Journal: Nature Communications

Article Title: Neuropeptide SP protects against colitis and linked anxiety-like behavior through the putative roles of gut microbiota and metabolite inositol

doi: 10.1038/s41467-025-67904-0

Figure Lengend Snippet: a , b Heat maps revealing differential gene expression in FMT-DSS vs FMT-CON, FMT-DSS + SP vs FMT-DSS groups. n = 4 mice/group. The pathway-related genes were selected (log2 fold change at least > 1, p < 0.05). c , d KEGG analysis of total-, up- and down-regulated genes in the FMT-DSS group compared with the FMT-CON group (Top 20). n = 4 mice/group. e , f KEGG analysis of total-, up- and down-regulated genes in the FMT-DSS + SP group vs the FMT-DSS group (Top 20). n = 4 mice/group. g Quantitative real-time PCR analysis of mRNA expressions of NF-κB signaling pathway genes ( p65 and IKBα ) in the hippocampus. n = 4 mice/group. The whiskers indicate the minimum and maximum values observed within the range of Q1 − 1.5 × IQR to Q3 + 1.5 × IQR. The box reveals the interquartile range (IQR) between the 25th (Q1) and 75th (Q3) percentiles, and the line inside the box represents the median (50th percentile). h The protein expression of p-p65 and p-IKBα in hippocampus tissue, as determined by western blotting. n = 3 independent experiments. i Quantitative real-t i me PCR analysis of mRNA expressions of GABA receptor and Ca 2+ signaling genes ( Gabra1 , Gabra3 , Gabrg2 , Gabrb2 and Camk2d ). n = 4 mice/group. The whiskers indicate the minimum and maximum values observed within the range of Q1 − 1.5 × IQR to Q3 + 1.5 × IQR. The box reveals the interquartile range (IQR) between the 25th (Q1) and 75th (Q3) percentiles, and the line inside the box represents the median (50th percentile). j Protein expressions of GABA A R, GABA B R, GAD65 and CaMKII were measured in hippocampus tissue by western blot. n = 3 independent experiments. k Representative immunofluorescence images of double-labeling for p-IKBα (red)/Iba1 (green) in hippocampus tissues. Scale bar = 50 μm. n = 3 independent experiments. I Representative immunofluorescence images of double-labeling for GABA A R (green)/GFAP (red) in hippocampus tissues. Scale bar = 50 μm. n = 3 independent experiments. m Double immunofluorescence staining for CaMKII (green)/GFAP (red) in hippocampus tissues and statistical analysis. Scale bar = 50 μm. n = 3 independent experiments. n Differential gene expressions were presented by the heatmap in microglia of three groups, and GSEA analysis of microglia from FMT-DSS + SP group vs the FMT-DSS group. n = 4 mice/group. o Heat maps of differential gene expression in astrocytes from three groups and GSEA analysis of astrocytes from FMT-DSS + SP group vs the FMT-DSS group. n = 4 mice/group. Data were presented as means ± SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Source data are provided as a file.

Article Snippet: Next, primary antibody against GFAP (1:1000, A8335, 1:200, NB100-53809, USA), GABA A Rα1 (1:1000, 12708-1-AP, Proteintech, China), GABA B R2 (1:1000, 27567-1-AP, Proteintech, China), CaMKII (1:1000, 12716, Cell Signaling Technology, USA), GAD65 (1:1000, ab239372, Abcam, USA), CD206 (1:1000, ab64693, Abcam, USA), iNOS (1:1000, 18985-1-AP, Proteintech, China), CD80 (1:1000, 66406-1, Proteintech, China), p-p65 (1:1000, A00284-1, Boster, China), p-IKBα (1:1000, WLH3930, Wanleibio, China), and β-actin (50201, 1:1000, Kemei Borui Science and Technology, China) were incubated with the membranes overnight at 4 °C.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Labeling, Double Immunofluorescence Staining

Journal: Nature Communications

Article Title: Neuropeptide SP protects against colitis and linked anxiety-like behavior through the putative roles of gut microbiota and metabolite inositol

doi: 10.1038/s41467-025-67904-0

Figure Lengend Snippet: a Diagram illustrating the mouse model of colitis employed in this study, and inositol treatments are indicated (Some schematic elements were created by Figdraw.com). b Daily body weight changes. n = 8 mice/group. c Diseases activity index. n = 6 mice/group. d Macroscopic pictures of colons. e H&E-stained colon sections. Scale bar = 100 μm. n = 5 independent experiments. f Immunofluorescence staining for E-cadherin (red)/DAPI (blue) in colon tissues. Scale bar = 100 μm. n = 4 independent experiments. g The mRNA level of inflammatory cytokines ( IL-1β , IL-6 and TNF-α ) in the colon. n = 4 mice/group. h , i Representative movement tracks in the open field test and related bar graphs (Distance traveled, distance of center region and speed). n = 6 mice/group. j , k Track plot of the elevated plus maze, and statistical analysis including percentage of time spent in the open arms and percentage of times entering the open arms. n = 6 mice/group. l Representative H&E-stained hippocampus sections of three groups. Scale bar = 100 μm. n = 6 independent experiments. m Photomicrographs of Nissl staining in the hippocampus. Scale bar = 100 μm. n = 6 independent experiments. n The mRNA expressions of downstream cytokines ( IL-6 , IL-1β , TNF-α and IL-10 ) in the hippocampus tissue. n = 5 mice/group. o Representative immunohistochemistry images of Iba-1 in hippocampus, Scale bar = 100 μm. n = 3 independent experiments. p The concentration of GABA in hippocampus tissue. n = 6 mice/group. q The mRNA expression of GABA A Rα1 in hippocampus. n = 5 mice/group. r The protein level of GABA B R detected by western blot. n = 3 independent experiments. s Double immunofluorescence staining for GFAP (red)/GAT1 (green) in the DG and CA1 regions of hippocampus tissue. Scale bar = 50 μm. n = 3 independent experiments. Data were presented as means ± SD. For body weight change, two-way repeated-measures ANOVA was performed and the rest of the statistics was analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Source data are provided as a file.

Article Snippet: Next, primary antibody against GFAP (1:1000, A8335, 1:200, NB100-53809, USA), GABA A Rα1 (1:1000, 12708-1-AP, Proteintech, China), GABA B R2 (1:1000, 27567-1-AP, Proteintech, China), CaMKII (1:1000, 12716, Cell Signaling Technology, USA), GAD65 (1:1000, ab239372, Abcam, USA), CD206 (1:1000, ab64693, Abcam, USA), iNOS (1:1000, 18985-1-AP, Proteintech, China), CD80 (1:1000, 66406-1, Proteintech, China), p-p65 (1:1000, A00284-1, Boster, China), p-IKBα (1:1000, WLH3930, Wanleibio, China), and β-actin (50201, 1:1000, Kemei Borui Science and Technology, China) were incubated with the membranes overnight at 4 °C.

Techniques: Activity Assay, Staining, Immunofluorescence, Immunohistochemistry, Concentration Assay, Expressing, Western Blot, Double Immunofluorescence Staining

Journal: Nature Communications

Article Title: Neuropeptide SP protects against colitis and linked anxiety-like behavior through the putative roles of gut microbiota and metabolite inositol

doi: 10.1038/s41467-025-67904-0

Figure Lengend Snippet: a Schematic illustration of experimental design (Some schematic elements were created by Figdraw.com). b , c Track plot of the elevated plus maze, and statistical analysis including percentage of time spent in the open arms, percentage of distance traveled in the open arms and percentage of times entering the open arms. n = 6 mice/group. d , e Track plot of the elevated zero maze, and statistical analysis including percentage of time spent in the open zones and percentage of times entering the open zones. n = 6 mice/group. f Statistical analysis, including percentage of distance traveled in the light area, percentage of time spent in the light area, and times of transitions in the light-dark box test. n = 6 mice/group. g Representative images of H&E-staining for the hippocampus sections. Scale bar = 100 μm. n = 6 independent experiments. h Representative images of Nissl staining for the hippocampus sections. Scale bar = 100 μm. n = 4 independent experiments. i The content of inositol in hippocampus tissue tested by ELISA. n = 8 mice/group. j The content of inositol in serum tested by ELISA. n = 8 mice/group. k–n The mRNA expression of TNF-α , IL-1β , IL-6 and iNOS in BV-2 cells. n = 3 mice/group. o, p The mRNA expression of Gabra3 and Camk2d in C8D1A cells. n = 3 mice/group. q The protein level of GABA A R, CaMKII, GABA B R detected by western blot. n = 3 independent experiments. Data were presented as means ± SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Source data are provided as a file.

Article Snippet: Next, primary antibody against GFAP (1:1000, A8335, 1:200, NB100-53809, USA), GABA A Rα1 (1:1000, 12708-1-AP, Proteintech, China), GABA B R2 (1:1000, 27567-1-AP, Proteintech, China), CaMKII (1:1000, 12716, Cell Signaling Technology, USA), GAD65 (1:1000, ab239372, Abcam, USA), CD206 (1:1000, ab64693, Abcam, USA), iNOS (1:1000, 18985-1-AP, Proteintech, China), CD80 (1:1000, 66406-1, Proteintech, China), p-p65 (1:1000, A00284-1, Boster, China), p-IKBα (1:1000, WLH3930, Wanleibio, China), and β-actin (50201, 1:1000, Kemei Borui Science and Technology, China) were incubated with the membranes overnight at 4 °C.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a The diagram of the calyx of Held synapse. b Strategy for selective deletions of all neurexins at the calyx of Held by crossing PV-Cre mice with triple Nrxn123 cKO mice (Chen et al. , Luo et al. ). c Representative traces of EPSC before and after application of 20 µM SKF-97541 (SKF), a potent and selective GABA B -receptor agonist, recorded in acute slices from littermate control and neurexin123 TKO mice at P12–P14. The normalized EPSCs before and after SKF are shown in inset. d Summary graphs of EPSC1 amplitudes before and after SKF for control and Nrxn123 TKO mice. P = 2.3E−7, P = 0.072, paired two-sided t -test. P = 0.0062, unpaired two-sided t -test. e Summary graphs of EPSC1 remaining unblocked by SKF application. P = 1.79E−7, unpaired two-sided t -test. f Summary graphs of the paired-pulse ratio (PPR) before and after SKF in control and Nrxn123 TKO mice. P = 2.81E−5, P = 0.09, paired two-sided t -test. P = 0.0038, unpaired two-sided t -test. Data are means ± SEM. Number of cells (from at least three mice per group) analyzed are indicated in the bars ( d – f ); Statistical differences were assessed by Student’s t -test. (* P < 0.05; ** P < 0.01; *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Control

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Representative confocal microscopy images of MNTB-containing brainstem slice with specific labeling of VGluT1 (green) and GABA B -receptor subunit 2 (GABA B2 , red) from both littermate control and Nrxn123 TKO mice at P12. Scale bar, 10 µm. b Summary of VGluT1 and GABA B2 immunostaining intensity (normalized to control). P = 1, P = 1.35E−9, unpaired two-sided t -test. c , d Same as a and b except for specific labeling of VGluT1 (green) and GABA B -receptor subunit 1 (GABA B1 , red). P = 1, P = 0.031, unpaired two-sided t -test. Data are means ± SEM. Number of sections/animals for immunostaining are indicated in the bars ( b , d ). Statistical differences were assessed by Student’s t -test. (* P < 0.05; *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Confocal Microscopy, Labeling, Control, Immunostaining

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Representative dSTORM images of MNTB-containing brainstem slice with dual labeling of Homer1 (magenta) and GABA B -receptor subunit 2 (GABA B2 , yellow) from both littermate control and Nrxn123 TKO mice. b Summary of the number of GABA B2 clusters normalized to the number of Homer1 clusters. P = 0.0051, unpaired two-sided t -test. c Distribution of Homer1 and GABA B2 localizations. d – f Similar as a – c , except for GABA B1 antibody used. P = 0.0057, unpaired two-sided t -test. Data are means ± SEM. Number of sections/animals for immunostaining are indicated in the bars ( b , e ). Statistical differences were assessed by Student’s t -test. (** P < 0.01). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Labeling, Control, Immunostaining

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Schematic of experimental approach for virus injection and electro-physiology recording (Top); GFP expression in AAV-transfected CA3 region of hippocampus (bottom). b Representative traces of EPSCs evoked by fiber stimulations with increased intensity, recorded in acute hippocampus slices from Nrxn123 cKO mice injected with AAV-ΔCre or AAV-Cre. c The input–output curve of EPSC amplitudes in relation to the stimulation intensity. P = 0.0113, unpaired two-sided t -test. d Example traces of paired-pulse EPSCs before and after addition of GABA B -receptor agonist SKF, the normalized EPSCs before and after SKF are shown in inset. The intensity of fiber stimulation was tuned to evoke EPSC1 at similar amplitude for each cell. e Summary graphs of EPSC1 amplitudes before and after SKF for control (ΔCre) and Nrxn123 TKO synapses (Cre). P = 0.0001, P = 0.0001, paired two-sided t -test. P = 0.99, unpaired two-sided t -test. f Summary graphs of EPSC1 rise time (left) and decay time constants (right). P = 0.0008, P = 0.6136, unpaired two-sided t -test. g Summary graphs of EPSC1 remaining unblocked by SKF application. P = 0.0013, unpaired two-sided t -test. h Summary graphs of the PPR before and after SKF in control and Nrxn123 TKO synapses. P = 0.0001, P = 0.0984, paired two-sided t -test. P = 0.2772, unpaired two-sided t -test. i Summary graphs of the CV of EPSC1 amplitude before and after SKF in control and Nrxn123 TKO synapses. P = 0.0385, P = 0.99, paired two-sided t -test. P = 0.4664, unpaired two-sided t -test. Data are means ± SEM. Number of cells (from at least three mice per group) analyzed are indicated in the graph ( c ) or bars ( e – i ); Statistical differences were assessed by Student’s t -test. (* P < 0.05; ** P < 0.01, *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Virus, Injection, Expressing, Transfection, Control

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Schematic of experimental setup for virus injection and optogenetic stimulation of PV + interneurons (Top); PV-Cre dependent expression of Chief-TdTomato in AAV-transfected CA1 region of hippocampus (bottom). b Example traces of paired-pulse blue light-evoked IPSCs before and after addition of GABA B -receptor agonist SKF, recorded in acute hippocampus slices from PVCre + mice (control) or PVCre + /Nrxn123 cKO mice (TKO) injected with AAV-DIO-Chief-TdTomato. The normalized IPSCs before and after SKF are shown in inset. c Summary graphs of IPSC1 amplitudes before and after SKF for control and Nrxn123 TKO synapses. P = 0.0001, P = 0.0026, paired two-sided t -test. P = 0.6, unpaired two-sided t -test. d Summary graphs of IPSC1 amplitude remaining unblocked by SKF. P = 0.0047, unpaired two-sided t -test. e Summary graphs of the PPR ratio before and after SKF in control and Nrxn123 TKO synapses. P = 0.0022, P = 0.6165, paired two-sided t -test. P = 0.5114, unpaired two-sided t -test. f Summary graphs of the CV of IPSC1 amplitude. P = 0.0143, P = 0.99, paired two-sided t -test. P = 0.99, unpaired two-sided t -test. g Summary graphs of IPSC1 rise time (left) and decay time constant (right). P = 0.4974, P = 0.063, unpaired two-sided t -test. Data are means ± SEM. Number of cells (from at least three mice per group) analyzed are indicated in the bars ( c – g ); Statistical differences were assessed by Student’s t -test. (* P < 0.05; ** P < 0.01; *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Virus, Injection, Expressing, Transfection, Control

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Schematic of cerebellum circuits and experimental approach for stimulating basket cell (BC)-Purkinje cell (PC) inhibitory synapse. b Example traces of paired-pulse IPSCs before and after addition of GABA B -receptor agonist SKF, recorded in acute cerebellar slices from littermate control and Nrxn123 TKO mice P35-49. The normalized IPSCs before and after SKF are shown in inset. c Summary graphs of IPSC1 amplitudes before and after SKF for control and Nrxn123 TKO synapses. P = 0.0016, P = 0.051, paired two-sided t -test. P = 0.0389, unpaired two-sided t -test. d Summary graphs of IPSC1 amplitude remaining unblocked by SKF for control and Nrxn123 TKO mice. P = 0.0028; unpaired two-sided t -test. e Summary graphs of the PPR before and after SKF in control and Nrxn123 TKO mice. P = 0.000101, P = 0.097, paired two-sided t -test. P = 0.762, unpaired two-sided t -test. f Summary graphs of the CV of IPSC1 amplitude before and after SKF in control and Nrxn123 TKO mice. P = 0.00073, P = 0.066, paired two-sided t -test. P = 0.348, unpaired two-sided t -test. g Summary graphs of IPSC1 rise time (left) and decay time constant (right). P = 0.000953, P = 0.977; unpaired two-sided t -test. Data are means ± SEM. Number of cells (from at least three mice per group) analyzed are indicated in the bars ( c – g ); Statistical differences were assessed by Student’s t -test. (* P < 0.05; ** P < 0.01; *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Control