New Results

β-arrestin-dependent ERK signaling positively correlates with reduced anxiety-like and conditioned fear-related behavior in mice

Mee Jung Ko, Terrance Chiang, Arbaaz M. Mukadam, Grace E. Mulia, Anna M. Gutridge, Julia A. Chester, Richard M. van Rijn
doi: https://doi.org/10.1101/790568

Summary

Exposure to anxiety- or fear-invoking stimuli initiate a convergence of executive actions orchestrated by multiple proteins and neurotransmitters across the brain. Dozens of G protein-coupled receptors (GPCRs) have been linked to regulation of fear and anxiety. GPCR signaling involves canonical G protein pathways but may also engage downstream kinases and effectors through β-arrestin scaffolds. Here, we investigate whether β-arrestin signaling is critical for the emotional regulation of anxiety-like and fear-related behavior. Using the δ-opioid receptor (δOR) as a model GPCR, we found that β-arrestin 2-dependent activation of extracellular signal–regulated kinases (ERK1/2) in the dorsal hippocampus and the amygdala are critical for δOR-induced anxiolytic-like effects. In contrast, G protein-mediated δOR signaling was associated with decreased ERK1/2 activity and increased fear-related behavior. Our results also suggest a potential contribution of β-arrestin 1 in fear-reducing effects. Overall, our findings highlight the significance of non-canonical β-arrestin signaling in the regulation of emotions.

Introduction

Anxiety- and fear-related behaviors are evolutionary adaptive behaviors important for human survival. However, excessive stimulation of the neural circuits that regulate anxiety- and fear-related behaviors can lead to harmful psychiatric disorders. The expression of anxiety- and fear-related behaviors is regulated by complex integration of both internal and external physiological and sensory cues that influence reflexive behavior, cognitive control, and executive functions. Accordingly, behavioral correlates of anxiety and fear are regulated by a harmonious activity of neurotransmitters and cellular actions across many overlapping and distinct neural circuits (Tovote et al., 2015).

With regard to cellular actions underlying (patho-)physiological behavior, G protein-coupled receptors (GPCRs) play an important role in neuronal signaling. GPCRs bind neurotransmitters and initiate intracellular signal transduction pathways which ultimately affect neuronal excitability, neurotransmitter release and synaptic plasticity. GPCRs, including serotonergic (Akimova et al., 2009), dopaminergic (de la Mora et al., 2010), adrenergic (Kindt et al., 2009), opioidergic (Land et al., 2009) and corticotropin-releasing factor receptors (Takahashi, 2001) have well-documented roles in the modulation of anxiety and fear.

Traditionally, drug development at GPCRs has focused on the canonical G protein pathways; however, the prior two decades have introduced arrestin-dependent signaling as a new concept of GPCR signal transduction. In particular, the ‘non-visual’ arrestins 2 and 3, referred here as β-arrestin 1 and 2, respectively, have been associated with specific and unique drug effects. The primary role of β-arrestins is to desensitize GPCRs, for example, HIV1-tat infection increases β-arrestin 2 expression in mice amygdala, leading to significant reduction in morphine efficacy in this region (Hahn et al., 2016). Beyond desensitization, β-arrestin 2 may also partake in receptor signaling by scaffolding with various kinases. For example, β-arrestin 2 p38 MAP kinase signaling has been linked to the aversive effects of κ-opioid receptor (κOR) agonists (Bruchas et al., 2006; Land et al., 2009), whereas a β-arrestin 2-GSK3β/AKT scaffold appears to be driving the antipsychotic effects of dopamine D2 receptors agonists (Allen et al., 2011; Beaulieu et al., 2005). Currently, few studies have investigated how signaling scaffolds involving the β-arrestin isoforms may influence anxiety- and fear-like behavior. It is important to begin to address this gap in our current knowledge of the GPCR modulation of psychiatric behavior, especially since the majority of medications that target GPCRs were developed without consideration of the potential adverse or therapeutic effects of β-arrestin signaling. Yet, it is now possible to develop molecules that preferentially activate or avoid β-arrestin signaling and thus have the potential to treat psychiatric disorders more effectively and with a wider therapeutic window.

Here, we describe our efforts to elucidate the roles of β-arrestin isoforms in mediating GPCR signaling in relation to the modulation of anxiety and fear-like behavior. We chose to utilize the δ-opioid receptor (δOR) as a model GPCR. Previous studies have shown that the δOR selective agonist SNC80 is an efficacious recruiter of β-arrestin 1 and 2 proteins (Chiang et al., 2016; Pradhan et al., 2016; Vicente-Sanchez et al., 2018), and has anxiolytic-like (Saitoh et al., 2004; Saitoh et al., 2018) and fear-reducing effects (Li et al., 2009; Saitoh et al., 2004). Moreover, the δOR-selective agonist TAN67, which is a poor β-arrestin 2 recruiter does not reduce anxiety-like behavior in naïve mice (van Rijn et al., 2010), providing further support for the correlation between β-arrestin 2 signaling and anxiety-like behavior.

Mitogen activated protein kinases (MAPKs) have been implicated with mood disorders and can scaffold with β-arrestin (Coyle and Duman, 2003; Lefkowitz and Shenoy, 2005). Studies have suggested that MAPK signaling, specifically ERK1/2, in the hippocampus and the basolateral amygdala is required for the acquisition and extinction of fear memory (Atkins et al., 1998; Herry et al., 2006). Therefore, we hypothesized that β-arrestin-dependent MAPK signaling may contribute to anxiety-like and fear-related behavior. To test our hypothesis, we assessed the degree to which β-arrestin isoforms and MAPK activation were involved in δOR agonist-mediated modulation of unconditioned anxiety-related behavior and cued-induced fear-related behavior. Our results suggest that ERK1/2 activity is differentially modulated by G protein and β-arrestin signaling and is correlated with anxiety-like and fear-related responses in C57BL/6 mice. We noted that different β-arrestin isoforms were involved in the activation of ERK1/2 across various brain regions, including the striatum, hippocampus and amygdala.

Results

Involvement of β-arrestin 2 in the modulation of anxiety-like behavior

In 2016, Astra Zeneca revealed that their novel δOR selective agonist AZD2327 (Fig. 1a, Left) was capable of reducing anxiety-like behavior in mice (Richards et al., 2016). AZD2327 is not commercially available, but is structurally similar to SNC80, a commercially available δOR selective agonist (Fig. 1a, Right). SNC80 is a known super-recruiter of β-arrestin 2 (Chiang et al., 2016) (Fig. 1b) and similar to AZD2327 exhibits anxiolytic-like effects in rodents (Saitoh et al., 2004; van Rijn et al., 2010). These previous findings led us to hypothesize that β-arrestin 2 may be required for the anxiolytic effects of SNC80 and AZD2327. Using two models of anxiety-like behavior, the elevated plus maze (EPM) test and dark-light box transition test (Fig. 1c), we measured the behavioral effects of SNC80 in β-arrestin 2 KO mice, at a dose known to produce anxiolytic-like effects in wild-type (WT) mice (van Rijn et al., 2010). As expected, systemic administration of SNC80 (20 mg/kg, s.c.) significantly increased the time WT mice spent in the open arm (elevated plus maze test: Fig. 1d; two-way ANOVA, Drug effect: F1, 68= 8.781, p=0.004) and the light chamber (Dark-light box test: Fig. 1e; Drug effect: F1, 59 = 4.978, p=0.03). As we predicted, the anxiolytic effects of SNC80 were attenuated in β-arrestin 2 KO mice (Elevated plus maze test: Fig. 1d; Genotype effect: F1, 68 = 3.15, p=0.08; Dark-light box test: Fig. 1e; Genotype effect: F1, 59 = 1.039, p=0.312). Although the total movement in the elevated plus maze was slightly lower in β-arrestin 2 KO mice than WT mice, no drug effects were observed in both genotypes (elevated plus maze test: Fig. 1f; two-way ANOVA, Drug vs. Genotype effect: F1, 68 = 0.339, p=0.56; Genotype effect: F1, 68 = 20.7, p<0.0001; Drug effect: F1, 68 = 0.796, p=0.38). Likewise, no statistical difference in total transition was observed in the dark light transition box test (dark-light box test: Fig. 1g; two-way ANOVA, Drug vs. Genotype effect: F1, 57 = 1.754, p=0.19; Genotype effect: F1, 57 = 0.1222, p<0.0001; Drug effect: F1, 57 = 0.037, p=0.85); however, as previously described, SNC80 produced hyperlocomotive behavior in mice ((Chiang et al., 2016), Fig. S1).

Figure 1.
Figure 1. Beneficial role for β-arrestin 2 in reducing anxiety-like behavior

(a) Chemical similarity between AZD2327, a δOR agonist used in phase II clinical trials for anxious major depressive disorder and SNC80. (b) Scheme highlighting that SNC80 is δOR selective agonist, that activates Gi proteins but also strongly recruits β-arrestin 2. (c) Schematic diagram of the elevated plus maze test and dark-light box test. (d) The β-arrestin-biased δOR agonist, SNC80 (20 mg/kg, s.c.), significantly increased percentage of time spent in open arms in WT mice (n=15). but not β-arrestin 2 KO mice (n=21). (e) SNC80 increased percentage of time spent in light box in WT mice (control: n=12, SNC80: n=11), but not in β-arrestin 2 KO mice (n=20). (f) The total time of movements were equal between drug treatments. (g) No statistical significance was observed in the total transitions between light and dark chambers. (Significance was calculated by two-way ANOVA followed by a Sidak’s multiple comparison; *p<0.05; all values are shown as individaul data points ± S.E.M.).

The β-arrestin recruiting δOR agonist SNC80 strongly activates ERK1/2 in vitro and in vivo

Activation of κOR has been associated with β-arrestin 2-mediated p38 phosphorylation (Bruchas et al., 2006). To determine if δOR agonism similarly stimulates mitogen-activated protein kinases (MAPKs), we measured p38, JNK, and ERK1/2 activation in Chinese Hamster Ovarian cells stably expressing δOR and β-arrestin 2 (CHO-δOR-βArr2) following stimulation with 10 µM SNC80, a concentration that will fully activate G-protein signaling and induce β-arrestin 2 recruitment (Robins et al., 2018b). We found that SNC80 led to a rapid increase in ERK1/2 phosphorylation within 3 minutes in CHO-δOR-βArr2 cells, which lasted until 60 minutes, in agreement with previous δOR-mediated ERK activation in CHO cells (Rozenfeld and Devi, 2007). We did not observe strong activation of p38 and JNK by SNC80 (Fig. 2a). The δOR mediated ERK1/2 signaling in these cells were not an artifact of the recombinant overexpression of δOR and β-arrestin 2 in the CHO cells as we observed a similar profile for ERK1/2 activation in NG108-15 neuroblastoma cells endogenously expressing δOR and β-arrestin (Cen et al., 2001; Eisinger et al., 2002; Klee et al., 1982) (Fig. 2b; one-way ANOVA, F4,30=6.958, p<0.0001). We similarly found ERK1/2 activation in several mouse brain regions, known to express δORs, including the dorsal hippocampus, the amygdala and the striatum (Chu Sin Chung et al., 2015; Erbs et al., 2015) (Fig. 2c-e). The SNC80-induced ERK1/2 activation in these regions was confirmed and quantified by the Western blot analysis of flash-frozen tissue punches upon collection (Fig. 2f). Here, we observed that SNC80 (20 mg/kg, i.p.) significantly increased ERK1/2 phosphorylation at the 10-minute time-point in all tested brain regions except for the ventral hippocampus of WT mice (Fig. 2g-k; see Table S1 for one-way ANOVA and post-hoc multiple comparison), and these activations returned to basal levels 30 minutes after the SNC80 administration.

Figure 2.
Figure 2. The anxiolytic agonist SNC80 activates ERK1/2 in vitro and in the limbic and striatal regions of the brain

(a) 10 uM SNC80 strongly increased ERK1/2 phosphorylation, compared to p38 and JNK, in CHO cells stably expressing δOR and β-arrestin 2. A representative Western blot image is presented to the right. (b) SNC80 increased ERK1/2 phosphorylation in a time-dependent manner, in NG-108-15 cells endogenously expressing δOR. A representative Western blot image is shown on the right. Representative immunoflouresence staining images of the limbic regions (c) and striatal regions (d) from mice 10 minute post administration of saline or SNC80 (20 mg./kg i.p.; Green: ERK; Red: pERK; Blue: DAPI; Image was stitched at 10x magnification and the scale is provided in the image). (e) 20x magnification images of the hippocampal/amgdalar and striatal slices (The corresponding location is marked in white squre in 10x images). (f) Representative images of brain sections with micropunctures of five specified brain regions for the Western blot analysis. (g-k) Activation of ERK1/2 protein activity following systemic adminstration of SNC80 in five wild-type mice brain regions. Representative Western blot images are depicted below the respective bar graph (For (a), significance was measured by two-way ANOVA followed by a Dunnett’s multiple comparison. For (c, i-m), significance was analyzed by one-way ANOVA followed by a Tukey’s multiple comparison; *p < 0.05, **p<0.01, ****p < 0.0001; all values are shown as individual data points ± S.E.M.).

β-arrestin 2 is required to activate ERK1/2 signaling in the limbic structures of the brain

To determine if β-arrestin 2 is responsible for the ERK1/2 activation in the tested brain regions (Fig. 2g-k), we injected 20 mg/kg of SNC80 in the β-arrestin 2 KO mice (Fig. 3a) and measured levels of ERK1/2 activation in the five brain regions tested in Figure 2. While, SNC80 still strongly activated ERK1/2 in the striatum and the nucleus accumbens of the β-arrestin 2 KO mice (Fig. 3b-c; see Table S1 for one-way ANOVA and post-hoc multiple comparison), we did not observe significant SNC80-induced ERK1/2 activation in the amygdala, the ventral hippocampus and the dorsal hippocampus of these KO mice (Fig. 3d-f; see Table S1 for one-way ANOVA and post-hoc multiple comparison).

Figure 3.
Figure 3. ERK1/2 activation in the amygdala and the dorsal hippocampus are β-arrestin 2 dependent

(a) Schematic diagram of the cellular context in β-arrestin 2 genetic KO mice. SNC80 (20 mg/kg, i.p.) induced ERK1/2 activation in the dorsal striatum (b) and nucleus accumbens (c). β-arrestin 2 KO ablated the SNC80-induced ERK1/2 activation in the dorsal hippocampus (d) and the amygdala (e) with no effects in the ventral hippocampus. (f) Representative Western blot images are shown tothe right of the related bar graph. (Significance was analyzed by one-way ANOVA followed by a Tukey’s multiple comparison; *p < 0.05, **p<0.01; all values are shown as individual data points ± S.E.M.).

ERK1/2 signaling plays a key role in the SNC80-mediated anxiolytic-like effects

We next assessed if the anxiolytic effects of SNC80 were dependent on ERK1/2 activation. We administered wild-type mice with SL327, a MEK1/2 inhibitor that indirectly prevents ERK1/2 activation, (Fig. 4a) (Tohgo et al., 2002). We found that SL327 (50 mg/kg, s.c.) ablated the anxiolytic-like effects of SNC80 (20 mg/kg, i.p.) in WT mice (Fig. 4b, one-way ANOVA, F3,34=12.35, p<0.0001). Thus, SNC80 anxiolytic-like behavior relied on the presence of β-arrestin 2 (Fig. 1d,e) and MEK/ERK1/2 activation (Fig. 2g-k). The hippocampus is a brain region associated with anxiety-like behavior in the elevated plus maze (File et al., 2000) 10837844 and δOR-agonism in the dorsal hippocampus, as well as the amygdala reduces anxiety-like behavior in the open field test (Saitoh et al., 2018; Solati et al., 2010). These published findings agree with our observation of SNC80-induced β-arrestin 2-dependent ERK1/2 activity specifically in these two brain regions (Fig. 3d,e). Therefore, if the β-arrestin-mediated ERK1/2 signaling in these two regions was critical for the anxiolytic-like effects of SNC80, we would expect ERK1/2 activity to be abolished in these regions in the mice co-treated with SNC80 and SL327. Indeed, we found that SL327 effectively decreased SNC80-induced ERK1/2 activity in the dorsal hippocampus and the amygdala (Fig. 4c,d, see Table S2 for detailed statistics).

Figure 4.
Figure 4. ERK1/2 is required for the anxiolytic-like effects induced by SNC80

(a) A schematic diagram of SL327 induced inhibition of SNC80 mediated ERK1/2 signaling. (b) SL327 (50 mg/kg, s.c.), attenuated the anxiolytic-like effects of SNC80 (20 mg/kg i.p.) in WT mice (control: n=8, SNC80: n=12, SNC+SL: n=12, SL327: n=12). (c-d) SL327 significantly inhibited SNC80-induced ERK1/2 phosphorylation in the dorsal hippocampus and the amygdala (Significance was calculated by one-way ANOVA followed by a Sidak’s or Tukey’s multiple comparison; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, #p<0.05; all values are shown as individual data points ± S.E.M.; SNC+SL means SNC80+SL327 and SL means SL327).

Fear-potentiated startle behavior is correlated with ERK1/2 activity but is not mediated by β-arrestin 2

Besides reducing anxiety-like behavior, δOR activation can also alleviate conditioned fear-related behavior (Saitoh et al., 2004; Sugiyama et al., 2019). Based on our results and previous studies, we hypothesized that SNC80 may similarly reduce fear-related behavior through a mechanism that involves β-arrestin 2. To measure conditioned fear, we utilized a mouse behavior paradigm of fear-potentiated startle (FPS) (Fig. 5a). In WT mice (Fig. 5b), we noted that SNC80 (20 mg/kg, i.p.) significantly reduced startle responses to the unconditioned ‘noise’ cue as well as to the conditioned ‘light+noise’ cue (Fig. 5c; two-way ANOVA, stimuli x drug effects: F2,120=20.42, p<0.0001, stimuli effects: F2,120 = 92.8, p<0.0001, drug effects: F1,120= 63.99, p<0.0001), resulting in a significant reduction in % FPS response (Fig. 5d; unpaired t-test, p=0.0085). To our surprise, we found that SNC80 was equally effective in reducing % FPS responses in β-arrestin 2 KO mice (Fig. 5e-f; two-way ANOVA, stimuli x drug effects: F2,42=20.22, p<0.0001, stimuli effects: F2,42 = 51.52, p<0.0001, drug effects: F1,42= 40.4, p<0.0001; noise: p=0.01, light+noise: p<0.0001 after Bonferroni’s multiple comparison; Fig. 5g; unpaired t-test, p=0.0003; Fig. 5d,g; comparison between WT and β-arrestin 2 KO via two-way ANOVA, Genotype x Drug effect: F1,53=1.306, p=2.583; Genotype effect: F1,53=5.055, p=0.029; Drug effect: F1,53=18.98, p<0.0001). While SNC80 is a very efficacious recruiter of β-arrestin, it still also fully activates Gi protein signaling (Chiang et al., 2016; Robins et al., 2018b). Thus, we next hypothesized that the observed fear-reducing effects of SNC80 could be mediated through Gi protein signaling. To address this hypothesis, we utilized a δOR selective agonist, TAN67, which is a poor recruiter of β-arrestin (Fig. 5f), and considered Gi protein-biased (Chiang et al., 2016; Robins et al., 2018b). However, when we administered TAN67 (25 mg/kg, i.p.) to our WT mice (Fig. 5h), we found that TAN67 did not significantly change startle to the noise or to the light+noise stimuli (Fig. 5i; two-way ANOVA, stimuli x drug effects: F2,42=0.725, p=0.491, stimuli effects: F2,42 = 25.06, p<0.0001, drug effects: F1,42 = 0.275, p=0.603), but it did produce a significant increase in %FPS (Fig. 5j; unpaired t-test, p<0.0001). The lack of a direct effect of TAN67 on noise-alone startle is in agreement with our previous finding that TAN67 did not change basal anxiety-like behavior in the elevated plus maze and dark-light transition test (van Rijn et al., 2010). Interestingly, our Western blot analysis of ERK1/2 activities in WT mice revealed that this Gi protein-biased agonist, TAN67, decreased ERK1/2 phosphorylation in the dorsal striatum, the nucleus accumbens, the dorsal hippocampus and the amygdala (Fig. 6a-d). In the ventral hippocampus, TAN67 did not alter ERK1/2 activity (Fig. 6e), which was similar to the lack of ERK1/2 modulation by SNC80 in this region (Fig. 2m) and may be indicative of low δOR expression in the ventral region as previously described (Mansour et al., 1987).

Figure 5.
Figure 5. Unique modulation of fear-potentiated startle (FPS) by a β-arrestin-biased and G protein-biased δOR agonist

(a) Schematic representation of the three-day experimental paradigm of the fear potentiated startle test; drugs were administered prior to the tests on the third day (See Figure S4 for Day 1 acoustic startle test). (b-d) SNC80 (n=21) reduced raw startle amplitudes to noise or light+noise and % FPS during the FPS test in WT mice. (e-g) SNC80 also reduced raw startle amplitudes to noise or light+noise and % FPS in β-arrestin 2 KO mice (n=8). (h-j) Yet, G protein-biased agonist, TAN67, increased % FPS in WT mice (n=8) (Significance was measured by two-way ANOVA followed by a Tukey’s multiple comparison or unpaired t-test; *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001; all values are shown as individual data points ± S.E.M.).

Figure 6.
Figure 6. Differential roles for G protein and β-arrestin 1 in δOR agonist-induced ERK1/2 activation

(a-e) The G protein-biased δOR agonist TAN67 (25 mg/kg, i.p.), decreased the basal ERK1/2 activity in all tested brain regions of wild-type mice. Representative Western blot images are depicted next to each related bar graph. (f-j). Systemic administration of SNC80 (20 mg/kg, i.p.) did not activate ERK1/2 in the striatal regions of β-arrestin 1 KO mice but resulted in persistent ERK1/2 activation in the dorsal and ventral hippocampus and amygdala (Significance was analyzed by one-way ANOVA followed by a Tukey’s multiple comparison; *p < 0.05, **p<0.01; all values are shown as individual data points ± S.E.M.).

Potential roles for ERK1/2 and β-arrestin 1 in the modulation of conditioned-fear behavior

Our results suggest that SNC80 reduces conditioned fear through a mechanism that does not involve β-arrestin 2 or G protein signaling. Therefore, we next hypothesized that the effect may be mediated by β-arrestin 1 instead. However, SNC80 is known to induce severe seizures in β-arrestin 1 KO mice (Vicente-Sanchez et al., 2018), preventing us from testing the FPS response of SNC80 in this strain. Instead, we measured SNC80-induced ERK1/2 phosphorylation in the β-arrestin 1 KO mice. In comparison to WT mice (Fig. 2g-k), genetic knockout of β-arrestin 1 prevented SNC80-mediated ERK1/2 phosphorylation in the dorsal striatum and nucleus accumbens (Fig. 6f-g; see Table S3 for one-way ANOVA and post-hoc multiple comparison). In the amygdala and dorsal hippocampus of β-arrestin 1 KO mice, SNC80 did increase ERK1/2 phosphorylation (Fig. 6h-i), but in contrast to the response observed in WT mice, the activation was sustained for at least 30 minutes. Additionally, we observed the same trend in the ventral hippocampus (Fig. 6j), a region where we had observed no δOR agonist-mediated modulation of ERK1/2 in WT mice (Fig. 2k and Fig. 6e). Thus, we hypothesized that the increased ERK1/2 in these regions is most likely a result of seizure activity and not necessarily a result of δOR-mediated effects.

Discussion

Here, we investigated the hypothesis that β-arrestin can modulate anxiety- and conditioned fear-related behavior via downstream MAPK activation. By utilizing G protein- and β-arrestin-biased δOR agonists together with β-arrestin-isoform selective knockout mice, we discovered that G protein, β-arrestin 1, and β-arrestin 2 uniquely modulated ERK1/2 activity resulting in differential outcomes in mouse models of anxiety/fear-related behavior. Our results suggest that the reduction in anxiety-like behavior by SNC80 required the presence of β-arrestin 2 as well as activation of ERK1/2. Distinctly in the dorsal hippocampus, we found that ERK1/2 activation was β-arrestin 2-dependent (Fig. 7a). Notably, G protein-biased signaling by TAN67 reduced ERK1/2 phosphorylation and was correlated with increased FPS (Fig. 7b). We found that SNC80-induced ERK1/2 activation in the nucleus accumbens and the dorsal striatum required β-arrestin 1, which may be part of the mechanism for SNC80 to decrease FPS (Fig. 7c).

Figure 7.
Figure 7. Graphical summary

δOR agonists differentially induce ERK activation in a β-arrestin isoform specific manner to modulate anxiety- and conditioned fear-related behaviors. SNC80 induced β-arrestin 2-mediated ERK1/2 activation (Cellular Makeup) in the hippocampus and amygdala (Localization) decreased anxiety-like behavior (a), whereas an increase in conditioned fear-related behavior (Behavior) by TAN67 can be linked to decreased ERK1/2 activity in all tested brain regions except for the ventral hippocampus (b). Decreased FPS could not be correlated with G protein or β-arrestin 2 signaling, but may involve β-arrestin −1 dependent ERK1/2 signaling in the dorsal striatum and nucleus accumbens (c).

Differential roles for β-arrestin isoforms in neuropsychiatric behavior

The β-arrestin proteins were discovered in quick succession (Attramadal et al., 1992; Lohse et al., 1990). Surprisingly, despite the availability of genetic KO mice for each isoform (Bohn et al., 1999; Conner et al., 1997). Studies investigating β-arrestin in the CNS have largely focused on β-arrestin 2 and have generally neglected β-arrestin 1 (Latapy and Beaulieu, 2013; Whalen et al., 2011). A potential reason for the preference of studying β-arrestin 2 may be that when the β-arrestin 2 knockout mice were generated their first utilization was to highlight the proteins’ involvement with the CNS-mediated adverse effects of µ-opioid receptor agonism (Bohn et al., 2000; Raehal et al., 2005). Researchers have only recently begun to utilize β-arrestin 1 KO mice to study various neurological disorders. In 2016, Pradhan et al. found that different δOR agonists either preferentially recruited β-arrestin 1 leading to δOR degradation or recruited β-arrestin 2 causing δOR resensitization (Pradhan et al., 2016). A study in 2017 found that amphetamine-induced hyperlocomotion was amplified in β-arrestin 1 KO mice, but attenuated in β-arrestin 2 KO mice (Zurkovsky et al., 2017), further emphasizing the importance of studying both β-arrestin isoforms.

As mentioned, our study further identified that SNC80-induced anxiolytic-like effects were β-arrestin 2-dependent. However, β-arrestin 2 KO mice still exhibited the fear-reducing effect of SNC80, which could suggest a potential role for β-arrestin 1 in the modulation of fear-related behavior. Particularly, we also noted that β-arrestin 1 KO abolished SNC80-induced ERK1/2 activation in the striatum and nucleus accumbens. Our findings were in line with the extensive in-situ hybridization studies on differential β-arrestin isoform levels in neonatal and postnatal rats (Gurevich et al., 2002, 2004) and studies showing relatively high β-arrestin 1 and low β-arrestin 2 expressions in the striatal regions (Attramadal et al., 1992; Bjork et al., 2008; Gurevich et al., 2002). In contrast, SNC80-induced ERK1/2 activation in the dorsal hippocampus and amygdala was β-arrestin 2-dependent, which agreed with reports of stronger expression of this isoform in those areas (Attramadal et al., 1992; Bjork et al., 2008). Unfortunately, we were limited in our ability to assess whether SNC80-induced reduction in FPS would be attenuated in β-arrestin 1 KO mice, as SNC80 produces severe seizures in these mice (Vicente-Sanchez et al., 2018), a phenomenon we have also observed ourselves and found to be accompanied by strong and persistent ERK1/2 activation in the dorsal hippocampus. Further investigation of the roles of β-arrestin 1 in neuropsychiatric behavior may be feasible using a conditional knockout approach; currently conditional β-arrestin 2 knockout mice already exist (Huang et al., 2018), but conditional β-arrestin 1 knockout mice have not yet been reported.

A unique role for Gi protein signaling and ERK1/2 signaling in conditioned fear-related behavior

In contrast to the β-arrestin-mediated activation of ERK1/2, we found that selectively activating the G protein pathway of the δOR using TAN67, a known weak recruiter of β-arrestin 1 and 2 (Chiang et al., 2016) (Fig. S2) decreased ERK1/2 activation, including in the striatum and the amygdala, and was associated with increased FPS. This result is in agreement with the observation and that blocking Gi/o protein signaling using pertussis toxin in the basolateral amygdala reduced FPS (Melia et al., 1992) and parallels finding that TAN67 did not reduce unconditioned anxiety-like behavior in naïve mice (van Rijn et al., 2010). Additionally, a study in ovariectomized mice found that estradiol benzoate, an anxiogenic estradiol prodrug (Morgan and Pfaff, 2002), decreased ERK in the hippocampus (Anchan et al., 2014), which supports our finding that decreased ERK1/2 is correlated with increased fear. It is noteworthy that both Gi protein and β-arrestin can activate ERK1/2 albeit via different mechanisms (Goldsmith and Dhanasekaran, 2007; Gutkind, 2000). One explanation for our observation is that TAN67 competes with the endogenous δOR agonist Leu-enkephalin, which is a much more efficacious recruiter of β-arrestin (Chiang et al., 2016) (Fig. S3). Importantly, based on observations of enhanced anxiety-like behavior in preproenkephalin KO mice, Leu-enkephalin has anxiolytic-like effects by itself (Kung et al., 2010; Ragnauth et al., 2001), which is in line with reports that the δOR antagonist, naltrindole, is anxiogenic (Narita et al., 2006). As a weak β-arrestin recruiter, TAN67 would attenuate any baseline Leu-enkephalin-induced β-arrestin-mediated ERK activity (Fig. S3). In contrast, because SNC80 is a stronger β-arrestin recruiter than Leu-enkephalin, it will elevate basal ERK1/2 activity produced by endogenous opioids. This hypothesis would also explain why SNC80-induced ERK1/2 activation is quite variable and produces on average only a two-fold increase.

β-arrestin serves as a scaffold for a range of kinases and effectors

In our study, we found that δOR agonism strongly activates ERK1/2 compared to the other tested MAPKs, p38 and JNK, and that the ERK1/2 activity induced by SNC80 was negatively correlated with FPS. Other GPCRs, besides the δOR, may also require β-arrestin-dependent ERK1/2 signaling for modulation of fear. Specifically, in the infralimbic prefrontal cortex, β-adrenergic receptor activation can promote the extinction of contextual fear memory (Do-Monte et al., 2010). In a recent study, it was shown that in this same brain region β-arrestin 2-dependent ERK1/2 activation was required for β-adrenergic receptors agonists to stimulate extinction learning of cocaine-induced reward memories (Huang et al., 2018). β-arrestin 2-mediated signaling in the CNS is not exclusive to ERK1/2 signaling; following κOR activation, β-arrestin 2 can scaffold with p38 as part of a potential mechanism for the aversive effects of κOR agonists (Bruchas et al., 2007). A β-arrestin 2 scaffold of AKT, GSK3β and PP2A has been proposed as a mechanism for stabilizing mood (O’Brien et al., 2011), highlighting that β-arrestin signaling is also not limited to MAPKs. In fact, because β-arrestin 1, in contrast to β-arrestin 2, contains a nuclear translocation sequence (Hoeppner et al., 2012), this enables it to enter the nucleus and regulate gene transcription (Kang et al., 2005; Shi et al., 2007). While in this study we report δORs require β-arrestin-dependent ERK signaling for reduction in anxiety-like behavior, it is certainly possible that other GPCRs may engage different intracellular signaling pathways following β-arrestin recruitment.

Fear- and anxiety-like behaviors rely on shared but distinct neural circuits

In our study, we observed that β-arrestin 2-dependent ERK1/2 activity in the dorsal hippocampus was associated with reduced anxiety-like behavior. Generally, CA1 regions of the ventral hippocampus are associated with responding to contextually-conditioned anxiogenic stimuli than dorsal CA1 regions (Fanselow and Dong, 2010; Jimenez et al., 2018), whereas the dorsal hippocampus is involved with cognitive functions, including exploration, navigation and memory (Kheirbek et al., 2013). Still, our finding that δOR signaling in the dorsal hippocampus is connected to the anxiolytic-like effects of SNC80, agrees with a study showing that intra-dorsal CA1 injection of the δOR antagonist naltrindole is anxiogenic (Solati et al., 2010). Additionally, the anxiolytic-like effects of SNC80 may also involve β-arrestin 2-dependent ERK1/2 signaling in the amygdala, a region more commonly associated innate anxiety-like behavior (Felix-Ortiz et al., 2013; Tye et al., 2011). The basolateral amygdala (BLA) also plays an important role in fear conditioning (Janak and Tye, 2015), including FPS (Terburg et al., 2018). The BLA receives dopaminergic inputs from the ventral tegmental area and projects to the nucleus accumbens. It has been proposed that dopaminergic signaling in the BLA is important for cue-dependent fear-conditioning, such as FPS (Fadok et al., 2009) and that the BLA to nucleus accumbens projection is critical for consolidation of memories associated with aversive effects such as foot shock (Fadok et al., 2010; LaLumiere et al., 2005).

Our finding that ERK1/2 activation in the striatal regions was ablated in β-arrestin 1 KO mice points to a role for striatal β-arrestin 1-mediated ERK1/2 signaling in the modulation of the expression of conditioned fear-related behavior. Processing and executing emotional behaviors in tasks such as the elevated plus maze and FPS tests engages multiple overlapping, yet distinct, brain regions and circuits involved in memory retrieval, locomotion, decision making, reward, and mood (Janak and Tye, 2015). Further studies with circuit-based approaches are necessary to assess the role of biased signaling pathways in the acquisition and expression of conditioned fear-related behavior.

Beneficial roles of β-arrestin signaling

For the longest time, β-arrestin 2 has been associated solely with adverse effects of opioid activation, including tolerance, constipation, respiratory depression, aversion and alcohol use (Raehal and Bohn, 2011; Raehal et al., 2005; van Rijn et al., 2010). These studies fueled a drive to develop G protein-biased opioids to treat pain and other disorders with an improved therapeutic window (Manglik et al., 2016; Mores et al., 2019). Yet, recently a number of studies have started to push back against this narrative (Austin Zamarripa et al., 2018; Hill et al., 2018; Kliewer et al., 2019). Clearly, β-arrestin signaling is not inherently negative as the therapeutic effects of lithium and fluoxetine seem to depend on β-arrestin 2 (David et al., 2009). The increased propensity for β-arrestin 1 KO mice to experience SNC80-induced seizure points to a potential beneficial role for this isoform in maintaining seizure threshold, which could be of use in the treatment of epilepsy. In this study, we provide additional insights regarding potential therapeutic benefits of β-arrestin signaling in reducing anxiety-like behavior. Providing adequate relief of chronic pain is not trivial, partly because it is often associated with negative affect (Corder et al., 2019; Massaly et al., 2019) including anxiety, which may exacerbate pain (al Absi and Rokke, 1991). δOR agonists have been proposed as potential treatment for chronic pain disorders (Pradhan et al., 2011), partly because they have the ability to not only provide analgesia, but also treat comorbid anxiety and depression (Perrine et al., 2006; Saitoh et al., 2004; van Rijn et al., 2010). However, our results would argue that developing G protein-biased δOR agonists may produce drugs that are suboptimal for the treatment of complex chronic pain; our findings suggest such a drug would not alleviate co-morbid fear and anxiety, but potentially even worsen these symptoms. Thus, our study results argue in favor of a reassessment of drug development efforts that seek solely to identify G protein-biased drugs. Instead, we propose that efforts should be directed towards the development of drugs with finely tuned bias and, if possible, towards development of molecules that are biased against a single β-arrestin isoform rather than both isoforms.

Conclusion

Overall, our results begin to reveal the complex- and context-specific nature of GPCR biased signaling in modulation of fear-related and anxiety-like behavior. These results expand our current understanding of therapeutic effects of β-arrestin signaling in mood disorders, which ultimately may aid development of more efficacious pharmacological treatment options for these disorders.

Author Contributions

MJ.K.: formal analysis, investigation, writing – original draft, writing – review & editing, visualization; T.C.: formal analysis, investigation, writing – review & editing; G.E.M.: formal analysis, investigation, writing – review & editing; A.M.M. formal analysis, investigation; A.M.G. formal analysis, investigation, writing – review & editing; J.A.C.: formal analysis, writing – review & editing, supervision; R.M.vR.: Conceptualization, formal analysis, investigation, writing – original draft, writing – review & editing, visualization, supervision, project administration, funding acquisition

Declaration of Interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

STAR Methods

Animals

Wild-type (WT) C57BL/6 male mice were purchased from Envigo (Indianapolis, IN), and β-arrestin 1 or 2 global knockout (KO) mice were bred in our facility (Chiang et al., 2016; Robins et al., 2018a). Adult mice (8-10 weeks, 24 ± 2g) were group housed (3-5 mice) in a single ventilated Plexiglas cage. Mice were maintained at ambient temperature (21°C) in an animal housing facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care and animals were kept on a reversed 12-hour dark-light cycle (lights off at 10:00, lights on at 22:00). Food and water were provided ad libitum. Purchased mice were acclimated for one week prior to the experiments. All animal protocols (#1305000864 by RMvR) were preapproved by Purdue Animal Care and Use Committee and were in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

Drug preparation and administration

SNC80 (#076410, Tocris, Thermo Fisher Scientific, Waltham, MA) was diluted in slightly acidic saline pH5-6. TAN67 (#092110, Tocris, Thermo Fisher Scientific) was diluted in saline, and SL327 (#19691, Tocris, Thermo Fisher Scientific) was diluted in 5% DMSO, 10% Cremophore (Millipore Sigma, Burlington, MA) and 85% saline. 20 mg/kg SNC80 was subcutaneously administered 30 minutes prior to the tests in Fig. 1d-g, Fig. 4b and Fig. S1, and 50 mg/kg SL327 was subcutaneously administered 60 minutes prior to the administration of SNC80 for Fig. 4. For Fig. 2-6 and Fig. S4, either 20 mg/kg SNC80 or 25 mg/kg TAN67 was intraperitoneally administered at the indicated timepoint prior to the testing. The dose of SNC80 and TAN67 was determined based on our previous study (van Rijn et al., 2010) and our preliminary studies (data not shown). Separate batches of mice with no prior history of drug injection were used for the brain collection and behavioral tests to test earlier time-points of ERK1/2 signaling (such as 10 minutes).

Elevated-plus maze test

The elevated-plus maze test was performed as previously described (van Rijn et al., 2010). Mice were allowed to explore the maze for 5 minutes, and arm entries and time spent in each arm were recorded with a camera positioned above the maze.

Dark–light transition box test

The test was performed based on previously established protocols (Robins et al., 2018a; van Rijn et al., 2010) Testing was conducted without a habituation session to the boxes and a 1/2 area dark insert was placed in the locomotor boxes, leaving the remaining 1/2 of the area lit as described previously (Bourin and Hascoet, 2003)(Bourin and Hascoet, 2003). Two LED lights were inserted above the light portion of the testing chamber where the lux of the light region ranged from 390-540 lumens and dark chamber lux ranged from 0-12 lumens. For testing, animals were placed in the light portion of the chamber and testing began upon animal entry. Time spent in the dark and light chambers as well as their locomotor activity was recorded for 5 minutes with a photobeam-based tracking system.

Fear potentiated startle (FPS) test

Startle reflexes of mice were recorded in the startle reflex chambers (25.8 × 25 × 26.5 cm) using the Hamilton Kinder Startle Monitor system (Kinder Scientific, Poway, CA). Mice groups were counterbalanced, such that no significant differences between startle reflexes were observed between groups (Fig. S4). On the conditioning day, all subjects were conditioned with 40 conditioning trials by a fixed 2 minute inter-trial interval (ITI), and FPS responses were tested on the following day. The fear conditioning and FPS parameters were based on a previously established protocol (Barrenha and Chester, 2007).

Preparation of tissue homogenates

After drug injections, mice were euthanized by carbon dioxide asphyxiation and rapidly decapitated. Based on our previous studies, we have particularly chosen carbon dioxide asphyxiation over other euthanasia methods that may potentially increase basal ERK1/2 activity in the brain (Ko et al., 2019). The collected brains were first sliced as coronal sections (1.5-2.0 mm) with a brain matrix (#RBMS-205C, Kent scientific, Torrington, CT), and then flash-frozen in dry-ice-chilled 2-methylbutane (−40 °C) (#03551-4, Fisher Scientific). Regions of interest were collected from these slices using a 1 mm biopsy micropunch (#15110-10, Miltex, Plainsboro, NJ) as follows: dorsal striatum and nucleus accumbens (A/P: +0.5 mm to +1.5 mm), dorsal hippocampus and amygdala (A/P: −1 mm to −2 mm), and ventral hippocampus (A/P: −2 mm to −4 mm) (Paxinos and Franklin, 2004). The punches targeted a specific region and produced enough tissue to run several blots. However, it is noteworthy that the extracted tissue may contain small amounts of tissue from neighboring regions. For example, while the punches for the amygdala primarily consisted of the BLA, the tissue will also have included a small portion of the central amygdala. Collected tissues were further homogenized with a tissue grinder (#357535 & 357537, DWK Life Sciences, Millville, NJ) in RIPA buffer mixed Halt™ Protease and Phosphatase Inhibitor Cocktail (#1861280, Thermo Fisher Scientific). Samples were further prepared based on previously established protocols (Ko et al., 2019). Data depicted in Fig. 2i-m also includes the data collected for SNC80 at the 0 min or 10 min time points in the experiment depicted in Fig. 4c,dto represent the full range of observed SNC80 induced ERK1/2 activation in mice tested in separate cohorts at different occasions.

Cell culture

Chinese hamster ovarian CHO-δOR-βArr2 cells (DiscoverX, Fremont, CA) U2OS-δOR-βArr1 (DiscoverX), and NG-108-15 cells (HB-12317™, ATCC®, Manassas, VA) were cultured as recommended by the manufacturer and maintained at 37° C/5 % CO2. Cells were seeded in a clear 6 well plate (Corning™, Thermo Fisher Scientific) with 250,000 cells/2 mL/well. On the following day, all growth media was aspirated and changed into 1 mL serum-free Opti-MEM (#31985070, Gibco®, Thermo Fisher Scientific). The next day, cells were challenged with 10 µM drugs (SNC80) for a specific duration (0, 3, 6, 20, and 60 minutes). All drugs were diluted in Opti-MEM prior to administration. The media was aspirated following the challenge and 100 µL RIPA buffer was added to collect the samples on ice. Using cell scrapers (#353089, Thermo Fisher Scientific), all samples were dislodged from the 6 well plate, collected and stored at −30° C until usage. For the Western blot, the collected samples were quantified with the Bradford assay and samples were prepared with 4 x Laemmli and boiled at 95° C for 5 minutes. The CHO-δOR-βArr2 cells were also used to measure β-arrestin recruitment using the DiscoverX PathHunter Assay as previously described (Chiang et al., 2016).

SDS-Page and Western blot

Samples (20 µL containing 10 µg protein) were loaded per well of a NuPage 4-12 % Bis-Tris gradient gels (#NP0336BOX, Thermo Fisher Scientific), and the SDS-Page gel was subsequently transferred to nitrocellulose membranes (#1620115, BioRad) by the Western blot. Membranes were incubated following previously established protocols (Ko et al., 2019). For reproducibility, detailed information regarding the antibodies used in the study are listed in Table S4. Prepared samples were scanned using the LiCor Odyssey® CLx Scanner (Li-Cor, Lincoln, NE). By utilizing the Li-Cor secondary antibodies, we were able to detect the MAPK, pMAPK, and α-Tubulin on the same blot without the need of stripping/reblotting. In the same membrane, each band was cut based on their size. For instance, ERK1/2 and pERK1/2 bands were collected around 42/44 kDa and α-Tubulin band was collected around 50 kDa in the same membrane. For statistical analysis, we normalized the pMAPK/MAPK ratio to α-Tubulin in case drug treatment changed ERK1/2 levels.

Preparation of tissue for immunofluorescence

To preserve the intact ERK1/2 activity in vivo for fluorescence microscopy, mice were transcardially perfused before isolation of brain tissue. Thirty minutes prior to transcardiac perfusion, mice were administered with 100/10 mg/kg Ketamine/Xylazine. Ten minutes prior to perfusion, 20 mg/kg SNC80 (i.p.) or a corresponding volume of saline was administered to the mice. Mice were then perfused with 30 mL of cold PBS and 4% paraformaldehyde (#100503-916, VWR, Radnor, PA) and were immediately decapitated to collect the brains. The brains were fixated in 4% paraformaldehyde overnight, dehydrated in 30% sucrose, and then embedded in Frozen Section Compound (#3801480, Leica, Wetzlar, Germany). Frozen brains were sliced at a width of 30 μm using the Leica cryostat and permeabilized in 100% methanol at - 20 °C for 10 minutes. The slices were blocked in 5 % Normal goat serum (#S26-100ml, Millipore Sigma) for an hour then stained with primary antibodies as listed in Table S4. For immunofluorescence labeling, the sections were incubated in the secondary antibodies according to the previously established protocol (Kim et al., 2018) and as listed in Table S4. After the final washing, the nuclei of the sections were stained and the slices were mounted on a glass slide with Vectashield® (#H-1200, Vector lab, Burlingame, CA). Images were acquired with a Nikon confocal microscope and assembled in Adobe Photoshop CS6 (Adobe).

Statistical analysis

The maximum amplitude of the startle response was measured from the average of all responses for each trial type (12 noise-alone, 12 light+noise) of each mouse. Fear-potentiated startle response was analyzed using raw (maximum) startle amplitudes and proportional changes of each trial type (noise-alone, light+noise), which is shown as % FPS in the graphs. The proportional change score (% FPS) was calculated as follows: (startle response to light and noise – startle response to noise)/startle response to noise x 100. Thus, %FPS is a sensitive measure that adjusts for individual and group differences (e.g., possible non-specific effects of drug treatment) in startle response magnitude that may be observed on noise-alone and light + noise trials (Walker and Davis, 2002).

All data are presented as individual data points (or means) ± standard error of the mean (S.E.M.). Assays with one independent variable were analyzed for statistical significance using a one-way Analysis of Variance (ANOVA), whereas assays with two independent variables were analyzed using a two-way ANOVA. If a significant deviation of the mean was identified, an appropriate post-hoc analysis was performed as indicated. Gaussian distribution of our datasets was assessed using the D’augostino and Pearson analysis. We excluded one outlier in our wild-type cohort that received SNC80 and one that received TAN-67 in the FPS assay based on the Grubbs’ test (α = 0.05). In the dark-light and elevated plus maze tests we excluded subjects that were frozen/stationary for >95% of the experimental time. All statistical analysis was conducted using GraphPad Prism 7 (GraphPad Software, La Jolla, CA).

Acknowledgements

This work was funded by a NARSAD Young Investigator Award from the Brain and Behavior Research Foundation (#23603 to RMvR) and the National Institute on Alcohol Abuse and Alcoholism (AA025368, AA026949, AA026675) and Drug Abuse (DA045897). We also appreciate Dr. Marcus M. Weera for technical assistance with the fear-conditioning experiments. Included diagrams were created with BioRender.

Footnotes

  • ↵‡ Lead Contact

References

  1. ↵
    Akimova, E., Lanzenberger, R., and Kasper, S. (2009). The serotonin-1A receptor in anxiety disorders. Biol Psychiatry 66, 627–635.
    OpenUrlCrossRefPubMedWeb of Science
  2. ↵
    al Absi, M., and Rokke, P.D. (1991). Can anxiety help us tolerate pain? Pain 46, 43–51.
    OpenUrlCrossRefPubMedWeb of Science
  3. ↵
    Allen, J.A., Yost, J.M., Setola, V., Chen, X., Sassano, M.F., Chen, M., Peterson, S., Yadav, P.N., Huang, X.P., Feng, B., et al. (2011). Discovery of beta-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy. Proc Natl Acad Sci U S A 108, 18488–18493.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Anchan, D., Clark, S., Pollard, K., and Vasudevan, N. (2014). GPR30 activation decreases anxiety in the open field test but not in the elevated plus maze test in female mice. Brain Behav 4, 51–59.
    OpenUrlCrossRefPubMed
  5. ↵
    Atkins, C.M., Selcher, J.C., Petraitis, J.J., Trzaskos, J.M., and Sweatt, J.D. (1998). The MAPK cascade is required for mammalian associative learning. Nat Neurosci 1, 602–609.
    OpenUrlCrossRefPubMedWeb of Science
  6. ↵
    Attramadal, H., Arriza, J.L., Aoki, C., Dawson, T.M., Codina, J., Kwatra, M.M., Snyder, S.H., Caron, M.G., and Lefkowitz, R.J. (1992). Beta-arrestin2, a novel member of the arrestin/beta-arrestin gene family. J Biol Chem 267, 17882–17890.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Austin Zamarripa, C., Edwards, S.R., Qureshi, H.N., Yi, J.N., Blough, B.E., and Freeman, K.B. (2018). The G-protein biased mu-opioid agonist, TRV130, produces reinforcing and antinociceptive effects that are comparable to oxycodone in rats. Drug Alcohol Depend 192, 158–162.
    OpenUrl
  8. ↵
    Barrenha, G.D., and Chester, J.A. (2007). Genetic correlation between innate alcohol preference and fear-potentiated startle in selected mouse lines. Alcohol Clin Exp Res 31, 1081–1088.
    OpenUrlCrossRefPubMed
  9. ↵
    Beaulieu, J.M., Sotnikova, T.D., Marion, S., Lefkowitz, R.J., Gainetdinov, R.R., and Caron, M.G. (2005). An Akt/beta-arrestin 2/PP2A signaling complex mediates dopaminergic neurotransmission and behavior. Cell 122, 261–273.
    OpenUrlCrossRefPubMedWeb of Science
  10. ↵
    Bjork, K., Rimondini, R., Hansson, A.C., Terasmaa, A., Hyytia, P., Heilig, M., and Sommer, W.H. (2008). Modulation of voluntary ethanol consumption by beta-arrestin 2. FASEB J 22, 2552–2560.
    OpenUrlCrossRefPubMedWeb of Science
  11. ↵
    Bohn, L.M., Gainetdinov, R.R., Lin, F.T., Lefkowitz, R.J., and Caron, M.G. (2000). Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence. Nature 408, 720–723.
    OpenUrlCrossRefPubMed
  12. ↵
    Bohn, L.M., Lefkowitz, R.J., Gainetdinov, R.R., Peppel, K., Caron, M.G., and Lin, F.T. (1999). Enhanced morphine analgesia in mice lacking beta-arrestin 2. Science 286, 2495–2498.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Bourin, M., and Hascoet, M. (2003). The mouse light/dark box test. European journal of pharmacology 463, 55–65.
    OpenUrlCrossRefPubMedWeb of Science
  14. ↵
    Bruchas, M.R., Land, B.B., Aita, M., Xu, M., Barot, S.K., Li, S., and Chavkin, C. (2007). Stress-induced p38 mitogen-activated protein kinase activation mediates kappa-opioid-dependent dysphoria. J Neurosci 27, 11614–11623.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Bruchas, M.R., Macey, T.A., Lowe, J.D., and Chavkin, C. (2006). Kappa opioid receptor activation of p38 MAPK is GRK3- and arrestin-dependent in neurons and astrocytes. J Biol Chem 281, 18081–18089.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Cen, B., Yu, Q., Guo, J., Wu, Y., Ling, K., Cheng, Z., Ma, L., and Pei, G. (2001). Direct binding of beta-arrestins to two distinct intracellular domains of the delta opioid receptor. J Neurochem 76, 1887–1894.
    OpenUrlCrossRefPubMedWeb of Science
  17. ↵
    Chiang, T., Sansuk, K., and van Rijn, R.M. (2016). beta-Arrestin 2 dependence of delta opioid receptor agonists is correlated with alcohol intake. Br J Pharmacol 173, 332–343.
    OpenUrl
  18. ↵
    Chu Sin Chung, P., Keyworth, H.L., Martin-Garcia, E., Charbogne, P., Darcq, E., Bailey, A., Filliol, D., Matifas, A., Scherrer, G., Ouagazzal, A.M., et al. (2015). A novel anxiogenic role for the delta opioid receptor expressed in GABAergic forebrain neurons. Biol Psychiatry 77, 404–415.
    OpenUrlCrossRefPubMed
  19. ↵
    Conner, D.A., Mathier, M.A., Mortensen, R.M., Christe, M., Vatner, S.F., Seidman, C.E., and Seidman, J.G. (1997). beta-Arrestin1 knockout mice appear normal but demonstrate altered cardiac responses to beta-adrenergic stimulation. Circ Res 81, 1021–1026.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Corder, G., Ahanonu, B., Grewe, B.F., Wang, D., Schnitzer, M.J., and Scherrer, G. (2019). An amygdalar neural ensemble that encodes the unpleasantness of pain. Science 363, 276–281.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Coyle, J.T., and Duman, R.S. (2003). Finding the intracellular signaling pathways affected by mood disorder treatments. Neuron 38, 157–160.
    OpenUrlCrossRefPubMedWeb of Science
  22. ↵
    David, D.J., Samuels, B.A., Rainer, Q., Wang, J.W., Marsteller, D., Mendez, I., Drew, M., Craig, D.A., Guiard, B.P., Guilloux, J.P., et al. (2009). Neurogenesis-dependent and - independent effects of fluoxetine in an animal model of anxiety/depression. Neuron 62, 479–493.
    OpenUrlCrossRefPubMedWeb of Science
  23. ↵
    de la Mora, M.P., Gallegos-Cari, A., Arizmendi-Garcia, Y., Marcellino, D., and Fuxe, K. (2010). Role of dopamine receptor mechanisms in the amygdaloid modulation of fear and anxiety: Structural and functional analysis. Prog Neurobiol 90, 198–216.
    OpenUrlCrossRefPubMedWeb of Science
  24. ↵
    Do-Monte, F.H., Kincheski, G.C., Pavesi, E., Sordi, R., Assreuy, J., and Carobrez, A.P. (2010). Role of beta-adrenergic receptors in the ventromedial prefrontal cortex during contextual fear extinction in rats. Neurobiol Learn Mem 94, 318–328.
    OpenUrlCrossRefPubMed
  25. ↵
    Eisinger, D.A., Ammer, H., and Schulz, R. (2002). Chronic morphine treatment inhibits opioid receptor desensitization and internalization. J Neurosci 22, 10192–10200.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Erbs, E., Faget, L., Scherrer, G., Matifas, A., Filliol, D., Vonesch, J.L., Koch, M., Kessler, P., Hentsch, D., Birling, M.C., et al. (2015). A mu-delta opioid receptor brain atlas reveals neuronal co-occurrence in subcortical networks. Brain Struct Funct 220, 677–702.
    OpenUrlCrossRefPubMed
  27. ↵
    Fadok, J.P., Darvas, M., Dickerson, T.M., and Palmiter, R.D. (2010). Long-term memory for pavlovian fear conditioning requires dopamine in the nucleus accumbens and basolateral amygdala. PLoS One 5, e12751.
    OpenUrlCrossRefPubMed
  28. ↵
    Fadok, J.P., Dickerson, T.M., and Palmiter, R.D. (2009). Dopamine is necessary for cue-dependent fear conditioning. J Neurosci 29, 11089–11097.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Fanselow, M.S., and Dong, H.W. (2010). Are the dorsal and ventral hippocampus functionally distinct structures? Neuron 65, 7–19.
    OpenUrlCrossRefPubMedWeb of Science
  30. ↵
    Felix-Ortiz, A.C., Beyeler, A., Seo, C., Leppla, C.A., Wildes, C.P., and Tye, K.M. (2013). BLA to vHPC inputs modulate anxiety-related behaviors. Neuron 79, 658–664.
    OpenUrlCrossRefPubMedWeb of Science
  31. ↵
    File, S.E., Kenny, P.J., and Cheeta, S. (2000). The role of the dorsal hippocampal serotonergic and cholinergic systems in the modulation of anxiety. Pharmacol Biochem Behav 66, 65–72.
    OpenUrlCrossRefPubMedWeb of Science
  32. ↵
    Goldsmith, Z.G., and Dhanasekaran, D.N. (2007). G protein regulation of MAPK networks. Oncogene 26, 3122–3142.
    OpenUrlCrossRefPubMedWeb of Science
  33. ↵
    Gurevich, E.V., Benovic, J.L., and Gurevich, V.V. (2002). Arrestin2 and arrestin3 are differentially expressed in the rat brain during postnatal development. Neuroscience 109, 421–436.
    OpenUrlCrossRefPubMedWeb of Science
  34. ↵
    Gurevich, E.V., Benovic, J.L., and Gurevich, V.V. (2004). Arrestin2 expression selectively increases during neural differentiation. J Neurochem 91, 1404–1416.
    OpenUrlCrossRefPubMedWeb of Science
  35. ↵
    Gutkind, J.S. (2000). Regulation of mitogen-activated protein kinase signaling networks by G protein-coupled receptors. Sci STKE 2000, re1.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Hahn, Y.K., Paris, J.J., Lichtman, A.H., Hauser, K.F., Sim-Selley, L.J., Selley, D.E., and Knapp, P.E. (2016). Central HIV-1 Tat exposure elevates anxiety and fear conditioned responses of male mice concurrent with altered mu-opioid receptor-mediated G-protein activation and beta-arrestin 2 activity in the forebrain. Neurobiol Dis 92, 124–136.
    OpenUrlCrossRefPubMed
  37. ↵
    Herry, C., Trifilieff, P., Micheau, J., Luthi, A., and Mons, N. (2006). Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala. Eur J Neurosci 24, 261–269.
    OpenUrlCrossRefPubMedWeb of Science
  38. ↵
    Hill, R., Disney, A., Conibear, A., Sutcliffe, K., Dewey, W., Husbands, S., Bailey, C., Kelly, E., and Henderson, G. (2018). The novel mu-opioid receptor agonist PZM21 depresses respiration and induces tolerance to antinociception. Br J Pharmacol 175, 2653–2661.
    OpenUrlCrossRef
  39. ↵
    Hoeppner, C.Z., Cheng, N., and Ye, R.D. (2012). Identification of a nuclear localization sequence in beta-arrestin-1 and its functional implications. J Biol Chem 287, 8932–8943.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Huang, B., Li, Y., Cheng, D., He, G., Liu, X., and Ma, L. (2018). beta-Arrestin-biased beta-adrenergic signaling promotes extinction learning of cocaine reward memory. Sci Signal 11.
  41. ↵
    Janak, P.H., and Tye, K.M. (2015). From circuits to behaviour in the amygdala. Nature 517, 284–292.
    OpenUrlCrossRefPubMed
  42. ↵
    Jimenez, J.C., Su, K., Goldberg, A.R., Luna, V.M., Biane, J.S., Ordek, G., Zhou, P., Ong, S.K., Wright, M.A., Zweifel, L., et al. (2018). Anxiety Cells in a Hippocampal-Hypothalamic Circuit. Neuron 97, 670–683 e676.
    OpenUrl
  43. ↵
    Kang, J., Shi, Y., Xiang, B., Qu, B., Su, W., Zhu, M., Zhang, M., Bao, G., Wang, F., Zhang, X., et al. (2005). A nuclear function of beta-arrestin1 in GPCR signaling: regulation of histone acetylation and gene transcription. Cell 123, 833–847.
    OpenUrlCrossRefPubMedWeb of Science
  44. ↵
    Kheirbek, M.A., Drew, L.J., Burghardt, N.S., Costantini, D.O., Tannenholz, L., Ahmari, S.E., Zeng, H., Fenton, A.A., and Hen, R. (2013). Differential control of learning and anxiety along the dorsoventral axis of the dentate gyrus. Neuron 77, 955–968.
    OpenUrlCrossRefPubMedWeb of Science
  45. ↵
    Kim, J.W., Ko, M.J., Gonzales, E.L., Kang, R.J., Kim, D.G., Kim, Y., Seung, H., Oh, H.A., Eun, P.H., and Shin, C.Y. (2018). Social support rescues acute stress-induced cognitive impairments by modulating ERK1/2 phosphorylation in adolescent mice. Sci Rep 8, 12003.
    OpenUrl
  46. ↵
    Kindt, M., Soeter, M., and Vervliet, B. (2009). Beyond extinction: erasing human fear responses and preventing the return of fear. Nat Neurosci 12, 256–258.
    OpenUrlCrossRefPubMedWeb of Science
  47. ↵
    Klee, W.A., Simonds, W.F., Sweat, F.W., Burke, T.R., Jr.., Jacobson, A.E., and Rice, K.C. (1982). Identification of a Mr 58 000 glycoprotein subunit of the opiate receptor. FEBS Lett 150, 125–128.
    OpenUrlCrossRefPubMedWeb of Science
  48. ↵
    Kliewer, A., Schmiedel, F., Sianati, S., Bailey, A., Bateman, J.T., Levitt, E.S., Williams, J.T., Christie, M.J., and Schulz, S. (2019). Phosphorylation-deficient G-protein-biased mu-opioid receptors improve analgesia and diminish tolerance but worsen opioid side effects. Nat Commun 10, 367.
    OpenUrl
  49. ↵
    Ko, M.J., Mulia, G.E., and van Rijn, R.M. (2019). Commonly Used Anesthesia/Euthanasia Methods for Brain Collection Differentially Impact MAPK Activity in Male and Female C57BL/6 Mice. Front Cell Neurosci 13, 96.
    OpenUrl
  50. ↵
    Kung, J.C., Chen, T.C., Shyu, B.C., Hsiao, S., and Huang, A.C. (2010). Anxiety- and depressive-like responses and c-fos activity in preproenkephalin knockout mice: oversensitivity hypothesis of enkephalin deficit-induced posttraumatic stress disorder. J Biomed Sci 17, 29.
    OpenUrlCrossRefPubMed
  51. ↵
    LaLumiere, R.T., Nawar, E.M., and McGaugh, J.L. (2005). Modulation of memory consolidation by the basolateral amygdala or nucleus accumbens shell requires concurrent dopamine receptor activation in both brain regions. Learn Mem 12, 296–301.
    OpenUrlAbstract/FREE Full Text
  52. ↵
    Land, B.B., Bruchas, M.R., Schattauer, S., Giardino, W.J., Aita, M., Messinger, D., Hnasko, T.S., Palmiter, R.D., and Chavkin, C. (2009). Activation of the kappa opioid receptor in the dorsal raphe nucleus mediates the aversive effects of stress and reinstates drug seeking. Proc Natl Acad Sci U S A 106, 19168–19173.
    OpenUrlAbstract/FREE Full Text
  53. ↵
    Latapy, C., and Beaulieu, J.M. (2013). beta-Arrestins in the central nervous system. Prog Mol Biol Transl Sci 118, 267–295.
    OpenUrl
  54. ↵
    Lefkowitz, R.J., and Shenoy, S.K. (2005). Transduction of receptor signals by beta-arrestins. Science 308, 512–517.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    Li, Y., Li, H., Liu, X., Bao, G., Tao, Y., Wu, Z., Xia, P., Wu, C., Li, B., and Ma, L. (2009). Regulation of amygdalar PKA by beta-arrestin-2/phosphodiesterase-4 complex is critical for fear conditioning. Proc Natl Acad Sci U S A 106, 21918–21923.
    OpenUrlAbstract/FREE Full Text
  56. ↵
    Lohse, M.J., Benovic, J.L., Codina, J., Caron, M.G., and Lefkowitz, R.J. (1990). beta-Arrestin: a protein that regulates beta-adrenergic receptor function. Science 248, 1547–1550.
    OpenUrlAbstract/FREE Full Text
  57. ↵
    Manglik, A., Lin, H., Aryal, D.K., McCorvy, J.D., Dengler, D., Corder, G., Levit, A., Kling, R.C., Bernat, V., Hubner, H., et al. (2016). Structure-based discovery of opioid analgesics with reduced side effects. Nature 537, 185–190.
    OpenUrlCrossRefPubMed
  58. ↵
    Mansour, A., Khachaturian, H., Lewis, M.E., Akil, H., and Watson, S.J. (1987). Autoradiographic differentiation of mu, delta, and kappa opioid receptors in the rat forebrain and midbrain. J Neurosci 7, 2445–2464.
    OpenUrlPubMedWeb of Science
  59. ↵
    Massaly, N., Copits, B.A., Wilson-Poe, A.R., Hipolito, L., Markovic, T., Yoon, H.J., Liu, S., Walicki, M.C., Bhatti, D.L., Sirohi, S., et al. (2019). Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid System. Neuron 102, 564–573 e566.
    OpenUrl
  60. ↵
    Melia, K.R., Falls, W.A., and Davis, M. (1992). Involvement of pertussis toxin sensitive G-proteins in conditioned fear-potentiated startle: possible involvement of the amygdala. Brain Res 584, 141–148.
    OpenUrlPubMed
  61. ↵
    Mores, K.L., Cummins, B.R., Cassell, R.J., and van Rijn, R.M. (2019). A Review of the Therapeutic Potential of Recently Developed G Protein-Biased Kappa Agonists. Front Pharmacol 10, 407.
    OpenUrl
  62. ↵
    Morgan, M.A., and Pfaff, D.W. (2002). Estrogen’s effects on activity, anxiety, and fear in two mouse strains. Behav Brain Res 132, 85–93.
    OpenUrlCrossRefPubMedWeb of Science
  63. ↵
    Narita, M., Kaneko, C., Miyoshi, K., Nagumo, Y., Kuzumaki, N., Nakajima, M., Nanjo, K., Matsuzawa, K., Yamazaki, M., and Suzuki, T. (2006). Chronic pain induces anxiety with concomitant changes in opioidergic function in the amygdala. Neuropsychopharmacology 31, 739–750.
    OpenUrlCrossRefPubMedWeb of Science
  64. ↵
    O’Brien, W.T., Huang, J., Buccafusca, R., Garskof, J., Valvezan, A.J., Berry, G.T., and Klein, P.S. (2011). Glycogen synthase kinase-3 is essential for beta-arrestin-2 complex formation and lithium-sensitive behaviors in mice. J Clin Invest 121, 3756–3762.
    OpenUrlCrossRefPubMedWeb of Science
  65. ↵
    Paxinos, G., and Franklin, K.B. (2004). The mouse brain in stereotaxic coordinates (Gulf professional publishing).
  66. ↵
    Perrine, S.A., Hoshaw, B.A., and Unterwald, E.M. (2006). Delta opioid receptor ligands modulate anxiety-like behaviors in the rat. Br J Pharmacol 147, 864–872.
    OpenUrlCrossRefPubMedWeb of Science
  67. ↵
    Pradhan, A.A., Befort, K., Nozaki, C., Gaveriaux-Ruff, C., and Kieffer, B.L. (2011). The delta opioid receptor: an evolving target for the treatment of brain disorders. Trends Pharmacol Sci 32, 581–590.
    OpenUrlCrossRefPubMedWeb of Science
  68. ↵
    Pradhan, A.A., Perroy, J., Walwyn, W.M., Smith, M.L., Vicente-Sanchez, A., Segura, L., Bana, A., Kieffer, B.L., and Evans, C.J. (2016). Agonist-Specific Recruitment of Arrestin Isoforms Differentially Modify Delta Opioid Receptor Function. J Neurosci 36, 3541–3551.
    OpenUrlAbstract/FREE Full Text
  69. ↵
    Raehal, K.M., and Bohn, L.M. (2011). The role of beta-arrestin2 in the severity of antinociceptive tolerance and physical dependence induced by different opioid pain therapeutics. Neuropharmacology 60, 58–65.
    OpenUrlCrossRefPubMed
  70. ↵
    Raehal, K.M., Walker, J.K., and Bohn, L.M. (2005). Morphine side effects in beta-arrestin 2 knockout mice. J Pharmacol Exp Ther 314, 1195–1201.
    OpenUrlAbstract/FREE Full Text
  71. ↵
    Ragnauth, A., Schuller, A., Morgan, M., Chan, J., Ogawa, S., Pintar, J., Bodnar, R.J., and Pfaff, D.W. (2001). Female preproenkephalin-knockout mice display altered emotional responses. Proc Natl Acad Sci U S A 98, 1958–1963.
    OpenUrlAbstract/FREE Full Text
  72. ↵
    Richards, E.M., Mathews, D.C., Luckenbaugh, D.A., Ionescu, D.F., Machado-Vieira, R., Niciu, M.J., Duncan, W.C., Nolan, N.M., Franco-Chaves, J.A., Hudzik, T., et al. (2016). A randomized, placebo-controlled pilot trial of the delta opioid receptor agonist AZD2327 in anxious depression. Psychopharmacology (Berl) 233, 1119–1130.
    OpenUrlCrossRef
  73. ↵
    Robins, M.T., Chiang, T., Berry, J.N., Ko, M.J., Ha, J.E., and van Rijn, R.M. (2018a). Behavioral Characterization of beta-Arrestin 1 Knockout Mice in Anxiety-Like and Alcohol Behaviors. Front Behav Neurosci 12, 54.
    OpenUrl
  74. ↵
    Robins, M.T., Chiang, T., Mores, K.L., Alongkronrusmee, D., and van Rijn, R.M. (2018b). Critical Role for Gi/o-Protein Activity in the Dorsal Striatum in the Reduction of Voluntary Alcohol Intake in C57Bl/6 Mice. Front Psychiatry 9, 112.
    OpenUrl
  75. ↵
    Rozenfeld, R., and Devi, L.A. (2007). Receptor heterodimerization leads to a switch in signaling: beta-arrestin2-mediated ERK activation by mu-delta opioid receptor heterodimers. FASEB J 21, 2455–2465.
    OpenUrlCrossRefPubMedWeb of Science
  76. ↵
    Saitoh, A., Kimura, Y., Suzuki, T., Kawai, K., Nagase, H., and Kamei, J. (2004). Potential anxiolytic and antidepressant-like activities of SNC80, a selective delta-opioid agonist, in behavioral models in rodents. J Pharmacol Sci 95, 374–380.
    OpenUrlCrossRef
  77. ↵
    Saitoh, A., Suzuki, S., Soda, A., Ohashi, M., Yamada, M., Oka, J.I., Nagase, H., and Yamada, M. (2018). The delta opioid receptor agonist KNT-127 in the prelimbic medial prefrontal cortex attenuates veratrine-induced anxiety-like behaviors in mice. Behav Brain Res 336, 77–84.
    OpenUrl
  78. ↵
    Shi, Y., Feng, Y., Kang, J., Liu, C., Li, Z., Li, D., Cao, W., Qiu, J., Guo, Z., Bi, E., et al. (2007). Critical regulation of CD4+ T cell survival and autoimmunity by beta-arrestin 1. Nat Immunol 8, 817–824.
    OpenUrlCrossRefPubMedWeb of Science
  79. ↵
    Solati, J., Zarrindast, M.R., and Salari, A.A. (2010). Dorsal hippocampal opioidergic system modulates anxiety-like behaviors in adult male Wistar rats. Psychiatry Clin Neurosci 64, 634–641.
    OpenUrlPubMed
  80. ↵
    Sugiyama, A., Yamada, M., Furuie, H., Gotoh, L., Saitoh, A., Nagase, H., Oka, J.I., and Yamada, M. (2019). Systemic administration of a delta opioid receptor agonist, KNT-127, facilitates extinction learning of fear memory in rats. J Pharmacol Sci 139, 174–179.
    OpenUrl
  81. ↵
    Takahashi, L.K. (2001). Role of CRF(1) and CRF(2) receptors in fear and anxiety. Neurosci Biobehav Rev 25, 627–636.
    OpenUrlCrossRefPubMedWeb of Science
  82. ↵
    Terburg, D., Scheggia, D., Triana Del Rio, R., Klumpers, F., Ciobanu, A.C., Morgan, B., Montoya, E.R., Bos, P.A., Giobellina, G., van den Burg, E.H., et al. (2018). The Basolateral Amygdala Is Essential for Rapid Escape: A Human and Rodent Study. Cell 175, 723–735 e716.
    OpenUrlCrossRefPubMed
  83. ↵
    Tohgo, A., Pierce, K.L., Choy, E.W., Lefkowitz, R.J., and Luttrell, L.M. (2002). beta-Arrestin scaffolding of the ERK cascade enhances cytosolic ERK activity but inhibits ERK-mediated transcription following angiotensin AT1a receptor stimulation. J Biol Chem 277, 9429–9436.
    OpenUrlAbstract/FREE Full Text
  84. ↵
    Tovote, P., Fadok, J.P., and Luthi, A. (2015). Neuronal circuits for fear and anxiety. Nat Rev Neurosci 16, 317–331.
    OpenUrlCrossRefPubMed
  85. ↵
    Tye, K.M., Prakash, R., Kim, S.Y., Fenno, L.E., Grosenick, L., Zarabi, H., Thompson, K.R., Gradinaru, V., Ramakrishnan, C., and Deisseroth, K. (2011). Amygdala circuitry mediating reversible and bidirectional control of anxiety. Nature 471, 358–362.
    OpenUrlCrossRefPubMedWeb of Science
  86. ↵
    van Rijn, R.M., Brissett, D.I., and Whistler, J.L. (2010). Dual efficacy of delta opioid receptor-selective ligands for ethanol drinking and anxiety. J Pharmacol Exp Ther 335, 133–139.
    OpenUrlAbstract/FREE Full Text
  87. ↵
    Vicente-Sanchez, A., Dripps, I.J., Tipton, A.F., Akbari, H., Akbari, A., Jutkiewicz, E.M., and Pradhan, A.A. (2018). Tolerance to high-internalizing delta opioid receptor agonist is critically mediated by arrestin 2. Br J Pharmacol 175, 3050–3059.
    OpenUrl
  88. ↵
    Walker, D.L., and Davis, M. (2002). Quantifying fear potentiated startle using absolute versus proportional increase scoring methods: implications for the neurocircuitry of fear and anxiety. Psychopharmacology (Berl) 164, 318–328.
    OpenUrlCrossRefPubMed
  89. ↵
    Whalen, E.J., Rajagopal, S., and Lefkowitz, R.J. (2011). Therapeutic potential of beta-arrestin- and G protein-biased agonists. Trends Mol Med 17, 126–139.
    OpenUrlCrossRefPubMedWeb of Science
  90. ↵
    Zurkovsky, L., Sedaghat, K., Ahmed, M.R., Gurevich, V.V., and Gurevich, E.V. (2017). Arrestin-2 and arrestin-3 differentially modulate locomotor responses and sensitization to amphetamine. Neuropharmacology 121, 20–29.
    OpenUrl
Back to top
Posted October 02, 2019.
Download PDF

Supplementary Material

Email
β-arrestin-dependent ERK signaling positively correlates with reduced anxiety-like and conditioned fear-related behavior in mice
Mee Jung Ko, Terrance Chiang, Arbaaz M. Mukadam, Grace E. Mulia, Anna M. Gutridge, Julia A. Chester, Richard M. van Rijn
bioRxiv 790568; doi: https://doi.org/10.1101/790568
Reddit logo Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools

Subject Area