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 rodents17,32-36. 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 Figs. S1-S2).
When cumulative baseline locomotion was examined in βArr1 mice, activity was not differentiated by genotype or by the pre-assigned treatment condition (Supplementary Table S1). Following LSD injection, only treatment effects were found (Fig. 1a). 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 (p-values≤0.001). It should be emphasized that no sex effects were detected in any experiments in this manuscript.
An examination of cumulative baseline rearing and stereotypical activities in the βArr1 mice found these responses to be significantly lower in some pre-assigned treatment groups than in others (p-values≤0.001) (Supplementary Table S1). To correct for these baseline differences in the subsequent LSD-post injection analyses, the rearing and stereotypical data were submitted separately to ANCOVA. No significant effects of LSD were observed for rearing (Fig. 1b). By comparison for stereotypical activities, ANCOVA revealed a significant treatment effect 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. 1c). Collectively, these results indicate that LSD stimulates locomotor activities to similar extents in the WT and βArr1-KO animals, and the 5-HT2AR antagonist 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 S2). 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. 2a). 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. 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. 2b). In WT animals, rearing activities were increased with LSD over that of the vehicle and MDL controls (p-values<0.001). 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. 2c). 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). Together, these results indicate that LSD stimulates motor responses to similar extents in the WT βArr1 and WT βArr2 mice, and in the βArr1-KO animals. The 5-HT2AR antagonist blocks these LSD-stimulated activities. By striking comparison, LSD exerts minimal effects on these same responses in the βArr2-KO mice where none of their motor activities were significantly increased above that of controls.
LSD effects on additional behaviors. LSD modifies a number of behaviors in mice12,17,37-41 that 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 in the WT and βArr1-KO mice (p-values<0.001) (Fig. 3a). 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 to those of controls. Aside from HTRs, LSD augmented also grooming over that of the controls (p-values<0.001) (Fig. 3b). When 0.1 or 0.5 mg/kg MDL was given with LSD, both doses of the 5-HT2AR antagonist normalized the LSD stimulatory effects to those of controls (p-values<0.001).
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. 3c;). With LSD, both 0.1 and 0.5 mg/kg MDL depressed retrograde walking (p-values<0.001). Nose poking behaviors were examined also. Here, LSD increased nose-poking over that of controls (p-values<0.001) (Fig. 3d). When MDL was administered with LSD, both doses of the 5-HT2AR antagonist normalized the LSD stimulated nose-poking behaviors (p-values<0.001).
In contradistinction to βArr1 mice, genotype differences were identified between the βArr2 animals. HTRs were significantly increased with LSD in WT relative to βArr2-KO mice (p<0.001) (Fig. 4a). Genotype effects were noted also in the 0.05 mg/kg MDL plus LSD group (p<0.001). In WT mice, HTRs were stimulated by LSD and they were still enhanced when 0.05 MDL was 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 the βArr2-KO mice, the LSD (p-values≤0.023) and 0.05 mg/kg MDL plus LSD treatments (p-values≤0.006) increased HTRs compared to the vehicle and MDL controls. Only 0.5 mg/kg MDL was sufficient to normalize this LSD-stimulated response 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. 4b). In WT mice, LSD augmented grooming relative to the vehicle and MDL controls (p<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 (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 the group 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 perception42, we examined grooming in detail as it has a chained organization of responses in rodents43. Note, that since the WT βArr1 and WT βArr2 mice responded identically to the different treatment conditions, only one of the WT strains is represented. Analyses of the video-recordings 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 sharp comparison, the grooming sequence was complete and rarely perturbed 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 would finish 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 (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 area of grooming (Movie 9). Nevertheless, they usually completed the grooming sequence
Aside from abnormalities in the organization of grooming, LSD also induced retrograde walking and stimulated nose-poking behaviors. No significant genotype effects were obtained for retrograde walking (Fig. 4c). 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 0.5 mg/kg MDL suppressed this response (p-values<0.001). By contrast, LSD was without any significant effect on retrograde walking in the βArr2-KO animals. Similar to retrograde walking, no genotype effects were observed for nose poking behavior (Fig. 4d). In WT mice, LSD stimulated nose-poking behaviors relative to all other groups (p-values<0.001). 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 βArr1 genotypes and the 5-HT2AR antagonist reduced these responses to levels of the vehicle and MDL controls. Importantly, the WT mice responded quite differently than the βArr2-KO animals. HTRs and grooming to LSD were significantly higher in WT than in βArr2-KO mice. LSD did not significantly increase retrograde walking or nose poking behaviors in the βArr2-KO animals. Notably, LSD disrupted the sequences of grooming in WT and in βArr1-KO mice; the βArr2-KO animals were unaffected. Nonetheless, divergent responses to MDL alone or MDL plus LSD were observed among the genotypes, indicating actions required by 5-HT2AR activation.
LSD and MDL100907 effects on prepulse inhibition. LSD disrupts PPI in both rats and humans and the response can be restored with 5-HT2AR antagonists37,44. βArr1 mice were pre-treated with the vehicle or with 0.1 or 0.5 mg/kg MDL as controls. 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 Fig. S3a-b). In contrast, genotype effects were found in PPI where 0.1 and 0.5 mg/kg MDL normalized PPI in WT mice, whereby these same doses were ineffective in the βArr1-KO animals (p-values≤0.018) (Fig. 5a). As anticipated, LSD depressed PPI in both βArr1 genotypes relative to their MDL and vehicle controls (p-values≤0.002). Thus, LSD depressed PPI in both WT and βArr1-KO mice, while MDL only restored PPI in WT animals.
Since haloperidol can normalize PPI in mouse models45, we tested whether this antipsychotic drug could normalize the LSD-disrupted PPI in the βArr1-KO mice. For null activity, no genotype effects were evident (Supplementary Fig. S3c). Overall treatment effects were found in the βArr1 animals 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). An assessment of startle activity revealed that responses were lower overall in the WT relative to βArr1-KO mice (p=0.028) (Supplementary Fig. S3d). For PPI, responses were reduced overall in the βArr1-KO compared to the WT animals (p=0.008) (Fig. 5b). 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 in both WT and βArr1-KO mice.
PPI responses in the βArr2 mice were examined also. No significant genotype effects were reported for null or startle activities. 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 Fig. S4a). No significant effects were detected for startle activity (Supplementary Fig. S4b). Nevertheless, striking genotype differences were evident for PPI (Fig. 6). 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 suppressed PPI compared to the MDL and vehicle controls (p=0.001). PPI was normalized when 0.1 mg/kg MDL was given with LSD. By dramatic comparison, LSD was completely without effect in the βArr2-KO mice. Collectively, these findings show that LSD disrupts PPI in both genotypes of the βArr1 mice. PPI was aberrant also the WT βArr2 animals. 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 [3H]-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. 7a). We examined also 5-HT2AR immunofluorescence in βArr1 and βArr2 brain sections (Fig. 7b-e). Here, we detected no apparent alterations in the relative receptor distributions among the genotypes. Together, these results are consistent with the hypothesis that neither global Arrb1 nor global Arrb2 genetic deletion decreases 5-HT2A receptor expression.