Journal: bioRxiv

Article Title: Neural Basis for Regulation of Vasopressin Secretion by Anticipated Disturbances in Osmolality

doi: 10.1101/2021.01.27.428388

Figure Lengend Snippet: a, i, Schematic of SON AVP photometry experiment with hM4Di-mediated non-specific inhibition of neurons in the MnPO/OVLT (a) and SFO (i). b, j, Single-trial timecourses of SON AVP population activity in response to water bowl placement in saline and CNO trials of mice expressing hM4Di in the MnPO/OVLT (b) and SFO (j). Trials are sorted according to latency from water bowl placement to drinking onset (black ticks). n= 6 (MnPO), 5 (SFO) mice. c, k, Average SON AVP population activity in response to water bowl placement in saline and CNO trials of mice expressing hM4Di in the MnPO/OVLT (c) and SFO (k). n= 6 (MnPO), 5 (SFO) mice. d, l, Average of pre-ingestive responses in saline and CNO trials of mice expressing hM4Di in the MnPO/OVLT (k) and SFO (l). n= 6 (MnPO), 5 (SFO) mice. Values are means ± SEMs across trials. e, m, Data from panel c (e) and k (m) binned across drinking periods. ***p < 0.001; repeated measures (RM) 2-way ANOVA, n= 6 (MnPO), 5 (SFO) mice. f, n, Average pre- and post-ingestive responses in saline and CNO trials of mice expressing hM4Di in the MnPO/OVLT (f) and SFO (n). ns, p > 0.05; *p < 0.05; **p<0.01; paired t-test, n= 6 (MnPO), 5 (SFO) mice. g, o, Average latency to drinking onset and number of drinking bouts in saline and CNO trials of mice expressing hM4Di in the MnPO/OVLT (k) and SFO (l). ns, p > 0.05; *p < 0.05; **p<0.01; paired t-test, n= 6 (MnPO), 5 (SFO) mice. h, p, Average change in baseline activity in saline and CNO trials of mice expressing hM4Di in the MnPO/OVLT (h) and SFO (p). ns, p > 0.05; *p < 0.05; **p<0.01; paired t-test, n= 6 (MnPO), 3 (SFO) mice. Values are means ± SEMs across mice except for d, l. See also Figure1-—figure supplement 1,2.

Article Snippet: The following viral vectors were used in this study: AAV8-FLEX-TVA-mCherry (UNC Vector Core, Addgene 38044), AAV1-CAG-FLEX-RG (UNC Vector Core, Addgene 48333), SADΔG–EGFP (EnvA) rabies (Salk Gene Transfer Targeting and Therapeutics Core, Addgene 32635), AAV9-EF1α-DIO-ChR2(H134R)-mCherry (Penn Vector Core, Addgene 20297), AAV8-EF1a-DIO-synaptophysin-YFP (MIT Vector Core), AAV9-CAG-ChR2(H134R)-mCherry (Penn Vector Core, Addgene 100054), HSV-hEF1a-LS1L-GCaMP6s (MGH Gene Delivery Technology Core, RN507), AAV1-Syn-FLEX-GCaMP6s (Penn Vector Core, Addgene 100845), AAV1-Ef1a-DIO-EYFP (UNC Vector Core, Addgene 27056), AAV8-hSyn-hM4Di-mCherry (UNC Vector Core, Addgene 50479), AAV8-hSyn-DIO-hM4Di-mCherry (UNC Vector Core, Addgene 44362), AAV8-nEF-fDIO-hM4Di-mCherry (Boston Children’s Hospital Viral Core, Custom-made vector (hM4Di-mCherry was cut from AAV8-hSyn-DIO-hM4Di-mCherry (Addgene 44362) by AscI and NheI, and subcloned into pAAV-nEF Con/Fon hChR2(H134R)-EYFP (Addgene 55644) into AscI and XbaI))

Techniques: Inhibition, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Neural Basis for Regulation of Vasopressin Secretion by Anticipated Disturbances in Osmolality

doi: 10.1101/2021.01.27.428388

Figure Lengend Snippet: a, Representative images showing expression of ChR2-mCherry in the NTS (top), and their efferent projections in the PVH and SON (bottom). b, Number of PVH AVP and SON AVP neurons (left), and non-GFP PVH and SON neurons (right) receiving direct synaptic inputs from the NTS. c, Representative traces showing light-evoked responses. Black trace is an average of all traces (gray) in consecutive trials. d, Schematic of SON AVP photometry experiment with hM4Di-mediated inhibition of A1/C1 neurons. e, Average SON AVP population activity to hypotension induced by vasodilating drug HDZ in saline and CNO trials. f, Data from panel e binned every 5 min. ***p < 0.001; RM 2-way ANOVA, n= 9 mice. g, Average SON AVP population activity in response to feeding onset in saline and CNO trials. h, Data from panel g binned across feeding periods. ns, p > 0.05; RM 2-way ANOVA, n= 8 mice. Scale bar, 500µm. Values are means ± SEMs across mice. See also .

Article Snippet: The following viral vectors were used in this study: AAV8-FLEX-TVA-mCherry (UNC Vector Core, Addgene 38044), AAV1-CAG-FLEX-RG (UNC Vector Core, Addgene 48333), SADΔG–EGFP (EnvA) rabies (Salk Gene Transfer Targeting and Therapeutics Core, Addgene 32635), AAV9-EF1α-DIO-ChR2(H134R)-mCherry (Penn Vector Core, Addgene 20297), AAV8-EF1a-DIO-synaptophysin-YFP (MIT Vector Core), AAV9-CAG-ChR2(H134R)-mCherry (Penn Vector Core, Addgene 100054), HSV-hEF1a-LS1L-GCaMP6s (MGH Gene Delivery Technology Core, RN507), AAV1-Syn-FLEX-GCaMP6s (Penn Vector Core, Addgene 100845), AAV1-Ef1a-DIO-EYFP (UNC Vector Core, Addgene 27056), AAV8-hSyn-hM4Di-mCherry (UNC Vector Core, Addgene 50479), AAV8-hSyn-DIO-hM4Di-mCherry (UNC Vector Core, Addgene 44362), AAV8-nEF-fDIO-hM4Di-mCherry (Boston Children’s Hospital Viral Core, Custom-made vector (hM4Di-mCherry was cut from AAV8-hSyn-DIO-hM4Di-mCherry (Addgene 44362) by AscI and NheI, and subcloned into pAAV-nEF Con/Fon hChR2(H134R)-EYFP (Addgene 55644) into AscI and XbaI))

Techniques: Expressing, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: Neural Basis for Regulation of Vasopressin Secretion by Anticipated Disturbances in Osmolality

doi: 10.1101/2021.01.27.428388

Figure Lengend Snippet: a, Representative image showing rabies-labeled neurons in the ARC that are monosynaptically connected to magnocellular SON AVP neurons. b, Schematic of SON AVP photometry experiment with hM4Di-mediated inhibition of AgRP or POMC neurons. c, Representative images showing hM4Di expression in AgRP (top) and POMC neurons (bottom). d, Average SON AVP population activity binned across feeding periods in saline and CNO trials of AVP-IRES-Cre;Agrp-IRES-Cre (top) and AVP-IRES-Cre;Pomc-IRES-Cre (bottom) mice. e, i, k, Schematic of SON AVP photometry experiment with hM4Di-mediated non-specific inhibition of the ARC+VMH+DMH (e), VMH only (i), and DMH only (k). f, Single-trial timecourses of SON AVP population activity in response to food bowl placement in saline and CNO trials of ARC+VMH+DMH group. Trials are sorted according to latency from food bowl placement to feeding onset (black ticks). n= 5 mice. g, Average SON AVP population activity in response to feeding onset in saline and CNO trials of ARC+VMH+DMH group. n= 9 mice. h, j, l, Average SON AVP population activity binned across feeding periods in saline and CNO trials of ARC+VMH+DMH (h), VMH only (j), and DMH only (l) groups. ns, p > 0.05; *p < 0.05; RM 2-way ANOVA, n= 5 (ARC+VMH+DMH), 4 (VMH only), 7 (DMH only) mice. Scale bar, 200µm. Values are means ± SEMs across mice. See also - .

Article Snippet: The following viral vectors were used in this study: AAV8-FLEX-TVA-mCherry (UNC Vector Core, Addgene 38044), AAV1-CAG-FLEX-RG (UNC Vector Core, Addgene 48333), SADΔG–EGFP (EnvA) rabies (Salk Gene Transfer Targeting and Therapeutics Core, Addgene 32635), AAV9-EF1α-DIO-ChR2(H134R)-mCherry (Penn Vector Core, Addgene 20297), AAV8-EF1a-DIO-synaptophysin-YFP (MIT Vector Core), AAV9-CAG-ChR2(H134R)-mCherry (Penn Vector Core, Addgene 100054), HSV-hEF1a-LS1L-GCaMP6s (MGH Gene Delivery Technology Core, RN507), AAV1-Syn-FLEX-GCaMP6s (Penn Vector Core, Addgene 100845), AAV1-Ef1a-DIO-EYFP (UNC Vector Core, Addgene 27056), AAV8-hSyn-hM4Di-mCherry (UNC Vector Core, Addgene 50479), AAV8-hSyn-DIO-hM4Di-mCherry (UNC Vector Core, Addgene 44362), AAV8-nEF-fDIO-hM4Di-mCherry (Boston Children’s Hospital Viral Core, Custom-made vector (hM4Di-mCherry was cut from AAV8-hSyn-DIO-hM4Di-mCherry (Addgene 44362) by AscI and NheI, and subcloned into pAAV-nEF Con/Fon hChR2(H134R)-EYFP (Addgene 55644) into AscI and XbaI))

Techniques: Labeling, Inhibition, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Neural Basis for Regulation of Vasopressin Secretion by Anticipated Disturbances in Osmolality

doi: 10.1101/2021.01.27.428388

Figure Lengend Snippet: a. Representative image showing rabies-labeled neurons in the PNZ that are monosynaptically connected to magnocellular SQN AVP neurons. b. Number of SQN AVP neurons (left) and non-GFP SON neurons (right) receiving direct synaptic inputs from PNZ GABA neurons as identified by CRACM in AVP-GFP;Vgat­lRES-Cre . c. Schematic of PNZ GABA photometry experiment. d. Heatmap showing single-trial timecourses of PNZ GABA population activity in response to food bowl placement. Trials are sorted according to latency from food bowl placement to feeding onset (black ticks). n = 8 mice. e. Average PNZ GABA population activity in response to water/food bowl placement. n = 3 mice. f. Average PNZ GABA population activity binned across drinking/feeding periods. *p < 0.05; 2-way ANOVA, n = 3 mice. g. Schematic of SQNAVP photometry experiment with hM4Di-mediated inhibition of PNZGABA neurons. h. Average of SQNAVP population activity in response to feeding onset in saline and CNO trials. n= 6 mice. i. Average SQNAVP population activity binned across feeding periods in saline and CNO trials. ns, *p < 0.05; 2-way ANOVA, n= 6 mice. Scale bar, 200µm. Values are means ± SEMs.

Article Snippet: The following viral vectors were used in this study: AAV8-FLEX-TVA-mCherry (UNC Vector Core, Addgene 38044), AAV1-CAG-FLEX-RG (UNC Vector Core, Addgene 48333), SADΔG–EGFP (EnvA) rabies (Salk Gene Transfer Targeting and Therapeutics Core, Addgene 32635), AAV9-EF1α-DIO-ChR2(H134R)-mCherry (Penn Vector Core, Addgene 20297), AAV8-EF1a-DIO-synaptophysin-YFP (MIT Vector Core), AAV9-CAG-ChR2(H134R)-mCherry (Penn Vector Core, Addgene 100054), HSV-hEF1a-LS1L-GCaMP6s (MGH Gene Delivery Technology Core, RN507), AAV1-Syn-FLEX-GCaMP6s (Penn Vector Core, Addgene 100845), AAV1-Ef1a-DIO-EYFP (UNC Vector Core, Addgene 27056), AAV8-hSyn-hM4Di-mCherry (UNC Vector Core, Addgene 50479), AAV8-hSyn-DIO-hM4Di-mCherry (UNC Vector Core, Addgene 44362), AAV8-nEF-fDIO-hM4Di-mCherry (Boston Children’s Hospital Viral Core, Custom-made vector (hM4Di-mCherry was cut from AAV8-hSyn-DIO-hM4Di-mCherry (Addgene 44362) by AscI and NheI, and subcloned into pAAV-nEF Con/Fon hChR2(H134R)-EYFP (Addgene 55644) into AscI and XbaI))

Techniques: Labeling, Activity Assay, Inhibition

Journal: Cell reports

Article Title: Distinct subpopulations of ventral pallidal cholinergic projection neurons encode valence of olfactory stimuli

doi: 10.1016/j.celrep.2024.114009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: A Hamilton syringe was slowly lowered to the VP and mice were injected with ~0.3 μL of the following viral vectors dependent on experiment: AAV 9 .DIO.TRE.hM4Di.P2A.m.Cherry (ADCD; packaged at UNC viral vector core), AAV 9 .RAM.d2tTA.TRE.mCherry.NLS-FLAG (RAM; Addgene # 63931, packaged at UNC viral vector core), AAV 9 .Syn.Flex-GCaMP6f.WPRE.SV40 (GCaMP6f; Addgene # 100837), AAV 8 .hSyn.DIO.hM4Di (Gi).mCherry (hM4Di; Addgene # 44362), AAV 9 .eSyn.eGFP (Vector Biolabs # VB4870), AAV 9 .RAM.d2tTA.TRE.Flex.tdTomato (FLEX-RAM; Addgene # 84468, packaged at NINDS Viral Vector Core).

Techniques: Virus, Plasmid Preparation, Recombinant, Software

Journal: Scientific Reports

Article Title: Action inflexibility and compulsive-like behavior accompany neurobiological alterations in the anterior orbitofrontal cortex and associated striatal nuclei

doi: 10.1038/s41598-024-84369-1

Figure Lengend Snippet: Breeding mice for poor action flexibility results in compulsive-like behavior: Reversal by fluoxetine and inhibitory chemogenetics. ( a ) EB mice spent more time in repetitive behavior following amphetamine than control mice (ctrl n = 6, EB n = 9). ( b ) The same amphetamine-elicited repetitive behaviors are represented as difference score from saline baseline; again, EB mice generated more repetitive behaviors. ( c ) EB mice groomed more than control mice in a stimulus-elicited grooming test ( n = 8/group). ( d ) Experimental schematic and timeline. ( e ) Fluoxetine ameliorated stimulus-elicited grooming behavior in EB mice (ctrl saline n = 8, all other groups n = 9). ( f ) Experimental schematic and timeline. ( g ) Histological representation of Gq-DREADDs-expressing viral vectors in the VLO is depicted on images from The Mouse Brain Library . Black indicates the largest spread, and white the smallest. ( h ) Chemogenetic stimulation of excitatory VLO neurons had no impact on stimulus-elicited grooming, ( i ) though it did elicit c-fos at the infusion site (ctrl n = 7, Gq-DREADD n = 6). ( j ) Representative images. ( k ) Histological representation of Gi-DREADDs-expressing viral vectors is depicted on images from The Mouse Brain Library . Black indicates the largest spread, and white the smallest. ( l ) Chemogenetic inhibition of excitatory VLO neurons mitigated stimulus-elicited grooming (ctrl n = 10, Gi-DREADD n = 11). ( m ) To validate Gi-DREADDs, mice received a swim stressor (Created in BioRender. Allen, A. (2024) BioRender.com/g86t305). ( n ) Despite this stressor, Gi-DREADDs-expressing cells displayed less c-fos, as expected. ( o ) Representative images. Data shown as mean + SEM with symbols representing individual mice. Scale bar = 50 μm. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Mice were randomly assigned to receive either the inhibitory chemogenetic receptor (AAV5-CamKIIα-hM4Di-mCherry, 2.4 × 10 12 GC/mL, Addgene #50477, deposited by Bryan Roth), the excitatory chemogenetic receptor (AAV5- CamKIIα-hM3Dq-mCherry,1.7 × 10 13 GC/mL, Addgene #50476, deposited by Bryan Roth), or a control construct (AAV5-CamKIIα-mCherry, 4.9 × 10 12 GC/mL, UNC GTC Vector Core #AV4809E, deposited by Karl Deisseroth), which was infused bilaterally.

Techniques: Control, Saline, Generated, Expressing, Inhibition

Journal: bioRxiv

Article Title: Chemogenetic Manipulations of Ventral Tegmental Area Dopamine Neurons Reveal Multifaceted Roles in Cocaine Abuse

doi: 10.1101/246595

Figure Lengend Snippet: a. Midbrain tyrosine hydroxylase (TH) staining (green) and mCherry-tagged DREADD expression (red) in VTA is shown, with yellow indicating co-labeling. Inset shows schematic of AAV double-inverted open reading frame (DIO)-DREADD vectors used. b. Percentage of midbrain TH+ cells located within borders of substantia nigra pars compacta (SN; white bars), or VTA (black bars). Individual rat data shown with red diamond (Gq), yellow triangle (Gs) or blue square (Gi) symbols, c. Percentage of TH+ VTA neurons expressing Fos after Vehicle or CNO (lOmg/kg) in Gq-, Gs-, or Gi-coupled DREADD rats (individual rat data represented with symbols on top of bars), d. Firing rates are depicted for baseline and post-CNO periods, individual cells depicted with lines, e. CNO increases mean (SEM) firing rate of VTA DREADD+ neurons in vitro, relative to baseline firing, f. In anesthetized rats with hM3Dq DREADDs, more Type 1 cells were encountered per electrophysiological recording track after 10mg/kg CNO (colored bars, symbols) than pre-CNO (white bars, symbols). No significant effects on population activity were observed after CNO in Gs, Gi, or WT groups, f. Interspike interval before CNO (black lines, error bars=SEM) and after 10mg/kg CNO i.p. (red=Gq, yellow=Gs, blue=Gi, black=WT). *p<0.05.

Article Snippet: Immediately after catheter implantation, 1µl/hemisphere of an AAV2 vector containing a double-floxed, inverted open reading frame (DIO) sequence for the mCherry-tagged hM4Di (Gi-coupled), hM3Dq (Gq-coupled), or rM3Ds (Gs-coupled) DREADD, or mCherry control ( hSyn-DIO-hM4Di-mCherry, hSyn-DIO-hM3Dq-mCherry, hSyn-DIO-rM3Ds-mCherry, hSyn-DIO-mCherry: University of North Carolina Vector Core or AddGene) was pressure injected via Hamilton syringe (28ga) or glass micropipette bilaterally into the ventromedial midbrain over 2min (retracted 5min later) causing DREADD expression selectively in TH neurons of VTA (Coordinates relative to bregma: AP-5.5, ML+0.8, DV-8.15).

Techniques: Staining, Expressing, Labeling, In Vitro, Activity Assay

Journal: bioRxiv

Article Title: Chemogenetic Manipulations of Ventral Tegmental Area Dopamine Neurons Reveal Multifaceted Roles in Cocaine Abuse

doi: 10.1101/246595

Figure Lengend Snippet: a. In Gq-DREADD rats, CNO (1mg/kg: grey bars, 10mg/kg: black bars) increased cocaine seeking (relative to vehicle: white bars) in the absence (extinguished context) or presence of cues, and after a cocaine prime (no cues) or yohimbine (YOH+cues) test (10mg/kg). b. CNO failed to increase reinstatement in rM3Ds Gs-DREADD rats. c. In Gi-DREADD rats, CNO reduced the priming effects of cocaine and potentiation of cued responding by YOH (10mg/kg). d. No effects of CNO were observed in WT rats with no DREADD expression. Gq-stimulation increased, and Gi-stimulation decreased locomotor activity in a familiar environment. Light grey bars indicate IL presses for each test. *p<0.05 e. Effects of CNO (0,1,10mg/kg) on horizontal locomotion (beam breaks) and f. rearing behavior is shown for DREADD-expressing and WT animals. Individual rat data shown in .

Article Snippet: Immediately after catheter implantation, 1µl/hemisphere of an AAV2 vector containing a double-floxed, inverted open reading frame (DIO) sequence for the mCherry-tagged hM4Di (Gi-coupled), hM3Dq (Gq-coupled), or rM3Ds (Gs-coupled) DREADD, or mCherry control ( hSyn-DIO-hM4Di-mCherry, hSyn-DIO-hM3Dq-mCherry, hSyn-DIO-rM3Ds-mCherry, hSyn-DIO-mCherry: University of North Carolina Vector Core or AddGene) was pressure injected via Hamilton syringe (28ga) or glass micropipette bilaterally into the ventromedial midbrain over 2min (retracted 5min later) causing DREADD expression selectively in TH neurons of VTA (Coordinates relative to bregma: AP-5.5, ML+0.8, DV-8.15).

Techniques: Expressing, Activity Assay

Journal: bioRxiv

Article Title: Chemogenetic Manipulations of Ventral Tegmental Area Dopamine Neurons Reveal Multifaceted Roles in Cocaine Abuse

doi: 10.1101/246595

Figure Lengend Snippet: Active Lever (AL) pressing is represented for each rat during reinstatement tests in each DREADD group: a) hM3Dq, b) rM3Ds, c) hM4Di, d) WT control. From left to right, change from vehicle day data (1 or 10mg/kg CNO) are shown for extinguished context, cue-induced reinstatement, cocaine primed reinstatement, and YOH+cue reinstatement. In panels e-h , horizontal locomotion (left) and rearing (right) is shown for 1 and 10mg/kg CNO days (change from vehicle day) for each rat. Iines=mean, error bars=SEM.

Article Snippet: Immediately after catheter implantation, 1µl/hemisphere of an AAV2 vector containing a double-floxed, inverted open reading frame (DIO) sequence for the mCherry-tagged hM4Di (Gi-coupled), hM3Dq (Gq-coupled), or rM3Ds (Gs-coupled) DREADD, or mCherry control ( hSyn-DIO-hM4Di-mCherry, hSyn-DIO-hM3Dq-mCherry, hSyn-DIO-rM3Ds-mCherry, hSyn-DIO-mCherry: University of North Carolina Vector Core or AddGene) was pressure injected via Hamilton syringe (28ga) or glass micropipette bilaterally into the ventromedial midbrain over 2min (retracted 5min later) causing DREADD expression selectively in TH neurons of VTA (Coordinates relative to bregma: AP-5.5, ML+0.8, DV-8.15).

Techniques:

Journal: bioRxiv

Article Title: Chemogenetic Manipulations of Ventral Tegmental Area Dopamine Neurons Reveal Multifaceted Roles in Cocaine Abuse

doi: 10.1101/246595

Figure Lengend Snippet: Timecourse of AL (solid lines) and IL (dashed lines) are shown for a) hM3Dq, b) rM3Ds, c) hM4Di, and d) WT control rats after vehicle or CNO (1, 10mg/kg). Data are shown in 30min bins throughout the 120min session. Lines represent mean, error bars represent SEM. *p<0.05, interaction of dose X time.

Article Snippet: Immediately after catheter implantation, 1µl/hemisphere of an AAV2 vector containing a double-floxed, inverted open reading frame (DIO) sequence for the mCherry-tagged hM4Di (Gi-coupled), hM3Dq (Gq-coupled), or rM3Ds (Gs-coupled) DREADD, or mCherry control ( hSyn-DIO-hM4Di-mCherry, hSyn-DIO-hM3Dq-mCherry, hSyn-DIO-rM3Ds-mCherry, hSyn-DIO-mCherry: University of North Carolina Vector Core or AddGene) was pressure injected via Hamilton syringe (28ga) or glass micropipette bilaterally into the ventromedial midbrain over 2min (retracted 5min later) causing DREADD expression selectively in TH neurons of VTA (Coordinates relative to bregma: AP-5.5, ML+0.8, DV-8.15).

Techniques:

Journal: bioRxiv

Article Title: Chemogenetic Manipulations of Ventral Tegmental Area Dopamine Neurons Reveal Multifaceted Roles in Cocaine Abuse

doi: 10.1101/246595

Figure Lengend Snippet: a. hM3Dq DREADDs are robustly transported to targets including medial prefrontal cortex (mPFC) and nucleus accumbens (NAc). b. VTA TH(+) neuron terminals (labelled with mCherry (red) co-localized with tyrosine hydroxylase (TH; green) to a greater extent in NAc as compared to mPFC. c. In acute coronal NAc slices from rats with hM3Dq DREADDs in VTA DA neurons, DA release was evoked with local electrical stimulation (single pulse, 20Hz, or 40Hz) before or after bath application of CNO (1μM), measured using cyclic voltammetry. Bars depict % baseline (pre-CNO) DA release after CNO in Gq-, Gs- or Gi-DREADD rats, or WT rats. CNO facilitated DA release in Gq-DREADD rats at all stimulation parameters, and decreased DA release in Gi-DREADD rats. No effects of CNO were seen in Gs- DREADD or WT rats. Effects of CNO microinjection into mPFC (d,g) , NAc (e,h) , or BLA (f,i) on cocaine seeking in an extinguished context with (right bars) or without cues (left bars), or on 2hr chow intake in a separate test with the same rats (j,k,l) . Microinjection sites in j. mPFC, k . NAc, or I . BLA are depicted with red circles. *p<0.05, **p<0.01. Individual rat behavioral data shown in .

Article Snippet: Immediately after catheter implantation, 1µl/hemisphere of an AAV2 vector containing a double-floxed, inverted open reading frame (DIO) sequence for the mCherry-tagged hM4Di (Gi-coupled), hM3Dq (Gq-coupled), or rM3Ds (Gs-coupled) DREADD, or mCherry control ( hSyn-DIO-hM4Di-mCherry, hSyn-DIO-hM3Dq-mCherry, hSyn-DIO-rM3Ds-mCherry, hSyn-DIO-mCherry: University of North Carolina Vector Core or AddGene) was pressure injected via Hamilton syringe (28ga) or glass micropipette bilaterally into the ventromedial midbrain over 2min (retracted 5min later) causing DREADD expression selectively in TH neurons of VTA (Coordinates relative to bregma: AP-5.5, ML+0.8, DV-8.15).

Techniques:

Journal: bioRxiv

Article Title: Chemogenetic Manipulations of Ventral Tegmental Area Dopamine Neurons Reveal Multifaceted Roles in Cocaine Abuse

doi: 10.1101/246595

Figure Lengend Snippet: No consistent evidence for long-lasting effects of CNO in a) hM3Dq, b) rM3Ds, c) hM4Di, or d) WT control rats was found, as demonstrated with AL presses on the extinction re-training trials held 24hrs after injection of vehicle (white bars), 1mg/kg CNO (light red, yellow, blue, black bars), or 10mg/kg CNO (dark color bars; repeated measures ANOVA, p>0.05 for each test condition/DREADD group). Similarly, no persistent effects of prior-day CNO was observed on: e) demand elasticity (α) or f) free cocaine intake (Q 0 ). Bars in panels e-f represent percentage of pre-injection baseline, on cocaine self-administration training days following tests days with veh or CNO (1 or 10mg/kg).

Article Snippet: Immediately after catheter implantation, 1µl/hemisphere of an AAV2 vector containing a double-floxed, inverted open reading frame (DIO) sequence for the mCherry-tagged hM4Di (Gi-coupled), hM3Dq (Gq-coupled), or rM3Ds (Gs-coupled) DREADD, or mCherry control ( hSyn-DIO-hM4Di-mCherry, hSyn-DIO-hM3Dq-mCherry, hSyn-DIO-rM3Ds-mCherry, hSyn-DIO-mCherry: University of North Carolina Vector Core or AddGene) was pressure injected via Hamilton syringe (28ga) or glass micropipette bilaterally into the ventromedial midbrain over 2min (retracted 5min later) causing DREADD expression selectively in TH neurons of VTA (Coordinates relative to bregma: AP-5.5, ML+0.8, DV-8.15).

Techniques: Injection

Journal: bioRxiv

Article Title: Therapeutic Reversal of Chemically Induced Parkinson Disease by Converting Astrocytes into Nigral Neurons

doi: 10.1101/2020.04.06.028084

Figure Lengend Snippet: a , Schematic depiction of the chemogenetic approach to demonstrate converted neurons are directly responsible for the phenotypic recovery, emphasizing the rapid effect (40 min) of injected CNO to prevent action potentials of reprogrammed neurons and functional restoration after metabolism of the drug in 3 days. b , The results of the cylinder test, showing preferential ipsilateral touches in lesioned mice before and after injecting AAV-hM4Di-shPTB as well as treatment with CNO and 3 days after drug withdrawal. Unlesioned mice served as control. n=mice analyzed in each group. Dashed lines show the same mice at different treatment conditions. c , Quantification of striatal dopamine levels, showing significant restoration of striatal dopamine after reprogramming with AAV-shPTB in ipsilateral nigra. n=mice analyzed in each group. d , Screening for efficient antisense oligonucleotides that target PTB. PTB levels were examined by immunoblotting in mouse astrocytes treated with different ASOs. ASO 4# was chosen for the rest of experiments. e , PTB-ASO induced neurons from mouse cortical astrocytes in vitro, which were stained positively for Tuj1 and MAP2 (left), NSE and NeuN (middle), as well as the dopaminergic neuron marker TH (right). Note that as in shPTB-treated cells, a small percentage of TH-positive cells was also detected in PTB-ASO treated astrocytes. Mouse astrocytes treated with GFP-ASO showed no positive staining of any of the neuronal markers under same culture conditions. Scale bar: 20 μm. f , Schematic depiction of Tamoxifen (TAM)-induced tdTomato expression and ASO-induced neuronal conversion from tdTomato-labeled astrocytes in substantia nigra. g , A fraction of tdTomato-labeled cells became NeuN + 8 weeks after injection of PTB-ASO. Scale bar: 20 μm. h , A fraction of PTB-ASO converted tdTomato-positive neurons were TH + in 12 weeks. Scale bar: 10 μm. i , Schematic depiction of the experimental schedule for 6-OHDA-induced lesion followed by reprogramming with ASO and behavioral test. j , PTB-ASO, but not GFP-ASO, partially rescued the rotation behavior induced by apomorphine in 6-OHDA lesioned mice. Dashed lines show the same mice before and after ASO-mediated reprogramming. In panel b, c , and j : Statistical results are represented as mean+/- SEM; significant differences are indicated by the p-values based on ANOVA with post-hoc Tukey test (b and c) or paired t-test. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: The AAV-hM4Di-shPTB vector was constructed by replacing RFP in AAV-shPTB with the cDNA of hM4Di, which was sub-cloned from pAAV-CBA-DIO-hM4Di-mCherry vector (Addgene, #81008).

Techniques: Injection, Functional Assay, Western Blot, In Vitro, Staining, Marker, Expressing, Labeling