(A) The timeline of CMS, behavioral tests, and electrophysiology. (B) CMS significantly decreased body weight compared with non-stressed control mice (**p=0.004, control, n = 14 mice; CMS n = 12 …
Body weight and behavior following CMS in Figure 1B–G.
Figure 1 was reanalyzed to compare C57BL/6J (C57) and DAT-tdTomato (tdTomato) reporter mice for control and CMS-induced effects on body weight (A) and behavior in OPT (B) SPT (C) EPM (D) NSF (E) and …
(A) Sample traces of VTA dopamine neuron AP firing in control and CMS mice. Dopamine neurons were identified by a broad triphasic extracellular action potential of a width greater than 2 ms and a …
In vivo VTA dopamine neuron firing following CMS in Figure 2C–E.
(A) DAT-Cre mice were bred with Ai9 reporter mice, which express tdTomato in the presence of Cre, to produce DAT-tdTomato mice. TdTomato and TH (green) were completely co-localized, indicating that …
(A) Left: Voltage protocol for recording Ih current. Right: Representative Ih current recorded from NAc-projecting VTA dopamine neurons in control and CMS mice. (B) Compared with the control group, …
Ih amplitude and activation properties in VTA dopamine neurons following CMS in Figure 4B–G.
(A) The instantaneous inward current (Iins) was measured as indicated by the dashed lines. (B) A representative example for calculating resting membrane conductance. Iins was plotted against the …
(A) Ih currents recorded at −130 mV in NAc-projecting VTA dopamine neurons in both control (***p<0.001, n = 6 cells from three mice) and CMS groups (***p<0.001, n = 6 cells from three mice) were …
Effects of ZD7288 on Ih current and ex vivo AP firing in VTA dopamine neurons following CMS in Figure 5A,C,D.
(A,B), Immunofluorescence labeling showing the expression of AAV2-HCN2-shRNA-eGFP (green), TH (dopamine neuron marker, red) and NeuN (neuronal marker, blue) in the midbrain under low magnification (A…
Ih current and AP firing following shRNA-mediated HCN2 knockdown in Figure 6C–E.
(A) VTA-specific HCN2 knockdown did not significantly affect the body weight of mice (control, n = 7 mice; CMS, n = 7 mice; p=0.727). (B) HCN2 knockdown significantly decreased the center time …
Body weight and behavior following VTA HCN2 knockdown in Figure 7A–F.
(A) The maximal amplitude of Ih current in AAV2-HCN2-eGFP-infected VTA dopamine neurons (n = 15 from four mice) was significantly increased compared with that in AAV2-eGFP-infected VTA dopamine …
VTA HCN2 overexpression effects on Ih current and CMS-induced changes in body weight and behavior in Figure 8A–G.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Mus musculus) | male C57BL/6J mice | The Jackson Laboratory | Stock#: 000664 RRID:IMSR_JAX:000664 | |
Genetic reagent (Mus musculus) | male heterozygous Slc6a3Cre+/− (DAT-Cre) mice | The Jackson Laboratory | Stock#: 006660 RRID:IMSR_JAX:006660 | maintained on the C57BL/6J background |
Genetic reagent (Mus musculus) | male Ai9 reporter mice | The Jackson Laboratory | Stock#: 007909 RRID:IMSR_JAX:007909 | maintained on the C57BL/6J background |
Strain, strain background (Adeno-associated virus) | AAV2.shRNA.U6.ShRLuc. CMV.eGFP.SV40 | shRNA provided by Dr. Han-gang Yu (PMID: 19236845), packed into AAV2 at Penn Vector Core | ||
Strain, strain background (Adeno-associated virus) | AAV2.scramble.U6.ShRLuc. CMV.eGFP.SV40 | Penn Vector Core | ||
Strain, strain background (Adeno-associated virus) | AAV2.CMV.PI.HCN2. WPRE.eGFP.SV40 | HCN2 plasmid packed into AAV2 at Penn Vector Core | HCN2 plasmid provided by Dr. Dane Chetkovich at Northwestern University | |
Strain, strain background (Adeno-associated virus) | AAV2.CMV.PI.eGFP.WPRE.bGH | Penn Vector Core | ||
Antibody | Mouse anti-TH | Santa Cruz Biotechnology | SC-136100, Lot: G1309, RRID:AB_2287193 | monoclonal, 1:300 |
Antibody | Rabbit anti-TH | Santa Cruz Biotechnology | SC-14007, Lot: C2707, RRID:AB_671397 | polyclonal, 1:300 |
Antibody | Rabbit anti-NeuN | Millipore | ABN78, Lot: 2702139, RRID:AB_10807945 | polyclonal, 1:400 |
Antibody | anti-mouse IgG Alexa Fluor 555 | Cell Signaling | Stock #4409, RRID:AB_1904022 | Goat anti-mouse, 1:300 |
Antibody | anti-rabbit IgG Alexa Fluor 647 | Invitrogen | A21245, Lot: 1445259, RRID:AB_141775 | Goat anti-rabbit, 1:100 |
Antibody | anti-mouse IgG Alexa Fluor 488 | Cell Signaling | Stock #4408, RRID:AB_10694704 | Goat anti-mouse, 1:300 |
Antibody | anti-rabbit IgG Alexa Fluor 488 | Cell Signaling | Stock #4412, RRID:AB_1904025 | Goat anti-rabbit, 1:500 |
Other | neurobiotin tracer | Vector Laboratories | SP-1120, RRID:AB_2336606 | 1.5% |
Other | Texas Red avidin D | Vector Laboratories | A-2006, RRID:AB_2336751 | 1:100 |
Software (Sigmaplot 11.2) | RRID:SCR_003210 |
Week | Monday | Tuesday | Wednesday | Thursday | Friday | Saturday | Sunday |
---|---|---|---|---|---|---|---|
1 | Cold Wet bedding | Restraint No bedding | Light inversion Cage tilt | Cage rotation Strobe | Cold Food and water deprivation | Restraint Overcrowding | Light inversion Wet bedding |
2 | Cold Cage tilt | Cage rotation Food and water deprivation | Restraint Wet bedding | Rat bedding Strobe | Light inversion No bedding | Cage rotation Food and water deprivation | Cold Wet bedding |
3 | Rat bedding Strobe | Restraint Light inversion | Cage rotation No bedding | Light inversion Food and water deprivation | Cold Wet bedding | Cage tilt Strobe | Light inversion Overcrowding |
4 | Cold No bedding | Restraint Food and water deprivation | Cage rotation Strobe | Rat bedding Light inversion | Cold Cage tilt | Restraint Wet bedding | Cage rotation No bedding |
5 | Cold Food and water deprivation | Cage rotation Strobe | Light inversion Wet bedding | Cold Cage tilt | Cage rotation No bedding | Light inversion Overcrowding | Restraint Cage tilt |
Statistical results for Figure 1—figure supplement 1.
Abbreviations: CMS, chronic mild unpredictable stress; EPM, elevated plus maze; FST, forced swimming test; NSF, novelty-suppressed feeding; OFT, open field test; SPT, sucrose preference test