A couple of posts ago I reviewed a paper by Braida et al (2007). Previous literature on Salvia divinorum had shown mostly negative or aversive effects of the drug on animals, but those studies had mostly used very high doses of Salvinorin A. Braida and her colleagues, however, demonstrated unique positive effects of the drug on zebrafish at lower doses; doses where the psychedelic effects only just become apparent in humans. Scientists from the same group then went on to publish several more papers investigating Salvinorin A, and here I’ll look at two of them. In these papers the authors have moved onto rodents, using mice and rats to demonstrate some more unique properties of Salvia at lower, more relevant doses.

Animal ResearchDISCLAIMER: these studies are on rodents, and measures of ‘depression’ or ‘reward’ are unlikely to fully correlate with human response to Salvia. As such, do not take the findings of these studies to mean that taking Salvia will cure your depression or reduce your anxiety in the long-term. (Picture credit: sperlaghlab.hu)

Braida et al (2008)

Conditioned Place Preference

One typical method of measuring an animal’s like or dislike for the effects of a drug is the Conditioned Place Preference (CPP) test. I have already described this test in detail here, but briefly: the animal is given a drug repeatedly in one of two rooms, then after many sessions they are allowed to move freely between the two rooms. If the animal spends more time in the drug-associated room, it may indicate that the animal enjoyed the drug and is seeking out its effects. In this study, Salvinorin A produced a similar pattern of CPP in rats compared to the author’s zebrafish study; that is, at low doses (0.1-40µg/kg) the rats frequently returned to the drug-associated room, and at a very high dose (160µg/kg) rats avoided the drug-associated room (figure 1). This provides evidence that in rodents as well as zebrafish, low doses of Salvinorin A have the potential to have rewarding effects.

Figure 1: Conditioned Place Preference from Salvinorin A in rats. A-C show an explanation of the CPP procedure. The graph shows the difference in time rats spend in their drug-associated room before and after conditioning (out of 15 minutes in total). Adapted from Braida et al (2008).

Self-administration

The authors employed an additional method to determine the rewarding effects of low-dose Salvinorin A; they trained rats to self-administer the drug. This was achieved by first training rats to press either of two levers to receive water. Once the rat had a preference for one lever over another, the experimenters changed the setup; now the preferred lever delivered a harmless saline solution to the brain of the rat, and the non-preferred lever delivered increasing doses of Salvinorin A. Interestingly, the rats started pressing the (previously non-preferred) Salvinorin A lever more than the (initially preferred) saline lever, even at very small doses (0.1µg delivered directly into the brain). Additionally, as the dose of Salvinorin A increased, the rats pressed the lever even more. Only when doses were ten times higher than the initial dose (1µg directly administered into the brain) did rats start showing aversion to the drug, and went back to pressing the initially preferred saline lever (figure 2). The authors present this as further evidence that rats will seek out pleasant effects of low doses of Salvinorin A.

Figure 2: Self-administration of Salvinorin A in rats. Diagram shows the procedure for conditioning rats to prefer one of two identical levers, both delivering harmless saline directly to the brain. Once a preference for one lever has developed, the experimenters change the less-preferred lever to deliver Salvinorin A at increasing concentrations directly to the brain. The graph shows how the rats’ lever-pressing habits change over time, as the dose of Salvinorin A increases. Presses recorded over 1-hour sessions. Adapted from Braida et al (2008).

Dopamine levels

Dopamine is an important neurotransmitter involved in reward and addiction. In the previous blog post I talked about how Salvinorin A is thought to reduce dopamine levels in the brain, potentially causing an aversive reaction to the drug. Here, the authors look at dopamine levels in the nucleus accumbens of the rat brain; an area of the brain where dopamine levels are thought to increase after taking drugs of abuse potential. In contrast to previous studies on rodents (Carlezon et al, 2006, and Zhang et al, 2005), the authors of this study find that a relatively low dose (40µg/kg) of Salvinorin A causes dopamine levels in the nucleus accumbens to increase to nearly 150% of the typical baseline. This may be evidence to suggest that Salvinorin A has the potential for abuse through typical dopaminergic addiction pathways.

Conclusions

Although the authors suggest that Salvinorin A may be a potential drug of abuse, there have been no case-studies of individual human addiction to Salvia, and other studies report lowered dopamine levels after Salvinorin A administration. Reports from individuals also seem to point towards Salvia having low addictive properties. However, this paper does provide an important bridge between fish and mammal studies, showing that in both zebrafish and rats, Salvinorin A has a dose-dependent profile of reward and aversion.

Braida et al (2009)

Following previous research that pointed towards kappa-opioid receptors being pro-depressive, Braida and her team next decided to investigate the depression- and anxiety- inducing potential of low doses of Salvinorin A.

Elevated plus maze test

This maze test is a common measure of anxiety in rodents. The apparatus consists of a plus-shaped platform, where two arms have no surrounding walls, and the other two arms are enclosed with walls (figure 3). Rodents typically avoid open areas, and so the theory of this test is that a less anxious rodent will spend more time on the open arms. At Salvinorin A doses from 0.1 all the way to 160µg/kg, rats were found to spend more time on the open arms of the maze; and indication that the rats were less anxious, or more willing to explore.

Figure 3: Elevated plus maze test reveals anxiolytic effect of Salvinorin A. Picture on the left shows a typical elevated plus maze apparatus, with rodents usually more cautious of entering the open arms. Graph shows how Salvinorin A administration makes rats more likely to spend time on the open arms. Rats were monitored for 5 minutes in total. Adapted from Braida et al (2009).

Forced swim test

The forced swim test is a common measure of ‘depression’ in rodents. It may not accurately measure psychiatric depression, but is thought to be a potential indicator of ‘despair’ in rodents. Rats are placed in a tank of water and their behaviour observed; typically rats will struggle for a while, trying to find a way out of the tank, before eventually relaxing and floating in the tank, having given up attempts at escape. Some anti-depressive drugs, such as imipramine, will cause rats to spend more time attempting to escape compared to non-drugged rats. In this study, Salvinorin A administration seemed to cause rats to swim more than control mice, almost doubling the amount of time rats spent swimming compared to non-drugged rats (figure 4). The effect was significant at doses of 10, 40 and 80µg/kg. This, say the authors, suggests that Salvinorin A may have anti-depressive properties.

Figure 4: Forced swim test reveals anti-depressant properties of Salvinorin A. Diagram shows typical setup for forced swim test. During 5 minutes of immersion in the water, the amount of time the rat spends swimming is recorded. The graph shows that Salvinorin A administration causes rats to swim for many seconds more than their non-drugged controls. Adapted from Braida et al (2009).

Tail-suspension test

This test was performed with mice, and works under the assumption that mice held by their tails for several minutes will struggle less if they are depressed. Here, the authors find that at Salvinorin A doses from 0.001 to 1µg/kg, mice struggled more than non-drugged controls. This further shows, according to the authors, potential anti-depressive properties of Salvinorin A.

Conclusions

This study challenges previous literature which showed pro-depressive effects of Salvinorin A at high doses (Carlezon et al, 2006), by presenting evidence of anti-depressive properties at low doses in rodents. However, the evidence is far from convincing. Firstly, these tests are extremely crude and give no reliable insight into the psychiatric effect of Salvinorin A on these rodents. The forced-swim, elevated plus maze and tail-suspension tests have all been previously criticised for not providing an accurate measure of depression or anxiety, and have produced conflicting results with various anti-depressant drugs (e.g. Carobrez & Bertoglio 2005). One possibility is that Salvinorin A is only making the animals more active; this may not relate to psychiatric depression. Additionally, the effects are almost certainly short-lived, so the potential of Salvinorin A in any kind of therapeutic setting may be limited.

Summary

Braida et al, in their 2008 study, have shown that the dose-dependent rewarding effects of Salvinorin A are not only present in fish, but also in mammals. In their follow-up 2009 study, Braida and colleagues have shown that Salvinorin A may have anti-depressant properties. Clearly researchers are still just scratching the surface of the fascinating properties of Salvia’s main psychedelic constituent, Salvinorin A. Its effects can be drastically different depending on dose, and its potential anti-depressant effects clearly warrant further investigation. Research into this psychedelic compound may produce many more surprises!

References

Braida D, Limonta V, Pegorini S, Zani A, Guerini-Roxxo C, Gori E & Sala M (2007) Hallucinatory and rewarding effect of Salvinorin A in zebrafish: kappa-opioid and CB1-cannabinoid receptor involvement. Psychopharmacology190:441-448

Braida D, Limonta V, Capurro V, Fadda P, Rubino T, Mascia P, Zani A, Gori E, Fratta W, Parolaro D & Sala M (2008) Involvement of kappa-opioid and endocannabinoid system on Salvinorin A-induced reward. Biol Psychiatry, 63:286-292

Braida D, Capurro V, Zani A, Rubino T, Vigano D, Parolaro D & Sala M (2009) Potential anxiolytic- and antidepressant-like effects of Salvinorin A, the main active ingredient of Salvia divinorum, in rodents. British Journal of Pharmacology, 157:844-853

Carlezon WA, Beguin C, DiNieri JA, Baumann MH, Richards MR, Todtenkopf MS, Rothman RB, Ma Z, Lee DY & Cohen BM (2006) Depressive-like effects of the kappa-opioid receptor agonist Salvinorin A on behaviour and neurochemistry in rats. J Pharm Exp Therapeutics, 316(1):440-447

Carobrez AP & Bertoglio LJ (2005) Ethological and temporal analyses of anxiety-like behaviour: The elevated plus-maze model 20 years on. Neuroscience & Biobehavioral Reviews, 29(8):1193-1205

Zhang Y, Butelman ER, Schlussman SD, Ho A & Kreek MJ (2005) Effects of the plant-derived hallucinogen salvinorin A on basal dopamine levels in the caudate putamen and in a conditioned place aversion assay in mice: agonist actions at kappa opioid receptors.Psychopharmacology179:551-558