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ORIGINAL INVESTIGATION
Comparison of the discriminative stimulus effects
of salvinorin A and its derivatives to U69,593
and U50,488 in rats
Lisa E. Baker &John J. Panos &Bryan A. Killinger &
Mary M. Peet &Lisa M. Bell &Larissa A. Haliw &
Sheryl L. Walker
Received: 19 May 2008 /Accepted: 28 December 2008 / Published online: 20 January 2009
#Springer-Verlag 2009
Abstract
Background and rationale Research interests regarding the
psychopharmacology of salvinorin A have been motivated
by the recreational use and widespread media focus on the
hallucinogenic plant, Salvia divinorum. Additionally, kappa
opioid (KOP) receptor ligands may have therapeutic
potential in the treatment of some neuropsychiatric con-
ditions, including drug dependence and mood disorders.
Salvinorin A is a selective KOP agonist, but only a few
studies have explored the discriminative stimulus effects of
this compound.
Objective This study compared the discriminative stimulus
effects of salvinorin A and two synthetic derivatives of
salvinorin B to the KOP agonists, U69,593 and U50,488.
Materials and methods Sixteen male Sprague–Dawley rats
trained to discriminate U69,593 (0.13 mg/kg, s.c., N=8) or
U50,488 (3.0 mg/kg, i.p., N=8) under a fixed-ratio 20
schedule of food reinforcement were administered substitu-
tion tests with salvinorin A (0.125–3.0 mg/kg, i.p.). The
animals trained to discriminate U69,593 were also adminis-
tered substitution tests with salvinorin B ethoxymethyl ether
(0.005–0.10 mg/kg, i.p.) and salvinorin B methoxymethyl
ether (0.03–0.10 mg/kg, i.p.). Another eight rats were trained
to discriminate 2.0 mg/kg salvinorin A and tested with
U69,593 (0.04–0.32 mg/kg) and U50,488 (0.4–3.2 mg/kg).
Results Salvinorin A and both synthetic derivatives of
salvinorin B substituted completely for U69,593. Addition-
ally, cross-generalization was observed between salvinorin
A and both KOP agonists.
Conclusion These findings support previous reports indi-
cating that the discriminative stimulus effects of salvinorin
A are mediated by kappa receptors. Future studies may
assist in the development and screening of salvinorin A
analogs for potential pharmacotherapy.
Keywords Salvia divinorum .Salvinorin A .
Kappa receptors .U69,593 .U50,488 .Salvinorin B EOM .
Salvinorin B MOM .Drug discrimination .Rats
Salvia divinorum is a plant indigenous to Mexico that has
been used in shamanistic rituals by the Mazatec people of
Oaxaca for several centuries (Wasson 1962; Váldes et al.
1984,1993). More recently, recreational use of this plant
and its extracts has gained considerable popularity and
widespread media attention in the United States and
Europe. Indeed, recreational use of this substance is
currently a popular topic of You Tube videos. Despite the
growing popularity of this substance, its current legal status
in the U.S. is unscheduled, which means growing, buying, and
selling the plant are not regulated by the federal government.
S. divinorum leaves and highly potent fortified extracts of
this plant can be purchased legally over the Internet (Drug
Enforcement Administration 2008; Prisinzano 2005a). The
rising popularity of its recreational use has led to legal action
in several of the U.S. states to ban the sale and use of S.
divinorum and its extracts (Siebert 2007)andtheDrug
Enforcement Administration has placed it on the list of drugs
of concern (Drug Enforcement Administration 2008).
Salvinorin A, the active constituent of S. divinorum,isa
non-nitrogenous neoclerodane diterpene (Ortega et al.
1982; Váldes 1994). Currently the most potent naturally
occurring hallucinogen (Váldes et al. 1984; Siebert 1994),
salvinorin A is structurally distinct from all other known
Psychopharmacology (2009) 203:203–211
DOI 10.1007/s00213-008-1458-3
L. E. Baker (*):J. J. Panos :B. A. Killinger :M. M. Peet :
L. M. Bell :L. A. Haliw :S. L. Walker
Department of Psychology, Western Michigan University,
Kalamazoo, MI 49008, USA
e-mail: Lisa.Baker@wmich.edu
psychoactive compounds. Salvinorin A induces an intense
and short-lived hallucinogenic experience reported to be
qualitatively distinct from that induced by the classic
hallucinogens, lysergic acid diethylamide and mescaline
(Siebert 1994). Several studies have demonstrated that
salvinorin A is a highly selective and potent kappa opioid
(KOP) receptor agonist (Roth et al. 2002; Sheffler and Roth
2003; Chavkin et al. 2004; Yan and Roth 2004). Chavkin et
al. (2004) examined the agonist activity at KOP receptors
expressed in Xenopus oocytes with salvinorin A, U50,488,
U69,593, and dynorphin A. Activity was determined by
measuring potassium conductance through G protein-gated
K+ channels. Results indicated that salvinorin A was
significantly more efficacious than either U50,488 or
U69,593 in this assay and comparable in efficacy to
dynorphin A, the endogenous ligand for KOP receptors.
In addition to clinical and scientific interests in charac-
terizing the abuse liability of salvinorin A, the unique
pharmacological profile of this compound may lead to
exciting prospects in the development of pharmacothera-
peutics for neuropsychiatric disorders, including drug
dependence (Prisinzano 2005b; Shippenberg et al. 2001,
2007). Thus, recent research efforts have focused on
characterizing the behavioral effects of this compound in
preclinical screening assays. There is now a considerable
body of experimental research regarding the neurobehavio-
ral effects of salvinorin A in a variety of animal species,
including zebra fish, rodents, and rhesus monkeys. Many of
the existing studies have compared the behavioral effects of
salvinorin A with the synthetic KOP receptor agonist,
U69,593, and reported substantial similarities between these
compounds with regards to their effects on locomotor
activity and motor coordination, sedative effects, and
antinociceptive properties. For example, salvinorin A pro-
duced sedation and decreased motor coordination of mice in
an inverted screen task (Fantegrossi et al. 2005), increased
immobility and decreased swimming behaviors of rats in a
forced swim test (Carlezon et al. 2006), and produced
antinociceptive effects in mice in tail flick and hot plate tests
of nociception (John et al. 2006; McCurdy et al. 2006).
Despite the recent surge in behavioral studies of
salvinorin A, there is still insufficient scientific knowledge
regarding the abuse liability of salvinorin A at the present
time. In view of anecdotal reports and survey studies
indicating that this substance tends to produce dysphoria
(Gonzalez et al. 2006), the abuse liability of S. divinorum
and its extracts may be relatively low compared to other
recreational drugs. However, given the paucity of con-
trolled scientific research, this conclusion may be a bit
premature. A few investigators have recently examined
salvinorin A in animal models of drug-induced reward,
including conditioned place preference and intracerebro-
ventricular (i.c.v.) self-administration (Zhang et al. 2005;
Braida et al. 2007; Braida et al. 2008). The results of these
studies suggest that high doses of salvinorin A have
aversive effects, although low doses may exert rewarding
effects. Interestingly, Braida et al. (2008) reported that the
reinforcing effects of low salvinorin A doses were
attenuated by the CB1 antagonist rimonabant (1 mg/kg) as
well as the KOP antagonist, nor-BNI (10 mg/kg). Using in
vivo microdialysis techniques, Braida et al. (2008) also
observed that a low dose of salvinorin A (40 μg/kg)
increased extracellular dopamine levels by approximately
150% in the NAc shell. This finding is in contrast to
previous findings that higher doses of salvinorin A
decreased NAc DA levels (Zhang et al. 2005).
Further research is required to more fully characterize
the psychopharmacology of salvinorin A. Drug discrimina-
tion procedures are a well-established animal model for
classifying novel compounds with putative psychoactive
actions because they allow for a comparison between novel
substances and known psychoactive drugs (Schuster and
Johanson 1988; Balster 1991). Salvinorin A has not yet
been evaluated extensively in drug discrimination procedures,
although a few studies have tested salvinorin A in rhesus
monkeys (Butelman et al. 2004) or rats (Willmore-Fordham
et al. 2007) trained to discriminate the kappa agonist
U69,593 or monkeys trained to discriminate the serotonergic
hallucinogen 2,5-dimethoxy-4-methylamphetamine (DOM;
Li et al. 2008). Butelman et al. (2004) demonstrated stimulus
generalization to salvinorin A in three rhesus monkeys
trained to discriminate the kappa agonist, U69,593. More-
over, salvinorin A and U69,593 produced similar dose-
dependent and time-dependent functions, indicating similar
stimulus properties of these two drugs. In contrast, the N-
methyl-D-aspartic acid receptor antagonist, ketamine, failed
to substitute for U69,593, ruling out the possibility that
animals might respond similarly to a hallucinogen with
different neuropharmacological actions. The kappa receptor
antagonists, quadazocine and 5′-guanidinonaltrindole
(GNTI), blocked the substitution of salvinorin A, although
GNTI effectively blocked salvinorin A substitution in only
two of the three subjects tested.
In a more recent study, Willmore-Fordham et al. (2007)
trained ten male Sprague–Dawley rats to discriminate
0.56 mg/kg U69,593 (i.p., 10 min) and tested a range of
salvinorin A doses (1.0–3.0 mg/kg) for substitution. At all
doses tested, salvinorin A exhibited full substitution for
U69,593 and these effects were blocked by i.c.v. adminis-
tration of the kappa receptor antagonist, nor-BNI (4.5 nM),
administered 60 min prior to salvinorin A. These findings
are consistent with those of Butelman et al. (2004).
Together, these findings suggest that the discriminative
stimulus effects of salvinorin A are mediated by kappa
receptors. Only one published study to date has tested
salvinorin A in animals trained to discriminate another
204 Psychopharmacology (2009) 203:203–211
hallucinogen. Li et al. (2008) recently reported that
salvinorin A did not substitute for the discriminative
stimulus effects of the serotonergic hallucinogen DOM in
rhesus monkeys.
To date, there are no published reports of animals trained to
discriminate salvinorin A. This may be related to the limited
solubility of salvinorin A. In preliminary studies employing a
vehicle comprised of ethanol/Tween 80/sterile water (1:1:8
proportion, by volume), we experienced difficulties maintain-
ing adequate stimulus control with salvinorin A in rats.
Therefore, the current study used a different vehicle, 75%
dimethyl sulfoxide (DMSO) in sterile water and successfully
established discrimination with 2.0 mg/kg salvinorin A. The
main objective of the present study was to determine whether
stimulus generalization between salvinorin A and other kappa
receptor agonists is symmetrical. In the first two experiments,
salvinorin A was tested for substitution in animals trained to
discriminate either U69,593 (0.13 mg/kg) or U50,488
(3.0 mg/kg). In addition, two synthetic derivatives of
salvinorin B, the ethoxymethyl ether (salvinorin B EOM)
and the methoxymethyl ether (salvinorin B MOM) were tested
for substitution in the animals trained to discriminate U69,593.
The chemical structures of these test compounds are depicted
in Fig. 1. The third experiment represents the first study to
establish discrimination with salvinorin A (2.0 mg/kg) and
assess the effects of other kappa agonists for substitution.
Materials and methods
Subjects
Twenty-four male Sasco Sprague–Dawley rats (Charles
River, Portage, MI, USA) were individually housed in
polycarbonate cages with corn cob bedding in a colony
maintained with a 12-h light/dark cycle (lights on 0700 to
1900) and constant temperature (20± 2°C) and humidity
(50±5%). Water was freely available in the home cages and
commercial rodent diet was restricted to maintain body
weights at 80–85% of free-feeding levels, accounting for
age-related growth. Animals were maintained according to
the general principles of animal husbandry outlined by the
National Research Council (1996), and the experimental
protocol was approved by the Institutional Animal Care and
Use Committee of Western Michigan University.
Apparatus
Behavioral training and test sessions were conducted in 16
standard operant conditioning chambers (Med-Associates,
Georgia, VT, USA) equipped with three retractable levers
on the front panel, a food pellet delivery mechanism located
above the center lever, and a 28-V house light located at the
top of the rear panel. Experimental events and data
collection were computer-controlled using MED-PC (ver-
sion 4.0 for Windows) instrumentation and software.
Lever pressing was reinforced with dustless precision
food pellets (45 mg, product # F0021, Bioserv®, French-
town, NJ, USA).
Drugs
Salvinorin A was obtained from the National Institute on
Drug Abuse (Bethesda, MD, USA) and McLean Hospital
(Belmont, MA, USA). Salvinorin B EOM and MOM were
obtained from McLean Hospital. Due to limited solubility
of these compounds, they were initially dissolved in DMSO
and then diluted with sterile water to 75% DMSO. This
vehicle has been used in a previous study (Willmore-Fordham
et al. 2007) and was well-tolerated by the animals in the
current study. There were no obvious signs of tissue
necrosis following repeated injections with this vehicle.
U69,593 (National Institute on Drug Abuse, Bethesda, MD,
USA) was initially dissolved in a few drops of lactic acid
and then diluted in sterile water. U50,488 (National
InstituteonDrugAbuse,Bethesda,MD,USA)was
dissolved in sterile water. Drug or vehicle injections were
administered intraperitoneally (i.p.) or subcutaneously (s.c.)
in a volume of 1 mg/mL. All drug doses were determined
based on the weight of the salt.
Preliminary training
Prior to lever-press training, subjects were exposed to two
60-min sessions in which food pellets were delivered under
a fixed-time 60 s (FT60″) schedule to familiarize animals
with the sound and location of food reinforcers. During
Fig. 1 Structures of salvinorin A, salvinorin B MOM, and salvinorin
B EOM in comparison to U50,488 and U69,593
Psychopharmacology (2009) 203:203–211 205
these sessions, no levers were present in the chambers.
Animals were then trained to press the center lever for food
pellets on a fixed-ratio 1 (FR 1) schedule during a single
20-min training session. This was the only session in which
the center lever was used. Once animals were trained to
lever press, a series of four to six errorless training sessions
were conducted in which only the left lever or the right
lever was present. Thirty minutes before these training
sessions, animals were injected with the training drug (see
below) or its vehicle solution. For half the animals in each
group of eight rats, responses on the left lever were
reinforced following drug administration and responses on
the right lever were reinforced following vehicle adminis-
tration. Conditions were reversed for the remaining animals
in each group. An equal number of errorless sessions were
conducted with each training condition. During these
sessions, responding was initially reinforced under an FR
1 schedule and the response requirement was gradually
increased from an FR 1 up to an FR 20 depending on an
individual animal’s performance. The FR was programmed
to increment by two after every fifth reinforcer earned. The
number of reinforcers that could be earned during a training
session was limited only by the schedule and by the
duration of the session. Once animals were reliably
responding under both drug and vehicle conditions,
discrimination training sessions commenced.
Discrimination training
For discrimination training sessions, both left and right
levers were always present. These sessions lasted 20 min/
day and were conducted approximately the same time of
day 6 to 7 days/week. Drug and vehicle training sessions
were alternated to include at least three drug training
sessions and at least three vehicle training sessions per
week with no more than two consecutive sessions under the
same stimulus conditions. A resetting FR schedule of
reinforcement was in effect, requiring a fixed consecutive
number of correct responses for reinforcer delivery.
Incorrect responses reset the response counter. Similar to
the errorless training sessions, responding was initially
reinforced under an FR 1 schedule and the response
requirement was incremented by two after every fifth
reinforcer up to a final FR 20. Once animals were
responding reliably under a resetting FR 20 schedule under
both drug and vehicle conditions, this schedule remained in
effect for the remainder of training. Discrimination accura-
cy was determined by calculating the percentage of correct
lever presses prior to the first food pellet delivery during
each training session. When discrimination performance
was 80% or greater for at least eight out of ten consecutive
training sessions, an animal was said to have met the
discrimination criterion. Three different groups of eight rats
were trained to discriminate one of the following com-
pounds: U69,593 (0.13 mg/kg), U50,488 (3.0 mg/kg), or
salvinorin A (2.0 mg/kg). Specific details regarding the
training and testing of each group are described below in
three separate experiments.
Stimulus generalization tests
Once animals met the criterion for stimulus discrimination,
test sessions were conducted to determine substitution with
a range of doses of the training drug. Following determi-
nation of dose–response curves with each training drug,
three to four doses of each test compound were tested for
substitution to the training stimulus. For each compound
tested, the order of test doses was randomized among
animals in each group. For each test dose, approximately
half the animals were tested following a drug training
session and the other half were tested following a vehicle
training session. Test sessions were conducted once or
twice per week depending on the performance of individual
animals. Animals received a minimum of one drug training
session and one vehicle training session between tests. Tests
were conducted only when discrimination performance
during these training sessions was at least 80%. Test
sessions were conducted in a similar manner to training
sessions with the exception that no reinforcers were
delivered and the animal was removed from the operant
conditioning chamber immediately following completion of
20 consecutive responses on either lever.
Experiment 1
Eight rats were trained to discriminate 0.13 mg/kg
U69,593(s.c.,30min)andadose–response curve was
generated with the training drug following the procedures
described above. Subsequently, these animals were admin-
istered substition tests with salvinorin A (0.125–1.0 mg/kg,
i.p., 30 min) salvinorin B EOM (0.005–0.10 mg/kg, i.p.,
30 min), and salvinorin B MOM (0.03–0.10 mg/kg, i.p.,
30 min).
Experiment 2
Eight rats were trained to discriminate 3.0 mg/kg U50,488
(i.p., 30 min) according to the procedures described above.
Subsequently, dose–response tests were conducted with
U50,488 (0.375–3.0 mg/kg) and salvinorin A (0.25–
3.0 mg/kg, i.p., 30 min) in five of these animals.
Experiment 3
Eight rats were trained to discriminate salvinorin A. The
initial training dose selected was 0.5 mg/kg (i.p., 15 min).
206 Psychopharmacology (2009) 203:203–211
Only two of the eight animals met the discrimination
criteria in less than 50 training sessions. After 58 training
sessions, the training dose was increased to 1.0 mg/kg and
the injection interval was increased to 30 min, based on
observations that this dose substituted in the animals trained
to disriminate U69,593 in experiment 1. There was no
improvement in the discrimination after an additional 30
training sessions, so the training dose was subsequently
increased to 2.0 mg/kg. Stimulus control was established
under these conditions in seven of the eight animals.
Substitution tests were conducted with the following
compounds: salvinorin A (0.25–2.0 mg/kg, N=7),
U69,593 (0.04–0.32 mg/kg, N=5–6), and U50,488 (0.4–
3.2 mg/kg, N=5–6).
Data analyses
The mean (±SEM) number of sessions to criterion were
calculated for each training drug. For each training drug
and test compound, dose–response curves were plotted to
depict the percentage of responses made on the drug-
appropriate lever and the response rate (number of
responses per second) at each dose. Group means (±SEM)
were calculated and plotted in the dose–response curves.
The test data from animals that completed all doses of a
particular test compound were also analyzed statistically
using a repeated-measures analysis of variance followed by
Dunnett’s multiple comparison tests to determine if indi-
vidual doses were significantly different from a vehicle
control. Complete stimulus generalization at a particular
dose of a test compound was defined as a group mean of
80% or greater drug-appropriate responding. If a test dose
produced drug-appropriate responding that was less than
80% but significantly different from the amount of drug-
appropriate responding during vehicle tests, it was consid-
ered to produce partial substitution. Statistical analyses
were conducted and graphs were created using Prism
GraphPad (version 4.0) software (San Diego, CA, USA).
Results
Experiment 1
Rats trained to discriminate 0.13 mg/kg U69,593 met the
discrimination criterion within an average of 69 (±11.4,
SEM) training sessions (range 36–136). Figure 2depicts
the dose–response curves obtained from the results of
substitution tests with U69,593, salvinorin A, salvinorin B
EOM, and salvinorin B MOM in these animals. There was
a statistically significant effect of U69,593 dose on the
percentage of drug-appropriate responses (F
4,39
=12.57, p<
0.0001) and all except the lowest dose of U69,593 were
significantly different from vehicle (0.065 and 0.098 mg/kg,
p<0.05 and 0.13 mg/kg, p<0.01 compared to vehicle).
U69,593 increased response rate relative to vehicle, but this
effect was not statistically significant.
As expected, salvinorin A produced dose-dependent
increases in U69,593-appropriate responding and substitut-
ed fully for this training drug at 1.0 mg/kg. Salvinorin A
significantly increased the percentage of U69,593-appro-
priate responses (F
4,39
=34.90, p<0.0001) and the 0.5 and
1.0 mg/kg doses were significantly different from vehicle
00.003 0.01 0.03 0.1 0.3 1.0
0
20
40
60
80
100
Salvinorin A
MOM
EOM
U69,593
//
Mean (
±
S.E.M.) Percent U69,593-Lever Responses
00.003 0.01 0.03 0.1 0.3 1.0
0.0
0.5
1.0
1.5
2.0
Dose (mg/kg)
//
Mean (
±
S.E.M.) Number of Responses Per Second
Fig. 2 Dose–response functions for U69,593 (N=8), salvinorin A (N=
8), salvinorin B EOM (N=7–8), and salvinorin B MOM (N=8) in rats
trained to discriminate U69,593 (0.13 mg/kg, s.c., 30 min). Discrim-
ination accuracy data are displayed in the top graph and response rates
are displayed in the bottom graph. Data points represent group means
(±SEM)
Psychopharmacology (2009) 203:203–211 207
(p<0.01). Effects of salvinorin A on response rate were not
statistically significant. Salvinorin B EOM and salvinorin B
MOM produced complete substitution for U69,593 at
considerably lower doses compared to salvinorin A.
Statistical tests showed a significant effect of salvinorin B
EOM on drug-appropriate responses (F
4,19
=13.73, p<
0.001) with three doses (0.01, 0.03, and 0.10 mg/kg)
significantly different from vehicle (p<0.01). Full substitu-
tion was obtained with both 0.01 and 0.10 mg/kg, but not
0.03 mg/kg salvinorin B EOM. The effects of salvinorin B
MOM on drug-appropriate responding were also statistical-
ly significant (F
3,27
=15.16, p<0.0001); both 0.06 and
0.10 mg were significantly different from vehicle (p<
0.01) and produced full substitution for U69,593. Effects of
these compounds on response rate were not significantly
different from vehicle control rates.
Experiment 2
Rats trained to discriminate U50,488 met the criteria for
discrimination specified above within an average of 38.8
(±4.4, SEM) training sessions (range 27–65). Although all
eight rats met these criteria, only five of the eight animals
maintained reliable discrimination to complete all the
stimulus generalization tests. Figure 3depicts the dose–
response curves generated from stimulus generalization
tests with the U50,488 and salvinorin A in these five rats.
The effects of U50,488 doses on the percentage of drug-
lever responses were statistically significant (F
4,24
=3.69,
p<0.05). Only the training dose was significantly different
from vehicle control (p< 0.01). Salvinorin A produced
dose-dependent increases in drug-appropriate responding
and substituted fully for U50,488 at 3.0 mg/kg. One animal
did not respond at the highest dose of salvinorin A, so the
data for this animal were excluded from statistical analyses.
The effects of salvinorin A on the percentage of U50,488-
appropriate responding were statistically significant (F
5,23
=
4.12, p<0.05) and both 2.0 mg/kg (p< 0.05) and 3.0 mg/kg
(p<0.01) produced drug-appropriate responding that was
significantly different from vehicle. Response rates were
slightly increased by U50,488 and slightly reduced by
salvinorin A relative to vehicle control levels, although
neither of these drug’s effects on response rate were
statistically significant.
Experiment 3
The initial training dose of salvinorin A (0.5 mg/kg, i.p.,
15 min) established stimulus control in two animals within
44 and 47 training sessions, respectively. As noted above,
the training dose was increased to 1.0 mg/kg (i.p., 30 min)
after 58 training sessions and then to 2.0 mg/kg after
another 30 training sessions. Seven of the eight rats met the
criteria for discrimination within an average of 19.7 (±7.2,
SEM) additional training sessions (range 10–62) after the
dose was increased to 2.0 mg/kg. Figure 4depicts the dose–
response curves generated from stimulus generalization
tests with salvinorin A, U69,593, and U50,488 in these
animals. Salvinorin A produced dose-dependent increases
in discrimination accuracy and both 1.0 and 2.0 mg/kg
produced complete stimulus generalization. Statistical anal-
yses on the percentage of drug-appropriate responding
revealed a significant effect of salvinorin A dose (F
4,29
=
0 0.375 0.75 1.5 3.0
0
20
40
60
80
100
Salvinorin A
U50,488
//
Mean (± S.E.M.) Percent U50,488H-Lever Responses
0 0.375 0.75 1.5 3.0
0.0
0.5
1.0
1.5
2.0
//
Dose (mg/kg)
Mean (± S.E.M.) Number of Responses Per Second
Fig. 3 Dose–response functions for U50,488 (N=5) and salvinorin A
(N=5) in rats trained to discriminate U50,488 (3.0 mg/kg, i.p.,
30 min). See Fig. 1for additional details
208 Psychopharmacology (2009) 203:203–211
7.44, p<0.001). Response rates were not significantly
different among different doses of salvinorin A.
Both U69,593 (0.32 mg/kg) and U50,488 (3.2 mg/kg)
produced complete stimulus generalization to salvinorin A.
The effects of U69,593 (F
4,24
=5.22, p<0.01) and U50,488
(F
4,24
=5.35, p<0.01) on salvinorin A-appropriate
responses were statistically significant; 0.32 mg/kg
U69,593 (p<0.01) and two doses of U50,488 (0.8 mg/kg,
p<0.05 and 0.32 mg/kg, p<0.01) were significantly
different from vehicle control. The large error bars at some
doses are indicative of disparate results among subjects. For
example, U50,488 0.8 mg/kg produced 95% to 100%
salvinorin A-responding in four of the six animals tested,
and only 5% and 31% salvinorin A-responding in the other
two animals. Complete substitution was observed with
3.2 mg/kg U50,488 in all five animals tested at this dose.
Similarly, lower doses of U69,593 produced complete
substitution in some animals and vehicle-appropriate
responding in other animals. The 0.32-mg/kg dose of
U69,593 produced 100% salvinorin A-responding in five
of the six animals tested and vehicle-appropriate responding
in one animal. Interestingly, both U69,593 and U50,488
increased response rates in a dose-dependent manner, but
these effects were not significantly different than vehicle
control rates of responding.
Discussion
Previous studies have demonstrated that salvinorin A
substitutes in monkeys (Butelman et al. 2004) or rats
(Willmore-Fordham et al. 2007) trained to discriminate the
kappa agonist, U69,593, and that these effects are attenu-
ated by KOP antagonists. The present results are consistent
with these reports and extend these findings to a lower dose
of the U69,593 and to another kappa agonist, U50,488.
Willmore-Fordham et al. (2007) trained rats to discriminate
a considerably higher dose of U69,593 (0.56 mg/kg, i.p.,
10 min) than the one employed in the current study
(0.13 mg/kg, s.c., 30 min). In addition, all three doses of
salvinorin A that they tested (1.0, 1.9, and 3.0 mg/kg)
produced full substitution for U69,593 without significantly
reducing response rates. The current study tested a lower
range of salvinorin A doses (0.125–1.0 mg/kg) in order to
generate a complete dose–response curve. Only the 1.0-mg/
kg dose produced complete substitution in animals trained
to discriminate 0.13 mg/kg U69,593. Although these doses
did not significantly reduce response rates in the current
study, a higher dose of salvinorin A (2.0 mg/kg) produced
sedative effects and impaired responding in animals trained
to discriminate 0.13 mg/kg U69,593. The 2.0-mg/kg
salvinorin A test data are not graphed because none of the
animals administered this dose made enough responses to
be included in the analyses. This was not the case with
animals trained to discriminate 0.56 mg/kg U69,593 in the
Willmore-Fordham et al. (2007) study or in animals trained
to discriminate 3.0 mg/kg U50,488 in the present study.
The differences in the effects of salvinorin A on response
rates in these studies are likely a result of exposure to
different doses of the training compounds.
The current study also represents the first known
demonstration that rats can be trained to discriminate
salvinorin A and that both U69,593 and U50,488 substitute
for this compound, showing evidence for symmetrical
generalization between salvinorin A and other KOP
0 0.04 0.08 0.16 0.32 0.8 1.6 3.2
0
20
40
60
80
100
Salvinorin A
U50,488
U69,593
//
Mean (± S.E.M.) Percent Salvinorin A-Lever Responses
0 0.04 0.08 0.16 0.32 0.8 1.6 3.2
0.0
0.5
1.0
1.5
2.0
//
Dose (mg/kg)
Mean (± S.E.M.) Number of Responses Per Second
Fig. 4 Dose–response functions for salvinorin A (N=7), U69,593 (N=
5–6), and U50,488 (N=5–6) in rats trained to discriminate salvinorin
A (2.0 mg/kg, i.p., 30 min). See Fig. 1for additional details
Psychopharmacology (2009) 203:203–211 209
agonists. A more complete characterization of the discrim-
inative cue properties of salvinorin A will require tests of
stimulus generalization between this substance and a
variety of psychoactive compounds representative of
different pharmacological classes. Such studies are an
essential component to a thorough investigation of the
abuse liability of salvinorin A.
It has been suggested that salvinorin A, by virtue of its
potency, efficacy, and selectivity as a KOP receptor agonist,
may serve as an important tool for discovery regarding the
role of the dynorphin/kappa opioid (DYN/KOP) system in
neurological diseases and neuroadaptation (Chavkin et al.
2004; Prisinzano 2005b; Shippenberg et al. 2001,2007).
Relatively few synthetic compounds have been developed
that have a high selectivity for KOP receptors. Until
recently, the most specific of these compounds were
U69,593, U50,488 and their congeners spiradoline and
enadoline, which have limited efficacy following oral
administration (Béguin et al. 2008; Endoh et al. 1999). In
a recent report, Prisinzano and Rothman (2008) suggested
that analogs of the chemicals isolated from S. divinorum
may prove to be excellent research tools and give greater
insight into opioid receptor-mediated phenomena. Some
synthetic analogs of salvinorin A with altered pharmacol-
ogy have recently been reported (partial agonists and
antagonists). However, these analogs show reduced binding
affinity and retain the rapidly metabolized acetate which
may be responsible for salvinorin A’s brief duration of
action. The pharmacological properties of salvinorin B
MOM were recently characterized by Wang et al. (2008).
This compound was reported to bind to KOP receptors with
high selectivity and it displayed an approximately threefold
higher affinity compared to U50,488 and salvinorin A.
Salvinorin B MOM also acted as a full agonist at kappa
receptors in functional assays, being approximately fivefold
to sevenfold more potent than U50,488 and salvinorin A.
All three of these kappa agonists internalized or down-
regulated kappa receptors to similar extents, and salvinorin
B MOM displayed the greatest potency. In mice, salvinorin
B MOM (0.05–1.0 mg/kg, s.c.) caused immediate and
dose-dependent immobility lasting approximately 3 h,
which was blocked by the kappa antagonist, nor-BNI.
Salvinorin B MOM (0.5–5.0 mg/kg, i.p.) also produced
analgesia in the hot plate test and hypothermia in a dose-
dependent manner in rats. This compound was more potent
than U50,488 in both tests and more efficacious than
U50,488 in the hot plate test. These latter two in vivo
effects were also blocked by nor-BNI, indicating kappa
receptor-mediated actions. In contrast, salvinorin A (10 mg/
kg) elicited neither antinociception nor hypothermia 30 min
after administration to rats. The findings reported by Wang
et al. (2008) suggest that salvinorin B MOM is a potent and
efficacious KOP receptor agonist with longer lasting in
vivo effects than salvinorin A. The current findings that
salvinorin B MOM and salvinorin B EOM produce
substitution in rats trained to discriminate U69,593 support
previous reports that these agents are KOP agonists and that
they are more potent than salvinorin A.
The recent upsurge in the recreational use and misuse of
S. divinorum and the widespread availability of this plant
and its fortified extracts require a comprehensive evaluation
and characterization of the psychopharmacology and abuse
liability of salvinorin A. There is also considerable
evidence that alterations in KOP systems may underlie
some of the neuroadaptive changes associated with com-
pulsive drug seeking and relapse and the search for KOPs
as possible pharmacotherapeutic agents has led to the recent
development of synthetic analogs of salvinorin A with
greater potency and a longer duration of action (Munro et
al. 2008; Wang et al. 2008). Preclinical in vivo screening of
these novel compounds is a crucial step in developing
potential therapeutic agents for the treatment of substance
abuse and dependence. A key component of these assess-
ments includes screening these compounds for their
subjective effects and abuse liability. Future studies aimed
at further delineating the psychopharmacology of salvinorin
A and related compounds offer an opportunity for the
development of synthetic analogs of this novel naturally
occurring KOP agonist for pharmacotherapeutic efficacy.
Acknowledgement The authors acknowledge Dr. Thomas Munro,
Harvard Medical School, and McLean Hospital Corporation for their
generous contribution of salvinorin A, salvinorin B EOM, and
salvinorin B MOM.
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