fiber optic patch cable  (Thorlabs)

Journal: bioRxiv

Article Title: Dysregulated Microglial Synaptic Engulfment in Diffuse Midline Glioma

doi: 10.64898/2025.12.22.696064

Figure Lengend Snippet: Neuronal activity-dependent engulfment of excitatory synapses in the healthy cortex is abrogated in glioma. (A) Xenograft, synaptic labeling, and optogenetics paradigm in NOD scid gamma (NSG) mice which underwent cortical xenograft with DMG, diffuse intrinsic pontine glioma (patient-derived cell line “SU-DIPG-VI”), or saline injections (“Nontumor control”), followed by viral injections with hSyn-PSD95-mCherry-eGFP (“Excitatory”) AAV9 or hSyn-gephyrin-mCherry-eGFP (“Inhibitory”) AAV9 and optogenetic ferrule implantation in mouse premotor frontal cortex (M2). NSG mice used were either positive or negative for Thy1::ChR2 transgene. One acute session of optogenetic stimulation was performed within DMG-xenografted or saline-injected M2 cortical regions, according to previously-established methods, on Thy1::ChR2 +/− cohorts. Synaptic engulfment analysis was then performed. P, postnatal day; hSyn, human synapsin promoter. (B) Representative histological and corresponding 3D rendered images (C) of HNA+ glioma cells (SU-DIPG-VI, violet), Iba1+ microglia (white), mCherry+ gephyrin+ synapses (red), and GFP+ gephyrin+ synapses (green) in xenografted deep-layer premotor cortex of Thy1::ChR2 negative (“ChR2−“) mice. Scale bar, 10 um. (D) Volumetric engulfment analysis of microglial engulfment of mCherry+ PSD95+ synapses (“Excitatory”), represented as the % volume of mCherry+ synapse contained in Iba1+ microglia in saline injected (“Nontumor control”) mice without Thy1::ChR2 expression (“ChR2−”) or with Thy1::ChR2 expression (“ChR2+”). (E) Volumetric engulfment analysis of microglial engulfment of mCherry+ gephyrin+ synapses (“Inhibitory”), represented as the % volume of mCherry+ synapse contained in Iba1+ microglia in saline injected (“Nontumor control”) mice without Thy1::ChR2 expression (“ChR2−”) or with Thy1::ChR2 expression (“ChR2+”). (F) Volumetric engulfment analysis of microglial engulfment of mCherry+ PSD95+ synapses (“Excitatory”), represented as the % volume of mCherry+ synapse contained in Iba1+ microglia in glioma xenografted (“DMG xenograft”, SU-DIPG-VI) mice without Thy1::ChR2 expression (“ChR2−”) or with Thy1::ChR2 expression (“ChR2+”). (G) Volumetric engulfment analysis of microglial engulfment of mCherry+ gephyrin+ synapses (“Inhibitory”), represented as the % volume of mCherry+ synapse contained in Iba1+ microglia in glioma xenografted (“DMG xenograft”, SU-DIPG-VI) mice without Thy1::ChR2 expression (“ChR2−”) or with Thy1::ChR2 expression (“ChR2+”). Each individual point represents the average volumetric quantification from images analyzed in one mouse (B-C, F-G). Data are mean ± s.e.m. One-way ANOVA with Tukey’s post hoc analysis (B-C, F-G); **P < 0.01, ****P < 0.0001; NS, not significant.

Article Snippet: Fiber Optic Rotary Joint Patch Cables (Optogenetics) , Thorlabs , RJPSL2.

Techniques: Activity Assay, Labeling, Optogenetics, Derivative Assay, Saline, Control, Injection, Expressing

Journal: bioRxiv

Article Title: Dysregulated Microglial Synaptic Engulfment in Diffuse Midline Glioma

doi: 10.64898/2025.12.22.696064

Figure Lengend Snippet: Neuronal activity alters microglial populations, modulating circuit refinement-related pathways in glioma. (A) Repetitive superimposed traces of field potentials recorded from mice with CA1 hippocampal murine glioma MADR allografts (“DMG allograft”) or from control saline-injected mice (“Nontumor control”). (B) Input-output (I-O) curve was performed in the CA1 region of hippocampal slices. Field excitatory postsynaptic potentials (fEPSPs) were recorded from mice with CA1 hippocampal murine glioma MADR allografts (“DMG allograft”) or from control saline-injected mice (“Nontumor control”). Changes in amplitude were measured relative to baseline following a series of current injections ranging from 0 to 140 μA in steps of 10 μA. (C-D) Frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) were measured in mice with CA1 hippocampal murine glioma MADR allografts (“DMG allograft”) or from control saline-injected mice (“Nontumor control”). (E) Allograft and optogenetics paradigm in CD-1::C57BL/6J (CD1::BL6) mice which underwent cortical allograft with previously-established MADR (“Mosaic analysis by dual recombinase-mediated cassette exchange”) murine glioma (H3.3K27M+ murine-derived cell line), or saline injections (“Nontumor control”), followed by optogenetic ferrule implantation in mouse premotor frontal cortex (M2). CD1::BL6 mice used were either positive or negative for Thy1::ChR2 transgene. One acute session of optogenetic stimulation was performed within MADR-allografted or saline-injected M2 cortical regions, according to previously-established methods, on Thy1::ChR2 +/− cohorts. Single nuclei sequencing analysis was then performed. P, postnatal day. (F) Microglial subpopulation abundance (labeled by color) was calculated as the % of total microglia (“Proportion of Total Microglia”, y-axis) in either nontumor nonstimulated control mice or glioma allografted nonstimulated mice (x-axis). (G) Scatterplot demonstrating gene expression changes in microglia from stimulated, glioma-allografted mice, as compared to stimulated, nontumor controls. The x-axis demonstrates log2 (fold change of stimulated glioma- vs nontumor-associated microglia) and the y axis demonstrates Log10P of the gene expression level. Points shown in red represent genes showing statistically significant changes. (H) Complement-enriched microglial subpopulation abundance was calculated as the % of total microglia (“Proportion of Total Microglia”, y-axis) across all experimental cohorts (x-axis). (I) Complement-associated gene score (y-axis) quantifies the relative expression levels of a complement-associated gene set in microglia across all experimental conditions (x-axis). (J) Homeostasis-associated gene score (y-axis) quantifies the relative expression levels of a homeostasis-associated gene set in microglia across all experimental conditions (x-axis). Data are presented as mean ± s.e.m. Unpaired t test with Welch’s correction (D-E); **P < 0.01; NS, not significant.

Article Snippet: Fiber Optic Rotary Joint Patch Cables (Optogenetics) , Thorlabs , RJPSL2.

Techniques: Activity Assay, Control, Saline, Injection, Optogenetics, Derivative Assay, Sequencing, Labeling, Gene Expression, Expressing

Journal: bioRxiv

Article Title: Dysregulated Microglial Synaptic Engulfment in Diffuse Midline Glioma

doi: 10.64898/2025.12.22.696064

Figure Lengend Snippet: Glioma-infiltrated brain-derived and activity-regulated secreted factors affect microglial synaptic engulfment and activation state in vitro. (A) Paradigm demonstrating ex vivo slice generation of conditioned media to acutely incubate with human microglial (“iMGL”) and glutamatergic/ GABAergic neuron tricultures and subsequently quantify microglial synaptic engulfment. Ex vivo slices were either saline-injected (“Nontumor”) or glioma xenografted (SU-DIPG-VI, “DMG”), with active optogenetic stimulation (Thy1::ChR2+, active conditioned media, “ACM”) or nonstimulated blue light control (Thy1::ChR2-, conditioned media, “CM”). (B) Representative 3D rendered images from human microglial/glutamatergic/GABAergic neuron tri-cultures exposed to ex vivo slice generated conditioned media. Microglia (white), neurons (blue), PSD95+ excitatory synapses (red), and gephyrin+ inhibitory synapses (green) illustrate microglial synaptic engulfment in tri-culture. Tri-cultures were either treated with conditioned media generated from nonstimulated glioma-bearing slices (“DMG CM”) or from stimulated glioma-bearing slices (“DMG ACM”). Scale bar, 15 um. (C) Volumetric engulfment analysis of microglial engulfment of PSD95+ synapses in tri-culture (“Excitatory”), represented as the % volume of PSD95+ synapse contained in Iba1+ microglia in tri-cultures exposed to ex vivo conditioned media from nonstimulated nontumor slices (“Nontumor CM”) or from stimulated nontumor slices (“Nontumor ACM”). (D) Volumetric engulfment analysis of microglial engulfment of gephyrin+ synapses in tri-culture (“Inhibitory”), represented as the % volume of gephyrin+ synapse contained in Iba1+ microglia in tri-cultures exposed to ex vivo conditioned media from nonstimulated nontumor slices (“Nontumor CM”) or from stimulated nontumor slices (“Nontumor ACM”). (E) Volumetric engulfment analysis of microglial engulfment of PSD95+ synapses in tri-culture (“Excitatory”), represented as the % volume of PSD95+ synapse contained in Iba1+ microglia in tri-cultures exposed to ex vivo conditioned media from nonstimulated glioma-bearing slices (“DMG CM”) or from stimulated glioma-bearing slices (“DMG ACM”). (F) Volumetric engulfment analysis of microglial engulfment of gephyrin+ synapses in tri-culture (“Inhibitory”), represented as the % volume of gephyrin+ synapse contained in Iba1+ microglia in tri-cultures exposed to ex vivo conditioned media from nonstimulated glioma-bearing slices (“DMG CM”) or from stimulated glioma-bearing slices (“DMG ACM”). (G) Ex vivo slice generated conditioned media underwent LC/MS analysis, calculating the ratio of protein abundance (“Protein Abundance Ratio”, y-axis) normalized on total peptide amount, and comparing conditioned media from stimulated glioma-bearing slices (“DMG ACM”) to conditioned media from nonstimulated glioma-bearing slices (“DMG CM”). Individual proteins with abundance ratios >1 are labeled and described. Each individual point represents the average volumetric quantification from images analyzed in one tri-culture (C-F). Data are mean ± s.e.m. One-way ANOVA with Tukey’s post hoc analysis (E); *P<0.05, NS, not significant.

Article Snippet: Fiber Optic Rotary Joint Patch Cables (Optogenetics) , Thorlabs , RJPSL2.

Techniques: Derivative Assay, Activity Assay, Activation Assay, In Vitro, Ex Vivo, Saline, Injection, Control, Generated, Liquid Chromatography with Mass Spectroscopy, Quantitative Proteomics, Labeling