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Cited by 112Memory and Neural MechanismsNeuroscience and Neuropharmacology ResearchNeurotransmitter Receptor Influence on Behavior

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DREADD in Parvalbumin Interneurons of the Dentate Gyrus Modulates Anxiety, Social Interaction and Memory Extinction

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Zou, D., Chen, L., Deng, D., Jiang, D., Dong, F., McSweeney, C., Zhou, Y., Liu, L., Chen, G., Wu, Y., Mao, Y.·2016·Current Molecular Medicine
DOI 10.2174/1566524016666151222150024PMC PMC4997952
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Abstract

Parvalbumin (PV)-positive interneurons in the hippocampus play a critical role in animal memory, such as spatial working memory. However, how PV-positive interneurons in the subregions of the hippocampus affect animal behaviors remains poorly defined. Here, we achieved specific and reversible activation of PV-positive interneurons using designer receptors exclusively activated by designer drugs (DREADD) technology. Inducible DREADD expression was demonstrated in vitro in cultured neurons, in which co-transfection of the hM3D-Gq-mCherry vector with a Cre plasmid resulted in a cellular response to hM3Dq ligand clozapine-N-oxide (CNO) stimulation. In addition, the dentate gyrus (DG) of PV-Cre mice received bilateral injection of control lentivirus or lentivirus expressing double floxed hM3D-Gq-mCherry. Selective activation of PV-positive interneurons in the DG did not affect locomotor activity or depression-related behavior in mice. Interestingly, stimulation of PV-positive interneurons induced an anxiolytic effect. Activation of PVpositive interneurons appears to impair social interaction to novelty, but has no effect on social motivation. However, this defect is likely due to the anxiolytic effect as the exploratory behavior of mice expressing hM3DGq is significantly increased. Mice expressing hM3D-Gq did not affect novel object recognition. Activation of PV-positive interneurons in the DG maintains intact cued and contextual fear memory but facilitates fear extinction. Collectively, our results demonstrated that proper control of PV interneurons activity in the DG is critical for regulation of the anxiety, social interaction and fear extinction. These results improve our fundamental understanding of the physiological role of PV-positive interneurons in the hippocampus.

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Fig. (1) Selective expression of the DREADD receptor hM3Dq in PV-positive interneurons in the mouse hippocampus. (A) The lentiviral vector carrying the hM3D-Gq coding sequence under the control of the human elongation factor 1 α promoter (EF1αp) was designed for DIO-hM3D-Gq-mCherry. BGH-PA, bovine growth hormone-polyadenylation site. (B) CAD cells transfected with the DIO-hM3D-Gq-mCherry lentiviral vector alone did not exhibit mCherry fluorescence under a fluorescent microscope. However, co-transfection of the viral vector with Cre plasmid resulted in the expression of hM3D-Gq and mCherry (red). Nuclei were counterstained with DAPI. Scale bars = 10 μm. (C) Cre mice received bilateral injections of lentiviral DIO-hM3D-Gq-mCherry vectors into the hippocampus area. Two weeks after viral injection, brain slices were stained with TO-PRO dye to label nuclei (blue). Under a fluorescent microscope, the expression of hM3D-Gq (red) and PV (green) was detected in the hippocampus. Scale bars = 50 μm. (D) A full view of a hemisphere injected with lentivirus expressing mCherry shows the specific targeting. Scale bars = 200 μm. (Color figure available online).
Fig. (1) Selective expression of the DREADD receptor hM3Dq in PV-positive interneurons in the mouse hippocampus. (A) The lentiviral vector carrying the hM3D-Gq coding sequence under the control of the human elongation factor 1 α promoter (EF1αp) was designed for DIO-hM3D-Gq-mCherry. BGH-PA, bovine growth hormone-polyadenylation site. (B) CAD cells transfected with the DIO-hM3D-Gq-mCherry lentiviral vector alone did not exhibit mCherry fluorescence under a fluorescent microscope. However, co-transfection of the viral vector with Cre plasmid resulted in the expression of hM3D-Gq and mCherry (red). Nuclei were counterstained with DAPI. Scale bars = 10 μm. (C) Cre mice received bilateral injections of lentiviral DIO-hM3D-Gq-mCherry vectors into the hippocampus area. Two weeks after viral injection, brain slices were stained with TO-PRO dye to label nuclei (blue). Under a fluorescent microscope, the expression of hM3D-Gq (red) and PV (green) was detected in the hippocampus. Scale bars = 50 μm. (D) A full view of a hemisphere injected with lentivirus expressing mCherry shows the specific targeting. Scale bars = 200 μm. (Color figure available online).CC BY-NC · PubMed Central
Fig. (2) Patch-clamp recording of cells after CNO exposure. Hippocampal neurons were transfected with the lentiviral DIO-mCherry-hM3D-Gq vector alone (A) or in combination with Cre plasmid (B). Then 10 μM CNO (horizontal bar) was added to cell cultures, and the firing of action currents in mouse hippocampal neurons was recorded.
Fig. (2) Patch-clamp recording of cells after CNO exposure. Hippocampal neurons were transfected with the lentiviral DIO-mCherry-hM3D-Gq vector alone (A) or in combination with Cre plasmid (B). Then 10 μM CNO (horizontal bar) was added to cell cultures, and the firing of action currents in mouse hippocampal neurons was recorded.CC BY-NC · PubMed Central
Fig. (3) Animal behavior analysis in the open field test. PV-Cre mice were injected with the lentiviral control vector or lentiviral DIO-hM3D-Gq-mCherry vector. Thirty minutes before behavioral testing, mice were given CNO (0.5 mg/kg body weight) by intraperitoneal injection. During the open field test, the total distance traveled (cm) (A), cumulative duration in the center (s) (B), center entry frequency (C), duration spent mobile (s) (D), maximum velocity (cm/s) (E) or mean velocity (cm/s) (F) were recorded over 5 min. n = 26 for the control group; n = 27 for the hM3D-Gq group. NS, not significant.
Fig. (3) Animal behavior analysis in the open field test. PV-Cre mice were injected with the lentiviral control vector or lentiviral DIO-hM3D-Gq-mCherry vector. Thirty minutes before behavioral testing, mice were given CNO (0.5 mg/kg body weight) by intraperitoneal injection. During the open field test, the total distance traveled (cm) (A), cumulative duration in the center (s) (B), center entry frequency (C), duration spent mobile (s) (D), maximum velocity (cm/s) (E) or mean velocity (cm/s) (F) were recorded over 5 min. n = 26 for the control group; n = 27 for the hM3D-Gq group. NS, not significant.CC BY-NC · PubMed Central
Fig. (4) Elevated maze test. PV-Cre mice were injected with the lentiviral control vector or lentiviral DIO-hM3D-Gq-mCherry vector. Thirty minutes before behavioral testing, mice were given CNO (0.5 mg/kg body weight) by intraperitoneal injection. During the elevated maze test, the time the mice spent in the open arms (A), the number of entries into the open arms (B), the number of head dips (C), the number of vertical rears (D) and the number of wall leans (E) was recorded. n = 21 for the control group; n = 24 for the hM3D-Gq group. *p < 0.05. NS, not significant.
Fig. (4) Elevated maze test. PV-Cre mice were injected with the lentiviral control vector or lentiviral DIO-hM3D-Gq-mCherry vector. Thirty minutes before behavioral testing, mice were given CNO (0.5 mg/kg body weight) by intraperitoneal injection. During the elevated maze test, the time the mice spent in the open arms (A), the number of entries into the open arms (B), the number of head dips (C), the number of vertical rears (D) and the number of wall leans (E) was recorded. n = 21 for the control group; n = 24 for the hM3D-Gq group. *p < 0.05. NS, not significant.CC BY-NC · PubMed Central
Fig. (5) Comparison of the immobile time between control mice and mice with hM3D-Gq-expressing PV-positive interneurons in the tail suspension test. PV-Cre mice were injected with the lentiviral control vector or lentiviral DIO-hM3D-Gq-mCherry vector. Thirty minutes before behavioral testing, mice were given CNO (0.5 mg/kg body weight) by intraperitoneal injection. During the tail suspension test, the immotility time (sec) was recorded. n = 26 for each group. NS, not significant.
Fig. (5) Comparison of the immobile time between control mice and mice with hM3D-Gq-expressing PV-positive interneurons in the tail suspension test. PV-Cre mice were injected with the lentiviral control vector or lentiviral DIO-hM3D-Gq-mCherry vector. Thirty minutes before behavioral testing, mice were given CNO (0.5 mg/kg body weight) by intraperitoneal injection. During the tail suspension test, the immotility time (sec) was recorded. n = 26 for each group. NS, not significant.CC BY-NC · PubMed Central
Fig. (6) Social interaction test. PV-Cre mice were injected with the lentiviral control vector or lentiviral DIO-hM3D-Gq-mCherry vector. Thirty minutes before behavioral testing, mice were given CNO (0.5 mg/kg body weight) by intraperitoneal injection. (A) In the first session of the social interaction test, the average duration of time (sec) spent in the chamber with the Stranger 1 mouse or with the empty cup (empty) was recorded. Both of the cherry and hM3D-Gq group mice preferred the stranger 1 mouse. Statistical analysis was conducted using two-way analysis of variance (ANOVA). Treatment vs chamber F(1,84)=0.350, P=0.556; treatment F(1,84)=5.922, P=0.017; chamber F(1,84)=11.768, P=0.001. (B) In the second session, the average duration of time (sec) spent in the chamber with the Stranger 1 mouse or with a new unfamiliar mouse (Stranger 2) was recorded. n = 22 for the control group; n = 22 for the hM3D-Gq group. Control group, P=0.033; hM3D-Gq group, P=0.947. Treatment×chamber F(1,84)=2.016, P=0.159; F(1,84)=8.806, P=0.004; chamber F(1,84)=0.982, P=0.325.
Fig. (6) Social interaction test. PV-Cre mice were injected with the lentiviral control vector or lentiviral DIO-hM3D-Gq-mCherry vector. Thirty minutes before behavioral testing, mice were given CNO (0.5 mg/kg body weight) by intraperitoneal injection. (A) In the first session of the social interaction test, the average duration of time (sec) spent in the chamber with the Stranger 1 mouse or with the empty cup (empty) was recorded. Both of the cherry and hM3D-Gq group mice preferred the stranger 1 mouse. Statistical analysis was conducted using two-way analysis of variance (ANOVA). Treatment vs chamber F(1,84)=0.350, P=0.556; treatment F(1,84)=5.922, P=0.017; chamber F(1,84)=11.768, P=0.001. (B) In the second session, the average duration of time (sec) spent in the chamber with the Stranger 1 mouse or with a new unfamiliar mouse (Stranger 2) was recorded. n = 22 for the control group; n = 22 for the hM3D-Gq group. Control group, P=0.033; hM3D-Gq group, P=0.947. Treatment×chamber F(1,84)=2.016, P=0.159; F(1,84)=8.806, P=0.004; chamber F(1,84)=0.982, P=0.325.CC BY-NC · PubMed Central
Fig. (7) Novel object recognition test. PV-Cre mice were injected with the lentiviral control vector or lentiviral DIO-hM3D-Gq-mCherry vector. Thirty minutes before behavioral testing, mice were given CNO (0.5 mg/kg body weight) by intraperitoneal injection. During the novel object recognition test, the duration of contact (sec) over 5 min was recorded. n = 23 for the control group; n = 22 for the hM3D-Gq group. (A) Short-term memory (1 h). Statistical analysis was conducted using two-way analysis of variance (ANOVA). Treatment F(1,86)=2.762, P=0.100; object F(1,86)=2.050, P=0.156). (B) Long-term memory (24 h). Treatment F(1,86)=0.099, P=0.754; object F(1,86)= 0.078, P=0.780.
Fig. (7) Novel object recognition test. PV-Cre mice were injected with the lentiviral control vector or lentiviral DIO-hM3D-Gq-mCherry vector. Thirty minutes before behavioral testing, mice were given CNO (0.5 mg/kg body weight) by intraperitoneal injection. During the novel object recognition test, the duration of contact (sec) over 5 min was recorded. n = 23 for the control group; n = 22 for the hM3D-Gq group. (A) Short-term memory (1 h). Statistical analysis was conducted using two-way analysis of variance (ANOVA). Treatment F(1,86)=2.762, P=0.100; object F(1,86)=2.050, P=0.156). (B) Long-term memory (24 h). Treatment F(1,86)=0.099, P=0.754; object F(1,86)= 0.078, P=0.780.CC BY-NC · PubMed Central
Fig. (8) Fear conditioning test. PV-Cre mice were injected with the lentiviral control vector or lentiviral DIO-hM3D-Gq-mCherry vector. Thirty minutes before behavioral testing, mice were given CNO (0.5 mg/kg body weight) by intraperitoneal injection. A sketch of experimental paradigm is shown (A). During the fear conditioning test, the percentage of freezing behavior in different trials, including (B) contextual fear conditioning-extinction learning, (C) cued fear conditioning-acclimation testing, and (D) cued fear conditioning-interval freezing, was calculated. n = 23 for the control group; n = 19 for the hM3D-Gq group. *p < 0.05.
Fig. (8) Fear conditioning test. PV-Cre mice were injected with the lentiviral control vector or lentiviral DIO-hM3D-Gq-mCherry vector. Thirty minutes before behavioral testing, mice were given CNO (0.5 mg/kg body weight) by intraperitoneal injection. A sketch of experimental paradigm is shown (A). During the fear conditioning test, the percentage of freezing behavior in different trials, including (B) contextual fear conditioning-extinction learning, (C) cued fear conditioning-acclimation testing, and (D) cued fear conditioning-interval freezing, was calculated. n = 23 for the control group; n = 19 for the hM3D-Gq group. *p < 0.05.CC BY-NC · PubMed Central

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Authors
Zou, D., Chen, L., Deng, D., Jiang, D., Dong, F., McSweeney, C., Zhou, Y., Liu, L., Chen, G., Wu, Y., & Mao, Y.
Affiliations
  • Pennsylvania State University (US)
  • Guangxi Medical University (CN)
Corresponding author
  • Wu, Y.
  • Mao, Y.
ORCID
Venue
Current Molecular Medicine
Publisher
Bentham Science Publishers Ltd.
Published
2016-01-08
Volume
16
Issue
1
Pages
91-102
ISSN
1566-5240 (ISSN-L)
DOI
10.2174/1566524016666151222150024
PMC
PMC4997952
OpenAlex
W2202096406
Cited by
112
References
64
Citations by year
2026: 4 · 2025: 8 · 2024: 3 · 2023: 8 · 2022: 22 · 2021: 23 · 2020: 14 · 2019: 14 · 2018: 3 · 2017: 9 · 2016: 4
FWCI
6.36
Type
journal article
Language
en
Open access
Locations
  • Current Molecular Medicine (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
  • National Alliance for Research on Schizophrenia and Depression
  • National Natural Science Foundation of China (81160167, 81360201)
Topics
  • Life Sciences › Neuroscience › Cognitive Neuroscience › Memory and Neural Mechanisms
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neurotransmitter Receptor Influence on Behavior
Keywords
parvalbumin interneurons, dentate gyrus, DREADD, clozapine-N-oxide, optogenetic stimulation, anxiety, social interaction, fear extinction, fear memory, hippocampus, social motivation, novel object recognition
MeSH terms
Animals, Anxiety, Anxiety Disorders, Depression, Fear, Hippocampus, Humans, Interneurons, Interpersonal Relations, Male, Memory, Motor Activity, Parvalbumins, Dentate Gyrus, Mice, HEK293 Cells

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