Journal: bioRxiv

Article Title: Circuit directionality for motivation: lateral accumbens-pallidum, but not pallidum-accumbens, connections regulate motivational attraction to reward cues

doi: 10.1101/474387

Figure Lengend Snippet: Each coronal section represents a range of three sections relative to Bregma (B, in mm). Expression for each animal is plotted at 90% transparency and then per-animal expression is overlaid. Sections run anterior (top) to posterior (bottom). A) Expression map of AAV-hSyn-DIO-hM4D(Gi)-mCherry in VP following CAV2-Cre injections in NAcLSh (i.e., VP → NAcLSh inhibition animals; red). B) Expression map of AAV-hSyn-DIO-mCherry in VP (i.e., controls; black).

Article Snippet: The inhibition group received 0.6 µl of AAV-hSyn-DIO-hM4D(Gi)-mCherry (n = 12, AAV5, UNC Vector Core; n = 4, AAV8, Addgene) in the VP and 1.0 µl CAV-Cre or CAV-Cre-GFP in the NAcLSh.

Techniques: Expressing, Inhibition

Journal: Cerebral Cortex (New York, NY)

Article Title: Differential Contributions of Glutamatergic Hippocampal→Retrosplenial Cortical Projections to the Formation and Persistence of Context Memories

doi: 10.1093/cercor/bhy142

Figure Lengend Snippet: 2 populations of vGlut1+ and vGlut2+ DH neurons project to RSC. (a) Schematic of Cre-dependent AAV-DIO-hM4D(Gi)-mCherry infusion in DH of vGlut1-Cre and vGlut2-Cre mice (left). Labeling of vGlut1+ (middle) and vGlut2+ (right) DH neurons 6 weeks after virus expression. The mCherry labeling patterns were similar to the one of endogenous transporters (Supplementary Fig. 2). (b) Immunofluorescent staining for mCherry RSC (top) in vGlut1-Cre mice and vGlut2-Cre mice revealing terminal fields in RSC layers 1 and 3. Immunostaining in DH is shown below. (c) Retrograde labeling of DH neurons projecting to RSC using Fluoro-Gold (top and bottom left) or CTB (top and bottom right). Fluoro-Gold injected into RSC (top left) was detected predominantly in the subiculum (SUB, bottom left). Sparse signals were also found in dorsomedial CA1. Similarly, injection of CTB (blue) in RSC labels SUB neurons of vGlut1-Cre mice expressing Cre-dependent retrograde AAV-flex-dtTomato (red) (bottom left).

Article Snippet: The viral vector carrying a construct coding for the Cre-independent inhibitory DREADD (AAV8-hSyn-HA-hM4D(Gi)-mCherry, Addgene 44 362) or Cre-dependent inhibitory DREADD (AAV8-hSyn-DIO-hM4D(Gi)-mCherry, Addgene 50 475) was bilaterally infused into the dorsal hippocampus (1.8 mm posterior, ±1.0 mm lateral, 2.25 mm ventral to bregma).

Techniques: Labeling, Expressing, Staining, Immunostaining, Injection

Journal: Cerebral Cortex (New York, NY)

Article Title: Differential Contributions of Glutamatergic Hippocampal→Retrosplenial Cortical Projections to the Formation and Persistence of Context Memories

doi: 10.1093/cercor/bhy142

Figure Lengend Snippet: Chemogenetic silencing of DH→ RSC terminals impairs memory encoding of CFC. (a) Schematic of virus infusions and cannula implantations. The Cre-independent viral vector AAV-hM4D(Gi)-mCherry was injected into the DH 6 weeks before behavioral experiments. During the same surgery, cannulae were implanted into the RSC bilaterally. (b) Immunostaining for mCherry showing expression of hM4D(Gi) in DH and its projection throughout ventral RSC (RSCv) but not in ventral hippocampus (VH). (c) Effect of pretraining infusion of CNO on activity (cm/s) during context and shock exposure at training (left) and freezing during context test (right). Pretraining infusions of Veh and CNO did not affect locomotor activity (Veh: 14.7 ± 2.33; CNO: 16.6 ± 1.48) or activity burst (Veh: 71.5 ± 7.52; CNO: 70.1 ± 5.69) to the footshock (activity before shock: t = 0.71, P = 0.49; activity during shock: t = 0.15, P = 0.89; Veh n = 6, CNO n = 7). However, CNO significantly impaired freezing at test when compared to vehicle (Veh)-injected controls (t = 5.843, P 0.001; Veh: 62.3 ± 3.03; CNO: 25.3 ± 5.30).

Article Snippet: The viral vector carrying a construct coding for the Cre-independent inhibitory DREADD (AAV8-hSyn-HA-hM4D(Gi)-mCherry, Addgene 44 362) or Cre-dependent inhibitory DREADD (AAV8-hSyn-DIO-hM4D(Gi)-mCherry, Addgene 50 475) was bilaterally infused into the dorsal hippocampus (1.8 mm posterior, ±1.0 mm lateral, 2.25 mm ventral to bregma).

Techniques: Plasmid Preparation, Injection, Immunostaining, Expressing, Activity Assay

Journal: Cerebral Cortex (New York, NY)

Article Title: Differential Contributions of Glutamatergic Hippocampal→Retrosplenial Cortical Projections to the Formation and Persistence of Context Memories

doi: 10.1093/cercor/bhy142

Figure Lengend Snippet: Chemogenetic silencing of vGlut1+ DH→ RSC terminals impair encoding and retrieval of contextual fear conditioning. (a) Experimental design similar to Figure ​Figure11 except for infusion of a Cre-dependent AAV8-DIO-hM4D(Gi)-mCherry. Freezing during the context test was significantly reduced in vGlut1-Cre mice injected with CNO when compared to Veh (Veh: 61.5 ± 4.68; CNO: 37.13 ± 6.99; t = 2.87, P < 0.05 (n = 8/group)), but not in vGlut2-Cre mice (Veh: 49.48 ± 5.32; CNO: 45.66 ± 6.41; t = 0.45, P = 0.65; Veh n = 12, CNO n = 14). (b) Within-subject design was used to determine the effect of pretraining CNO on recent and remote memory. Significant treatment effects were found for each genotype (vGlut1-Cre: F1,16 = 43.78, P < 0.001; n = 9/group; vGlut2-Cre: F1,16 = 10.91, P < 0.01; Veh n = 12, CNO n = 14). However, vGlut1-Cre mice receiving CNO before training showed reduced freezing both at recent (Veh: 54.3 ± 6.36; CNO: 7.56 ± 2.29; P < 0.05) and remote memory tests (Veh: 55.8 ± 6.09; CNO: 22.7 ± 5.79; P < 0.01), whereas similarly treated vGlut2-Cre mice showed freezing deficits only at the remote (Veh: 61.3 ± 6.35; CNO: 30.9 ± 5.96; P < 0.01), but not recent test (Veh: 58.2 ± 3.86; CNO: 57.7 ± 5.06). (c) Infusion of CNO before the recent memory test impaired freezing to the conditioning context in vGlut1-Cre mice (Veh: 64.6 ± 3.08; CNO: 37.2 ± 6.98; t = 3.14, P < 0.01; n = 9/group) without affecting freezing in vGlut2-Cre mice (Veh: 47.5 ± 7.73; CNO: 53.9 ± 8.46; t = 0.56, P = 0.58; n = 7/group). Infusion of CNO before the remote memory test was ineffective in either mouse line (vGlut1-Cre, Veh: 36.2 ± 5.04; CNO: 38.2 ± 6.32; t = 0.61, P = 0.54; n = 15/group; vGlut2-Cre: Veh: 49.9 ± 8.62; CNO: 54.8 ± 8.78; t13 = 0.39, P = 0.7; n = 7/group).

Article Snippet: The viral vector carrying a construct coding for the Cre-independent inhibitory DREADD (AAV8-hSyn-HA-hM4D(Gi)-mCherry, Addgene 44 362) or Cre-dependent inhibitory DREADD (AAV8-hSyn-DIO-hM4D(Gi)-mCherry, Addgene 50 475) was bilaterally infused into the dorsal hippocampus (1.8 mm posterior, ±1.0 mm lateral, 2.25 mm ventral to bregma).

Techniques: Injection

Journal: Nature

Article Title: Flexible scaling and persistence of social vocal communication

doi: 10.1038/s41586-021-03403-8

Figure Lengend Snippet: LPOAEsr1+ GCaMP activity during natural social behavior. a, top: experimental design for LPOAEsr1 fiber photometry recordings. Bottom: sample image showing fiber optic track and viral expression of GCaMP6s. Scale bar = 200μm. N=9 animals, >3 sections/animal collected. In addition to evoking USV calling, the presence of a female also dramatically alters male behavior (arousal, social sniffing, locomotion, sexual mounting) potentially confounding interpretation of the observed neural activity. We observed that following the removal of the female, males often generate intermittent USV calls, perhaps to lure her back, without the behavioral noise of mounting or social sniffing. We leveraged this post-female period to observe increases in LPOAEsr1/GCaMP6s activity with a rise shortly before the onset of post-female USVs, clearly suggesting that endogenous LPOAEsr1 neural activity correlates with emission of USVs. b, representative USV production and GCaMP fiber photometry of male LPOAEsr1 neurons as he behaves alone (pre-female), with a behaving female, and after female is removed (post-female). Dashed line indicates when the female was added and removed. Top: mean USV power, blue dots indicate USV syllable detection. Bottom: dF/F of LPOAEsr1 GCaMP6s signals was calculated by MATLAB GUI described previously34. c, dark green line: mean z-score of GCaMP6 signals before and after initiation of USV with behaving female phase, light green shading indicates 95% confidence interval. Grey shading: 95% confidence interval of the mean of the scrambled data (N=9 animals). d, mean average z-score of GCaMP signals during all USV syllables evoked with a behaving female compared to scrambled datapoints during pre-female behavior. Mean ± s.e.m. N=9 animals, unpaired t-test, two sided,*** p=0.0003. e, dark green line: mean z-score of GCaMP6s signals before and after initiation of USV during post-female stage, light green shading indicates 95% confidence interval. Grey shading: 95% confidence interval of the mean of the scrambled data. (N=9 animals) f, mean average z-score of GCaMP6s signal of all USV syllables during post-female stage compared to scrambled datapoints during pre-female stages. Mean ± s.e.m. N=9 animals, unpaired t test, two sided, *** p=2.2e−4. g-i, to determine whether the increased hypothalamic activity is involved in odor-evoked USV calling, we targeted chemogenetic inhibition to the LPOA, which is a largely unstudied heterogeneous region that has been implicated in sleep, thirst, and reward behavior39–43, and quantified USV production during natural interactions with an awake female. g, non-specific chemogenetics. left: AAV-hM4Di virus injection in LPOA of wildtype mice. Right: experimental assay; following expression of hM4Di virus, males were IP injected with CNO-saline-CNO-saline (every other day for 4 days total) and allowed to interact with a freely moving female to evoke USV calling. h, number of USV syllables emitted following injection with CNO (purple) or saline (black). Mean ± s.e.m., N=10 animals. Paired two-tailed Wilcoxon test, N.S.: p=0.11. i, number of USVs emitted across four sequential test days. Overall, the manipulation did not produce a significant effect on behavior, however half of this group (solid lines, N=5 animals) did show a constant reduction in USVs while the other half (dashed lines, N=5 animals) continued to emit USVs in the presence of CNO. This experiment suggests the potential for a functional role of the hypothalamic neurons in social vocal communication and a need for a more specific viral labelling method. j, average number of USVs between all Saline and CNO injected days as showed in Fig 1d. N=6 animals, Wilcoxon test, two sided, * p=0.03. k, sample image of hM4Di expression in Esr1-Zsgreen animals. Scale bar = 500μm. N=6 animals, >3 sections/animal collected. l, quantification of total time performing social behaviors observed by male LPOAEsr1/hM4Di during a 4 min interaction with live females on CNO and Saline injection days. N=6 animals. Paired t test was performed, two-sided,*** p<0.001, * p=0.02. Note: we observed an unexpected increase in the female’s anti-social defensive behavior (kicking, running away) which reduced his ability to direct sniffing to her anogenital region. This observation is consistent with male USVs serving to enhance female courtship behavior44,45.m, experiment design of expressing control AAV-TdTomato virus into LPOAEsr1 cells. n, number of USVs emitted with behaving female over 5 test days, alternating injections of either CNO or saline. o, average number of USVs between Saline and CNO injected days. N=5 animals. Wilcoxon test, two sided, p>0.05.

Article Snippet: For DREADD inhibition in wildtype mice, AAVdj-CAG-FLEX-hM4Di-GFP (Addgene plasmid # 52536, a gift from Scott Sternson) mixed with AAVdj/1-EF1α-FLEX-hM4Di-mCherry (Addgene plasmid # 50461, a gift from Bryan Roth) and AAV9-CRE (UPenn AV-9-PV1090) injected bilaterally at 4x10 12 GC/mL in LPOA.

Techniques: Activity Assay, Expressing, Inhibition, Injection, Two Tailed Test, Functional Assay

Journal: Nature

Article Title: Flexible scaling and persistence of social vocal communication

doi: 10.1038/s41586-021-03403-8

Figure Lengend Snippet: a, representative USVs emitted during scaling (ascending/descending frequency) of photostimulation in LPOAEsr1/ChR2 male demonstrate USVs persist after photostimulation ceases (blue bar). b, raster plot of USV syllables emitted during 10Hz photostimulation of PAGvGluT2/ChR2 neurons. USVs are locked to light stimulus (blue bars). Each row is a single trial. N=3 animals. 32 trials total. c, Number of syllables from photostimulation of either LPOAEsr1/ChR2 (blue, N=26 animals, 102 trials) or PAGvGluT2/ChR2 neurons (orange, N=3 animals, 32 trials) during light on (top) or light off (bottom). ND = not done. d, experimental design to simultaneously express ChR2 in LPOAvGat and hM4Di in PAGvGat cells. e-f, USVs emitted during optostimulation of LPOAvGAT/ChR2/PAGvGat/hM4Di following injection of saline (grey) or CNO (purple) on alternate days. USV number during e) 25Hz stimulation. f) raster plot of USVs emitted during and following photostimulation (blue bars). Each row is 30” of a 230” trial aligned to light stimuli applied every 40”. Mean ± s.e.m. N=3, 6 trials/animal per test day. Paired Wilcoxon test, two sided, ****p<0.0001. ns: p>0.05. g, Ex vivo cell-attached recording of PAGvGat neurons during photostimulating (blue shading) of LPOAvGat/ChR2 axon terminals. N=4 animals, total 23 cells. Left bars group the same cell during multiple trials. h, model of disinhibition circuitry for USV calling. LPOA disinhibition to scale USV power and bout duration enables flexible response to social/sensory context.

Article Snippet: For DREADD inhibition in wildtype mice, AAVdj-CAG-FLEX-hM4Di-GFP (Addgene plasmid # 52536, a gift from Scott Sternson) mixed with AAVdj/1-EF1α-FLEX-hM4Di-mCherry (Addgene plasmid # 50461, a gift from Bryan Roth) and AAV9-CRE (UPenn AV-9-PV1090) injected bilaterally at 4x10 12 GC/mL in LPOA.

Techniques: Injection, Ex Vivo

Journal: Nature

Article Title: Flexible scaling and persistence of social vocal communication

doi: 10.1038/s41586-021-03403-8

Figure Lengend Snippet: a-b, USVs emitted during photostimulation of LPOAvGAT/ChR2/ PAGvGat/hM4Di following injection of saline (grey) or CNO (purple) on alternate days. USV number during a) 1Hz stimulation. b) raster plot of USVs emitted during and following photostimulation (blue bars). Each row is 30” of a 230” trial aligned to light stimuli applied every 40”. Mean ± s.e.m. N=3, 6 trials/animal per test day. Paired Wilcoxon test, two sided, ****p<0.0001. ns: p>0.05. c, representative image of co-expression of hM4Di-tdt with vGat-Zsgreen cells in PAG. Scale bar = 200μm. N=2 animals, 2 sections/animal collected. d, number of USVs emitted during increasing frequency of photostimulation of LPOA in LPOAvGAT/ChR2/ PAGvGat/hM4Di males with CNO or saline. N=3, 36 trials per condition. Mean ± s.e.m. Wilcoxon test,two sided. **** = p<0.0001. e, experimental design to express control virus (tdt) in PAG of vGat-Cre mice (to control Main text Fig. 4e–f). f, number of USVs emitted by photostimulating LPOAvGat cells either under CNO or saline conditions in animals expressing FLEX-tdt (control). N=3, 18 trials/condition. Mean ± s.e.m. Wilcoxon test, two sided. ns= p>0.05. g, raster plot of USV bouts emitted during either CNO or saline test days under different stimulation frequency. Note: drosophila courtship songs similarly show feature separation across the circuit with songs evoked by pIP10 neurons tightly locked to stimulation (like the PAG USV-gate neurons) compared to calling generated by P1 neurons which persists beyond stimulation (as with the LPOAEsr1 neurons)53. This suggests diverse social species utilize general circuit strategies to maintain persistent auditory responses outlasting the detection of sensory information.

Article Snippet: For DREADD inhibition in wildtype mice, AAVdj-CAG-FLEX-hM4Di-GFP (Addgene plasmid # 52536, a gift from Scott Sternson) mixed with AAVdj/1-EF1α-FLEX-hM4Di-mCherry (Addgene plasmid # 50461, a gift from Bryan Roth) and AAV9-CRE (UPenn AV-9-PV1090) injected bilaterally at 4x10 12 GC/mL in LPOA.

Techniques: Injection, Expressing, Generated

Journal: eLife

Article Title: The anterior cingulate cortex and its role in controlling contextual fear memory to predatory threats

doi: 10.7554/eLife.67007

Figure Lengend Snippet:

Article Snippet: For the pharmacogenetic inhibition, ACA was injected bilaterally with 150 nl of AAV5-hSyn-HA-hM4D(Gi)-IRES-mCitrine (Dr. Bryan Roth; Addgene plasmid #50464) or AAV5-hSyn-eGFP (titer≥7×1012 vg/ml; Addgene viral prep #50465-AAV5) as control.

Techniques: Transfection, Construct, Virus, Plasmid Preparation, Software