HCN2 channels in the ventral tegmental area regulate behavioral responses to chronic stress

  1. Peng Zhong
  2. Casey R Vickstrom
  3. Xiaojie Liu
  4. Ying Hu
  5. Laikang Yu
  6. Han-Gang Yu
  7. Qing-song Liu  Is a corresponding author
  1. Medical College of Wisconsin, United States
  2. West Virginia University, United States
8 figures, 2 tables and 2 additional files

Figures

Figure 1 with 1 supplement
CMS produced depressive- and anxiety-like behaviors.

(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 …

https://doi.org/10.7554/eLife.32420.002
Figure 1—source data 1

Body weight and behavior following CMS in Figure 1B–G.

https://doi.org/10.7554/eLife.32420.004
Figure 1—figure supplement 1
C57BL/6J and DAT-tdTomato mice do not significantly differ in body weight or behaviors at baseline or in response to CMS.

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 …

https://doi.org/10.7554/eLife.32420.003
CMS decreased single-unit AP firing in VTA dopamine neurons in vivo.

(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 …

https://doi.org/10.7554/eLife.32420.005
Figure 2—source data 1

In vivo VTA dopamine neuron firing following CMS in Figure 2C–E.

https://doi.org/10.7554/eLife.32420.006
Retrobead labeling of VTA dopamine neurons that project to the lateral shell of the NAc.

(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 …

https://doi.org/10.7554/eLife.32420.007
Figure 4 with 1 supplement
CMS decreased Ih currents in VTA dopamine neurons that project to the lateral shell of the NAc (LAcbSh).

(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, …

https://doi.org/10.7554/eLife.32420.008
Figure 4—source data 1

Ih amplitude and activation properties in VTA dopamine neurons following CMS in Figure 4B–G.

https://doi.org/10.7554/eLife.32420.010
Figure 4—figure supplement 1
The measurement of resting membrane conductance.

(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 …

https://doi.org/10.7554/eLife.32420.009
, CMS decreased AP firing in VTA dopamine neurons in midbrain slices.

(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 …

https://doi.org/10.7554/eLife.32420.011
Figure 5—source data 1

Effects of ZD7288 on Ih current and ex vivo AP firing in VTA dopamine neurons following CMS in Figure 5A,C,D.

https://doi.org/10.7554/eLife.32420.012
Figure 6 with 1 supplement
AAV-mediated shRNA knockdown of HCN2 in the VTA.

(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

https://doi.org/10.7554/eLife.32420.013
Figure 6—source data 1

Ih current and AP firing following shRNA-mediated HCN2 knockdown in Figure 6C–E.

https://doi.org/10.7554/eLife.32420.015
Figure 6—figure supplement 1
The resting conductance (Gresting) was significantly decreased in the HCN-shRNA group (n = 14) compared with the scrambled group (n = 15, *p=0.022).
https://doi.org/10.7554/eLife.32420.014
ShRNA knockdown of HCN2 in the VTA produced anxiety- and depressive-like behaviors.

(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 …

https://doi.org/10.7554/eLife.32420.016
Figure 7—source data 1

Body weight and behavior following VTA HCN2 knockdown in Figure 7A–F.

https://doi.org/10.7554/eLife.32420.017
Overexpression of HCN2 in the VTA prevented the development of CMS-induced depressive-like behavior.

(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 …

https://doi.org/10.7554/eLife.32420.018
Figure 8—source data 1

VTA HCN2 overexpression effects on Ih current and CMS-induced changes in body weight and behavior in Figure 8A–G.

https://doi.org/10.7554/eLife.32420.019

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background
(Mus musculus)
male C57BL/6J miceThe Jackson LaboratoryStock#: 000664
RRID:IMSR_JAX:000664
Genetic reagent
(Mus musculus)
male heterozygous
Slc6a3Cre+/− (DAT-Cre) mice
The Jackson LaboratoryStock#: 006660
RRID:IMSR_JAX:006660
maintained on the
C57BL/6J background
Genetic reagent
(Mus musculus)
male Ai9 reporter miceThe Jackson LaboratoryStock#: 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.bGHPenn Vector Core
AntibodyMouse anti-THSanta Cruz BiotechnologySC-136100, Lot: G1309,
RRID:AB_2287193
monoclonal, 1:300
AntibodyRabbit anti-THSanta Cruz BiotechnologySC-14007, Lot: C2707,
RRID:AB_671397
polyclonal, 1:300
AntibodyRabbit anti-NeuNMilliporeABN78, Lot: 2702139,
RRID:AB_10807945
polyclonal, 1:400
Antibodyanti-mouse IgG
Alexa Fluor 555
Cell SignalingStock #4409,
RRID:AB_1904022
Goat anti-mouse, 1:300
Antibodyanti-rabbit IgG
Alexa Fluor 647
InvitrogenA21245, Lot: 1445259,
RRID:AB_141775
Goat anti-rabbit, 1:100
Antibodyanti-mouse IgG
Alexa Fluor 488
Cell SignalingStock #4408,
RRID:AB_10694704
Goat anti-mouse, 1:300
Antibodyanti-rabbit IgG
Alexa Fluor 488
Cell SignalingStock #4412,
RRID:AB_1904025
Goat anti-rabbit, 1:500
Otherneurobiotin tracerVector LaboratoriesSP-1120,
RRID:AB_2336606
1.5%
OtherTexas Red avidin DVector LaboratoriesA-2006,
RRID:AB_2336751
1:100
Software (Sigmaplot 11.2)RRID:SCR_003210
Table 1
Experimental schedule for the chronic mild stress (CMS) procedure in mice
https://doi.org/10.7554/eLife.32420.020
WeekMondayTuesdayWednesdayThursdayFridaySaturdaySunday
1Cold
Wet bedding
Restraint
No bedding
Light inversion
Cage tilt
Cage rotation
Strobe
Cold
Food and water deprivation
Restraint
Overcrowding
Light inversion
Wet bedding
2Cold
Cage tilt
Cage rotation
Food and water deprivation
Restraint
Wet bedding
Rat bedding StrobeLight inversion
No bedding
Cage rotation
Food and water deprivation
Cold
Wet bedding
3Rat bedding
Strobe
Restraint
Light inversion
Cage rotation
No bedding
Light inversion
Food and water deprivation
Cold
Wet bedding
Cage tilt
Strobe
Light inversion
Overcrowding
4Cold
No bedding
Restraint
Food and water deprivation
Cage rotation
Strobe
Rat bedding
Light inversion
Cold
Cage tilt
Restraint
Wet bedding
Cage rotation
No bedding
5Cold
Food and water deprivation
Cage rotation
Strobe
Light inversion
Wet bedding
Cold
Cage tilt
Cage rotation
No bedding
Light inversion
Overcrowding
Restraint
Cage tilt

Additional files

Supplementary file 1

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

https://doi.org/10.7554/eLife.32420.021
Transparent reporting form
https://doi.org/10.7554/eLife.32420.022

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