Novus Biologicals) and mouse anti-GlyR α1 antibody (1:200; #146111; Synaptic System), a mixture of rabbit anti-GlyR α1 (1:200; #146003; Synaptic System) and mouse phosphotyrosine (PY) antibody (1:200; #P4110; Sigma-Aldrich) or a mixture of mouse anti-PTP1B antibody (1:200; #PH01; Calbiochem) and rabbit anti-GlyR α1 (1:200; #146003; Synaptic System).

Techniques: Phospho-proteomics, MANN-WHITNEY, Expressing, shRNA, Injection

Journal: Communications Biology

Article Title: Enhanced expression of spinal PTP1B contributes to peripheral inflammation- induced glycinergic disinhibition and mechanical pain

doi: 10.1038/s42003-026-10318-5

Figure Lengend Snippet: a TC-G 1008 (50 pmol) enhanced the ability of Flag-GPR39 to displace PTP1B from Myc-α1 complex in HEK293T cells (left). Graphs summarized the ratio of precipitated PTP1B to Myc-α1 (middle; ** P = 0.005, *** P < 0.001) or the ratio of precipitated Flag-GPR39 to Myc-α1 (right; * P = 0.013, ** P = 0.007). One-way ANOVA and post hoc Bonferroni test. n = 6 experiments. Total cell lysates (TCL) were immunoblotted for PTP1B, Flag and Myc. b Changes of GlyR α1 complex with GPR39 and PTP1B after intrathecal TC-G 1008 (50 pmol) treatment for 30 min in CFA-injected mice (left). Graphs showed the ratio of precipitated GPR39 to GlyR α1 (middle; # P = 0.038, * P = 0.026) or the ratio of precipitated PTP1B to GlyR α1 (right; ** P = 0.005, ## P = 0.008). One-way ANOVA and post hoc Bonferroni test, n = 6 experiments. c PLA dete c tion of PTP1B-GlyR α1 complex in spinal sections from control or CFA-injected SOM-Cre mice that were pretreated with intrathecal TC-G 1008 (50 pmol). *** P < 0.001 (one-way ANOVA and post hoc Bonferroni test). n = 29 neurons from three mice per group. Scale bar, 10 μm. All data were shown as mean ± SEM.

Article Snippet: After the antigen retrieval, the sections were permeabilized with PBS containing 0.25% Triton X-100 overnight, blocked for 1 h at 37 °C with the Duolink blocking solution, and incubated at 4 °C overnight with a mixture of rabbit anti-GPR39 (1:500; #NLS142; Novus Biologicals) and mouse anti-GlyR α1 antibody (1:200; #146111; Synaptic System), a mixture of rabbit anti-GlyR α1 (1:200; #146003; Synaptic System) and mouse phosphotyrosine (PY) antibody (1:200; #P4110; Sigma-Aldrich) or a mixture of mouse anti-PTP1B antibody (1:200; #PH01; Calbiochem) and rabbit anti-GlyR α1 (1:200; #146003; Synaptic System).

Techniques: Injection, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Polyclonal glycine receptor aAbs: a challenge for personalized epitope characterization

doi: 10.3389/fnmol.2026.1747209

Figure Lengend Snippet: Overview of GlyR α 1/α3 constructs. (A) Overall structure of the heteromeric GlyR pentamer (side and top views), with α-subunits in blue and the β subunit in dark gray (PDB: 7MLY; ). (B) The subunit interface between two GlyR α-subunits (indicated by different blue colors, light blue and blue) is depicted. Within the darker blue α-subunit (right subunit), the epitopes of commercial antibodies are labeled in red, and residues different between GlyRα1 and GlyRα3 are marked in yellow (as in C ) and labeled by residue numbers in the enlarged extracellular domain (ECD) of a single subunit. (C) Sequence alignment of ECD of GlyRα1 (UniProt P23415-1) and GlyRα3L (UniProt O75311-1), including the N-terminal domain (NTD), transmembrane 2–3 loop (TM2-3 loop), and the C-terminal domain (CTD). Residues differing between GlyRα1 and GlyRα3 are displayed in bold letters, and missing residues in the alignment are marked by a dot. Residues or small domains exchanged between GlyRα1 and α3 are labeled with a yellow background. The commonly proposed aAb-binding epitope is marked by a gray box. The blue box indicates the location of the neutralizing peptides used. The epitopes of the commercial antibodies, mAb2b (GlyRα1-specific) and mAb4a (pan-α), are depicted in red. Amino acid residue numbers refer to mature proteins. Secondary elements are labeled above the sequence, including two α-helices and 10 β-sheets. (D) Sequences of the neutralizing peptides: a mAb4a-like epitope identified in a patient with aAbs against GlyRβ; the mAb4a epitope N-terminally elongated; and the mAb4a N-terminally shortened combined with C-terminal elongation. (E) Scheme of GlyRα1/α3L Constructs. Exchanges of amino acids between GlyRα1 (blue) and GlyRα3 (orange) are marked, as well as existing epitopes for commercial antibodies mAb2b and mAb4a (red dotted box).

Article Snippet: For live-cell staining, transfected cells were incubated with patient sera diluted 1:50 or a commercial monoclonal antibody against GlyR α 1 (mAb2b, mouse: 146111 or rabbit: 146118, Synaptic Systems, Göttingen, Germany) diluted 1:500 in cell medium for 2 h. After fixation for 10 min using 4% paraformaldehyde (PFA) with 4% sucrose in phosphate-buffered saline (PBS; pH 7.4), the cells were blocked with 5% goat serum in PBS for 30 min.

Techniques: Construct, Labeling, Residue, Sequencing, Binding Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Polyclonal glycine receptor aAbs: a challenge for personalized epitope characterization

doi: 10.3389/fnmol.2026.1747209

Figure Lengend Snippet: Overview of GlyR α 1/α3 constructs. (A) Overall structure of the heteromeric GlyR pentamer (side and top views), with α-subunits in blue and the β subunit in dark gray (PDB: 7MLY; ). (B) The subunit interface between two GlyR α-subunits (indicated by different blue colors, light blue and blue) is depicted. Within the darker blue α-subunit (right subunit), the epitopes of commercial antibodies are labeled in red, and residues different between GlyRα1 and GlyRα3 are marked in yellow (as in C ) and labeled by residue numbers in the enlarged extracellular domain (ECD) of a single subunit. (C) Sequence alignment of ECD of GlyRα1 (UniProt P23415-1) and GlyRα3L (UniProt O75311-1), including the N-terminal domain (NTD), transmembrane 2–3 loop (TM2-3 loop), and the C-terminal domain (CTD). Residues differing between GlyRα1 and GlyRα3 are displayed in bold letters, and missing residues in the alignment are marked by a dot. Residues or small domains exchanged between GlyRα1 and α3 are labeled with a yellow background. The commonly proposed aAb-binding epitope is marked by a gray box. The blue box indicates the location of the neutralizing peptides used. The epitopes of the commercial antibodies, mAb2b (GlyRα1-specific) and mAb4a (pan-α), are depicted in red. Amino acid residue numbers refer to mature proteins. Secondary elements are labeled above the sequence, including two α-helices and 10 β-sheets. (D) Sequences of the neutralizing peptides: a mAb4a-like epitope identified in a patient with aAbs against GlyRβ; the mAb4a epitope N-terminally elongated; and the mAb4a N-terminally shortened combined with C-terminal elongation. (E) Scheme of GlyRα1/α3L Constructs. Exchanges of amino acids between GlyRα1 (blue) and GlyRα3 (orange) are marked, as well as existing epitopes for commercial antibodies mAb2b and mAb4a (red dotted box).

Article Snippet: The primary antibody mAb4a, which is a pan-α GlyR antibody (mAb4a, 146,011, Synaptic Systems), was used at a dilution of 1:250 in blocking solution for 1 h at RT.

Techniques: Construct, Labeling, Residue, Sequencing, Binding Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Polyclonal glycine receptor aAbs: a challenge for personalized epitope characterization

doi: 10.3389/fnmol.2026.1747209

Figure Lengend Snippet: Expression of GlyRα1/α3 Variants. Immunocytochemical staining using commercial antibodies mAb2b (α1-specific) and mAb4a (pan-α) and healthy control serum (magenta) on HEK-293 cells expressing eGFP (cyan) and GlyRα1 WT , GlyRα3 WT L, and Variants 1–10. Cell nuclei are labeled with DAPI (blue). The scale bar indicates 5 μm.

Article Snippet: The primary antibody mAb4a, which is a pan-α GlyR antibody (mAb4a, 146,011, Synaptic Systems), was used at a dilution of 1:250 in blocking solution for 1 h at RT.

Techniques: Expressing, Staining, Control, Labeling

Journal: Frontiers in Molecular Neuroscience

Article Title: Polyclonal glycine receptor aAbs: a challenge for personalized epitope characterization

doi: 10.3389/fnmol.2026.1747209

Figure Lengend Snippet: Peptides resembling a distinct sequence of aAb epitopes can eliminate binding to GlyRs. (A) Scheme of the neutralization assay using patient aAbs and purified peptides. (B–C) Immunocytochemical staining of (B) GlyRα1 hs and GFP (cyan) or (C) GlyRα1 dr and GlyRβ-myc (cyan) co-transfected HEK-293 cells together with patient aAbs or mAb4a staining (magenta). The commercial antibody mAb4a (upper panel) or Patient samples (lower panel) were pre-incubated with (A) the 91 DSIWKPDLFFANEK 104 peptide and a scrambled (scr) peptide or (B) the N-terminal elongated 93 LWKPDLFFANEKSAN 107 peptide, the C-terminal elongated 98 LFFANEKSANFHDVT 112 peptide, and a scr peptide for 30 min prior to cell staining. Nuclei are labeled with DAPI (blue). Scale bars indicate 20 μm (overview) or 5 μm (enlargement).

Article Snippet: The primary antibody mAb4a, which is a pan-α GlyR antibody (mAb4a, 146,011, Synaptic Systems), was used at a dilution of 1:250 in blocking solution for 1 h at RT.

Techniques: Sequencing, Binding Assay, Neutralization, Purification, Staining, Transfection, Incubation, Labeling

Journal: Brain Communications

Article Title: Induced epileptic seizures in larval zebrafish reveal a synaptic modulation associated with the post-ictal state

doi: 10.1093/braincomms/fcag323

Figure Lengend Snippet: Glycine receptors (GlyRs) in glycinergic synapses are more densely packed after seizures. ( A ) Two planes from an M cell in an experimental fish, identifiable via surrounding Synaptic Vesicle protein 2 (SV2) staining (grey) and the characteristic shape of the axon cap covering the M cell axon initial segment. The image is down-sampled to have equal resolution in x-y and z, as this was used for M cell segmentation. Overlaid is a segmentation mask (green) of the M cell cytoplasm. Scale bars in all figure panels—5 μm (corrected for an estimated expansion factor of 3.77). ( B ) Top—Another plane from the same M cell as in A, showing both SV2 (cyan) and GlyR (magenta) staining (left), or GlyR staining only (right). Bottom—Same images as above, overlaid with a segmentation mask of the M cell membrane (left) and of the GlyRs within the membrane (right). ( C ) Box plots describing the fraction of the M cell membrane covered by GlyRs in control fish (black box) and experimental fish that have previously undergone a PTZ-induced seizure (blue box). Group means were compared with a two-tailed t -test with unequal variances and did not significantly differ (t(degrees of freedom—df = 7.73) = 1.54, standard error of the difference—SE = 0.0061, P = 0.1625). ( D ) Box plots describing the ratio of GlyR intensity in synapses (segmented as in the bottom right panel in B) and in the rest of the M cell membrane (segmented as in the bottom left panel in B), in controls (black box) and experimental fish (blue box). * P < 0.05 according to a two-tailed t -test with unequal variances (t(df = 11.2) = 2.82, SE = 0.1694). ( E ) Box plots describing the GlyR intensity in segmented synapses (left), the rest of the M cell membrane (middle) and intracellularly—within the M cell (right), for controls (black boxes) and experimental (blue boxes) fish. A two-way ANOVA analysis showed that both the receptor location (F = 126.16; df = 2,36) and the experimental condition (F = 4.99; df = 1,36) affect GlyR signal intensities. *** P < 0.001 in post-hoc multiple comparisons using Tukey-Kramer’s honestly significant difference procedure comparing GlyR signal intensities within synapses between control and experimental larvae (differences in other locations were not significant). ( F ) Box plots describing the ratio of SV2 intensity in the GlyR mask and in the rest of the M cell membrane, in controls (black box) and experimental fish (blue box). ** P < 0.01 according to a two-tailed t -test with unequal variances (t(df = 11.96) = 3.06, SE = 0.04). ( G ) Box plots describing the SV2 intensity in the entire M cell membrane (left), and divided into ‘near GlyRs’—in the GlyR mask and ‘other’—in the rest of the M cell membrane (middle and right, respectively), for controls (black boxes) and experimental (blue boxes) fish. As in E, a two-way ANOVA analysis showed a significant effect on SV2 intensity for both the location (F = 12.39; df = 1,36) as well as the experimental condition (F = 22.07; df = 2,36). ** P < 0.01 according to Tukey-Kramer’s multiple comparisons procedure following the two-way ANOVA analysis.

Article Snippet: We used the primary antibodies anti-GlyR (146008, Synaptic Systems) and anti- Synaptic Vesicle Protein 2 (SV2, Developmental Studies Hybridoma Bank), and the secondary antibodies Alexa 546 goat anti-rabbit (Invitrogen, A1107) and Atto647N goat anti-mouse (Sigma-Aldrich, 40839).

Techniques: Staining, Membrane, Control, Two Tailed Test