Journal: bioRxiv

Article Title: Long-lasting, subtype-specific regulation of somatostatin interneurons during sensory learning

doi: 10.1101/2024.11.19.624383

Figure Lengend Snippet: ( A ) DiO-PSD95.FingR-Citrine was injected in barrel cortex of SST-Cre mice. ( B ) Schematic outlining injection and training timeline. Animals in fixed tissue analysis were time matched to control for viral expression time. ( C ) 10x image of SST-Cre mice expressing PSD95.FingR across the cortical column. Scale=100µm ( D ) 63x volumetric image of PSD95.FingR labeled somatostatin neurons. Scale=10µm ( E ) (Left) Zoomed image of PSD95.FingR puncta along a dendrite. (Right) Surface reconstruction of citrine signal. Scale=1µm ( F ) Immunohistochemical labeling of SST neurons (Left) PSD95.FingR-Citrine (Middle) calretinin expression (protein product of Calb2 gene) Scale=10µm (Right) merged channels. ( G ) Schematic indicating layer imaging field of view was captured (Layer 2/3). ( H ) Cumulative distribution of PSD95 puncta volume in layer 2/3 SST-O neurons. ( I ) Same as (H) for L2/3 SST-Calb2 neurons. ( J ) Within animal comparison of mean PSD95 puncta volume between SST-O (gray bars) and SST-Calb2 (pink bars) after experiencing acclimation (ACC), one, or five days of sensory association training (SAT). ( K-N ) Same as G-J but for L5 SST-O and SST-Calb2 SST neurons.

Article Snippet: For chemogenetic experiments designed to monitor changes in PSD95 puncta after suppressing activity, we coinjected pAAV8-hSyn-DIO-hM4Di-mCherry (Adgene #44362) with AAV-PHP.eB-ZFN-hSyn-DIO-PSD95.FingR-Citrine-reg.WPRE into S1BF using the stereotaxic injection procedures outlined above.

Techniques: Injection, Control, Expressing, Labeling, Immunohistochemical staining, Imaging, Comparison

Journal: bioRxiv

Article Title: Long-lasting, subtype-specific regulation of somatostatin interneurons during sensory learning

doi: 10.1101/2024.11.19.624383

Figure Lengend Snippet: ( A ) Region of interest (ROI) containing raw PSD95.FingR signal in confocal image stack (63x). Scale=5µm ( B ) Background subtraction prior surface reconstructions. ( C ) Semi-automated thresholding of 3D surface masks (gray) to optimize signal coverage while minimizing noise contained in background voxels. ( D ) A quality filter designating seed points (punctate signal) for splitting fused 3D surface masks. ( E ) 3D reconstructed fluorescence signal (multi-colored) after filtering structures to be ≥ 3 voxels to visualize all PSD95.FingR surface objects. ( F ) 3D surface mask of somatic PSD95.FingR signal. ( G ) 3D surface masks of PSD95.FingR signal within 0.15 um from reconstructed soma were removed. Remaining punctate PSD95.FingR signal (multi-colored) were included for whole field puncta analysis. ( H ) Zoomed out ROI from the same confocal image stack containing punctate PSD95.FingR signal for single cell puncta localization and subsequent comparisons between SST subtypes. Scale=15µm ( I ) Manually selected 3D surfaces (multi-colored) belonging to traceable dendrites emanating from soma in the top left corner. ( J ) Same as (I) but for soma in the bottom right corner. ( K ) Calb2-IR (purple) revealed for classifying puncta belonging to SST-Calb2 or SST-O. ( L ) Designated puncta belonging to SST-O (yellow) and SST-Calb2 (purple) neurons

Article Snippet: For chemogenetic experiments designed to monitor changes in PSD95 puncta after suppressing activity, we coinjected pAAV8-hSyn-DIO-hM4Di-mCherry (Adgene #44362) with AAV-PHP.eB-ZFN-hSyn-DIO-PSD95.FingR-Citrine-reg.WPRE into S1BF using the stereotaxic injection procedures outlined above.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Long-lasting, subtype-specific regulation of somatostatin interneurons during sensory learning

doi: 10.1101/2024.11.19.624383

Figure Lengend Snippet: ( A ) Representative field of view from PSD95.FingR labeled SST neurons in L2/3 of primary somatosensory cortex. Scale=20µm. ( B ) Cumulative distribution of reconstructed PSD95.FingR surface volume from whole field analysis in acclimated (black dashed line), one day (thin blue line), and five day trained (thick blue line) SST-Cre mice. ( C ) Mean puncta volume in individual animals across training conditions. (ACC N=11 mice, 60,500 puncta; SAT1 N=8 mice, 44,000 puncta; SAT5 N=9 mice, 49,500) puncta ( D-F ) Same as A-C but for L4 PSD95.FingR labeled SST neurons. (ACC N=11 mice, 79,420 puncta; SAT1 N=8 mice, 57,760 puncta; SAT5 N=9 mice, 64,980 puncta; ACC vs SAT1 p=.04, unpaired t-test) ( G-I ) Same as A-C but for L5 PSD95.FingR labeled SST neurons. (ACC N=11 mice, 98,900 puncta; SAT1 N=8 mice, 79,120 puncta; SAT5 N=9 mice, 89,010 puncta).

Article Snippet: For chemogenetic experiments designed to monitor changes in PSD95 puncta after suppressing activity, we coinjected pAAV8-hSyn-DIO-hM4Di-mCherry (Adgene #44362) with AAV-PHP.eB-ZFN-hSyn-DIO-PSD95.FingR-Citrine-reg.WPRE into S1BF using the stereotaxic injection procedures outlined above.

Techniques: Labeling

Journal: bioRxiv

Article Title: Long-lasting, subtype-specific regulation of somatostatin interneurons during sensory learning

doi: 10.1101/2024.11.19.624383

Figure Lengend Snippet: ( A ) Schematic outlining viral injection strategy. ( B ) Schematic outlining training timeline. ( C ) 10x image of PSD95.FingR labeled SST neurons merged with calretinin-IR. Scale = 100µm ( D ) 63x confocal image stack containing PSD95.FingR labeled SST neurons merged with calretinin-IR. Scale = 20µm ( E ) Zoomed image of SST-O neuron. PSD95.FingR-Citrine (left), calretinin-IR (middle), overlay, (right). Scale = 5µm ( F ) Same as (E) but for a SST-Calb2 neuron. ( G ) Schematic outlining stimulus and reward presentation probabilities after trial initiations in pseudo-training. ( H ) Within animal comparison between SST-O neurons (gray bars) and SST-Calb2 (pink bars) during acclimation or 5 days of pseudo-training (PSE5). (ACC N = 4mice, 800 puncta; PSE N = 5 mice, 1000 puncta). ( I ) Image showing naïve (left) and environmentally enriched cages (right). ( J ) Same as (H) but for naïve animals and animals experiencing five days of environmental enrichment (EE5). (Naive N = 5mice, 1000 puncta; EE5 N = 5 mice, 1000 puncta)

Article Snippet: For chemogenetic experiments designed to monitor changes in PSD95 puncta after suppressing activity, we coinjected pAAV8-hSyn-DIO-hM4Di-mCherry (Adgene #44362) with AAV-PHP.eB-ZFN-hSyn-DIO-PSD95.FingR-Citrine-reg.WPRE into S1BF using the stereotaxic injection procedures outlined above.

Techniques: Injection, Labeling, Comparison

Journal: bioRxiv

Article Title: Long-lasting, subtype-specific regulation of somatostatin interneurons during sensory learning

doi: 10.1101/2024.11.19.624383

Figure Lengend Snippet: Chemogenetic suppression of SST neuron activity reduces PSD95 puncta size on both SST-O and SST-Calb2 neurons . ( A ) Schematic of Viral injection. ( B ) Schematic of experimental timeline. ( C ) 63x somatic ROI of volumetric confocal image stack containing from left to right, PSD95.FingR-Citrine (yellow), calretinin-IR (purple), and hM4Di-mCherry (pseudo-colored cyan) fluorescence channels. ( D ) Dendrite example from an SST-O neuron (left) overlay between PSD95.FingR (yellow) and calretinin-IR (purple). (right) overlay between PSD95.FingR and hM4Di-mCherry (cyan). ( E, F ) same as in (C, D) but for an SST-O neuron transduced with hM4Di-mCherry. ( G ) Cumulative distribution of PSD95 puncta volume in SST-O neurons with and without hM4Di expression (hM4Di-N=6 mice, 1200 puncta; hM4Di+ N=6 mice, 1200 puncta). ( H ) Within animal comparison of average PSD95 puncta size in SST-O cells with and without hM4Di expression. ( I, J ) same as in (C, D) but an SST-Calb2 neuron not transduced with hM4Di-mCherry. ( K, L ) same as in (C, D) but for an SST-Calb2 neuron transduced with hM4Di-mCherry. ( M, N ) Same as in (G,H) but for SST-Calb2 neurons (hM4Di-N=6 mice, 1200 puncta; hM4Di+ N=6 mice, 1200 puncta).

Article Snippet: For chemogenetic experiments designed to monitor changes in PSD95 puncta after suppressing activity, we coinjected pAAV8-hSyn-DIO-hM4Di-mCherry (Adgene #44362) with AAV-PHP.eB-ZFN-hSyn-DIO-PSD95.FingR-Citrine-reg.WPRE into S1BF using the stereotaxic injection procedures outlined above.

Techniques: Activity Assay, Injection, Fluorescence, Transduction, Expressing, Comparison

Journal: eLife

Article Title: Asymmetric retinal direction tuning predicts optokinetic eye movements across stimulus conditions

doi: 10.7554/eLife.81780

Figure Lengend Snippet: ( A ) Confocal images of exemplar Superior (left) and Inferior (right) oDSGCs filled with dye. Convex polygons are drawn around the tips of their dendrites. (Bottom) Side views of different Superior and Inferior oDSGCs filled and stained for acetylcholinesterase (ChAT) bands. Both cell types have dendrites that stratify in the ON and OFF ChAT bands, with the majority of dendrites in the ON sublamina. ( B ) Convex polygon areas across all filled cells. ( C ) Sholl analysis indicating the number of dendritic crossings as a function of radial distance from the soma (mean ± SEM). ( D, G ) Ganglion cells with immunostaining for ( D ) excitatory postsynaptic scaffolding protein PSD-95 or ( G ) inhibitory postsynaptic scaffolding protein gephyrin. (Bottom) Magnification of a stretch of dendrites with labeled puncta. ( E, H ) Total number of puncta within each ganglion cell for ( E ) PSD-95 and ( H ) gephyrin. ( F, I ) Quantification of average puncta volume for ( F ) PSD-95 and ( I ) gephyrin. For box plots, horizontal line represents median, box boundaries are IQR, and whiskers represent the most extreme observation within 1.5× IQR. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The following protocol was used to enhance for the cell fills (anti-Lucifer yellow [Life Technologies A5750] and/or streptavidin-488 [Thermo Fisher S11223]), label synaptic puncta (anti-postsynaptic density [PSD-95, UC Davis NeuroMab 75-028], anti-Gephyrin [Synaptic Systems 147 111]), and stain for cholinergic starburst amacrine cells (anti-choline acetyltransferase [ChAT, Millipore AB144P]): blocking serum (1 day), primary antibody incubation (5 days), rinse 3× in PBS, secondary antibody (Jackson ImmunoResearch) incubation (1 day), rinse 3× in PBS.

Techniques: Staining, Immunostaining, Scaffolding, Labeling