LSD

LSD-stimulated behaviors in mice require β-arrestin 2 but not β-arrestin 1

This mice study finds that, using mice with specific receptor deficiencies, the signals are β-arrestin-2 (βArr; type of protein important in signalling) mediated, but not βarr1 mediated. This line of evidence points towards the requirement of βArr2 for LSD's psychedelic effects.

Authors

Chiu, Y-T.
Means, C. R.
Nadkarni, V.

Published

September 3, 2021

Scientific Reports

individual Study

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Abstract

Recent evidence suggests that psychedelic drugs can exert beneficial effects on anxiety, depression, and ethanol and nicotine abuse in humans. However, their hallucinogenic side-effects often preclude their clinical use. Lysergic acid diethylamide (LSD) is a prototypical hallucinogen and its psychedelic actions are exerted through the 5-HT2A serotonin receptor (5-HT2AR). 5-HT2AR activation stimulates Gq- and β-arrestin- (βArr) mediated signaling. To separate these signaling modalities, we have used βArr1 and βArr2 mice. We find that LSD stimulates motor activities to similar extents in WT and βArr1-KO mice, without effects in βArr2-KOs. LSD robustly stimulates many surrogates of psychedelic drug actions including head twitches, grooming, retrograde walking, and nose-poking in WT and βArr1-KO animals. By contrast, in βArr2-KO mice head twitch responses are low with LSD and this psychedelic is without effects on other surrogates. The 5-HT2AR antagonist MDL100907 (MDL) blocks the LSD effects. LSD also disrupts prepulse inhibition (PPI) in WT and βArr1-KOs, but not in βArr2-KOs. MDL restores LSD-mediated disruption of PPI in WT mice; haloperidol is required for normalization of PPI in βArr1-KOs. Collectively, these results reveal that LSD’s psychedelic drug-like actions appear to require βArr2.

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Research Summary of 'LSD-stimulated behaviors in mice require β-arrestin 2 but not β-arrestin 1'

Introduction

Rodriguiz and colleagues frame their study around the observation that psychedelic drugs such as LSD produce profound alterations in perception and cognition and have shown therapeutic promise for conditions including anxiety, depression, and substance-use disorders. LSD is known to act at many serotonin G protein-coupled receptors (GPCRs) and its hallucinogenic actions have been attributed primarily to activation of the 5-HT2A receptor (5-HT2AR). At the molecular level, 5-HT2AR engagement can initiate both Gq-mediated G protein signalling and arrestin-dependent (β-arrestin, βArr) pathways, and earlier work indicates LSD shows bias towards β-arrestin-mediated signalling at this receptor. The introduction notes that both non-visual arrestins, βArr1 and βArr2, are expressed in 5-HT2AR-containing neurons and that global knockout mouse lines for Arrb1 and Arrb2 are available, creating an opportunity to dissect the roles of each arrestin isoform in LSD-evoked behaviours. The present study set out to determine whether behavioural surrogates of psychedelic drug actions in mice depend on βArr1 or βArr2. Using global βArr1-KO and βArr2-KO mice and their wild-type littermates, the investigators compared LSD effects across a battery of behaviours commonly used as rodent proxies for psychedelic action (locomotion, head-twitch response, grooming and its organisation, retrograde walking, nose-poking) and on sensorimotor gating measured by prepulse inhibition (PPI). They also assessed 5-HT2AR expression by radioligand binding and immunofluorescence to check whether any behavioural differences could be explained by receptor-level changes. The work aims to clarify whether β-arrestin signalling is necessary for LSD-stimulated behaviours and, if so, which arrestin isoform is required.

Methods

The study used adult male and female mice on a C57BL/6J background: wild-type (WT) and global Arrb1 (βArr1) knockout littermates, and WT and global Arrb2 (βArr2) knockout littermates. Heterozygote matings produced the WT and KO animals; mice were housed under standard conditions and experiments followed institutional and ARRIVE guidelines. The investigators report an approximately equal mix of mutant and relevant WT littermates in each experiment and state that no sex effects were detected, so sex was collapsed in analyses. Pharmacological agents included (+)-LSD (source: NIDA), the selective 5-HT2AR antagonist MDL100,907 (MDL), haloperidol (D2 antagonist), and DOI for binding assays. MDL was tested at multiple doses that appear in the results and figures (reported as 0.05, 0.1, and 0.5 mg/kg). Haloperidol was used in PPI tests (methods list 0.1 mg/kg). Drugs were administered intraperitoneally in a 5 mL/kg volume; the vehicle composition is described. In the behavioural paradigms animals typically received vehicle or MDL, were returned to the home cage for 30 min, then received vehicle or LSD and were tested immediately. The extracted text does not clearly report the LSD dose used for all behavioural assays; however, for PPI the methods and results specify 0.3 mg/kg LSD. Behavioural testing included open-field monitoring of locomotion (distance), rearing (vertical beam breaks), and stereotypy (repetitive beam breaks <1 s) in 5-min bins over a 90 min post-LSD period; video recordings allowed scoring of head-twitch responses (HTRs), grooming duration and organisation, and retrograde walking during the first 30 min post-injection. Nose-poking was measured in a five-choice serial reaction-time apparatus without reward during 30 min sessions. Prepulse inhibition of acoustic startle (PPI) used a standard protocol with a 64 dB background, 120 dB pulse trials, and prepulse levels 4, 8, and 12 dB above background; %PPI was calculated as [1 - (pre-pulse trials / startle-only trials)] * 100. Observers scoring videos were blinded to sex, genotype and treatment. To examine receptor expression, radioligand binding used 2.3 nM [3H]-ketanserin with DOI competition and 75 μg brain protein per assay to estimate Ki values, analysed in GraphPad Prism. Immunofluorescence used a validated anti-5-HT2AR antibody on 40 μm brain sections, with standard fixation, blocking, primary (1:250) and secondary (Alexa 594) incubations and imaging at 20X. Statistical analysis employed ANOVA (one- or two-way), repeated measures ANOVA, or ANCOVA as appropriate, followed by post-hoc tests; data were checked for normality and presented as means ± SEM. Group sizes in key analyses are reported as N = 8–12 mice per group.

Results

Across multiple behavioural assays, LSD produced robust stimulant and psychedelic-like surrogate behaviours in WT mice and in βArr1-KO animals but elicited markedly reduced or absent responses in βArr2-KO mice. Baseline (pre-injection) locomotor, rearing and stereotypy did not differ between genotypes. In the βArr1 cohort, LSD increased locomotor activity relative to vehicle or MDL controls (p ≤ 0.001) and both 0.1 and 0.5 mg/kg MDL blocked this hyperlocomotion. Rearing was not significantly affected by LSD in these mice; stereotypy showed a treatment effect (ANCOVA p = 0.024) but only a trend in post-hoc comparisons. By contrast, βArr2-KO mice showed attenuated locomotor responses: LSD stimulated cumulative locomotion to a greater extent in WT than in βArr2-KO animals (p < 0.001). In WT βArr2-strain mice LSD increased locomotion and rearing (treatment effects and genotype-by-treatment interactions reported: locomotion F(5,96)=18.578, p<0.001 and genotype × treatment F(5,96)=5.273, p<0.001; rearing F(5,96)=7.150, p<0.001 with genotype × treatment F(5,96)=3.437, p=0.007). While LSD produced some increase in stereotypical activity (treatment effect F(5,96)=4.242, p=0.002), 0.5 mg/kg MDL reduced these effects to control levels. In βArr2-KO mice, LSD did not produce statistically significant increases over controls for locomotion or rearing. For head-twitch responses (HTRs), grooming, retrograde walking and nose-poking, LSD stimulated these behaviours in WT and βArr1-KO animals (p < 0.001 for many comparisons) and MDL co-administration (0.1 or 0.5 mg/kg) generally restored responses to control levels. In βArr2-KO mice HTRs were strongly blunted relative to WT (p < 0.001) and grooming duration was not significantly different from controls after LSD. Retrograde walking and nose-poking were increased by LSD in WT but not in βArr2-KO animals; MDL reduced these responses in genotypes that showed an LSD effect. Analyses of video recordings showed that LSD disrupted the normal sequential organisation of grooming (face → body → feet/tail) in WT and βArr1-KO mice, producing abbreviated or focal grooming, whereas βArr2-KO mice typically retained an intact grooming sequence under LSD. Prepulse inhibition (PPI) findings were complex. In the βArr1 cohort LSD disrupted PPI in both WT and βArr1-KO mice relative to vehicle (p ≤ 0.002). MDL at 0.1 and 0.5 mg/kg restored PPI to control levels in WT animals but failed to normalise PPI in βArr1-KO mice; however, haloperidol normalised LSD-disrupted PPI in both WT and βArr1-KO mice. In the βArr2 cohort, LSD disrupted PPI in WT mice but had no effect on PPI in βArr2-KO animals; MDL normalised PPI in WT βArr2-strain mice. Thus, βArr2-KO mice were largely resistant to LSD-induced PPI disruption. Radioligand competition binding with [3H]-ketanserin and DOI yielded similar Ki values across WT and KO brain membranes, and immunofluorescence showed comparable 5-HT2AR distribution with prominent cortical staining in all genotypes. The authors report no apparent reductions in 5-HT2AR expression in either global Arrb1 or Arrb2 knockouts. Sample sizes are indicated as N = 8–12 mice per group in several experiments, and the investigators state that no sex differences were detected.

Discussion

Rodriguiz and colleagues interpret their results as evidence that LSD-evoked behaviours classically used as rodent surrogates for psychedelic action require βArr2 but not βArr1. The main pattern is clear: WT mice and βArr1-KO animals showed LSD-stimulated locomotion, HTRs, grooming alterations, retrograde walking, nose-poking and PPI disruption, whereas these effects were greatly attenuated or absent in βArr2-KO mice. Antagonism of the 5-HT2AR with MDL100,907 blocked many LSD effects in genotypes that were responsive, consistent with 5-HT2AR involvement; for PPI, MDL restored disruption in WT animals but failed to normalize PPI in βArr1-KO mice, where haloperidol (a D2 antagonist) was required for rescue. The authors place these findings in the context of receptor signalling, emphasising that Arrb1 or Arrb2 deletion would leave G protein signalling intact but remove isoform-specific arrestin-mediated signalling and desensitisation. They note prior evidence that βArr2-dependent signalling can be more efficacious than βArr1 in certain systems and propose that βArr2-mediated pathways may be essential for the behavioural expression of LSD actions at 5-HT2AR. The discussion acknowledges LSD's polypharmacology and that other GPCRs, including dopamine receptors, could contribute to some behaviours; this possibility is used to explain why haloperidol could normalise PPI in βArr1-KO animals and why global Arrb2 deletion blunted locomotor responses that may involve dopaminergic mechanisms. Limitations and uncertainties are acknowledged: species- and protocol-dependent differences in locomotor responses to LSD (rats sometimes show decreased ambulation), contextual effects of LSD in humans and animals (the authors note a 30 min habituation may have reduced emotionality and yielded predominantly stimulatory effects), and the likelihood that multiple receptor systems contribute to repetitive behaviours like grooming or nose-poking. The immunohistochemical and binding data argue against altered 5-HT2AR expression as the explanation for behavioural differences, but the authors recognise that arrestins act as scaffolds for diverse signalling molecules and that further mechanistic work is needed to define downstream pathways that underlie the βArr2-dependence of LSD effects. Overall, the investigators conclude that βArr2 plays a critical role in mediating many LSD-stimulated behaviours and that dissecting arrestin-dependent signalling could help explain how psychedelic ligands produce their characteristic actions.

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SECTION

shown the hallucinogenic actions of LSD are blocked with the 5-HT2AR preferring antagonist ketanserin. Thus, the hallucinogenic effects of LSD appear to be mediated through the 5-HT2AR. The 5-HT2AR is a rhodopsin family member of GPCRs that is coupled to G q protein and to non-visual arrestin mediated signaling. Recent experiments reveal the 5-HT2AR preferentially activates G q family members, with moderate activity at G z , and minimal activities at G i -, G 12/13 -, and G s -family members. However, the 5-HT2AR binds to both β-arrestin 1 (βArr1) and βArr2 proteins in vitro and is complexed with these βArrs in cortical neurons in vivo. Note, within the arrestin family the non-visual βArr1 and βArr2 are termed Arr2 and Arr3, respectively. While most GPCR agonists, like 5-HT, activate both G protein and βArr signaling, ligand binding can activate also G protein-dependent signaling while serving to activate or inhibit βArr-mediated signaling. Hence, a given ligand can act as an agonist at one pathway while inhibiting the other pathway or it can possess combinations of these actions. This property is termed functional selectivity or biased signalingand ligands have been developed to exploit these signaling features. Although LSD activates G protein signaling at many GPCRs, this psychedelic stimulates βArr-mediated responses at most tested biogenic amine GPCRs. Interestingly, LSD displays βArr-biased signaling at the 5-HT2AR. Most 5-HT2AR-containing neurons express both βArr1 and βArr2, and global βArr1 and βArr2 knockout (KO) mice have been generated. Since LSD is βArr biased at the 5-HT2AR, the present investigations were conducted to determine whether LSD produces behavioral effects that were differential among the wild-type (WT) and βArr1-KO, and WT and βArr2-KO mice.

RESULTS

The Arrb1 (βArr1 protein) or Arrb2 (βArr2 protein) genes were obtained from 129 libraries, the constructs were injected into ES cells that were microinjected into C57BL/6 blastocysts. These chimeric mice were backcrossed to C57BL/6J mice. Both the βArr1 and βArr2 mice are on a C57BL/6J genetic background and are maintained as separate strains. However, their behavioral responses are somewhat different between the strains. All experiments have an approximate equal mix of mutant and relevant WT littermates. No sex effects were detected in any experiments.

EFFECTS OF ARRB1 OR ARRB2 DELETION ON LSD-STIMULATED MOTOR ACTIVITIES. LSD HAS BEEN REPORTED

to stimulate, inhibit, or produce biphasic effects on a variety of motor activities in rodents. We examined responses to LSD in the global βArr1-KO and global βArr2-KO mice to determine whether disruption of either gene product could modify the behavioral responses to this hallucinogen and to test whether 5-HT2AR antagonism could block these effects. Locomotor, rearing, and stereotypical activities were monitored at 5-min intervals over the 120 min test in both the βArr1 and βArr2 genotypes (Supplementary Figures). When cumulative baseline locomotion was examined in βArr1 mice, activity was not differentiated by genotype or by the pre-assigned treatment condition (Supplementary Table). Following LSD injection, only treatment effects were found (Fig.). Here, locomotor activities were stimulated by LSD relative to control groups given the vehicle or 0.5 mg/kg MDL alone (p values ≤ 0.001). When administered with LSD, both doses of MDL blocked the locomotor-stimulating effects of this psychedelic. An examination of cumulative baseline rearing and stereotypical activities in the βArr1 mice found these overall responses to be significantly lower or higher in the vehicle, LSD, 0.1 mg/kg MDL plus LSD, and 0.5 mg/kg MDL plus LSD pre-assigned treatment groups than in the pre-assigned 0.5 mg/kg MDL group (p values ≤ 0.001) (Supplementary Table. To correct for these baseline differences in the subsequent LSD-post injection analyses for βArr1 mice, their rearing and stereotypical data were analyzed separately by ANCOVA. No significant effects of LSD were observed for rearing (Fig.). By comparison for stereotypical activities, ANCOVA revealed a significant main effect of treatment in βArr1 mice following LSD administration (p = 0.024). Nevertheless, Bonferroni post-hoc analyses only identified a trend between the group treated with LSD and the group given MDL alone (p = 0.062) (Fig.). Collectively, these results indicate that LSD stimulates locomotor activities to similar extents in the WT and βArr1-KO animals, and the 5-HT2AR antagonist MDL blocks these responses. Rearing and stereotypical activities are unaffected by LSD in either genotype. When baseline motor activities were evaluated in the βArr2 mice, no significant differences were found (Supplementary Table). Effects of LSD in the βArr2-KO mice were quite different from those of the WT animals. LSD was more potent in stimulating cumulative locomotor activities in the WT than in the βArr2-KO mice (p values < 0.001) (Fig.). When locomotion was analyzed within WT animals, the LSD-stimulated responses were higher than those in the vehicle and MDL controls, as well as in the treatment groups administered MDL with LSD (p values < 0.001). Hence, all three doses of the 5-HT2AR antagonist were efficacious in suppressing the LSD-induced hyperlocomotion to control levels. Although LSD increased locomotor activity in βArr2-KO mice, it was not significantly different from any other treatment group. Similar to locomotion, LSD also stimulated rearing activities to a greater extent in WT compared to βArr2-KO mice (p values < 0.001) (Fig.). In WT animals, vertical activities were increased with LSD over that of the vehicle and MDL controls (p values < 0.001). Rearing was higher also in mice given 0.05 mg/kg MDL plus LSD than the controls (p values ≤ 0.029). When 0.1 or 0.5 mg/kg MDL was given with LSD, both doses reduced the LSD-stimulated rearing activities to control levels (p values ≤ 0.001). By comparison, LSD was without effect in the βArr2-KO mice. An assessment of stereotypical activities failed to find any genotype differences between the βArr2 mice (Fig.). Nonetheless, treatment effects were evident with LSD stimulating stereotypical activities over that of the vehicle and MDL controls (p values ≤ 0.013). Notably, 0.5 mg/kg MDL abrogated the LSD effects (p = 0.003) by bringing levels to those of the controls. Together, these results indicate that LSD stimulates locomotor responses in the WT and βArr1-KO animals. LSD stimulated also locomotor, rearing, and stereotypical activities in WT mice from the βArr2 strain. The 5-HT2AR antagonist blocks these LSD-stimulated activities. By LSD effects on additional behaviors. LSD modifies a number of behaviors in micethat include, at least, HTRs, grooming, and retrograde walking. When these responses were examined in the βArr1 mice, no genotype differences were noted, although overall treatment effects were evident. Relative to the vehicle and MDL controls, LSD stimulated HTRs, grooming, and nose-poking behaviors in the WT and βArr1-KO mice (p values < 0.001) (Fig.). When 0.1 or 0.5 mg/kg MDL was administered with LSD, both doses of the 5-HT2AR antagonist blocked the LSD effects by restoring the numbers of HTRs, the duration of grooming, and nose-poking behaviors to those of the controls. Besides HTRs and grooming, LSD was efficacious in potentiating retrograde walking in the WT and βArr1-KO mice compared to the vehicle and MDL controls (p values < 0.001) (Fig.). Responses were higher also with 0.1 mg/kg MDL plus LSD than the controls (p ≤ 0.018). Nonetheless, both 0.1 and 0.5 mg/kg MDL decreased retrograde walking to control levels when administered with LSD (p values < 0.001). In contradistinction to βArr1 mice, genotype differences were present for βArr2 animals. HTRs were significantly increased in the LSD and 0.05 mg/kg MDL plus LSD groups of WT relative to βArr2-KO mice (p values < 0.001) (Fig.). In WT mice, HTRs were stimulated by LSD and they were still enhanced when 0.05 or 0.1 mg/kg MDL were given with LSD relative to the vehicle and MDL controls (p values < 0.001). Notably, both 0.1 and 0.5 mg/kg MDL significantly reduced the LSD-stimulated responses (p values ≤ 0.002)-with the higher MDL dose being the more efficacious in suppressing HTRs to control levels (p < 0.001). In βArr2-KO mice, the LSD and the 0.05 and 0.1 mg/kg MDL plus LSD treatments increased HTRs compared to the vehicle and MDL controls (p values ≤ 0.023). Only 0.5 mg/kg MDL was sufficient to normalize the LSD-stimulated response to control levels in the βArr2-KO mice (p = 0.019). For grooming, the durations of responding were higher in WT than in the βArr2-KO groups administered LSD alone, 0.05 mg/kg MDL plus LSD, or 0.5 mg/kg MDL with LSD (p values ≤ 0.016) (Fig.). In WT mice, grooming was augmented in the LSD and the 0.05 mg/kg MDL plus LSD groups relative to the vehicle and MDL controls (p values < 0.001). While 0.05 mg/kg MDL failed to block the LSD effects, both of the 0.1 and 0.5 mg/ kg doses were efficacious in normalizing the responses to that of the controls (p values < 0.001). In βArr2-KO animals, the duration of grooming to LSD was not significantly different from the vehicle and MDL controls. Nevertheless, grooming was enhanced in mice administered 0.05 mg/kg MDL plus LSD compared to all groups (p values ≤ 0.013), except those given LSD alone. Since LSD can induce alterations in tactile perception, we examined grooming in detail as it has a chained organization of responses in rodents. Note, since in our video recordings the WT mice in the βArr1 and βArr2 strains responded similarly to the vehicle and MDL controls, as well as to LSD and the 0.5 mg/kg MDL plus LSD treatments for grooming, recordings from only one of the WT strains is presented. Analyses of the videorecordings confirmed that all genotypes engaged in a normal sequence of grooming beginning with the face, progressing down the body, and ending at the feet or tail (Movie 1). When LSD was administered, the sequence of grooming in the WT and βArr1-KO mice became abbreviated, non-sequential, and/or restricted to one area of the body (Movies 2-3). By comparison, the grooming sequence was complete and rarely perturbed with LSD in the βArr2-KO animals (Movie 4). When the 5-HT2AR antagonist MDL was administered alone, the organization of grooming was intact in the WT and βArr1-KO mice (Movie 5). By comparison, with MDL the βArr2-KO animals often paused in grooming bouts and/or displayed twitching of the neck and back muscles; however, they typically finished the grooming sequence (Movie 6). The patterns of grooming among the genotypes administered MDL plus LSD were divergent. In WT mice given MDL plus LSD, the organization of grooming was restored but with some focus initially on facial groming (Movie 7). When the βArr1 mutants received the same treatment, they began the grooming sequence, engaged in focal grooming of a part of the body, and then completed the sequence (Movie 8). When this same drug combination was administered to βArr2-KO mice, they usually began the sequence appropriately, but at some mid-or later-point they would become focused on one body area of grooming and sometimes did not complete the grooming sequence (Movie 9). Aside from abnormalities in the organization of grooming, LSD also induced retrograde walking and stimulated nose-poking behaviors. Incidences of retrograde walking were increased significantly in WT mice in the groups given LSD or 0.05 MDL plus LSD compared to the βArr2-KO groups (p values ≤ 0.040) (Fig.). In WT mice, LSD potentiated the incidences of retrograde walking compared to the MDL and vehicle controls (p < 0.001). Although 0.05 mg/kg MDL was ineffective in decreasing this LSD-stimulated behavior, both 0.1 and. Two-way ANOVAs failed to identify any significant effects for baseline locomotion, rearing, or stereotypy. (a) LSD-stimulated locomotor activities in WT and βArr2-KO subjects. A two-way ANOVA reported a significant treatment effect [F(5,96) = 18.578, p < 0.001] and a significant genotype by treatment interaction [F(5,96) = 5.273, p < 0.001]. (b) LSD-stimulated rearing activities in βArr2 animals. A two-way ANOVA observed a significant treatment effect [F(5,96) = 7.150, p < 0.001] and a significant genotype by treatment interaction [F(5,96) = 3.437, p = 0.007]. (c) LSD-stimulated stereotypical activities in βArr2 mice. A two-way ANOVA identified a significant treatment effect [F(5,96) = 4.242, p = 0.002]. N = 8-12 mice/group. ***p < 0.001, WT vs. KO; +++ p < 0.001, LSD vs. designated groups within genotype; ^p < 0.05, 0.05 MDL + LSD vs. controls within genotype. Bonferroni corrected post-hoc tests for stereotypy for treatment effects: p < 0.05, LSD vs. vehicle and MDL controls; p < 0.01, LSD vs. 0.5 MDL + LSD. 0.5 mg/kg MDL suppressed this response to that of controls (p values < 0.001). By contrast, LSD was without any significant effect on retrograde walking in the βArr2-KO animals compared to its vehicle and MDL controls. Similar to retrograde walking, nose-poking behavior was increased by LSD in WT relative to βArr2-KO mice (p < 0.001) (Fig.). In WT mice, LSD stimulated nose-poking behaviors relative to all other groups (p values < 0.007). All doses of the 5-HT2AR antagonist reduced the LSD-stimulated nose poking to the levels of the vehicle and MDL controls. No treatment effects were noted among the βArr2-KO animals. In summary, responses to LSD across these LSD-stimulated behaviors were similar between the WT and βArr1-KO mice and the 5-HT2AR antagonist reduced these responses to levels of the vehicle and MDL controls. By contrast, the WT mice responded quite differently from the βArr2-KO animals. HTRs, grooming, retrograde waking, and nose-poking to LSD were significantly higher in WT than in βArr2-KO mice. Notably, LSD disrupted the sequences of grooming in the WT and βArr1-KO mice; βArr2-KO animals were unaffected. Nonetheless, divergent responses to MDL alone or to MDL plus LSD were observed among the genotypes. LSD and MDL100907 effects on prepulse inhibition. LSD disrupts PPI in both rats and humans and the response can be restored with 5-HT2AR antagonists. βArr1 mice were pre-treated with the vehicle or with 0.1 or 0.5 mg/kg MDL. Subsequently, they were administered the vehicle or 0.3 mg/kg LSD and tested in PPI. No significant genotype or treatment effects were observed for null activity or in response to the 120 dB startle stimulus (Supplementary Figure). In contrast, genotype effects were found in PPI where responses in the WT groups that received 0.1 or 0.5 mg/kg MDL plus LSD were higher than those in βArr1-KO animals (p values ≤ 0.018) (Fig.). As anticipated, LSD disrupted PPI in both βArr1 genotypes relative to their vehicle and 0.5 mg/kg MDL controls (p values ≤ 0.002). Both 0.1 and 0.5 mg/kg MDL normalized PPI in WT mice to control levels. In βArr1-KO animals, PPI was still significantly disrupted in mice administered 0.1 mg/kg MDL with LSD relative to controls (p values ≤ 0.001). Although PPI responses in the 0.5 mg/kg MDL plus LSD group were not significantly different from these controls, they were also not significantly different from the LSD group. Hence, LSD disrupted PPI in both WT and βArr1-KO mice, while MDL restored PPI only in WT animals. Since haloperidol can normalize PPI in mouse models, we tested whether this antipsychotic drug could normalize the LSD-disrupted PPI in the βArr1-KO mice. Overall treatment effects were found where null activities were higher in the 0.1 mg/kg haloperidol plus LSD group than in mice treated with the vehicle or haloperidol alone (p values = 0.009) (Supplementary Figure). An assessment of startle activity revealed that responses were lower overall in the WT relative to βArr1-KO mice (p = 0.028) (Supplementary Figure). For PPI, genotype effects were found where responses were reduced overall in the βArr1-KO compared to the WT animals (p = 0.008) (Fig.). Treatment effects were observed also, where LSD suppressed PPI relative to all other treatment conditions (p values ≤ 0.002). Here, haloperidol normalized the LSD-disrupted PPI to control levels in both WT and βArr1-KO mice. PPI responses in the βArr2 mice were examined also. Overall null activity was decreased in the 0.1 mg/kg MDL plus LSD group compared to the vehicle control and the LSD group (p values ≤ 0.003) (Supplementary Figure). No significant effects were detected for startle activity (Supplementary Figure). Nevertheless, genotype differences were evident for PPI (Fig.). Here, responses to LSD and to the 0.05 MDL plus LSD treatments were reduced in WT relative to the βArr2-KO mice (p values ≤ 0.001). In WT animals, LSD disrupted PPI compared to the MDL and vehicle controls (p values = 0.001). PPI remained disrupted in the 0.05 mg/kg MDL plus LSD group relative to the MDL control (p = 0.050). However, PPI was normalized to controls with 0.1 mg/ kg MDL. By comparison, LSD was without effect in the βArr2-KO mice. Collectively, these findings show that LSD disrupts PPI in both genotypes of the βArr1 mice. PPI was disrupted also with LSD in the WT animals from the βArr2 strain. The 5-HT2AR antagonist restored PPI in both WT strains, whereby haloperidol was required to normalize it in βArr1-KO mice. By contrast, PPI in βArr2-KO mice was unaffected by LSD.

EFFECTS OF ARRB1 OR ARRB2 DELETION ON 5-HT2AR EXPRESSION. WE EXAMINED WHETHER DELETION OF ARRB1

or Arrb2 could alter 5-HT2AR expression by radioligand binding with brains from WT and βArr1-KO, and WT and βArr2-KO littermates. When [ 3 H]-ketanserin competition binding was examined, displacement with DOI and Ki values were found to be very similar with membranes from the WT and βArr1-KO and the WT and βArr2-KO brains (Fig.). We examined also 5-HT2AR immunofluorescence in βArr1 and βArr2 brain sections (Fig.). Here, we detected no apparent alterations in the relative receptor distributions among the genotypes, with prominent 5-HT2AR immunostaining in the cortex. Together, these results are consistent with the hypothesis that neither global Arrb1 nor global Arrb2 genetic deletion decreases 5-HT2AR expression.

DISCUSSION

In the present study, we analyzed whether global deletion of Arrb1 or Arrb2 was involved in LSD-stimulated responses in mice. In many cases, we found that LSD modified behaviors in both βArr strains of WT mice, as well as in the βArr1-KO animals. By contrast, LSD exerted little effect on βArr2-KO responses. Collectively, these results suggest the LSD-stimulated responses require βArr2. In this regard, βArr2 is reported to play a similar role in morphine-stimulated hyperlocomotionand amphetamine-stimulated locomotor and rearing activities in βArr2 mice. While we found LSD stimulates locomotion in mice, in rats it has been reported to decrease ambulation 35 or increase locomotion. While an inhibitory response to 0.2 mg/kg LSD was observed in rats, we only saw stimulatory effects with 0.3 mg/kg LSD and in pilot studies, doses of 0.1-0.5 mg/kg LSD were all stimulatory. An absence of LSD inhibitory effects could be attributed to differences in species tested, test environment and apparatus, and/or test procedure. In humans LSD's behavioral effects can be context specific 1,2 and our 30 min habituation to the open field prior to LSD administration may have reduced emotionality in our mice, such that only the stimulatory effects of LSD were evident. To determine whether the locomotor-stimulating effects of LSD were due to 5-HT2AR activation, MDL was used as an antagonist. When used alone, this antagonist exerted no effects on motor performance in either βArr mouse strain. Importantly, 0.1 and 0.5 mg/kg MDL blocked the locomotor-stimulating effects of LSD in both WT strains and in the βArr1-KO animals. A similar antagonist effect has been observed in rats. Hence, the present results indicate that the LSD-induced hyperactivity in βArr mice is promoted through the 5-HT2AR. Despite this finding, LSD binds to other GPCRs including dopamine receptors. Since various drugs of abuse are known to stimulate dopamine neurotransmission, it is likely these receptors are involved in the observed LSD-stimulated response. Notably, global deletion of Arrb2 blunts locomotor responses to amphetamine in the open field. Thus, the reduced response to LSD by the βArr2-KO mice may be due actions mediated not only through the 5-HT2AR, but also through a dopamine receptor mechanism. Besides motor activity, we examined the effects of LSD on HTRs, grooming, retrograde walking, and nosepoking behaviors. LSD and other psychedelics are well-known to stimulate HTRs in miceand this behavior has been proposed as a proxy for hallucinations in humans. Compared to vehicle, LSD stimulated HTRs to similar extents in WT and βArr1-KO mice. In βArr2-KO animals, this response was severely blunted compared to the WT controls. These results were unexpected since the individual competition binding curves could be superimposed among the different genotypes. Regardless, in both βArr1 and βArr2 mice, MDL reduced HTRs to levels of the vehicle controls. These findings are consistent not only with the known action of MDL on blocking HTRs to various hallucinogens, but also on the inability of LSD and other psychedelics to induce this response in the htr2A homozygous mutant mice. Aside from HTRs in rodents, LSD accentuates grooming behaviors in catsand it can stimulate or inhibit grooming in mice. In our investigations, LSD augmented grooming in both WT strains, and in βArr1-KO animals. By comparison, this psychedelic was ineffective in βArr2-KO mice. In both WT strains and in βArr1-KO animals, 0.1 and 0.5 mg/kg MDL returned the LSD-stimulated grooming to control levels. Thus, antagonism of the 5-HTR2A was sufficient to restore LSD-induced grooming to baseline. Effects of LSD were examined also for the organization of grooming behavior. Under vehicle treatment, all mice displayed similar patterns of grooming that began with the face, progressed to the flanks, and ended with the feet or tail. LSD disturbed this sequence of events in both WT strains and in βArr1-KO mice. By comparison, grooming in the βArr2-KO mice was largely unaffected by LSD. MDL did not alter grooming in the WT and βArr1-KO mice, whereas it prolonged grooming and promoted twitching of the neck and back muscles in βArr2-KO animals. This 5-HT2AR antagonist blocked the LSD-disrupting effects on the organization of grooming in WT mice and it mostly restored it in βArr1-KO animals. The MDL-LSD combination in βArr2-KO animals produced some disturbances, but the mice typically completed the grooming sequence. Together, these results suggest that additional receptor systems may be involved in the LSD-induced grooming responses. The effects of LSD on retrograde walking and nose-poking responses were also examined. We found LSD to stimulate these behaviors in WT animals from both strains, as well as in the βArr1-KO mice. However, LSD promoted neither response in βArr2-KO animals. Nevertheless, in the other genotypes MDL restored retrograde walking and nose-poking to the levels of vehicle controls. Hence, this 5-HT2AR antagonist normalized these LSD-stimulated behaviors. It should be emphasized that repetitive behaviors like grooming or nose-poking may be mediated by several receptor systems. For instance, these repetitive responses can be modified at least through alterations in serotonergic, dopaminergic, and glutamatergic neurotransmission. Given the polypharmacology of LSD, it is likely the LSD disruptive effects on these behaviors are mediated through additional receptor systems in our experiments. LSD-induced states share many similarities with the early acute phases of psychosis. PPI is abnormal in individuals diagnosed with schizophreniaand LSD disrupts PPI in rats. In βArr1 mice, LSD disrupted PPI in both genotypes without affecting startle or null activities. Both 0.1 and 0.5 mg/kg MDL restored the LSDdisrupted PPI, but only in WT mice; an effect consistent with the action of the 5-HT2AR antagonist MDL11939 in rats. By comparison, MDL was ineffective in blocking the LSD effects in βArr1-KO animals. Since LSD activates human dopamine D2 receptors, we used haloperidol as a D2 antagonist. We found this antagonist to restore the LSD-disrupted PPI in the βArr1-KO mice. Parenthetically, both 0.1 and 0.2 mg/kg haloperidol failed to rescue PPI in rats given 0.1 mg/kg LSD (s.c.); the possible reasons for this discrepancy in mice versus rats are unclear. When βArr2 mice were tested, LSD disrupted PPI selectively only in WT mice. Notably, βArr2-KO mice were completely unresponsive to this psychedelic. As with WT animals from the βArr1 strain, MDL also normalized the LSD-disrupted PPI in the WT βArr2 mice. Thus, the LSD effects on PPI in the βArr mice are complex, with restoration of PPI with MDL in both strains of WT mice, normalization of PPI with haloperidol in βArr1-KO animals, and without any discernable effect in βArr2-KO subjects. LSD and other psychedelics are well-known for their hallucinogenic actions 1 and these responses have been attributed to 5-HT2AR agonism. We observed LSD to stimulate motor activity, head twitches, grooming, retrograde walking, and nose-poking in both βArr strains of WT mice and in βArr1-KO animals. LSD also disrupted PPI in these same genotypes. The LSD-elicited responses in βArr2-KO mice were either significantly attenuated or completely absent. In conditions where LSD produced changes in behavior, these alterations were blocked with the 5-HT2AR antagonist MDL. While these results suggest that the 5-HT2AR is an essential component for all these responses, it should be recalled that LSD exerts a plethora of actions at many GPCRs 8-10 and, aside from HTRs, other behaviors are inconsistently affected by hallucinogens. Hence, it is likely that LSD's effects on the 5-HT2AR are involved in a cascade of many GPCR-signaling events mediating these varied responses. In this regard, arrestins are known to serve as scaffolds for many signal transduction molecules. Future work will examine some of these mechanisms across our behavioral tests. Our immunohistochemical results show that the 5-HT2AR protein is expressed in several areas of the brain, especially the cortex. In this brain region the 5-HT2ARs are localized primarily to pyramidal cells and, to some extent, to interneurons. Since the glutamatergic neurons project to multiple subcortical brain areas, actions on 5-HT2ARs in these neurons could exert varied effects on behavior. Within 5-HT2AR-containing neurons, agonist actions at this receptor can lead to G protein-dependent and -independent signaling, the latter of which involves βArr. Disruption of the Arrb1 or Arrb2 genes would leave G protein signaling intact, while affecting respective βArr2 or βArr1 mediated signaling and desensitization. Zurkovsky and colleagueshave proposed a model of arrestin actions that may apply to our results with LSD. Both βArr1 and βArr2 are co-expressed, with few exceptions throughout the adult rodent brain. However, expression of βArr1 mRNA is much higher than that for βArr2--except in selected brain areas. While the 5-HT2AR binds to both βArr proteins in vitro and is complexed with these βArrs in cortical neurons in vivo, there is some evidence that the affinities of βArr1 and βArr2 for different GPCRs can vary in vitro. Moreover, in the few systems that have been studied, signaling in the presence of βArr2 is more efficacious than with βArr1. In our experiments, the LSD-elicited responses were largely intact in the βArr1-KO than in the βArr2-KO mice, because in the βArr1-KO animals βArr2-mediated signaling is still retained. In this regard, it is especially intriguing that LSD-induced HTRs were much more robust in both WT strains and in the βArr1-KO animals, than in the βArr2-KO mice. Our results with LSD suggest that βArr2 may be essential for the expression of hallucinogenic-like actions at the 5-HT2AR.

METHODS

Subjects. Adult male and female WT and βArr1-KO, and WT and βArr2-KO mice were used in these experiments. All mice had been backcrossed onto a C57BL/6J genetic background. Heterozygotes were used to generate the respective WT and KO animals. The mice were housed 3-5/cage in a temperature-and humiditycontrolled room on a 14:10 h (lights on at 0600 h) light-dark cycle with food and water provided ad libitum. All experiments were conducted with an approved protocol from the Duke University Institutional Animal Care and Use Committee and all experiments and methods were performed in accordance with the relevant regulations and ARRIVE guidelines. Drugs. The drugs consisted of (+)-LSD-(+)-tartrate (NIDA Drug Supply Program, Bethesda, MD), MDL 100,907 (Bio-Techne Corp., Minneapolis, MN), haloperidol (Sigma-Aldrich, St. Louis, MO), and (-)-1-(2,5-diethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI; Sigma-Aldrich). The vehicle was composed of N,Ndimethyllacetamide (final volume 0.5%; Sigma-Aldrich) that was brought to volume with 5% 2-hydroxypropoylβ-cyclodextrin (Sigma-Aldrich) in water (Mediatech Inc., Manassas, VA). All drugs were administered (i.p.) in a 5 mL/kg volume. All studies used groups that were administered the vehicle and the 5-HT2AR antagonist, MDL100907 as controls. Open field activity. Motor activities were assessed in an open field (21 × 21 × 30 cm; Omnitech Electronics, Columbus, OH) illuminated at 180 lux. All behaviors were filmed. Mice were injected with the vehicle or different doses of MDL and placed into the open field. Thirty min later, they were administered the vehicle or LSD and were immediately returned to the open field for 90 min. Motor activity was monitored using Fusion Versamax 5.3 Edition software (Omnitech Electronics, Columbus, OH) for locomotor activity (distance traveled), rearing (vertical beam-breaks), and stereotypical activities (repetitive beam-breaks less than 1 s) in 5-min blocks or as cumulative activities. Head twitch, grooming, and retrograde walking. These behaviors were filmed during assessment of motor activity. The responses were scored over the first 30 min following injection of the vehicle or LSD after collection of baseline activity. Observers who were blinded to the sex, genotype, and treatment conditions in the experiments scored the video recordings. The data are expressed as the numbers of head twitches, duration of grooming, and incidences of retrograde walking. Nose-poking responses. Nose-pokes were monitored in a 5-choice serial reaction-time apparatus (Med Associates Inc., St. Albans, VT). Each chamber had five LED-illuminated 1.24 cm 2 nose-poke apertures with infrared diodes to register nose pokes. No food or liquid reward was available. Mice were injected with the vehicle or different doses of MDL and returned to their home-cages. Thirty min later, the animals were injected with the vehicle or LSD and were placed immediately into the operant chambers for 30 min. The data are depicted as the numbers of head pokes.

PREPULSE INHIBITION (PPI).

PPI of the acoustic startle response was conducted using SR-LAB chambers (San Diego Instruments, San Diego, CA) as reported. Mice were injected with vehicle or different doses of MDL or with 0.1 mg/kg haloperidol and returned to their home cages. Fifteen min later the animals received the vehicle or LSD and were placed into the apparatus. After 10 min of habituation to a white noise background (64 dB), testing began. Each test consisted of 42 trials with 6 null trials, 18 pulse-alone trials, and 18 prepulse-pulse trials. Null trials comprised the white noise background, pulse trials consisted of 40 ms bursts of 120 dB white-noise, and prepulse-pulse trials were composed of 20 ms pre-pulse stimuli that were 4, 8, or 12 dB above the whitenoise background (6 trials/dB), followed by the 120 dB pulse stimulus 100 ms later. Testing commenced with 10 pulse-alone trials followed by combinations of the prepulse-pulse and null trials, and it terminated with 10 pulse-alone trials. PPI responses were calculated as %PPI = [1 -(pre-pulse trials/startle-only trials)]*100. Radioligand binding and immunohistochemistry of the 5-HT2AR. Binding experiments on mouse brains were conducted as described using 2.3 nM [ 3 H]-ketanserin (NEN Life Sciences, Wellesley, MA) as the radioligandwith varying concentrations of unlabeled DOI (Sigma-Aldrich) and 75 μg protein from brain. Binding was analyzed by GraphPad Prism (San Diego, CA). The 5-HT2AR immunofluorescence study was performed as describedwith a validated 5-HT2AR-specific antibody. Mice were intracardially perfused with PBS followed by 4% paraformaldehyde (PFA) in PBS. Brains were harvested, post-fixed overnight in 4% PFA, and dehydrated in 30% sucrose. Brains were sectioned at 40 µm by cryostat. Brain sections were washed 3X with 0.4% Triton X-100 in PBS (TX-100/PBS) before incubating for 1 h with blocking buffer (5% normal donkey serum in 0.4% TX-100/PBS). Next, they were incubated for 48 h at 4 °C with the anti-5-HT2AR antibody (1:250, #RA24288; Neuromics, Edian, MN). Subsequently sections were washed 3X with 0.1% TX-100/PBS and incubated for 2 h with the secondary antibody (1:1000, donkey anti-rabbit, Alexa Fluor 594; Jackson Immunoresearch, West Grove, PA). The sections were imaged under a 20X objective using an Olympus VS120 virtual slide microscope (Olympus, Tokyo, Japan).

STATISTICS.

All statistical analyses were performed with IBM SPSS Statistics 27 programs (IBM, Chicago, IL). The data are presented as means and standard errors of the mean. No sex effects were detected in any experiments. Hence, this variable was collapsed. All data were normally distributed. One-or two-way ANOVA, repeated measures ANOVA (RMANOVA), or analyses of covariance (ANCOVA) were used to analyze the data, followed by Tukey or Bonferroni post-hoc analyses. A p < 0.05 was considered significant. All results were plotted using GraphPad Prism.