Chemogenetic and Optogenetic Activation of Gαs Signaling in the Basolateral Amygdala Induces Acute and Social Anxiety-Like States

Abstract

Anxiety disorders are debilitating psychiatric illnesses with detrimental effects on human health. These heightened states of arousal are often in the absence of obvious threatening cues and are difficult to treat owing to a lack of understanding of the neural circuitry and cellular machinery mediating these conditions. Activation of noradrenergic circuitry in the basolateral amygdala is thought to have a role in stress, fear, and anxiety, and the specific cell and receptor types responsible is an active area of investigation. Here we take advantage of two novel cellular approaches to dissect the contributions of G-protein signaling in acute and social anxiety-like states. We used a chemogenetic approach utilizing the Gαs DREADD (rM3Ds) receptor and show that selective activation of generic Gαs signaling is sufficient to induce acute and social anxiety-like behavioral states in mice. Second, we use a recently characterized chimeric receptor composed of rhodopsin and the β2-adrenergic receptor (Opto-β2AR) with in vivo optogenetic techniques to selectively activate Gαs β-adrenergic signaling exclusively within excitatory neurons of the basolateral amygdala. We found that optogenetic induction of β-adrenergic signaling in the basolateral amygdala is sufficient to induce acute and social anxiety-like behavior. These findings support the conclusion that activation of Gαs signaling in the basolateral amygdala has a role in anxiety. These data also suggest that acute and social anxiety-like states may be mediated through signaling pathways identical to β-adrenergic receptors, thus providing support that inhibition of this system may be an effective anxiolytic therapy.

Figure 1

Gαs DREADD signaling in the BLA induces anxiety-like behavior. (a) Bilateral viral injection sites in the BLA. (b) AAV5-CaMKIIα-HA-GSD-IRES-mCitrine (rM3Ds; green) expression in BLA (DAPI=red; scale bar=50 μm). (c) Behavior diary. Representative open field traces of (d) control and (e) rM3Ds^BLA/CaMKIIα mice. (f) rM3Ds^BLA/CaMKIIα mice (green) show cumulatively less time in the center of the open field as compared with controls (black) following CNO administration (1 mg/kg, i.p.) (**P<0.01, multiple unpaired Student's t-tests). (g) Distance traveled of rM3Ds^BLA/CaMKIIα and control mice.

Figure 2

Gαs DREADD signaling in the BLA alters social interaction. (a) Behavior diary. Representative traces of (b) control and (c) rM3Ds^BLA/CaMKIIα mice in the absence (top) and presence (bottom) of a conspecific. (d) Control animals (black) spend more time in the social zone in the presence of a conspecific (solid bars) than in its absence (empty bars) (**P<0.01, paired Student's t-test), while rM3Ds^BLA/CaMKIIα (green;) animals spend less time in the social zone in the presence of a conspecific (**P<0.01, paired Student's t-test). (e) Both control (black) and rM3Ds^BLA/CaMKIIα (green) mice travel less distance in the presence of a novel mouse (solid bars) than in its absence (empty bars) (****P<0.0001, paired Student's t-test). (f) Both control (black) and rM3Ds^BLA/CaMKIIα (green) mice travel less distance in the presence of a novel mouse (solid bars) than in its absence (empty bars) (****P<0.0001, paired Student's t-test). (f) Control (black; P=0.084) and rM3Ds^BLA/CaMKIIα (green; **P<0.01 paired Student's t-test) mice make fewer entries into the social zone in the presence of a novel mouse (solid bars) than in its absence (empty bars).

Figure 3

Photoactivation of Opto-β₂AR in the BLA promotes anxiety-like behavior. (a) Bilateral viral injection sites and optic fiber implants. (b) Viral expression of lenti-CaMKIIα-opto-β₂AR-mCherry in the BLA (scale bar=50 μm). (c) Behavior diary. Representative traces of control (d) and opto-β₂AR^BLA/CaMKIIα (e) mice in OFT. (f) Cumulative time course shows opto-β₂AR^BLA/CaMKIIα (orange) mice spend less time in the center of an open field compared with controls (black) while receiving light stimulation (473 nm, 5 s off/on; **P<0.01, via multiple unpaired Student's t-tests). (g) In the OFT, viral control (black) and opto-β₂AR^BLA/CaMKIIα (orange) mice do not differ in total distance traveled.

Figure 4

Photoactivation of Opto-β₂AR does not alter RPTA or CPA. (a) Behavior diary and (b) chamber schematic. (c) Opto-β₂AR^BLA/CaMKIIα (orange) and control mice (black) spend similar amounts of time in the photostimulation-paired chamber in real-time place aversion (RTPA) and show no differences in total distance traveled (d). (e) Behavior diary and (f) chamber schematic. (g) Opto-β₂AR^BLA/CaMKIIα (orange) do not show an aversive response to the condition-photostimulation chamber. (h) Opto-β₂AR^BLA/CaMKIIα (orange) and control animals (black) show no differences in distances traveled during conditioning day 1 (CD1) and or conditioning day 2 (CD2) in CPA.

Figure 5

Photoactivation of Opto-β₂AR in the BLA alters social behavior. (a) Behavior diary. Representative traces of (b) control and (c) opto-β₂AR^BLA/CaMKIIα in the absence (top) and presence (bottom) of a novel conspecific. (d) Control animals (black) spend more time in the social zone in the presence of a novel conspecific (solid bars) than in its absence (empty bars) (*P<0.05, paired Student's t-test), while opto-β₂AR^BLA/CaMKIIα (orange) animals show no increased time spent in the social zone in the presence of a conspecific. (e) Both control (black; *P<0.05 paired Student's t-test) and opto-β₂AR^BLA/CaMKIIα (orange; ****P<0.0001, paired Student's t-test) mice travel less distance in the presence of a novel mouse (solid bars) than in its absence (empty bars). (f) Both control (black) and opto-β₂AR^BLA/CaMKIIα (orange) animals show similar number of entries into the social zone in the presence (solid bars) or absence (empty bars) of a novel mouse.