The 5HT2A receptor is a serotonin receptor that is normally found on the surface of cells as a G protein-coupled receptor (Wikipedia contributors, 2023a). This means that the ligand, which is nominally serotonin, binds to the receptor which in turn activates second messengers within the cell, such as cAMP. Other potential ligands for this receptor include LSD and psilocybin mushrooms, which are often termed “serotonergic” psychedelics. According to the article discussing the recent work by Olson and colleagues (Lanese, 2023), the are several of these receptors that are also located within the cell, near the Golgi body. This would make sense, since the Golgi body is responsible for maturation of proteins, and GPCRs would finish “assembly” in the Golgi and exit from the Golgi would be regulated (Wu, 2013). The work being discussed here (Olson et al., 2023) looks into the activation of intracellular 5HT2A receptors by serotonergic psychedelics, which have the ability to pass through the cell membrane, in contrast to serotonin, which is more polar and thus unable to cross the membrane.

Serotonin is derived from tryptophan (Wikipedia contributors, 2023b), which is an essential amino acid that must be obtained through the diet. Serotonin looks like tryptophan, but without the carboxylic acid group and with the addition of a hydroxy group on the aromatic ring. Serotonin has often been implicated in depression due to the moderate success of antidepressants targeting serotonin reuptake. The “chemical imbalance” or “serotonin” hypothesis is still a view subscribed to by many practitioners and 80% or more of the general public (Moncrieff et al., 2022). However, the recent systematic review finds no evidence of association between serotonin and depression, as well as no support for low serotonin causing depression. This being said, there is still varying success with the use of serotonergic drugs which will be discussed below, and use of serotonergic psychedelics suggests an increase in plasticity of rat neurons (Olson et al., 2023).

LSD, DMT, and Psylocibin are mentioned in the article as psychedelics that could cross the cell membrane and attach to 5HT2A receptors. LSD is synthesized from lysergic acid, which is derived from a fungus (Wikipedia contributors, 2023c). It targets the 5HT2A receptor and can cause visual and auditory hallucinations. DMT can be extracted from several different plants and occurs endogenously in many animals, including humans (Wikipedia contributors, 2023d). It targets the 5HT2A receptor and produces geometric hallucinations. Psylocibin is found in several species of fungi, most commonly in the genus Psilocybe, which are termed “magic mushrooms” (Wikipedia contributors, 2023e). It targets the 5HT2A receptor and causes visual hallucinations and perceived spiritual experiences. Looking at the structure of each of these three compounds, as well as serotonin, similarity can be seen regarding the indole structure present.

Neuroplasticity is the ability of the nervous system to change the way it is connected. This occurs through several avenues, including how neurons are connected, how many connections there are, and strengthening of the connections themselves (Wikipedia contributors, 2024f). Recent studies have connected the success of antidepressants not to the increase of serotonin itself, but on the increase in neuroplasticity that is associated with it (Pittinger & Duman, 2008). This hypothesis suggests that synaptic deficits are caused by high levels of stress, which in turn contribute to depression and PTSD (Holmes et al., 2019). Consequently, drugs that increase neuroplasticity should decrease symptoms of depression and PTSD. If the psychedelic drugs used in this study increased neuroplasticity, more robust connections should be seen, which could result in lessening of depression symptoms.

 As mentioned earlier, this study compared several different drugs, both polar and nonpolar, that would act on the 5HT2A receptor. The main hypothesis being tested was that plasticity, and concurrent reduction in depression, was driven by activating the intracellular, rather than extracellular, 5HT2A receptors. Researchers first tested this by increasing N-methylation on tryptamine (making NMT with one methyl group and DMT with two) and measuring dendrite morphology, or increase in spines. As tryptamine gains more methyl groups, it becomes more hydrophobic and able to enter the cell through the membrane, allowing it to act on intracellular receptors. In Figure 1D, the neurons associated with each treatment can be seen: the control and tryptamine both have 6 spines, NMT has 8, and DMT has at least 10 and shows branching. This increase in number of spines and complexity is confirmed in Fig 1B, the Sholl analysis, which shows a quantitative measure of dendritic crossings and complexity. The amount of crossings increases as N-methylation increases. This means that an increase in hydrophobicity of the ligand is associated with neuroplasticity.

Next the researchers looked at where the 5HT2A receptors were located in the neuron versus kidney cells (Wikipedia contributors, 2023g). The results of this are seen in Figure 2, which shows the 5HT2AR, β2AR (known to be localized in the cell membrane), and cytoplasm (as an intracellular control) tagged with fluorescent proteins. It looks like the 5HT2A receptor may be more intracellular than the β2AR, but it is hard to tell visually. However, it is confirmed in the paper that the 5HT2AR was more correlated with the Golgi apparatus in neurons than kidney cells (in a supplementary figure, S4).

With these two correlations in mind, membrane permeable psychoactive drugs (DMT and psilocin) and their charged analogs (TMT and psilocybin) were used to treat neurons. Electroporation, which allows charged molecules to enter the cell through temporary membrane openings, was used to observe its effect on the polar analogs. The results are seen in Figure 3, which shows that the nonpolar, membrane permeable drugs resulted in more dendritic spines than the polar charged drugs. With electroporation however, the polar drugs resulted in about the same amount of spines. For example, TMT in Fig. 3B has 7 weak spines without electroporation, but 9 strong spines and branching with electroporation. Fig. 3C confirms that this is statistically significant in the graph. This finding further shows that it is activation of the intracellular receptors that results in the growth of new spines.

For the final part of the study, the researchers wanted to see if serotonin would result in new spine growth when able to enter the cell membrane. First, they did this through electroporation, which resulted in significantly more crossings, seen in Figure 4A. Next, they added serotonin transporters (SERTs) to the neurons and measured the number of crossings under a variety of conditions. As seen in Figure 4F, serotonin only produced more spines when SERTs were present, while DMT established growth with or without SERT presence. Two conditions were used to block the ligands, one with citalopram (an antidepressant, which inhibits SERT) and KTSN (which is membrane permeable and inhibits 5HT2AR). As expected, the citalopram condition resulted in no growth for serotonin, since it could not enter the cell, and no effect for DMT, which is able to enter the cell without use of SERTs. In the KTSN condition, neither serotonin nor DMT resulted in growth since the intracellular receptors were inhibited.

To test the effects of SERTs in vivo, the researchers induced SERT production in mice and flooded the neurons with serotonin using PCA. Figure 5D shows that mice expressing SERTs had more dendritic spine growth than those without. Behavioral tests were also done with these mice, notably the forced swim test (FST) seen in Figure 5G. The mice expressing SERTs spent significantly less time immobile than controls, which suggests that they are in a less depressive state.

The compounds that were used should have an effect on depression because they activate the intracellular 5HT2A receptor, which is associated with new dendritic spine growth and neuroplasticity. Neuroplasticity in turn is associated with reduction in depression, as discussed above. The mechanism of these compounds is essentially the opposite of traditional antidepressants. Whereas antidepressants, namely SSRIs, increase extracellular serotonin by blocking reuptake, the study increased intracellular serotonin by increasing intake (or by using drugs that bypass a need for transporters).

This study established the presence of intracellular 5HT2A receptors in neurons, as well as the connection between their activation and neuroplasticity. These findings were also used in an animal model, which suggested a reduction of depressive symptoms in mice who had increased neuroplasticity due to intracellular 5HT2A activation. The work done in this study opens up many avenues for future research, especially in creating more effective depression treatments and understanding the cellular basis of depression. There are still more connections that need to be made, but this is a good bridge between the serotonin hypothesis of depression and current models involving neuroplasticity.

References

1. Wikipedia contributors. (2023a). 5-HT2A receptor, https://en.wikipedia.org/wiki/5-HT2A_receptor

2.  Lanese, N. (2023). Psychedelics may treat depression by invading brain cells. LiveScience, https://www.livescience.com/psychedelics-may-treat-depression-by-invading-brain-cells

3. Wu, G. (2012). Regulation of Post-Golgi Traffic of G Protein-Coupled Receptors. Subcell Biochem 63, 83-95.

4. Vargas, M., Dunlap, L., Dong, C., Carter, S., Tombari, R., Jami, S., Cameron, L., Patel, S., Hennessey, J., Saeger, H., McCorvy, J., Gray, J., Tian, L., Olson, D. (2023). Psychedelics promote neuroplasticity through the activation of intracellular 5-HT2A receptors. Science 379, 700-706.

5. Wikipedia contributors. (2023b). Serotonin, https://en.wikipedia.org/wiki/Serotonin

6. Moncrieff, J., Cooper, R., Stockmann, T., Amendola, S., Hengartner, M., Horowitz, M., The serotonin theory of depression: a systematic umbrella review of the evidence. Molecular Psychiatry, https://www.nature.com/articles/s41380-022-01661-0

7. Wikipedia contributors. (2023c). LSD, https://en.wikipedia.org/wiki/LSD

8. Wikipedia contributors. (2023d). N,N-Dimethyltryptamine, https://en.wikipedia.org/wiki/N,N-Dimethyltryptamine

9. Wikipedia contributors. (2023e). Psilocybin, https://en.wikipedia.org/wiki/Psilocybin

10. Wikipedia contributors. (2023f). Neuroplasticity, https://en.wikipedia.org/wiki/Neuroplasticity

11. Pittenger, C., Duman, R. (2008). Stress, Depression, and Neuroplasticity: A Convergence of Mechanisms. Neuropsychopharmacology 33, 88-109.

12. Holmes, S., Scheinost, D., Finnema, S., Naganawa, M., Davis, M., DellaGioia, N., Nabulsi, N., Matuskey, D., Angarita, G., Pietrzak, R., et al. Lower synaptic density is associated with depression severity and network alterations. Nat. Commun. 10, 1529.

13. Wikipedia contributors. (2023g). HEK 293 cells,