Journal: Cell reports

Article Title: Cerebellar nuclei neurons projecting to the lateral parabrachial nucleus modulate classical fear conditioning.

doi: 10.1016/j.celrep.2023.112291

Figure Lengend Snippet: Figure 1. DCN neurons project to the lPBN (A) Viral injection of AAV-hSyn-EYFP into the DCN. Left: EYFP expression in the DCN. Right: EYFP signals in the lPBN. Scale bars: 500 mm (left) and 200 mm (right). (B) Retrograde tracing from the lPBN to DCN. Left: strategy for labeling lPBN-projecting DCN neurons. Right: representative images of retro bead injection into the lPBN and retrogradely labeled DCN neurons. FN, fastigial nucleus; IpN, interpositus nucleus; DN, dentate nucleus. Scale bars: 1 mm (top) and 500 mm (bottom). (C) Number of labeled cells along the rostro-caudal axis (n = 3 mice). (D) Whole-cell patch-clamp recordings of optogenetically evoked EPSCs (oEPSCs) in the lPBN via stimulation of ChR2-expressing cerebellar axons. Of 49 cells recorded, 37 cells exhibited time-locked synaptic responses to blue laser stimulation. (E) Example recording traces of oEPSCs with pharmacological treatment. (F) oEPSCs recorded in the lPBN treated with TTX, 4-AP, and NBQX (n = 5 slices from 5 mice; ACSF vs. +TTX, two-tailed paired t test, ***p = 0.0002; +TTX vs. +4- AP, two-tailed paired t test, **p = 0.0036; +4-AP vs. +NBQX, two-tailed paired t test, **p = 0.0088). (G) Anterograde labeling from the DCN to lPBN. AAV8-EF1a-mCherry-IRES-WGA-Cre and AAV1-EF1a-DIO-EYFP were injected into the DCN and the lPBN, respectively. The anterograde transneuronal tracer wheat germ agglutinin (WGA) fused to Cre recombinase in the DCN permits EYFP expression in lPBN neurons receiving input from the DCN. (H) Representative images of DCN-connected lPBN projections. BLA, basolateral amygdala; BNST, bed nucleus of the stria terminalis; CeA, central amygdala; LH, lateral hypothalamic area; PAG, periaqueductal gray; PVH, paraventricular nucleus of the hypothalamus; GPi, globus pallidus internus; ZI, zona incerta; 3V, third ventricle. Scale bar: 500 mm. (I) Anterograde labeling from the DCN to lPBN using synaptophysin-mRuby. AAV1-hSyn-Cre and AAV1-hSyn-DIO-synaptophysin-mRuby were injected into the DCN and the lPBN, respectively. (J) Representative images of synaptophysin-mRuby signals of the DCN-connected lPBN projections. Scale bar: 500 mm. Data are presented as mean ± SEM. See also Figures S1 and S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal anti-c-Fos (9F6) Cell Signaling Technology Cat# 2250; RRID: AB_2247211 Rabbit polyclonal anti-PACAP-38 Peninsula Laboratories Cat# T-4473; RRID: AB_519166 Sheep polyclonal anti-FoxP2 R&D Systems Cat# AF5647; RRID: AB_2107133 Mouse monoclonal anti-CGRP Abcam Cat# ab81887; RRID: AB_1658411 Alexa Fluor 568-conjugated donkey anti-mouse Invitrogen Cat# A10037; RRID: AB_2534013 Alexa Fluor 594-conjugated donkey anti-sheep Abcam Cat# ab150180; RRID: AB_2716768 Alexa Fluor 568-conjugated goat anti-rabbit Invitrogen Cat# A11011; RRID: AB_143157 Alexa Fluor 647-conjugated donkey anti-rabbit Invitrogen Cat# A31573; RRID: AB_2536183 Bacterial and virus strains AAV-EF1ɑ-DIO-EYFP Addgene Cat# 27056-AAV1; RRID: Addgene_27056 AAV-EF1ɑ-DIO-eNpHR3.0-EYFP Addgene Cat# 26966-AAV1; RRID: Addgene_26966 AAV-EF1ɑ-double floxedhChR2(H134R)-EYFPWPRE-HGHpA Addgene Cat# 20298-AAV1; RRID: Addgene_20298 AAVrg-EF1ɑ-mCherry-IRES-Cre Addgene Cat# 55632-AAVrg; RRID: Addgene_55632 AAV-hSyn-double floxedhChR2(H134R)-EYFP Addgene Cat# 26973-AAV1; RRID: Addgene_26973 AAV-hSyn-eNpHR3.0-EYFP Addgene Cat# 26972-AAV5; RRID: Addgene_26972 AAV-CAG-FLEX-GCaMP6s-WPRE-SV40 Addgene Cat# 100842-AAV1; RRID: Addgene_100842 AAVrg-hSyn-EGFP Addgene Cat# 50465-AAVrg; RRID: Addgene_50465 AAV-hSyn-EGFP Addgene Cat# 50465-AAV5; RRID: Addgene_50465 pENN.AAV-hSyn-Cre-WPRE-hGH Addgene Cat# 105553-AAV1; RRID: Addgene_105553 AAV-hSyn-FLEx-mGFP-2ASynaptophysin-mRuby Addgene Cat# 71760-AAV1; RRID: Addgene_71760 rAAV8-CAG-mCherry-IRES-WGA-Cre UNC vector Core Cat# AV5901FG Chemicals, peptides, and recombinant proteins Normal Donkey Serum Sigma Cat# D9663 Triton X-100 Sigma Cat# X100 DAPI mounting solution Vectorlab Cat# H-1200 Retrobead (red) LumaFluor N/A (Continued on next page) 14 Cell Reports 42, 112291, April 25, 2023

Techniques: Injection, Expressing, Retrograde Tracing, Labeling, Patch Clamp, Two Tailed Test

Journal: Molecular psychiatry

Article Title: Cocaine- and stress-primed reinstatement of drug-associated memories elicit differential behavioral and frontostriatal circuit activity patterns via recruitment of L-type Ca 2+ channels

doi: 10.1038/s41380-019-0513-2

Figure Lengend Snippet: Cav1.2 within the PrL→NAcC projection is required for reinstatement, a) cacan1c-floxed mice were injected with a Flp-dependent AAV expressing Cre recombinase (AAV-fDIO-Cre-GFP) into bilateral PrL and a retrograde AAV expressing Flp (retro-AAV-Flp) into bilateral NAcC to knockdown cacna1c expression selectively in PrL→NAcC cells, b) Experimental timeline for surgery and behavioral experiments, c) Representative images of RNAscope in situ hybridization showing cacna1c mRNA (red) and GFP-tagged cells (green) in the PrL of control mice injected with AAV-GFP (Top- PrL-NAcC Cav1.2 control) and experimental cacna1c floxed mice injected with AAV-fDIO-Cre-GFP into the PrL and retro-AAV-Flp into the NAcC (bottom-PrL-NAcC Cav1.2 KO). Scale bar = 50μm d) Quantitative analysis of cacna1c mRNA transcripts. Mice with Cav1.2 knocked out in PrL cells projecting to the NAcC have significantly less cacna1c mRNA in GFP+ cells in the PrL compared to controls (**p< 0.01, unpaired t-test; GFP, n = 2 mice, 26-30 cells/mouse, Cre, n = 3 mice, 30 cells/mouse), e) Control mice (PrL-NAcC Cav1.2 Control) and mice with knockdown of Cav1.2 within the PrL→NAcC projection (PrL-NAcC Cav1.2 KO) acquired (****p < 0.0001, **p < 0.01) and extinguished (****p < 0.0001, ***p < 0.001) cocaine CPP. CPR significantly increased preference score in PrL-NAcC Cav1.2 control mice (***p < 0.001, n = 10) but not in PrL-NAcC Cav1.2 KO mice (n = 13). Similarly, SPR significantly increased preference score in PrL-NAcC Cav1.2 control mice (**p < 0.01) but not in PrL-NAcC Cav1.2 KO mice. Data are presented as mean + SEM.

Article Snippet: In Ca v 1.2 +/− mice, a Cre-dependent AAV expressing the hM3Dq excitatory DREADD (AAV2-hSyn-DIO-hM3DGq-mCherry) was injected into bilateral PrL (AP: +2.00 mm, ML: ±0.25 mm, and DV: −2.25 mm) and a retrograde AAV expressing Cre recombinase (rAAV-CAG-Cre) was injected into bilateral NAcC (AP: +1.0 mm, ML: ±1.20 mm, and DV: −4.75 mm). pAAV-hSyn-DIO-hM4D(Gi)-mCherry (Addgene viral prep # 44362-AAV2; http://n2t.net/addgene:44362 ; RRID:Addgene_44362) and pAAV-hSyn-DIO-hM3D(Gq)-mCherry (Addgene viral prep # 44361-AAV2; http://n2t.net/addgene:44361 ; RRID:Addgene_44361), gifts to Addgene from Bryan Roth, were purchased from Addgene, Watertown, MA.

Techniques: Injection, Expressing, In Situ Hybridization

Journal: Molecular psychiatry

Article Title: Cocaine- and stress-primed reinstatement of drug-associated memories elicit differential behavioral and frontostriatal circuit activity patterns via recruitment of L-type Ca 2+ channels

doi: 10.1038/s41380-019-0513-2

Figure Lengend Snippet: Cocaine- and stress-primed reinstatement recruit the PrL→NAcC projection, a) C57BL/6J mice were injected with a Cre-dependent AAV expressing GCaMP6s into unilateral PrL and a retrograde AAV expressing Cre recombinase was injected into ipsilateral NAcC. An optic fiber was implanted above the PrL, which could be attached to a patch cord to record Ca2+ signals, b) Representative image of GCaMP6s expressing in the PrL. Optic fiber location is outlined in white. MO, medial orbitofrontal cortex; Cg, cingulate cortex, c) Experimental timeline for fiber photometry recording during cocaine CPP. Ca2+ imaging was conducted during the baseline test, acquisition test, extinction test, and CPR/SPR. Mice were treated with vehicle or isradipine to block LTCCs prior to CPR/SPR recording, d) Representative fiber photometry trace of a vehicle-treated mouse during CPR showing Ca2+ transients prior to cocaine-paired entries (red) but not saline-paired entries (blue). Grey, middle CPP chamber, e) The average amplitude (% ΔF/F) of PrL→NAcC fiber photometry signal 5 seconds prior to entry into the cocaine-paired chamber was significantly higher as compared to entries into the saline-paired chamber in vehicle-treated mice (**p < 0.01, linear mixed effects model, number of entries = 454, number of mice = 7). f) Representative fiber photometry trace of an isradipine-treated mouse during CPR. g) The average amplitude of PrL→NAcC fiber photometry signal 5 seconds prior to entry did not differ between cocaine-paired entries and saline-paired entries in isradipine-treated mice (linear mixed effects model; number of entries = 468, number of mice = 6). h) There was no change in the number of events per minute observed during any day of CPP testing (vehicle, n = 7; isradipine, n = 7). i) The average amplitude of PrL→NAcC fiber photometry signal was significantly positively correlated with the average cocaine-paired duration during CPR (**p < 0.01, n = 13). j) The number of events per minute was not correlated with the average cocaine-paired duration during CPR (n = 13). k) Representative fiber photometry trace of a vehicle-treated mouse during SPR. 1) The average amplitude of PrL→NAcC fiber photometry signal 5 seconds prior to entry did not differ between cocaine-paired entries and saline-paired entries during SPR (linear mixed effects model; number of entries = 840, number of animals = 6). m) Representative fiber photometry traces of a vehicle-treated mouse during extinction and SPR. Red boxes indicate significant calcium events, n) SPR significantly increased the events per minute in vehicle-treated mice (****p < 0.0001, Bonferroni post-hoc extinction vs. SPR; n = 6) but not in isradipine-treated mice (n = 5). o) The number of events per minute was significantly positively correlated with the number of cocaine-paired entries during SPR (****p < 0.0001, n = 11). p) The average fiber photometry signal 5 seconds prior to entry into the cocaine-paired chamber was not correlated with the number of entries into the cocaine-paired chamber (n = 11). Data are presented as mean + SEM.

Article Snippet: In Ca v 1.2 +/− mice, a Cre-dependent AAV expressing the hM3Dq excitatory DREADD (AAV2-hSyn-DIO-hM3DGq-mCherry) was injected into bilateral PrL (AP: +2.00 mm, ML: ±0.25 mm, and DV: −2.25 mm) and a retrograde AAV expressing Cre recombinase (rAAV-CAG-Cre) was injected into bilateral NAcC (AP: +1.0 mm, ML: ±1.20 mm, and DV: −4.75 mm). pAAV-hSyn-DIO-hM4D(Gi)-mCherry (Addgene viral prep # 44362-AAV2; http://n2t.net/addgene:44362 ; RRID:Addgene_44362) and pAAV-hSyn-DIO-hM3D(Gq)-mCherry (Addgene viral prep # 44361-AAV2; http://n2t.net/addgene:44361 ; RRID:Addgene_44361), gifts to Addgene from Bryan Roth, were purchased from Addgene, Watertown, MA.

Techniques: Injection, Expressing, Imaging, Blocking Assay

Journal: Molecular psychiatry

Article Title: Cocaine- and stress-primed reinstatement of drug-associated memories elicit differential behavioral and frontostriatal circuit activity patterns via recruitment of L-type Ca 2+ channels

doi: 10.1038/s41380-019-0513-2

Figure Lengend Snippet: Activation of PrL→NAcC projections is sufficient to induce cocaine- and stress-primed reinstatement in Cav1.2+/− mice, a) Cav1.2+/− mice were injected with mCherry-tagged Cre-dependent AAV expressing the excitatory DREADD hM3Dq into bilateral PrL and a retrograde AAV expressing Cre recombinase into bilateral NAcC. b) Representative image of mCherry staining in bilateral PrL. c) Experimental timeline of viral injection and cocaine CPP. Following 21 days for viral expression, mice were run through the cocaine CPP protocol and prior to reinstatement, mice were injected with CNO to activate the PrL→NAcC projection, d) Control (hM3Dq + Sham) and hM3Dq-expressing mice acquired (***p < 0.001, **p < 0.01, Bonferroni post-hoc baseline vs. acquisition) and extinguished (####p < 0.0001, #p < 0.05, Bonferroni post-hoc acquisition vs. extinction) cocaine CPP. CPR had no effect on preference score in control Cav1.2+/− mice (n = 8), but significantly increased preference score in Cav1.2+/− mice expressing hM3Dq treated with CNO (^p < 0.05, Bonferroni post-hoc extinction vs. CPR; n = 7). e) CPR significantly increased average cocaine-paired duration in Cav1.2+/− mice expressing hM3Dq treated with CNO (***p < 0.001, Bonferroni post-hoc extinction vs. CPR; n = 7) but not in Cav1.2+/− control mice (n = 8). f) CPR had no effect on number of cocaine-paired entries in either group. g) Control (hM3Dq + Sham) and hM3Dq-expressing mice acquired (***p < 0.001, Bonferroni post-hoc baseline vs. acquisition) and extinguished (###p < 0.001, ##p < 0.01, Bonferroni post-hoc acquisition vs. extinction) cocaine CPP. SPR had no effect on preference score in control Cav1.2+/− mice (n = 8), but induced a trending increase in preference score in Cav1.2+/− mice expressing hM3Dq treated with CNO (p = 0.09, Bonferroni post-hoc extinction vs. CPR; n = 8). h) SPR had no effect on average cocaine-paired duration in either group. i) SPR significantly increased the number of cocaine-paired entries in Cav1.2+/− mice expressing hM3Dq treated with CNO (**p < 0.01, Bonferroni post-hoc extinction vs. SPR; n = 8) but not in Cav1.2+/− control mice (n = 8). Data are presented as mean + SEM.

Article Snippet: In Ca v 1.2 +/− mice, a Cre-dependent AAV expressing the hM3Dq excitatory DREADD (AAV2-hSyn-DIO-hM3DGq-mCherry) was injected into bilateral PrL (AP: +2.00 mm, ML: ±0.25 mm, and DV: −2.25 mm) and a retrograde AAV expressing Cre recombinase (rAAV-CAG-Cre) was injected into bilateral NAcC (AP: +1.0 mm, ML: ±1.20 mm, and DV: −4.75 mm). pAAV-hSyn-DIO-hM4D(Gi)-mCherry (Addgene viral prep # 44362-AAV2; http://n2t.net/addgene:44362 ; RRID:Addgene_44362) and pAAV-hSyn-DIO-hM3D(Gq)-mCherry (Addgene viral prep # 44361-AAV2; http://n2t.net/addgene:44361 ; RRID:Addgene_44361), gifts to Addgene from Bryan Roth, were purchased from Addgene, Watertown, MA.

Techniques: Activation Assay, Injection, Expressing, Staining

Journal: Molecular psychiatry

Article Title: Cocaine- and stress-primed reinstatement of drug-associated memories elicit differential behavioral and frontostriatal circuit activity patterns via recruitment of L-type Ca 2+ channels

doi: 10.1038/s41380-019-0513-2

Figure Lengend Snippet: PrL→NAcC projections are necessary but not sufficient for cocaine- and stress-induced reinstatement, a) C57BL/6J mice were injected with a mCherry-tagged Cre-dependent AAV expressing the inhibitory DREADD hM4Di into bilateral PrL and a retrograde AAV expressing Cre recombinase into bilateral NAcC. b) Representative image of mCherry staining in bilateral PrL. c) Experimental timeline of DREADD injection and cocaine CPP. Following 21 days for viral expression, mice were run through the cocaine CPP protocol and prior to reinstatement, mice were injected with CNO to inhibit the PrL→NAcC projection, d) Both sham and hM4Di-expressing mice acquired (****p < 0.0001, Bonferroni post-hoc baseline vs. acquisition) and extinguished (####p < 0.0001, Bonferroni post-hoc acquisition vs. extinction) cocaine CPP. CPR significantly increased preference score in control mice (^^^^p < 0.0001, Bonferroni post-hoc extinction vs. CPR; n = 6) but not in hM4Di-expressing mice injected with CNO (n = 7). e) CPR significantly increased the average cocaine-paired duration in control mice (**p < 0.01, Bonferroni post-hoc extinction vs. CPR; n = 7) but not in hM4Di-expressing mice treated with CNO (n = 7). f) CPR had no effect on number of cocaine-paired entries, g) Both sham and hM4Di-expressing mice acquired (***p < 0.001, *p < 0.05, Bonferroni post-hoc baseline vs. acquisition) and extinguished (####p < 0.0001, #p < 0.05, Bonferroni post-hoc acquisition vs. extinction) cocaine CPP. SPR significantly increased preference score in control mice (^^^^p < 0.0001, Bonferroni post-hoc extinction vs. SPR; n = 11) but not in hM4Di-expressing mice injected with CNO (n = 8). h) SPR had no effect on average cocaine-paired duration, i) SPR significantly increased the number of cocaine-paired entries in control mice (**p < 0.01, Bonferroni post-hoc extinction vs. SPR; n = 11) but not in hM4Di-expressing mice treated with CNO (n = 8). j) C57BL/6J mice were injected with an mCherry-tagged Cre-dependent AAV expressing the excitatory DREADD hM3Dq into bilateral PrL and a retrograde AAV expressing Cre recombinase into bilateral NAcC. k) Representative image of mCherry staining in bilateral PrL. 1) All mice acquired (*p < 0.05, Bonferroni post-hoc baseline vs. acquisition) and extinguished (#p < 0.05, Bonferroni post-hoc acquisition vs. extinction) cocaine CPP. Treatment with CNO in hM3Dq-expressing mice had no effect on preference score (n = 7). Data are presented as mean + SEM.

Article Snippet: In Ca v 1.2 +/− mice, a Cre-dependent AAV expressing the hM3Dq excitatory DREADD (AAV2-hSyn-DIO-hM3DGq-mCherry) was injected into bilateral PrL (AP: +2.00 mm, ML: ±0.25 mm, and DV: −2.25 mm) and a retrograde AAV expressing Cre recombinase (rAAV-CAG-Cre) was injected into bilateral NAcC (AP: +1.0 mm, ML: ±1.20 mm, and DV: −4.75 mm). pAAV-hSyn-DIO-hM4D(Gi)-mCherry (Addgene viral prep # 44362-AAV2; http://n2t.net/addgene:44362 ; RRID:Addgene_44362) and pAAV-hSyn-DIO-hM3D(Gq)-mCherry (Addgene viral prep # 44361-AAV2; http://n2t.net/addgene:44361 ; RRID:Addgene_44361), gifts to Addgene from Bryan Roth, were purchased from Addgene, Watertown, MA.

Techniques: Injection, Expressing, Staining

Journal: Nature Communications

Article Title: Ventral tegmental area GABA neurons mediate stress-induced blunted reward-seeking in mice

doi: 10.1038/s41467-021-23906-2

Figure Lengend Snippet: a Left: experimental design. Right: event-related potential of NAc LFP. Bars indicate 10 ms laser pulse onset. b Left: representative NAc spectra with no stimulation (black) and rhythmic low-frequency stimulation (blue). Center: relationship between max 4 Hz NAc power during light off and stimulation, with a line of equality for reference. Right: summary of data from the center panel. ChR2 stimulation increased peak 4 Hz NAc power (* P = 0.031 two-tailed signed-rank test, n = 6 mice). Data are mean ± s.e.m. c Left: experimental design. Right: rhythmic ChR2 stimulation impaired subsequent reward retrieval latency and anticipatory lick rate (* P = 0.031 and * P = 0.031, two-tailed signed-rank test, n = 6 mice). Data are mean ± s.e.m. d Left: experimental design. Right: immunofluorescent image of retrograde ChR2 expression (green) in a Vgat-ires-Cre mouse VTA, with tyrosine hydroxylase counterstain (blue). The scale bar is 30 µm. Representative of five experiments. e Left: representative NAc spectra of no stimulation and rhythmic low-frequency stimulation. Center: relationship between max 4 Hz NAc power during light off and stimulation, with the line of equality for reference. Right: Summary of data from the center panel. Stimulating NAc-projecting VTA GABA neurons increased peak 4 Hz NAc power (* P = 0.040 two-tailed paired t test, n = 5 mice). Data are mean ± s.e.m. f Left: experimental design. Right: rhythmic VTA illumination impaired subsequent reward retrieval latency and anticipatory lick rate (** P = 0.0091 and ** P = 0.0057 two-tailed paired t test, n = 5 mice). Data are mean ± s.e.m.

Article Snippet: For projection-specific experiments, mice were injected with retrogradely transported Cre-inducible ChR2 (rAAV2-EF1a-DIO-hChR2(H134R)-EYFP-WPRE-HGHpA, Addgene) at eight locations within the NAc bilaterally (±1.8 mm ML, 0.98 mm AP, −4.12 mm DV; ±0.9 mm ML, 1.5 mm AP, −4.0 mm DV; ±0.6 mm ML, 1.5 mm AP, −3.4 mm DV; ±1.0 mm ML, 1.0 mm AP, −3.4 mm DV; 0.4 μL virus per injection).

Techniques: Two Tailed Test, Expressing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: rAAV2-Retro Enables Extensive and High-Efficient Transduction of Lower Motor Neurons following Intramuscular Injection

doi: 10.1016/j.omtm.2019.11.006

Figure Lengend Snippet: Comparison of the Transduction Efficiency after the Intramuscular Delivery of Eight Serotypes of rAAV (A–H) Native GFP-expressing cells in transverse cervical spinal cord sections following retrograde transport with recombinant adeno-associated virus (rAAV)-CMV-GFP injection to extensor carpi muscle of the right forelimb of P4 mice (scale bars, 500 μm). (A) rAAV1, (B) rAAV2, (C) rAAV5, (D) rAAV6, (E) rAAV7, (F) rAAV8, (G) rAAV9, and (H) rAAV serotype retro (rAAV-retro). (I) Magnification of (H) (scale bar, 100 μm). (J) Total number of native GFP-expressing cells in the ipsilateral cervical spinal cord. rAAV2-retro demonstrates significantly more efficient retrograde infection than do other serotypes. Statistical analysis of fluorescence measurements is shown in the bar graphs. **p < 0.01, ***p < 0.001, one-way ANOVA followed by the Bonferroni correction (error bars indicate mean ± SEM; n = 4/group). n.s., not significant.

Article Snippet: rAAV serotypes (rAAV1, rAAV2, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, and rAAV2-retro) were purchased from Vigene Biosciences (Jinan, China).

Techniques: Comparison, Transduction, Expressing, Recombinant, Virus, Injection, Infection, Fluorescence