It’s no secret that plasticity is becoming a buzzword in the psychedelic field. Anyone and everyone points to plasticity as being the cause for behavioral change. But there is still not a full understanding of this concept, as evidence to support this notion is still being collected! Further, it’s important to understand the nuances in changes in plasticity that scientists refer to. Recently, a paper titled “5-MeO-DMT modifies innate behaviors and promotes structural neural plasticity in mice” was published by Jefferson et al., and actually measures changes in structural plasticity in response to serotonergic psychedelic administration.
What Is 5-MeO-DMT?
5-MeO-DMT is a serotonergic psychedelic that is found naturally occurring on the Sonoran desert toad (yes the one people say they lick to ingest the psychedelic, please don’t though). Despite being naturally occurring in the toad, it is most commonly inhaled and has a rapid onset of about a minute, with its psychoactive effects lasting for only about 20 minutes. Interestingly, this drug is a more selective agonist of the 5-HT1A receptor as opposed to 5-HT2A receptor, which is common in tryptamine psychedelics. Clinical research suggests that it is safe with only few recorded adverse effects and has been shown an effective treatment for anxiety and depression. As with everything, this needs to be investigated further and more controlled clinical trials are necessary.
Question and Purpose
So now, let’s dive into the paper! With the knowledge that 5-MeO-DMT results in decreases in measures of psychopathology in humans, this lab wanted to test whether they could visualize structural plasticity changes in response to 5-MeO-DMT administration in a live behaving animal overtime. Further, they tested if these changes occurred in conjunction with a change in the animal’s behavior.
Methods
This lab is known for using some really cool and complex imaging techniques and the paper showcased this for us once again. They used both male and female mice and characterized the psychedelic onboarding by observing the head twitch response, which if you’ve been following along you know is a high frequency movement of the head which correlates with drug receptor occupancy, effects in the brain, and is our only measure of whether a psychedelic drug is activating 5-HT2A or not in rodents.
The lab employed two-photon imaging to visualize changes in cell morphology. They did this by conducting a surgery in which a piece of the skull was removed from the animal and replaced with a glass screen through which the animal could be imaged over time. Cells 400 microns below the dura were imaged while the animal was head fixed in an apparatus and anesthetized. Just want us to stop here and think about how incredible it is that we can follow the same neurons over time like this!
As their measure of behavior, they used ultrasonic vocalizations between the mice to note changes in socialization behavior. Male mice were injected with saline and then placed in a chamber with a female mouse and the interaction vocalizations were recorded, this was repeated for 3 days. One the fourth day the male mice were injected with a drug and this same interaction observation occurred.
What did they actually find?
After conducting HTR analysis, the authors decided to use 20 mg/kg 5-MeO-DMT, 1 mg/kg psilocybin, and 10 mg/kg ketamine. Ketamine is the positive control, as it has been shown to produce increases in dendritic spines in neurons. As a reminder, spines refer to dendritic spines on neuronal processes. These little growths are very important for creating functional connections with other cells and an increase in the number and size of these would suggest an increase in structural plasticity.
Imaging: 10 mice (4 females; 6 males) were imaged two days before 5-MeO-DMT administration followed by two day intervals for one week and a final session 1 month later. They found that spine density and formation of spines was significantly increased, but spine size remained unchanged. They noted that there was actually a decrease in spine density for the vehicle (control) group.
Behavior: With 15 male mice, they characterized 11 different types of vocalizations and found that psychedelic administration seems to significantly suppress vocalization behaviors, 5-MeO-DMT more so than psilocybin and ketamine. Both male and females produce vocalizations when they are removed from their mothers at weaning or earlier and males produced female-induced vocalizations during mating. This study assessed male vocalization during introduction to female mates, which is thought to convey an “emotional state.” To our knowledge, vocalizations have not been studied using psychedelics until this study.
What do these results mean?
When we think about results, it’s important to think critically. Therefore, it’s important to note the WHOPPING dose of 5-MeO-DMT these animals were given. This study compared 1 mg/kg of psilocybin to 20 mg/kg of 5-MeO-DMT, in which their rationale was that the HTR experiments produced an equivalent amount of head twitches (see the figure from the text on the right). While this rationale is not uncommon it’s usually decided based on determining the maximal amount of effect produced by a drug (Emax) or 50% of the maximal effect (ED50) of the drugs following a log dose response curve, which was not reported in this publication. It’s said that “the dose makes the poison” when it comes to all drugs, and in rodents, this large of a dose of 5-MeO-DMT may produce a behavioral threshold where the animal is unable to behave normally in other measures. There is concern of off-target receptor effects because of the large amount of drug in the system. Something to keep in mind as we proceed.
In the imaging experiments they noted increases in spine density and formation rate, however, they note that spine size remained unchanged. It is likely that if they averaged spine size across all the spines, by including newly formed spines which will naturally have a smaller spine head, they pulled their spine head average down. These two groups should have been analyzed separately for a more accurate result. Further, they note that in the vehicle group spine density actually decreases. In figure 3D, they have a well-powered two-way repeated measures ANOVA (a common statistical analysis in this type of research) and the effect of 5-MeO-DMT is far from being statistically different from the basal (dotted line at Y=1). Interestingly, there are more differences between the vehicle group and Y=1 at day 34 than between 5-MeO-DMT and Y=1. One possible explanation is that when imaging neurons, the light can bleach the cells, causing issues with
quantification, or perhaps the vehicle (in some cases ethanol + saline) itself was causing decreases. It is difficult to determine an effect of a drug treatment if the vehicle control is producing its own effects.
Finally in the behavior experiments with vocalizations, they used only males due to their rationale that males tend to vocalize more than females. Interestingly, they do use males and females in the HTR experiments, but not enough per group to analyze sex as an independent variable. This is a large issue considering sex differences with tryptamine and phenethylamine psychedelics have been previously reported by multiple labs. Regardless, they found that ultrasonic vocalizations were significantly suppressed in males and in the plots you can note that for the 5-MeO-DMT group they are completely gone. This is unsurprising considering the massive dose they were given. The use of other behavioral tasks such as measures of locomotion would have provided more information about the doses used and their potential to obliterate normal behavior or if this was a specific effect on this social vocalization behavior.
Additionally, the publication did not include any statistical analyses representation on their graphs. Statistical tests are not shown in the figure legends, and differences (asterisks, for examples) are not shown either. The only reporting of statistical analyses is within the text, which while a stylistic choice, makes it difficult to read/interpret results.
Conclusion
Overall, the methodology of two-photon imaging is an incredible way to visualize structural plasticity in real time, which is important to understanding psychedelics effects. It is important to follow up on these studies, especially in behavioral assays, to make sure to include sex as an independent variable and be sure that the effects seen are psychedelic-specific.