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

View Article: PubMed Central - PubMed

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 PV-positive 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 hM3D-Gq 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.

No MeSH data available.


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).
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Figure 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).

Mentions: To specifically activate PV-positive interneurons in the dentate gyrus (DG), hM3Dq, which has high binding capacity for CNO but not endogenous ligand, was fused with mCherry red fluorescence protein (RFP) and subcloned into a Cre-inducible lentiviral construct. The hM3Dq-mCherry DNA fragment was flanked with double loxp sites such that only when the Cre recombinase is present will hM3Dq be expressed (Fig. 1A). The lentiviral DIO-mCherry vector was used as a negative control. To confirm the inducibility, the DIO-hM3D-Gq-mCherry construct plasmid was transfected into neuroblastoma Cath.a-differentiated (CAD) cells without the Cre-expressing construct. We did not observe any mCherry expression (Fig. 1B), suggesting that there is no leakage of expression. When this transfection was combined with the Cre plasmid, notable expression of mCherry was observed in cells (Fig. 1B), confirming that the expression of hM3Dq in cells was Cre-dependent.


DREADD in Parvalbumin Interneurons of the Dentate Gyrus Modulates Anxiety, Social Interaction and Memory Extinction
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).
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Figure 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).
Mentions: To specifically activate PV-positive interneurons in the dentate gyrus (DG), hM3Dq, which has high binding capacity for CNO but not endogenous ligand, was fused with mCherry red fluorescence protein (RFP) and subcloned into a Cre-inducible lentiviral construct. The hM3Dq-mCherry DNA fragment was flanked with double loxp sites such that only when the Cre recombinase is present will hM3Dq be expressed (Fig. 1A). The lentiviral DIO-mCherry vector was used as a negative control. To confirm the inducibility, the DIO-hM3D-Gq-mCherry construct plasmid was transfected into neuroblastoma Cath.a-differentiated (CAD) cells without the Cre-expressing construct. We did not observe any mCherry expression (Fig. 1B), suggesting that there is no leakage of expression. When this transfection was combined with the Cre plasmid, notable expression of mCherry was observed in cells (Fig. 1B), confirming that the expression of hM3Dq in cells was Cre-dependent.

View Article: PubMed Central - PubMed

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 PV-positive 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 hM3D-Gq 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.

No MeSH data available.