Journal: Scientific reports

Article Title: Optogenetic targeting of astrocytes restores slow brain rhythm function and slows Alzheimer's disease pathology.

doi: 10.1038/s41598-023-40402-3

Figure Lengend Snippet: Figure 2. Optogenetic targeting of astrocytes allows manipulation of slow oscillations. (A) Experimental design. (B) Low resolution wide-field image of Voltron fluorescence. Scale bar, 500 μm. (C) High resolution wide-field image of a neuron expressing Voltron. Scale bar, 10 μm. (D) Representative traces of voltage sensor signal in NTG and APP mice. (E) Representative traces of voltage sensor signal acquired spontaneously (Spon), during light activation of mCherry lacking ChR2 at 1.2 Hz (mCherry 2XRx), or during optogenetic activation of ChR2 at 1.2 Hz (ChR2 2XRx) in NTG mice. Light pulse stimulations are shown in blue. (F) Representative traces of voltage sensor signal acquired spontaneously (Spon), during light activation of mCherry lacking ChR2 at 0.6 Hz (mCherry 1XRx), or during optogenetic activation of ChR2 at 0.6 Hz (ChR2 1XRx) in APP mice. Light pulse stimulations are shown in blue. (G–I) Power spectral density plots of slow oscillations in NTG (H) and APP (I) mice across conditions. Mean ± SEM. (J) Bar graph comparing the average power of slow oscillations in NTG and APP mice. (K) Bar graph comparing the average power of slow oscillations in NTG mice across conditions. (L) Bar graph comparing the average power of slow oscillations in APP mice across conditions. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: In addition, viral vectors encoding Cre recombinase that targeted neurons (0.75 μl AAV9-hSyn-Cre, titer ≥ 1 × 1013 vg/mL, Addgene) and Cre-dependent voltage sensor Voltron (0.75 μl AAV1-hSyn-flex-Voltron-ST, titer ≥ 2 × 1012 vg/mL, Addgene) were injected into the right hemispheres of the same mice (AP − 3 mm, ML − 2 mm, DV − 0.2 mm) to monitor slow oscillations.

Techniques: Fluorescence, Expressing, Activation Assay

Journal: bioRxiv

Article Title: Direct interoceptive input to the insular cortex shapes learned feeding behavior

doi: 10.1101/2025.05.13.653896

Figure Lengend Snippet: (A) Two-color fluorescent in situ hybridization for LepR mRNA (magenta) and Vglut1 mRNA (cyan) in the insula-claustrum region. A 1-3 (inset from A) show LepR mRNA and Vglut1 mRNA in deep layers of the insula. White arrows point to colocalized neurons. (B) Scheme for Cre-dependent mCherry virus injected bilaterally into the insula of LepR-Cre mice. (C) mCherry (magenta) expression in LepR+ cells located the insula. (D) LepR+ cell number across the anterior/posterior (AP) axis of the insular cortex (n = 6-8/group, two-way ANOVA followed by Turkey post-hoc test, F (4,160) = 125.6, **** p < 0.0001. Layer I vs. Layer6 VI, Layer II/III vs. Layer6 VI, Layer IV vs. Layer VI at all coordinates in the graph, **** p < 0.0001. Layer I vs. Layer V, Layer II/III vs. Layer V, Layer IV vs. Layer V, Layer V vs. Layer VI at different coordinates, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, NS: No significance). (E) Scheme for whole cell patch-clamp recording of LepR+ cells. (F) Firing rate (expressed in hertz, Hz) triggered by a series of currents before and after leptin administration (n = 7-10 cells from 7 mice in each group, two-way ANOVA, F (1,345) = 25.7, **** p < 0.0001). (G) Maximum firing rate (expressed in hertz, Hz) triggered by current injection before and after leptin (Lep.) administration (n = 10 from 7 mice, Wilcoxon matched-pairs signed rank test, ** p = 0.002). (H) Afterhyperpolarization (AHP) in the first action potential triggered by the minimum current, before and after leptin (Lep.) administration (n = 11, Wilcoxon matched-pairs signed rank test, *** p = 0.001). (I) Adaptation ratio (inter spike interval (ISI) of the 5 th and 6 th spikes/ISI of 1 st and 2 nd spikes) before and after leptin (Lep.) administration (n = 9, Wilcoxon matched-pairs signed rank test, * p = 0.0117; excluded one outlier). (J) Scheme for cannula implantation and local infusion of PBS or leptin (120ng) into the insula. (K) Food intake measurements, expressed in cumulative grams (s) consumed following infusion of PBS or leptin (120ng) into the insula (n = 16 DPBS, n = 16 leptin, two-way ANOVA followed by Turkey post-hoc test, F (1,150) = 31.06, **** p < 0.0001. PBS vs Leptin, at 12-hour, ** p = 0.0014; PBS vs Leptin, at 24-hour, **** p < 0.0001). (L-M) Food intake, expressed as grams (g) per day, and body weight measurements, expressed as the grams (g) change (Δ) in body weight from baseline (-2 days) before and after the injection of PBS or leptin (120ng) into the insula-claustrum region (For L , n = 15-16 PBS, n = 16 leptin, two-way ANOVA, PBS vs. leptin, F (1,179) = 18.71, **** p < 0.0001; at 1 st , *** p = 0.0003; at 2 nd , *** p = 0.0006. For M , n = 16 PBS, n = 16 leptin (Days -2 to 2, 4) n = 8 PBS, n = 8 leptin (Day 3); DPBS vs. leptin, two-way ANOVA, F (1,164) = 3.025, p = 0.0838; at 1 st , *** p < 0.0005; at 2 nd , * p = 0.0407). (N) Breakpoint, represented as the maximum # of nose pokes required to get a food pellet in the progressive ratio test, 1 day after PBS or leptin infusion (n = 14 PBS, n = 14 leptin, two-tailed unpaired t test, * p = 0.027). (O) Scheme for chronic local infusion of PBS or leptin (5ng/hour for ∼14 days) using osmotic pumps. (P-Q) Food intake and body weight, normalized to the average daily food intake and body weight in the week prior to osmotic pump implantation, expressed as the % increase in grams (g) from starting body weight, after chronic PBS and leptin infusion to the insula (For P , n = 15-16 PBS, n = 15-16 leptin, two-way ANOVA followed by Turkey post-hoc test, PBS vs. leptin, F (1,356) = 5.54, * p = 0.0191. For Q , n = 15-16 PBS, n = 15-16 leptin, PBS vs. leptin, two-way ANOVA, F (1,450) = 290.6, **** p < 0.0001. *** p < 0.001, ** p < 0.01). For more information, see Figures S1 and S2 .

Article Snippet: Similar to Nissl staining, LepR-Cre mice were injected with cre-dependent mCherry (pAAV-hSyn-DIO-mCherry, Addgene, 50459-AAV5, titer 2.3×10 GC/mL) into the insular cortex.

Techniques: In Situ Hybridization, Virus, Injection, Expressing, Patch Clamp, Two Tailed Test

Journal: bioRxiv

Article Title: Direct interoceptive input to the insular cortex shapes learned feeding behavior

doi: 10.1101/2025.05.13.653896

Figure Lengend Snippet: (A) Morphology of LepR+ (magenta) and LepR- (cyan) cells in the insula, across cortical layers. (B) Sholl analysis for the morphological comparison between LepR+ (magenta) and LepR- (cyan) cells. Data is represented as the number of intersections at different distances from the soma, measured in μm. (n = 13 LepR+, n = 11 LepR-, two-way ANOVA followed by Turkey post-hoc test, F (1,616) = 0.0108; at 40 μm, * p = 0.0268; at 60 μm, ** p = 0.002). (C) Percentage of connected cells around the patched cells. 22.3% of cells around the patched cells (4 cells from E ) can be activated. (D) Scheme for the method of viral injection and GRIN lens implantation to image calcium activity in the general insular cortex population following saline or leptin treatment. (E) Trajectory of activity during feeding or drinking bouts 12 hours after PBS (control) or leptin (5 μg/kg) administration using principal component analysis (PCA) . Data is shown as activity over time from 5 seconds before to 20 seconds after the bout begins. (F) Similarity of insula activity trajectories at feeding/drinking between DPBS- and leptin-treated group. Top: Procustes analysis (feeding, magenta; drinking, blue). Bottom: Subspace angle analysis (feeding, magenta; drinking, blue). (G) Similarity of insula activity trajectories in DPBS- and leptin-treated group between feeding and drinking behavior. Top: Procustes analysis (leptin, magenta; DPBS, blue). Bottom: Subspace angle analysis (leptin, magenta; DPBS, blue). (H) Representative images of terminal projection mapping from mCherry expression in LepR+ cells in the insular cortex injection site (left) and in axon terminals in the basolateral amygdala (right). (I) Scheme for the method of studying functional connectivity by using whole-cell patch-clamp recording. (J) Excitatory postsynaptic current (EPSC) is inducted by 2 ms of blue light (Opto-EPSC). (K) Scheme for viral injection and optical fiber cannula implantation for optogenetic stimulation of BLA-projecting LepR+ cells in the insula. (P) Scheme for pre-session optogentic stimulation. (Q) Pellet number consumed over one testing session with pre-session stimulation (magenta) or control (blue)) (n = 6 Stim, n = 6 No stim, two-tailed paired t test, p = 0.2098). (R) Scheme for the protocol of post-nosepoke stimulation. (S) Latency to pellet retrieval following nose poke with stimulation (magenta) or control (blue) (n = 6 Stim, n = 6 No stim, two-tailed paired t test, * p = 0.0276). For more information, see Figures S9-S10 .

Article Snippet: Similar to Nissl staining, LepR-Cre mice were injected with cre-dependent mCherry (pAAV-hSyn-DIO-mCherry, Addgene, 50459-AAV5, titer 2.3×10 GC/mL) into the insular cortex.

Techniques: Comparison, Injection, Activity Assay, Saline, Control, Expressing, Functional Assay, Patch Clamp, Two Tailed Test

Journal: bioRxiv

Article Title: Control of flight responses by a non-canonical corticolimbic pathway

doi: 10.1101/2022.05.19.492688

Figure Lengend Snippet: A , Schematic of the intersectional strategy used to record DP-to-CeA projector activity. B , Implanted mice were subjected to a fear conditioning paradigm designed to elicit high fear states. C , Left , Representative GCaMP6f expression with lens placement in the DP (scale bar = 500 µm). Right , miniscope field-of-view (raw and post cell extraction). D , Freezing and flight scores in the high-threat context. N = 5 E , Freezing and flight scores in the low-threat context. N = 5 F , Zscores of active neurons and speed in the high-threat context (N = 148 neurons). G , Zscores of active neurons and speed in the low-threat context (N = 109 neurons). Inset , representative Ca 2+ traces (dF/F) extracted from a representative mouse. H , Heatmap of Z-scores from active neurons during the last trial in the high-threat context (148 neurons from 5 mice, left ), and from the last trial in the low-threat context, organized by response profile (109 neurons from 5 mice, right ). I , Fraction of active cells and non-responsive cells during different sessions, categorized by activity profile. J , Percentages of neurons activated (neurons with peaks ≥3 s.d. above baseline) during different cue periods in the high-( left ) and low-( right ) threat contexts. K , Z-score of preSCS, tone and WN and post-shock (post) of the entire DP-to-CeA population in the high-threat context (from all trials, Ordinary one-way ANOVA, effect of stimuli F (3,16) = 19.4, p < 0.0001, followed by Bonferroni’s post-hoc test, *P < 0.05, ***P < 0.001). Data shown as means ± s.e.m. L , Z-score of preSCS, tone and WN and post-SCS (post) of the entire DP-to-CeA population in the low-threat context (from all trials, Ordinary one-way ANOVA, effect of stimuli F (3,16) = 0.815, p = 0.504, followed by Bonferroni’s post-hoc test, n.s.). Data shown as means ± s.e.m. M , Repeated measure correlation of Z-score of active neuronal populations (N = 5 mice) with speed or freezing in the high-threat context ( r rm = repeated measures correlation coefficient). N , Repeated measure correlation of Z-score of active neuronal populations (N = 5 mice) with speed or freezing in the low-threat context ( r rm = repeated measures correlation coefficient).

Article Snippet: For Cre-dependent GCaMP6f expression, we used AAV5-CAG-Flex-GcaMP6f-WPRE-SV40 (Addgene 100835, a gift from Douglas Kim & GENIE Project).

Techniques: Activity Assay, Expressing, Extraction

Journal: bioRxiv

Article Title: POMC-specific knockdown of Tril reduces body adiposity and increases hypothalamic leptin responsiveness

doi: 10.1101/2020.06.25.172379

Figure Lengend Snippet: POMC-Cre mice were submitted to an intracerebroventricular injection with Cre-dependent AAV-FLEX-EGFP-mir30 carrying either a non-target sequence (rAAVshNonTarget) or a Tril targeting sequence (rAAVshTril) (A). Evidence for the efficiency of the adenoviral incorporation into cells of the arcuate nucleus (ARH) was obtained by immunofluorescence evaluation of sections of the mediobasal hypothalamus (B). The protocol employed in the experiments is depicted in C. Body mass (D and E) was determined throughout the experimental period. Relative fat (F) and lean (G) mass as well as the absolute (H) and relative (I) epididymal fat mass were determined at the end of the experimental period. The expressions of inflammatory genes were determined in the epididymal adipose tissue at the end of the experimental period (J). In D–J, n=6–14; *p<0.05 vs. shNonTarget.

Article Snippet: With regard to the POMC-specific knockdown of Tril, referred to here as POMC rAAV miTRIL , a TRIL-based miRNA construct was constructed by modifying the Cre-dependent AAV-FLEX-EGFP-mir30 (Scn9a) (Addgene plasmid # 79672) ( , ).

Techniques: Injection, Sequencing, Immunofluorescence

Journal: bioRxiv

Article Title: POMC-specific knockdown of Tril reduces body adiposity and increases hypothalamic leptin responsiveness

doi: 10.1101/2020.06.25.172379

Figure Lengend Snippet: POMC-Cre mice were submitted to an intracerebroventricular injection with Cre-dependent AAV-FLEX-EGFP-mir30 carrying either a non-target sequence (rAAVshNonTarget) or a Tril targeting sequence (rAAVshTril) and then submitted to the same protocol as in . The cumulative consumption of diet was determined throughout the experimental period (A). At the end of the experimental period, mice were submitted to a determination of spontaneous food intake after a period of 24 h fasting (B). Leptin-induced phosphorylation of STAT3 was determined by calculating the proportion of phospho-STAT3 per POMC-positive cells in the arcuate nucleus (C) and retrochiasmatic hypothalamus (D) employing immunofluorescence staining; an illustrative image obtained from the mediobasal hypothalamus is depicted in E. Immunofluorescence staining was employed to determine the density of aMSH (F, upper panels and G) and AgRP (F, lower panels and H) fiber projections to the paraventricular hypothalamus. In A, n=11–13; in B–H, n=4–5; *p<0.05 vs. shNonTarget.

Article Snippet: With regard to the POMC-specific knockdown of Tril, referred to here as POMC rAAV miTRIL , a TRIL-based miRNA construct was constructed by modifying the Cre-dependent AAV-FLEX-EGFP-mir30 (Scn9a) (Addgene plasmid # 79672) ( , ).

Techniques: Injection, Sequencing, Phospho-proteomics, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: POMC-specific knockdown of Tril reduces body adiposity and increases hypothalamic leptin responsiveness

doi: 10.1101/2020.06.25.172379

Figure Lengend Snippet: In A–D, POMC-Cre mice were submitted to an intracerebroventricular injection with Cre-dependent AAV-FLEX-EGFP-mir30 carrying either a non-target sequence (rAAVshNonTarget) or a Tril targeting sequence (rAAVshTril) and then left to recover for 2 weeks followed by another 2 weeks fed on chow; at the end of the experimental period, mice were submitted to determination of O 2 consumption (A), CO 2 production (B) and energy expenditure (C and D). In E–P, POMC-Cre mice were submitted to an intracerebroventricular injection with Cre-dependent AAV-FLEX-EGFP-mir30 carrying either a non-target sequence (rAAVshNonTarget) or a Tril targeting sequence (rAAVshTril) and then submitted to the same protocol as in . At the end of the experimental period, mice were submitted to determination of O 2 consumption (E), CO 2 production (F), energy expenditure (G and H), determination of interscapular brown adipose tissue total (I) and relative (J) mass, determination of interscapular temperature (K–M) and determination of interscapular brown adipose transcript expression of thermogenic genes (N). In addition, blood glucose levels were determined in fasting mice (O) and whole-body glucose tolerance was determined by means of an intraperitoneal glucose tolerance test (P). In A–H, K–M, O and P, n=5–7; in I, J and N, n=12–14. In all, *p<0.05 vs. shNonTarget.

Article Snippet: With regard to the POMC-specific knockdown of Tril, referred to here as POMC rAAV miTRIL , a TRIL-based miRNA construct was constructed by modifying the Cre-dependent AAV-FLEX-EGFP-mir30 (Scn9a) (Addgene plasmid # 79672) ( , ).

Techniques: Injection, Sequencing, Expressing

Journal: bioRxiv

Article Title: POMC-specific knockdown of Tril reduces body adiposity and increases hypothalamic leptin responsiveness

doi: 10.1101/2020.06.25.172379

Figure Lengend Snippet: POMC-Cre mice were fed for 14 weeks on a high-fat diet and then submitted to an intracerebroventricular injection with Cre-dependent AAV-FLEX-EGFP-mir30 carrying either a non-target sequence (rAAVshNonTarget) or a Tril targeting sequence (rAAVshTril); after 2 weeks of recovery, mice were fed a high-fat diet for another 8 weeks (experimental protocol depicted in A). Body mass (B) and food intake (C) were recorded throughout the experimental period. Relative fat (F) and lean (G) mass as well as the absolute (H) and relative (I) epididymal fat mass were determined at the end of the experimental period. Absolute (D) and relative (E) epididymal fat mass as well as absolute (F) and relative (G) interscapular brown adipose tissue mass were determined at the end of the experimental period. The interscapular temperature (H–J) and the determination of interscapular brown adipose transcript expression of thermogenic genes were determined at the end of the experimental period. In all experiments, n=4–5; *p<0.05 vs. shNonTarget.

Article Snippet: With regard to the POMC-specific knockdown of Tril, referred to here as POMC rAAV miTRIL , a TRIL-based miRNA construct was constructed by modifying the Cre-dependent AAV-FLEX-EGFP-mir30 (Scn9a) (Addgene plasmid # 79672) ( , ).

Techniques: Injection, Sequencing, Expressing

Journal: bioRxiv

Article Title: POMC-specific knockdown of Tril reduces body adiposity and increases hypothalamic leptin responsiveness

doi: 10.1101/2020.06.25.172379

Figure Lengend Snippet: POMC-Cre mice were fed a high-fat diet for 14 weeks and then submitted to an intracerebroventricular injection with Cre-dependent AAV-FLEX-EGFP-mir30 carrying either a non-target sequence (rAAVshNonTarget) or a Tril targeting sequence (rAAVshTril); after 2 weeks of recovery, mice were fed a high-fat diet for another 8 weeks (according to the protocol depicted in ). Immunofluorescence staining was employed to determine the number of cells expressing POMC (A and B) and cleaved caspase-3 (C and D). In all experiments, n=3. In C, arrows depict cells expressing cleaved caspase-3.

Article Snippet: With regard to the POMC-specific knockdown of Tril, referred to here as POMC rAAV miTRIL , a TRIL-based miRNA construct was constructed by modifying the Cre-dependent AAV-FLEX-EGFP-mir30 (Scn9a) (Addgene plasmid # 79672) ( , ).

Techniques: Injection, Sequencing, Immunofluorescence, Staining, Expressing