Journal: eLife

Article Title: A robust activity marking system for exploring active neuronal ensembles

doi: 10.7554/eLife.13918

Figure Lengend Snippet: Unless indicated otherwise, cultured mouse hippocampal neurons were transfected with luciferase reporter constructs on DIV5 and stimulated with 35 mM KCl for 6 hr on DIV 7 or 8 (See for details). The relative luciferase value is the absolute luciferase value normalized against the internal control. The fold induction is the ratio of the relative luciferase values in stimulated and unstimulated conditions. ( a ) P RAM has higher fold induction than promoters in which the NRE/AP-1 element is replaced by other elements found enriched in the 11,830 activity-regulated enhancers (See ‘Methods’). CME is not regulated by activity. Each construct contains four enhancer modules (EM) inserted upstream of the FOS minimal promoter (left). The relative luciferase values are shown in . n = 5–7 separate experiments per condition, one-way ANOVA, Tukey’s post-hoc test. ( b ) The robust activity response of P RAM is a result of the combination of the four repeated RAM EM and FOS minimal promoter, which alone are respectively weakly responsive or non-responsive to neuronal activity. n = 5–10 separate experiments per condition, one-way ANOVA, Tukey’s post-hoc test. ( c ) Comparison of P RAM to various activity-dependent promoters. Promoter size (Kb) is shown in brackets. The relative luciferase values are shown in . n = 4–10 separate experiments per condition, Student’s t-test. ( d ) Comparison of P RAM and ESARE. n = 6–8 separate experiments per condition, Student’s t-test. ( e ) P RAM can be driven by overexpression of FOS and NPAS4 individually or in combination. n = 9–10 separate experiments per condition, one-way ANOVA, Dunnett’s post-hoc test. ( f ) Top, schematic outline of Tet-OFF system with P RAM driving tTA expression. Binding of tTA protein to the TRE promoter is prevented by Dox administration (+Dox); withdrawal of Dox (-Dox) allows downstream effector gene transcription. Bottom, schematic diagram of the AAV-RAM construct with critical genetic elements outlined. ( g ) Comparison of P RAM -d2tTA and P RAM -tTA by assaying pTRE-luciferase activity with and without Dox (+Dox, −Dox). The relative luciferase values are shown in . n = 3 separate experiments per condition, Student’s t-test. ( h ) Representative images of hippocampal neurons grown on a glia monolayer and infected with AAV-RAM-tdTomato (red). Neurons are identified by MAP2 staining (green). Cultures were either left undisturbed (No Stim) or stimulated with bicuculline and 4AP (+Bic/4AP), either with (+Dox) or without (-Dox) doxycycline added. The scale bar is 150 μm and applies to all images. ( i ) Quantification of h . Percentages of neurons (MAP2+) that are RAM+ (%RAM+) are plotted. n = 3 separate experiments per condition, one-way ANOVA, Tukey’s post-hoc test. All data in a – e , g and i are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. DOI: http://dx.doi.org/10.7554/eLife.13918.003

Article Snippet: Primary antibodies used: FOS (rabbit, 1:1000, Santa Cruz sc-52), NPAS4 (rabbit, 1:10,000) , Glutamate Receptor 2&3 (GLUR2/3, rabbit, 1:200, Millipore AB1506), Somatostatin (SST, rat, 1:200, Millipore MAB354), Parvalbumin (PV, mouse, 1:1000, Millipore MAB1572), α-mKate2 (tRFP antibody recognizing mKate2, rabbit, 1:200, Evrogen AB223) and GAD67 (mouse, 1:1000 or 1:500 Millipore MAB5406).

Techniques: Cell Culture, Transfection, Luciferase, Construct, Activity Assay, Over Expression, Expressing, Binding Assay, Infection, Staining

Journal: eLife

Article Title: A robust activity marking system for exploring active neuronal ensembles

doi: 10.7554/eLife.13918

Figure Lengend Snippet: ( a ) Schematic drawing of the hippocampus with the viral injection site (DG) highlighted in blue. ( b ) Timeline of the experimental procedure. Animals were injected with AAV-RAM-NLS-mKate2 and kept on a Dox diet (+Dox) for at least 7 days after surgery. Two days after Dox removal (−Dox), animals were exposed to context A (for RAM labeling) and shocked, and then exposed to either context A again or a new context B 24 hr later (for IEG labeling). Animals were sacrificed 1.5 hr after the second context exposure. ( c , d ) Representative images of DG after A−A ( c ) and A−B ( d ) exposure showing DAPI staining (cyan), FOS staining (green), RAM labeling (red), and the merged image. ( e , f ) As panels c and d , except staining with NPAS4 (white) instead of FOS. ( g ) Freezing behavior observed during re-exposure to context A or B. n = 4–5 animals per condition, Student’s t-test. ( h ) Percentage of all DAPI-labeled cells (i.e. all neurons) in the DG stratum granulosum labeled with RAM (red) and FOS (green; n = 9 animals per condition), and the percentage overlap between the RAM and FOS labeled cells following A-A exposure (n = 4 animals) and A-B exposure (n = 5 animals). Student’s t-test. ( i ) As h , except with NPAS4 (white) instead of FOS. The scale bar is 50 μm for all images. Data in g – i are mean ± SEM. *p<0.05, ***p<0.001. DOI: http://dx.doi.org/10.7554/eLife.13918.015

Article Snippet: Primary antibodies used: FOS (rabbit, 1:1000, Santa Cruz sc-52), NPAS4 (rabbit, 1:10,000) , Glutamate Receptor 2&3 (GLUR2/3, rabbit, 1:200, Millipore AB1506), Somatostatin (SST, rat, 1:200, Millipore MAB354), Parvalbumin (PV, mouse, 1:1000, Millipore MAB1572), α-mKate2 (tRFP antibody recognizing mKate2, rabbit, 1:200, Evrogen AB223) and GAD67 (mouse, 1:1000 or 1:500 Millipore MAB5406).

Techniques: Injection, Labeling, Staining

Journal: Neuron

Article Title: The forebrain thirst circuit drives drinking through negative reinforcement

doi: 10.1016/j.neuron.2017.11.041

Figure Lengend Snippet: (A) Colocalization of Agtr1a-GFP and Fos induced by salt challenge in the OVLT of BAC transgenic mice (left; scale bar, 100 μm). Quantification (right; n=3). (B) Schematic for generation of Agtr1a-2A-Cre knock-in mouse line by CRISPR-mediated homologous recombination. (C) Histology showing GFP expression in the OVLT of Agtr1a-2A-Cre mice crossed to a GFP reporter line. (D) Cumulative drinking (left) and total drinking during photostimulation (middle) by ChR2 (n=8) and GFP (n=4) mice. Total drinking during photostimulation by ChR2 mice (n=5) at different photostimulation frequencies (right). (E) Total drinking during photostimulation by ChR2 mice (n=3) with ad libitum access to various NaCl solutions. (F) Total drinking during photostimulation by ChR2 mice (n=5) with access to water and 300mM NaCl. (G) Change in mean arterial blood pressure over time (left) and average photostimulation-induced change (right) of ChR2 (n=5) and GFP (n=4) mice. (H) Schematic of instrumental responding for water access experiment (left). Cumulative lever presses by ChR2 mice (middle). Total lever presses during photostimulation by ChR2 (n=3) and GFP (n=3) mice (right). (I) Cumulative lever presses by ChR2 mice in the negative-reinforcement experiment (left). Total lever presses by ChR2 (n=8) and GFP (n=4) mice (right). (J) RNAscope in situ hybridization showing colocalization of GFP, Vglut2, and Vgat mRNA in the OVLT of Agtr1a-2A-Cre mice crossed to a GFP reporter line (left; scale bars, 100 μm). Quantification (right; n=201 cells from 2 mice). Values are reported as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. See also Figure S2.

Article Snippet: Primary antibodies used were: anti-GFP (Abcam, ab13970, 1:1000), anti-FOS (SCB, sc-52 and sc-52-G), and anti-RFP (Chromotek 5F8, 1:1000).

Techniques: Transgenic Assay, Knock-In, CRISPR, Homologous Recombination, Expressing, In Situ Hybridization

Journal: Neuron

Article Title: The forebrain thirst circuit drives drinking through negative reinforcement

doi: 10.1016/j.neuron.2017.11.041

Figure Lengend Snippet: (A) Colocalization of Fos with Adcyap1 (n=4), Vglut2 (n=3), and Agtr1a (n=3) in the MnPO following salt challenge. Scale bar, 30 μm. (B) RNAscope in situ colocalization of Adcyap1, Vglut2, and Vgat. Scale bars, 200 μm. (C) Monosynaptic retrograde rabies tracing from MnPOAdcyap1 neurons. Scale bars, 200 μm. (D) Injection site and fiber placement (dashed line) in MnPOAdcyap1 ChR2 mice. Scale bar, 1 mm. (E) Cumulative drinking (left) and total drinking during photostimulation (right) by ChR2 (n=10) and GFP (n=9) mice. (F) Cumulative drinking (left) and total drinking during photostimulation (right) by ChR2 mice (n=5) during 2-bottle choice assay with water and 0.3 M NaCl. (G) Change in mean arterial blood pressure over time (left) and average photostimulation-induced change (right) in ChR2 (n=9) and GFP (n=6) mice. (H) Cumulative lever pressing for water by ChR2 mice (n=5) (left) and total lever presses during photostimulation by ChR2 (n=5) and GFP (n=3) mice (right). (I) RTPP assay with location heatmaps of individual ChR2 and GFP mice from pre-conditioning and third session of conditioning (left). Percent time spent in photostimulation-paired chamber by ChR2 (n=6) and GFP (n=6) mice (right). (J) Cumulative lever pressing to pause photostimulation by ChR2 mice (n=8) (left). Lever presses by ChR2 (n=8) and GFP (n=4) mice (right). Values are reported as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also Figure S3.

Article Snippet: Primary antibodies used were: anti-GFP (Abcam, ab13970, 1:1000), anti-FOS (SCB, sc-52 and sc-52-G), and anti-RFP (Chromotek 5F8, 1:1000).

Techniques: In Situ, Injection