Journal: bioRxiv

Article Title: GABA-induced Ca 2+ signaling in the primary cilium of neurons

doi: 10.1101/2025.05.26.656109

Figure Lengend Snippet: A. Immunofluorescence images from naïve or 5HT6-GGECO1-expressing cultured mouse neurons (from cortex and CA1) showing the distribution of GABA-B1 receptors (magenta) in primary cilia positive for AC3 (green). Intensity profiles from lines drawn along the cilia are shown to the right and further show the confinement of GABA-B1 receptors to the cilia base of cultured neurons. B. Quantification of GABA-B1 receptor enrichment at the cilia base in naïve cultured neurons or cultured cortical and hippocampal neurons expressing 5HT6-GGECO1 (naïve 2,76±0,37; Cx 8,18±0,91; Hipp 4,94±0,44 arbitrary units). GABA-B1 positive segment length was measured for each group and found significantly different (naïve, Cx and Hipp, in µm: 2,78±0,37; 8,18±0,91; 5,03±0,39; mean±SEM; Kruskal-Wallis ANOVA followed by multiple comparisons, naïve n=12, Cx n=22, Hipp n=38, for A & B). C. Simultaneous recording of cytoplasmic (grey) and ciliary (pink) calcium activities from a neuron before and after stimulation with 100nM baclofen. Activation of metabotropic GABA receptors was without effect on the frequency of somatic events while ciliary signaling was increased by the agonist. Positive deviations from baseline were computed over time to produce ciliary activity count traces (black, lower panel). D. Averaged activity counts corresponding to neurons in control (blue) baclofen (100nM, black) and TTX (1 µM) + baclofen (100nM, grey) showing an increase of activity after stimulation with baclofen. E. Quantification of activity changes was performed in a pair wise fashion, comparing the averaged activity during initial vs final period (control) and baseline vs baclofen (either in the absence of presence of TTX). Time lacked an effect on activity (control P=0,4332) while baclofen stimulation led to a significant increase in both - /+ TTX conditions (respectively P=0,0002 and P=0,0039; two tailed Wilcoxon matched-pairs signed rank test) F. To test whether abolition of action potentials had an impact on spontaneous ciliary activity, recordings in control and TTX (initial 20 minutes both groups) were compared. Statistical analysis showed no difference (P=0,1468 Mann-Whitney two tailed t-test). G. Immunofluorescence images showing the distribution of primary cilia (AC3; green) and synapses (Synapsin II; magenta) in cultured cortical neurons. Boxed areas are magnified to the right. Scatter plot to the left shows Synapsin II fluorescence intensity at primary cilia and random locations within the same sample (P=0,3696 Wilcoxon matched pairs signed rank test).

Article Snippet: The following primary antibodies (1/300 dilutions) were used: ARL13b (Abcam ab136648), AC3 (Abcam ab277619), AC3 (Alomone AAR-043), NeuN (Sigma-Aldrich ABN90), GFAP (Synaptic System 173–004), Synapsin II (Alomone ANR-015), GABA-B1 (Alomone AGB-001), MCHR1 (Thermo Fischer PA5-77492), Patched (Abcam ab53715), EP4 (Santa Cruz Biotechnology sc-55596), ANKS6 (Sigma HPA008355), NPHP3 (Proteintech 22026-1-AP), NEK8 (kind gift from Prof. David R. Beier, Seattle Children’s Hospital, USA).

Techniques: Immunofluorescence, Expressing, Cell Culture, Activation Assay, Activity Assay, Control, Two Tailed Test, MANN-WHITNEY, Fluorescence

Journal: Neural Regeneration Research

Article Title: Nerve root magnetic stimulation regulates the synaptic plasticity of injured spinal cord by ascending sensory pathway

doi: 10.4103/NRR.NRR-D-24-00628

Figure Lengend Snippet: NRMS upregulates transcriptional levels of genes related to synaptic plasticity in damaged region of SCI rats. (A) The functional classification of different genes in the SCI + NRMS and SCI + SS groups by GO enrichment analysis. (B) The functional classification of different genes between the SCI + NRMS and SCI + SS groups by KEGG database. (C–F) The relative levels of Syn2 , Syp , GAP43 , and MAP2 mRNA normalized by the Sham group. The bold lines, upper boundaries, and lower boundaries represent the medians, 75 th percentiles, and 25 th percentiles, respectively. Whiskers extend 1.5 times interquartile range ( n = 4 per group). * P < 0.05, vs . Sham group; # P < 0.05, vs . SCI + SS group. GAP43: Growth-associated protein 43; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; MAP2: microtubule-associated protein 2; NRMS: nerve root magnetic stimulation; SCI: spinal cord injury; SS: sham stimulation; Syn2: synapsin II; Syp: synaptophysin.

Article Snippet: Moreover, the major functions of Synapsin II are focused on the regulation of Ca 2+ -dependent plasticity and neuronal outgrowth in glutamatergic neurons (Matos et al., 2019).

Techniques: Functional Assay

Journal: Neural Regeneration Research

Article Title: Nerve root magnetic stimulation regulates the synaptic plasticity of injured spinal cord by ascending sensory pathway

doi: 10.4103/NRR.NRR-D-24-00628

Figure Lengend Snippet: NRMS alters expression of proteins associated with synaptic plasticity in the injured spinal cord of rats. (A) Representative western blots show the expression levels of PSD95, GAP43, Synapsin I and Synapsin II collected from the damaged area of spinal cord on day 22 after SCI. (B–E) The semi-quantified results of PSD95, GAP43, Synapsin I, and Synapsin II protein levels. All experiments were repeated three times. Data are expressed as mean ± SD ( n = 5 per group). ** P < 0.01, *** P < 0.001, vs. Sham group; # P < 0.05, ## P < 0.01, vs . SCI + SS group. GAP43: Growth-associated protein 43; PSD95: postsynaptic density protein-95; NRMS: nerve root magnetic stimulation; SCI: spinal cord injury; SS: sham stimulation.

Article Snippet: Moreover, the major functions of Synapsin II are focused on the regulation of Ca 2+ -dependent plasticity and neuronal outgrowth in glutamatergic neurons (Matos et al., 2019).

Techniques: Expressing, Western Blot

Journal: Neural Regeneration Research

Article Title: Nerve root magnetic stimulation regulates the synaptic plasticity of injured spinal cord by ascending sensory pathway

doi: 10.4103/NRR.NRR-D-24-00628

Figure Lengend Snippet: NRMS upregulates transcriptional levels of genes related to synaptic plasticity in damaged region of SCI rats. (A) The functional classification of different genes in the SCI + NRMS and SCI + SS groups by GO enrichment analysis. (B) The functional classification of different genes between the SCI + NRMS and SCI + SS groups by KEGG database. (C–F) The relative levels of Syn2 , Syp , GAP43 , and MAP2 mRNA normalized by the Sham group. The bold lines, upper boundaries, and lower boundaries represent the medians, 75 th percentiles, and 25 th percentiles, respectively. Whiskers extend 1.5 times interquartile range ( n = 4 per group). * P < 0.05, vs . Sham group; # P < 0.05, vs . SCI + SS group. GAP43: Growth-associated protein 43; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; MAP2: microtubule-associated protein 2; NRMS: nerve root magnetic stimulation; SCI: spinal cord injury; SS: sham stimulation; Syn2: synapsin II; Syp: synaptophysin.

Article Snippet: The following antibodies were used: postsynaptic density protein-95 (PSD95; rabbit, 1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3450S, RRID: AB_2292883), Synapsin I (rabbit, 1:1000, Abmart, Shanghai, China, Cat# 334286), GAP43 (mouse, 1:400, Santa Cruz, Biotechnology, Dallas, TX, USA, Cat# sc-17790, RRID: AB_627660), Synapsin II (rabbit, 1:1000, Abclonal, Wuhan, China, Cat# A19542), and GAPDH (mouse, 1:20 000, Sigma-Aldrich, Cat# G8795, RRID: AB_1078991).

Techniques: Functional Assay

Journal: Neural Regeneration Research

Article Title: Nerve root magnetic stimulation regulates the synaptic plasticity of injured spinal cord by ascending sensory pathway

doi: 10.4103/NRR.NRR-D-24-00628

Figure Lengend Snippet: NRMS alters expression of proteins associated with synaptic plasticity in the injured spinal cord of rats. (A) Representative western blots show the expression levels of PSD95, GAP43, Synapsin I and Synapsin II collected from the damaged area of spinal cord on day 22 after SCI. (B–E) The semi-quantified results of PSD95, GAP43, Synapsin I, and Synapsin II protein levels. All experiments were repeated three times. Data are expressed as mean ± SD ( n = 5 per group). ** P < 0.01, *** P < 0.001, vs. Sham group; # P < 0.05, ## P < 0.01, vs . SCI + SS group. GAP43: Growth-associated protein 43; PSD95: postsynaptic density protein-95; NRMS: nerve root magnetic stimulation; SCI: spinal cord injury; SS: sham stimulation.

Article Snippet: The following antibodies were used: postsynaptic density protein-95 (PSD95; rabbit, 1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3450S, RRID: AB_2292883), Synapsin I (rabbit, 1:1000, Abmart, Shanghai, China, Cat# 334286), GAP43 (mouse, 1:400, Santa Cruz, Biotechnology, Dallas, TX, USA, Cat# sc-17790, RRID: AB_627660), Synapsin II (rabbit, 1:1000, Abclonal, Wuhan, China, Cat# A19542), and GAPDH (mouse, 1:20 000, Sigma-Aldrich, Cat# G8795, RRID: AB_1078991).

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Effects of serotonergic psychedelics on synaptogenesis and immediate early genes expression – comparison with ketamine, fluoxetine and lithium

doi: 10.1101/2024.08.07.606965

Figure Lengend Snippet: The effect of selected psychoplastogens on synaptogenesis. Quantification of presynaptic density ( A , synapsin puncta), postsynaptic density ( B , PSD-95 puncta) and their co-localization ( C , established synapses) after 24 h drug treatment of primary cortical neurons. n = 38–79 randomly chosen neurons per treatment from at least three biological replicates. One-way ANOVA followed by Dunnett’s post hoc test was performed, data are presented as mean fold change ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. D Representative images of primary dendrites of cortical neurons (DIV18) after 24 h treatment. Orange areas in the synapsin + PSD-95 merged images indicate colocalization. Green, synapsin I/II; red, PSD-95; cyan, MAP2.

Article Snippet: The coverslips were subsequently incubated with primary antibodies chicken anti-MAP2 (1:10,000, Abcam, ab5392), mouse anti-PSD-95 (1:500, Abcam, ab192757), and guinea-pig anti-synapsin I/II (1:1000, Synaptic systems, 106004) at 4°C overnight.

Techniques: