BACKGROUND
psychostimulants such as cocaine and methamphetamine  dramatically enhance transmission of monoamines, including serotonin .
it is clear that the 5-ht2c receptor subtype is involved in stimulant-mediated behaviors  <cit> .
for example, 5-ht2c receptor agonists decrease cue-induced reinstatement of cocaine-seeking  <cit>  .
these latter findings suggest that cue-associated seeking behavior is not dependent on 5-ht levels at synapses expressing 5-ht2c receptors ; nonetheless, the systems regulating seeking behavior are under negative control by activated 5-ht2c receptors .
the majority of behavioral studies focusing on 5-ht2c receptors have relied on agonist/antagonist relationships.
however, evidence indicates constitutive activity  of 5-ht2c receptors in vivo <cit>  and it currently is unknown how constitutively active 5-ht2c receptors impact stimulant-induced behaviors.
5-ht2c receptors are the only known g protein coupled receptor to undergo mrna editing by adenosine deaminases  <cit> ; a process that has important implications for the level of constitutive activity.
adenosine to inosine switches result in amino acid substitutions in the second intracellular loop of the receptor protein  <cit> .
the unedited 5-ht2c receptor displays high levels of constitutive activity  <cit> , and these receptor protein substitutions decrease constitutive activity.
constitutive activity can be attenuated pharmacologically by inverse agonists, these drugs stabilize the receptor in its inactive conformation  <cit> .
for example, systemic administration of the 5-ht2c inverse agonist sb  <dig>  enhances dopamine  efflux in the nucleus accumbens and striatum in rats  <cit> , whereas an agonist either decreases or has no effect on accumbal and striatal da  <cit> .
supporting a role of constitutive activity in this effect, the sb206-induced effects were verified to occur independent of endogenous 5-ht  <cit> .
we previously revealed that the atypical antidepressant, mirtazapine, attenuates methamphetamine -induced sensitization  <cit> , place conditioning  <cit> , and seeking  <cit>  in rodent models of human substance abuse.
mirtazapine historically has been considered to be an antagonist at 5-ht2c and other receptors  <cit> ; however, more recent studies indicate its action as an inverse agonist at constitutively active 5-ht2c receptors  <cit> .
these new observations raised the following questions: would a more selective 5-ht2c receptor inverse agonist, like mirtazapine, also reduce meth-seeking?
a related question is whether seeking for meth is similar to, or differs from, that for cocaine with regard to sensitivity to selective 5-ht2c inverse agonists and/or antagonists?
to answer the above questions, we used two 5-ht2c antagonists of different chemical structures, sb  <dig>  and sdz ser  <dig>  as well as the putative 5-ht2c receptor inverse agonist sb <dig>  dose–response evaluations on meth-seeking behaviors were made for these drugs in using cue reactivity  to meth-associated cues established during a self-administration protocol.
we have previously demonstrated that cr offers an effective means to study drug-seeking that, as is more typical in the human scenario, does not depend on prior extinction training  <cit> .
we also revealed that the ability of mirtazapine to alter seeking behavior is similar for cr and cue-induced reinstatement  protocols  <cit> .
finally, to ascertain whether 5-ht2c receptor compounds influenced motor activity, rats tested for cr also underwent motor assessments with acute 5-ht2c ligand pretreatment.
methods
subjects
fifty male sprague–dawley rats were purchased from harlan , acclimated to housing in our local vivarium for 5 days, and handled a minimum of  <dig> times prior to surgery.
food and water were provided ad libitum throughout the study.
rats were maintained in accordance with the guide for care and use of laboratory animals  and with the guidelines and approval of the rush university institutional animal care and use committee.
drugs
-methamphetamine hcl  was dissolved in sterile saline.
the stimulant was self-administered at  <dig>  mg/kg/ <dig>  ml infusion intravenously  and non-contingently administered intraperitoneally  for motor assessments as  <dig>  mg/kg.
sdz ser  <dig> , a 5-ht2c antagonist, was dissolved in saline and injected ip .
sb  <dig> , also a 5-ht2c antagonist, was dissolved in 8 % β-cyclodextran and 1 % citric acid in deionized water and injected ip .
sb  <dig>  , a 5-ht2c inverse agonist, was dissolved in 1 % lactic acid in deionized water and injected ip .
serotonin2c ligands were administered in volumes of  <dig>  ml/kg with the exception of  <dig>  mg/kg sb <dig>  which was administered at 2 ml/kg .
all drugs were administered as the base.
pki values of sdz, sb <dig> and sb <dig> for 5-ht <dig> receptor subtypes are provided in table  <dig>
pki values  for 5-ht <dig> receptor ligands in hek- <dig> or cho-k <dig> cells expressing human recombinant 5-ht <dig> receptors  <cit> .
surgical procedures
rats were instrumented with a jugular vein catheter under continuous isoflurane anesthesia.
custom built catheters were constructed with silastic tubing  and implanted into the right jugular vein.
the distal end of the catheter extended to the mid-scapular region with a metal guide canulae  and anchored to a plastic mesh.
rats were allowed to recover for a minimum of 5 days prior to beginning self-administration procedures.
during this time, rats were handled, inspected and weighed daily to ensure that surgical wounds were healing properly , that normal behavioral grooming/eating patterns were retained, and that preoperative weight was regained.
self-administration acquisition
rats were trained to self-administer meth 3 hr/day for  <dig> consecutive days in standard operant chambers enclosed in ventilated, sound attenuating cabinets .
each operant chamber contained two levers; the left lever assigned as the “active” lever and the right the “inactive” lever.
above each lever was a cue light and located on the opposite wall was an in-house light.
a cue light above the active lever was activated in association with the infusion pump and the in-house light was subsequently activated for 20s, indicating a time-out period during which responses had no programmed consequences.
responding on the inactive lever also had no programmed consequences.
on days 1– <dig>  rats self-administered meth for 3 hr/day on a fixed ratio   <dig> schedule of reinforcement.
on days 8– <dig>  rats self-administered on a fr <dig> for 3 hr/day  to enhance lever pressing behaviors and resistance to extinction  <cit> .
starting on day  <dig>  rats were acclimated to injection procedures via daily ip vehicle pretreatments .
the number of active lever presses, inactive lever presses, and infusions were recorded for all sessions.
stable self-administration behavior was operationally defined as <15 % infusion variability between days  <dig> and 14; failure to reach this criterion resulted in subject exclusion from the study.
cue reactivity testing
meth-seeking behavior was assessed via cr tests.
cr testing consisted of single, 1 hr extinction sessions during which cues  were contingently presented on an fr <dig> schedule.
during cr tests, infusion lines were filled with sterile saline and remained connected to saline-filled syringes .
meth was not accessible during cr testing.
the number of active and inactive lever presses was measured continuously and tallied in 15 min intervals.
on day  <dig>  rats were given a 30 min pretreatment of vehicle and tested for baseline meth-seeking ; baseline was used to equally distribute rats into treatment groups.
rats were tested for an additional four cr tests ; between each cr test, rats were allowed to self-administer meth for 3 hr/day on an fr <dig> schedule  to prevent extinction training.
rats failing to administer at least 50 % of the number of infusions administered on day  <dig>  for two consecutive days during intermittent self-administration sessions, were removed from the study.
rats were tested for cr after a 30 min pretreatment of  <dig> ,  <dig> ,  <dig>  mg/kg sdz and vehicle  or  <dig> ,  <dig> ,  <dig>  mg/kg sb <dig>  and vehicle .
the 5-ht2c receptor antagonist, sb <dig>  was tested against the 5-ht2c receptor inverse agonist sb <dig> in the same rats, wherein sb <dig>  was administered 45 min, and sb <dig>  was administered 30 min, prior to the onset of cr testing.
dose order was randomized for all treatment groups.
doses of sb <dig> and sb <dig> were guided by literature demonstrating significant neurochemical effects in the nucleus accumbens  <cit> .
doses of sdz were selected based on reports revealing an enhancement of the interoceptive cues of cocaine  <cit>  and cocaine-evoked motor activity in naïve rats  <cit> .
we have previously shown that the described cr paradigm is comparable to cue-induced reinstatement and that pharmacological intervention paradigms similar to those tested in the current report similarly reduce cr and cue-induced reinstatement  <cit> .
assessments were made at an early stage of withdrawal  based on our aforementioned work  <cit>  as well as evidence indicating that psychostimulant administration dysregulates neuronal function in the nucleus accumbens shell  as early as 1–3 days of withdrawal  <cit>  as well as biochemical indices indicating psychostimulant-induced plasticity in multiple brain regions during early phase withdrawal  using place conditioning paradigms  <cit>  and motor sensitization  <cit> .
motor assessments
a subset of rats tested for cr dose–response assessments was used to determine the motor effects of sdz  and sb <dig>  in the presence and absence of meth .
for this study, rats were withdrawn from meth and remained in their home cages during protocol days 28– <dig> and received no treatment .
after this 5 day period, rats were tested for motor activity for  <dig> consecutive days .
all motor assessments were conducted using automated small animal activity boxes  equipped with two banks of photobeams positioned at different heights to characterize motor activity in three dimensional space.
rats were habituated to activity chambers for 1 hr prior to each motor test.
on day  <dig>  rats were administered 1 ml/kg of the respective vehicle for each test drug .
the injected rats were immediately returned to motor boxes for 1 hr after which rats were injected with either sdz , or sb <dig>  and behavior was recorded for an additional 1 hr.
motor data collected 30 min post-injection were subsequently analyzed; this time frame reflected the one that was relevant to cr behaviors.
on day  <dig>  rats were administered a 30 min pretreatment of vehicle , then administered 1 mg/kg meth  and behavior recorded for 1 hr.
on day  <dig>  the procedure from day  <dig> was repeated using sdz , or sb <dig>   instead of respective vehicles.
peak meth effects occurred 15 min post meth injection; meth-evoked motor activity was therefore analyzed for the last 45 min of testing .
horizontal activity , vertical activity , and total distance  were recorded.
these assessments provide a reliable index of overall motor patterns evoked by this dose of meth  <cit> .
stereotypy  is a prominent component of meth-induced motor activity  <cit> ; therefore, stereotypy number  also was analyzed for meth-evoked motor activity.
rats tested for effects of sdz on motor function were also tested for sdz effects on cr; similarly, rats tested for effects of sb <dig> on motor function had prior exposure to sb <dig> during cr assessments.
statistical analysis
active lever presses, inactive lever presses, and number of infusions achieved during self-administration sessions were analyzed using a one-way rmanova with newman-keuls post-hoc analysis.
the differences in lever pressing during cr tests were detected using two-way rmanova with newman-keuls post-hoc analysis.
lever pressing behavior for days  <dig> and  <dig> of intermittent self-administration, and motor activity were analyzed using a paired t-test.
for all tests, α =  <dig> .
data are presented as mean ± sem.
data greater than two standard deviations from the mean were considered to be statistical outliers and were excluded from the analyses; for cr, outliers were determined from behavior collapsed across the first 30 min of the tests.
RESULTS
stable meth self-administration
fifty rats acquired the self-administration task; four rats were removed from the study as they displayed >15 % infusion variability between the last two training sessions ; three rats died after a self-administration session.
an additional  <dig> rats were removed due to failure to maintain stable self-administration behavior during periods of intermittent self-administration .
of the  <dig> rats that completed the study, there were no differences for active lever pressing , inactive lever pressing , or infusions  for days 11– <dig> .
meth-seeking behavior: cue reactivity
a within-subjects design was used wherein each rat was tested with three doses  of either the antagonist , inverse agonist  and the respective vehicles.
similarly, a within-subjects design was used to test for antagonist/inverse agonist interactions with  <dig>  mg/kg sb <dig> vs.  <dig>   and  <dig>  mg/kg  sb <dig>  and respective vehicles.
there were no significant differences among rats assigned to sdz , sb <dig> , or interaction studies  for active or inactive lever pressing during cr <dig> .
additionally, active lever pressing comparing cr after vehicle pretreatment was not different among the four treatment groups .
self-administration behavior was not altered by the interposed cr testing sessions; the first and last day of intermittent self-administration were not significantly different for active lever presses, inactive lever presses, or infusions comparing day  <dig> and day  <dig> .
in agreement with our prior report validating consistent self-administration and seeking  <cit> , these data verify that behavior is persistent throughout the paradigm.
independent of the cr test number or pretreatment, meth-seeking behavior occurred most intensely within the first 15 min of cr testing  as previously reported  <cit> ; by 30 min, levels emulated those expressed on the inactive lever .
it is also important to note that the number of lever presses achieved during self-administration sessions are far greater than the number during cr tests with a vehicle pretreatment.
this is a consequence of i) lever presses during cr tests are not reinforced with meth and ii) self-administration sessions are 3 hrs long whereas cr tests are shown in15min intervals.
sdz, the 5-ht2c antagonist, had no effect on number of active  or inactive lever pressing  at any dose tested.
two-way rmanova comparing active lever pressing revealed no significant treatment effect  =  <dig> ), a significant time effect  =  <dig> ), and no treatment x time interaction  =  <dig> ).
in contrast, administration of the 5-ht2c inverse agonist sb <dig> attenuated active lever pressing in the first 15 min at all doses tested  resulting in an approximately 25 %, 50 %, and 95 % reduction in active lever pressing by  <dig> ,  <dig> , and  <dig>  mg/kg, respectively.
two-way rmanova revealed a significant effect of treatment  =  <dig> ) and time  =  <dig> ), and a treatment x time interaction  =  <dig> ).
inactive lever pressing was attenuated by approximately  <dig>   <dig>  and 100 % for  <dig> ,  <dig> , and  <dig>  mg/kg sb <dig>  respectively, in the first 15 min of testing .
while the percentage change in inactive lever pressing appears dramatic, this only corresponds to approximately  <dig>   <dig>  and  <dig> fewer inactive lever presses by  <dig> ,  <dig> , and  <dig>  mg/kg, respectively.
similar to the effects seen with sdz,  <dig>  mg/kg of sb <dig>  had no effect on active  or inactive  lever pressing when administered alone.
pretreatment with  <dig>  mg/kg sb <dig> 15 min prior to administration of  <dig>  or  <dig>  mg/kg of sb <dig> however, did not attenuate the sb206-induced suppression of active  and inactive  lever pressing.
two-way rmanova revealed a significant treatment effect  =  <dig> ), time effect  =  <dig> ), and treatment x time interaction  =  <dig> ) comparing the effects of  <dig>  mg/kg sb <dig> and  <dig>  mg/kg sb <dig> as well as a significant treatment effect  =  <dig> ), time effect  =  <dig> ), and treatment x time interaction  =  <dig> ) comparing the effects of  <dig>  mg/kg sb <dig> and  <dig>  mg/kg sb <dig>  our antagonist data are consistent with the inability of 5-ht2c antagonism to alter cue-induced reinstatement of cocaine-seeking  <cit> .
however, because the sb <dig> did not prevent sb206-induced decreases in lever pressing, it is unclear whether sb <dig> effects are 5-ht2c dependent.
normal exploratory and meth-evoked motor activity
to assess the ability for the 5-ht2c ligands to impact non-operant-related motor activity, sdz and sb <dig> were tested for motor effects in the absence and presence of meth.
in the absence of meth, neither the sdz  nor sb <dig>  altered motor activity .
sdz  had no effect on meth-evoked motor activity, .
likewise, the lower dose of  <dig>  mg/kg sb <dig>  which was sufficient to attenuate meth-seeking, had no effect on meth-evoked motor activity .
the higher dose  only significantly attenuated meth-evoked vertical activity.
rats that had undergone repeated cue reactivity testing were withdrawn from methamphetamine and assessed for motor effects after pretreatment with  <dig>  mg/kg sdz ser  <dig> ,  <dig>  mg/kg sb  <dig> ,  <dig>  mg/kg sb <dig> and respective vehicles.
serotonin2c receptor antagonism and inverse agonism had no effect on motor activity.
rats that had undergone repeated cue reactivity testing were withdrawn from methamphetamine and assessed for methamphetamine-evoked  motor effects after a 30 min pretreatment with  <dig>  mg/kg sdz ser  <dig> ,  <dig>  mg/kg sb  <dig> ,  <dig>  mg/kg sb <dig> and respective vehicles.
serotonin2c receptor antagonism had no effect on methamphetamine-evoked motor activity whereas inverse agonism with  <dig>  mg/kg sb <dig> significantly attenuated vertical activity .
discussion
sb  <dig> attenuates methamphetamine-seeking
results from the current study are the first to investigate the role of 5-ht2c receptor activity in meth-mediated behaviors and the first to test sb <dig> in a rodent model of addiction.
we revealed that the putative inverse agonist robustly and dose-dependently attenuated meth-seeking without impacting exploratory motor activity at  <dig>  mg/kg and  <dig>  mg/kg.
the 5-ht2c antagonists had no effect on either meth-seeking or meth-induced motor activity.
these results suggest that our prior work with mirtazapine may reflect the ability of mirtazapine to act as an inverse agonist, not as a 5-ht2c antagonist.
the finding that the antagonist sb <dig> did not attenuate the effects of the inverse agonist sb <dig> was unexpected, and raised the question regarding the receptor selectivity of sb <dig>  sb <dig> has high affinity for both the 5-ht2c and 5-ht2b receptors   <cit>  with  <dig> fold or greater selectivity over other receptor targets including 5-ht2a receptors  <cit> .
in the current study, sb <dig> may antagonize 5-ht2b receptors, which are known to regulate behavioral and da-enhancing effects of amphetamine  <cit> .
however, the increase in accumbal da seen with  <dig>  mg/kg sb <dig> is not altered with coadministration of the 5-ht2b antagonist ly  <dig> indicating that  <dig>  mg/kg sb <dig> does not substantially antagonize 5-ht2b receptors  <cit> .
antagonism of 5-ht2a receptors are a third 5-ht receptor that may be contributing to our findings.
5-ht2a antagonism with m <dig> attenuates cue-induced reinstatement of cocaine-seeking  <cit>  and meth-seeking .
as mentioned above, sb <dig> demonstrates  <dig> fold selectivity over the 5-ht2a receptor.
5-ht2a antagonism by sb <dig>  particularly at  <dig>  mg/kg, is plausible but unlikely at  <dig>  and  <dig>  mg/kg, for amphetamine-evoked da concentrations in the nucleus accumbens and striatum are attenuated by 5-ht2a antagonism but  <dig>  mg/kg sb <dig> has no effect  <cit>  suggesting that at this dose, sb <dig> does not act on 5-ht2a receptors.
additionally,  <dig>  mg/kg sb <dig> and 5-ht2a antagonism with sr 46349b have oppositional effects demonstrated by striatal 11 c]raclopride binding lending further evidence to the selectivity of sb <dig> for 5-ht2c over 5-ht2a receptors  <cit> .
taken together, the available in vivo data suggests that sb <dig> does not antagonize 5-ht2a receptors at  <dig>  mg/kg.
sb <dig> may also act as a positive allosteric modulator of the α <dig> nicotinic acetylcholine receptor with an ec <dig> of  <dig>  μm for potentiation of responses evoked in gh4c <dig> cells by ec <dig> nicotine  <cit> .
dunlop et al., find that  <dig>   <dig>  and 30 mg/kg sb <dig> reverses mk-801-induced deficits in prepulse inhibition, a classical model of schizophrenia, but only confirm actions as a positive allosteric modulator using a nicotinic antagonist against  <dig>  mg/kg sb <dig>  it is unclear whether lower doses, such as  <dig>  and  <dig>  mg/kg sb <dig>  would also act as a positive allosteric modulator at α <dig> nicotinic receptors.
consistent with our findings, antagonism of α <dig> nicotinic receptors in the ventral tegmental area attenuates the reward-facilitating effect of cocaine  <cit>  whereas nicotine administration attenuates reinstatement of meth-seeking  <cit> .
the role of α <dig> nicotinic receptors and in particular, positive allosteric modulation of this receptor, in meth abuse is understudied.
future investigation are needed to determine if this receptor is engaged by sb <dig> in the dose range tested before actions at the α <dig> receptor can be ruled out.
debating receptor specificity based on our findings is difficult; however, it is important to note that a prior report showed that  <dig>  mg/kg sb <dig> attenuates  <dig>  mg/kg sb206-induced increases in striatal and accumbal da  <cit> .
reasons why spampinato and colleagues were able to antagonize sb <dig> effects and we were not may relate to the treatment history of rodent subjects.
in the aforementioned neurochemical study  <cit> , subjects were stimulant naïve, halothane anesthetized rats.
in our study, rats had an extensive history of meth self-administration.
this chronic meth history may have modified the 5-ht2c system, for we have observed a functionally upregulated response to systemic 5-ht2a/c receptor agonism in the ventral pallidum of rats chronically administered meth  <cit> .
repeated meth administration may result in changes in mrna editing profiles .
proof of concept for this hypothesis is seen with studies on stress, antidepressant, and antipsychotic administration where mrna editing of the 5-ht2c receptor is altered  <cit> .
in addition to changing constitutive activity, mrna editing of the 5-ht2c receptor also alters the binding affinity of ligands whereby increased editing most often results in decreased agonist affinity for the receptor with enhanced or no effect on antagonists and inverse agonist binding affinity  <cit> .
it is unknown how editing patterns affect the affinities of sb <dig> and sb <dig>  thus, the inability of sb <dig> to augment the effects of sb <dig> on cr may reflect changes in affinity for constitutively active isoforms and/or in the allosteric constant .
unfortunately ex vivo binding studies, which would greatly facilitate our studies, are hampered by a lack of available tritiated ligands with 5-ht2c specificity.
while future studies are needed to explore the mechanism of action for sb <dig> and determine receptor specificity in vivo, we speculate that despite a lack of antagonism by sb <dig>  5-ht2c receptors may still be involved.
moreover, continued study is necessary to determine the effects of 5-ht2c receptor ligands on neuron function; to provide further insights our laboratory is conducting patch clamp studies in ex vivo slice preparations to determine the effects of 5-ht2c ligands, including sb <dig>  on neurophysiology.
sb  <dig>  but not sdz ser  <dig>  attenuates methamphetamine-evoked motor activity
acute antagonism of 5-ht2c receptors enhances the motor effects of acute cocaine administration; however, this enhancement is lost in rats that are motorically sensitized by repeated injections of cocaine  <cit> .
this is comparable to our current findings wherein rats with a history of meth self-administration, 5-ht2c antagonism did not alter meth-evoked motor activity in rats.
a single study testing sb <dig> on cocaine-evoked motor activity finds an attenuation of cocaine-evoked motor activity by  <dig>  mg/kg sb <dig> but a potentiation with  <dig>  mg/kg  <cit> ; however, this was an acute study with a single injection of cocaine making it difficult to compare with the current investigations using meth self-administration.
in rats with a history of meth self-administration, only meth-evoked vertical activity was attenuated by  <dig>  mg/kg sb <dig>  both our findings and  <cit>  are in agreement in that sb <dig> does not alter normal exploratory motor behavior suggesting that sb <dig> is not sedating; differences in our study and  <cit>  regarding the ability to augment psychostimulant-evoked motor activity may reflect behavioral differences consequent to meth-induced plasticity.
this hypothesis is supported by  <cit>  and our current findings wherein 5-ht2c antagonism fails to augment cocaine-evoked and meth-evoked motor activity in rats with a history of repeated cocaine  <cit>  or meth administration.
sb  <dig> decreases inactive lever pressing
antagonists had no effect, whereas sb <dig> attenuated inactive lever pressing.
mirtazapine, also an inverse agonist at the 5-ht2c receptor  <cit> , similarly attenuates inactive lever pressing in a cue-induced reinstatement paradigm without effecting non-operant related motor activity in a small animal activity chamber in rats with a history of meth self-administration or rotorod performance in naïve rats  <cit> .
moreover, in naïve rats, administration of  <dig>   <dig>  and 4 mg/kg sb <dig> has no effect on motor activity   <cit> .
a similar pattern is found for gabab receptor activation.
the gabab agonist  decreases inactive lever pressing in an operant task  <cit> , yet has no effect on performance in an intracranial self-stimulation procedure  <cit> .
in summary, as sb <dig> had no effect on motor activity in in rats with a meth history  or in naïve rats  <cit> , and that mirtazapine, also an inverse agonist at 5-ht2c receptors  <cit> , does not impair motor activity in naïve rats  <cit> , we considered that decreased lever pressing of both active and inactive levers seen in the current study was not a consequence of decreased motor activity.
accordingly, we propose that sb <dig> blunted the salience of the levers, both active and inactive, rather than impairing the ability of the rats to perform the operant task.
this interpretation is supported by our prior demonstration that significance of meth-associated context cues are reduced by mirtazapine  <cit> .
additional studies aimed at exploring the effect of sb <dig> on non-drug reinforced behaviors may advance this hypothesis; however, as 5-ht2c receptors regulate food intake  <cit>  as well as intracranial self-stimulation behavior  <cit> , such evaluations may challenging to conduct and difficult to interpret.
CONCLUSIONS
the presented findings are the first to explore the effects of 5-ht2c antagonists and putative inverse agonist, sb <dig>  on meth-seeking and meth-induced motor activity.
serotonin2c antagonism had no effect on meth-seeking or motor activity ; in contrast, sb <dig>  attenuated meth-seeking.
this effect was selective, as sb <dig> agonist did not alter exploratory behavior, and only the higher dose reduced one motor parameter evoked by meth .
there is a growing interest in assessing the role of constitutively active 5-ht2c receptors in vivo.
it is currently unclear whether sb <dig> elicits the observed effects via 5-ht2c receptor inverse agonism; nonetheless, based on the dramatic reduction in seeking behavior, further investigation of this compound is merited as well as continued study of 5-ht2c regulation of psychostimulant-mediated behaviors.
abbreviations
meth, methamphetamine; da, dopamine; sdz, sdz ser 082; sb <dig>  sb 206553; sb <dig>  sb 242084; 5-ht, serotonin; cr, cue reactivity.
competing interests
the authors have no competing interests or financial disclosures to report.
authors’ contributions
experiments and data analysis were conducted by smg.
experimental design and manuscript writing were done by smg and tcn.
all authors read and approved the final manuscript.

 psychostimulants such as cocaine and methamphetamine dramatically enhance transmission of monoamines, including serotonin. it is clear that the 5-ht2c receptor subtype is involved in stimulant-mediated behaviors. for example, 5-ht2c receptor agonists decrease cue-induced reinstatement of cocaine-seeking. these latter findings suggest that cue-associated seeking behavior is not dependent on 5-ht levels at synapses expressing 5-ht2c receptors; nonetheless, the systems regulating seeking behavior are under negative control by activated 5-ht2c receptors. the majority of behavioral studies focusing on 5-ht2c receptors have relied on agonist/antagonist relationships. however, evidence indicates constitutive activity of 5-ht2c receptors in vivo and it currently is unknown how constitutively active 5-ht2c receptors impact stimulant-induced behaviors. 5-ht2c receptors are the only known g protein coupled receptor to undergo mrna editing by adenosine deaminases; a process that has important implications for the level of constitutive activity. adenosine to inosine switches result in amino acid substitutions in the second intracellular loop of the receptor protein. the unedited 5-ht2c receptor displays high levels of constitutive activity, and these receptor protein substitutions decrease constitutive activity. constitutive activity can be attenuated pharmacologically by inverse agonists, these drugs stabilize the receptor in its inactive conformation. for example, systemic administration of the 5-ht2c inverse agonist sb enhances dopamine efflux in the nucleus accumbens and striatum in rats, whereas an agonist either decreases or has no effect on accumbal and striatal da. supporting a role of constitutive activity in this effect, the sb206-induced effects were verified to occur independent of endogenous 5-ht. we previously revealed that the atypical antidepressant, mirtazapine, attenuates methamphetamine -induced sensitization, place conditioning, and seeking in rodent models of human substance abuse. mirtazapine historically has been considered to be an antagonist at 5-ht2c and other receptors; however, more recent studies indicate its action as an inverse agonist at constitutively active 5-ht2c receptors. these new observations raised the following questions: would a more selective 5-ht2c receptor inverse agonist, like mirtazapine, also reduce meth-seeking? a related question is whether seeking for meth is similar to, or differs from, that for cocaine with regard to sensitivity to selective 5-ht2c inverse agonists and/or antagonists? to answer the above questions, we used two 5-ht2c antagonists of different chemical structures, sb and sdz ser as well as the putative 5-ht2c receptor inverse agonist sb dose–response evaluations on meth-seeking behaviors were made for these drugs in using cue reactivity to meth-associated cues established during a self-administration protocol. we have previously demonstrated that cr offers an effective means to study drug-seeking that, as is more typical in the human scenario, does not depend on prior extinction training. we also revealed that the ability of mirtazapine to alter seeking behavior is similar for cr and cue-induced reinstatement protocols. finally, to ascertain whether 5-ht2c receptor compounds influenced motor activity, rats tested for cr also underwent motor assessments with acute 5-ht2c ligand pretreatment. methods subjects fifty male sprague–dawley rats were purchased from harlan, acclimated to housing in our local vivarium for 5 days, and handled a minimum of times prior to surgery. food and water were provided ad libitum throughout the study. rats were maintained in accordance with the guide for care and use of laboratory animals and with the guidelines and approval of the rush university institutional animal care and use committee. drugs -methamphetamine hcl was dissolved in sterile saline. the stimulant was self-administered at  mg/kg/  ml infusion intravenously and non-contingently administered intraperitoneally for motor assessments as  mg/kg. sdz ser, a 5-ht2c antagonist, was dissolved in saline and injected ip. sb, also a 5-ht2c antagonist, was dissolved in 8 % β-cyclodextran and 1 % citric acid in deionized water and injected ip. sb, a 5-ht2c inverse agonist, was dissolved in 1 % lactic acid in deionized water and injected ip. serotonin2c ligands were administered in volumes of  ml/kg with the exception of  mg/kg sb which was administered at 2 ml/kg. all drugs were administered as the base. pki values of sdz, sb and sb for 5-ht receptor subtypes are provided in table pki values for 5-ht receptor ligands in hek- or cho-k cells expressing human recombinant 5-ht receptors. surgical procedures rats were instrumented with a jugular vein catheter under continuous isoflurane anesthesia. custom built catheters were constructed with silastic tubing and implanted into the right jugular vein. the distal end of the catheter extended to the mid-scapular region with a metal guide canulae and anchored to a plastic mesh. rats were allowed to recover for a minimum of 5 days prior to beginning self-administration procedures. during this time, rats were handled, inspected and weighed daily to ensure that surgical wounds were healing properly, that normal behavioral grooming/eating patterns were retained, and that preoperative weight was regained. self-administration acquisition rats were trained to self-administer meth 3 hr/day for consecutive days in standard operant chambers enclosed in ventilated, sound attenuating cabinets. each operant chamber contained two levers; the left lever assigned as the “active” lever and the right the “inactive” lever. above each lever was a cue light and located on the opposite wall was an in-house light. a cue light above the active lever was activated in association with the infusion pump and the in-house light was subsequently activated for 20s, indicating a time-out period during which responses had no programmed consequences. responding on the inactive lever also had no programmed consequences. on days 1– rats self-administered meth for 3 hr/day on a fixed ratio schedule of reinforcement. on days 8– rats self-administered on a fr for 3 hr/day to enhance lever pressing behaviors and resistance to extinction. starting on day rats were acclimated to injection procedures via daily ip vehicle pretreatments. the number of active lever presses, inactive lever presses, and infusions were recorded for all sessions. stable self-administration behavior was operationally defined as <15 % infusion variability between days and 14; failure to reach this criterion resulted in subject exclusion from the study. cue reactivity testing meth-seeking behavior was assessed via cr tests. cr testing consisted of single, 1 hr extinction sessions during which cues were contingently presented on an fr schedule. during cr tests, infusion lines were filled with sterile saline and remained connected to saline-filled syringes. meth was not accessible during cr testing. the number of active and inactive lever presses was measured continuously and tallied in 15 min intervals. on day rats were given a 30 min pretreatment of vehicle and tested for baseline meth-seeking; baseline was used to equally distribute rats into treatment groups. rats were tested for an additional four cr tests; between each cr test, rats were allowed to self-administer meth for 3 hr/day on an fr schedule to prevent extinction training. rats failing to administer at least 50 % of the number of infusions administered on day for two consecutive days during intermittent self-administration sessions, were removed from the study. rats were tested for cr after a 30 min pretreatment of,,  mg/kg sdz and vehicle or,,  mg/kg sb and vehicle. the 5-ht2c receptor antagonist, sb was tested against the 5-ht2c receptor inverse agonist sb in the same rats, wherein sb was administered 45 min, and sb was administered 30 min, prior to the onset of cr testing. dose order was randomized for all treatment groups. doses of sb and sb were guided by literature demonstrating significant neurochemical effects in the nucleus accumbens. doses of sdz were selected based on reports revealing an enhancement of the interoceptive cues of cocaine and cocaine-evoked motor activity in naïve rats. we have previously shown that the described cr paradigm is comparable to cue-induced reinstatement and that pharmacological intervention paradigms similar to those tested in the current report similarly reduce cr and cue-induced reinstatement. assessments were made at an early stage of withdrawal based on our aforementioned work as well as evidence indicating that psychostimulant administration dysregulates neuronal function in the nucleus accumbens shell as early as 1–3 days of withdrawal as well as biochemical indices indicating psychostimulant-induced plasticity in multiple brain regions during early phase withdrawal using place conditioning paradigms and motor sensitization. motor assessments a subset of rats tested for cr dose–response assessments was used to determine the motor effects of sdz and sb in the presence and absence of meth. for this study, rats were withdrawn from meth and remained in their home cages during protocol days 28– and received no treatment. after this 5 day period, rats were tested for motor activity for consecutive days. all motor assessments were conducted using automated small animal activity boxes equipped with two banks of photobeams positioned at different heights to characterize motor activity in three dimensional space. rats were habituated to activity chambers for 1 hr prior to each motor test. on day rats were administered 1 ml/kg of the respective vehicle for each test drug. the injected rats were immediately returned to motor boxes for 1 hr after which rats were injected with either sdz, or sb and behavior was recorded for an additional 1 hr. motor data collected 30 min post-injection were subsequently analyzed; this time frame reflected the one that was relevant to cr behaviors. on day rats were administered a 30 min pretreatment of vehicle, then administered 1 mg/kg meth and behavior recorded for 1 hr. on day the procedure from day was repeated using sdz, or sb instead of respective vehicles. peak meth effects occurred 15 min post meth injection; meth-evoked motor activity was therefore analyzed for the last 45 min of testing. horizontal activity, vertical activity, and total distance were recorded. these assessments provide a reliable index of overall motor patterns evoked by this dose of meth. stereotypy is a prominent component of meth-induced motor activity; therefore, stereotypy number also was analyzed for meth-evoked motor activity. rats tested for effects of sdz on motor function were also tested for sdz effects on cr; similarly, rats tested for effects of sb on motor function had prior exposure to sb during cr assessments. statistical analysis active lever presses, inactive lever presses, and number of infusions achieved during self-administration sessions were analyzed using a one-way rmanova with newman-keuls post-hoc analysis. the differences in lever pressing during cr tests were detected using two-way rmanova with newman-keuls post-hoc analysis. lever pressing behavior for days and of intermittent self-administration, and motor activity were analyzed using a paired t-test. for all tests, α = . data are presented as mean ± sem. data greater than two standard deviations from the mean were considered to be statistical outliers and were excluded from the analyses; for cr, outliers were determined from behavior collapsed across the first 30 min of the tests.  stable meth self-administration fifty rats acquired the self-administration task; four rats were removed from the study as they displayed >15 % infusion variability between the last two training sessions; three rats died after a self-administration session. an additional rats were removed due to failure to maintain stable self-administration behavior during periods of intermittent self-administration. of the rats that completed the study, there were no differences for active lever pressing, inactive lever pressing, or infusions for days 11–. meth-seeking behavior: cue reactivity a within-subjects design was used wherein each rat was tested with three doses of either the antagonist, inverse agonist and the respective vehicles. similarly, a within-subjects design was used to test for antagonist/inverse agonist interactions with  mg/kg sb vs. and  mg/kg sb and respective vehicles. there were no significant differences among rats assigned to sdz, sb, or interaction studies for active or inactive lever pressing during cr. additionally, active lever pressing comparing cr after vehicle pretreatment was not different among the four treatment groups. self-administration behavior was not altered by the interposed cr testing sessions; the first and last day of intermittent self-administration were not significantly different for active lever presses, inactive lever presses, or infusions comparing day and day. in agreement with our prior report validating consistent self-administration and seeking, these data verify that behavior is persistent throughout the paradigm. independent of the cr test number or pretreatment, meth-seeking behavior occurred most intensely within the first 15 min of cr testing as previously reported; by 30 min, levels emulated those expressed on the inactive lever. it is also important to note that the number of lever presses achieved during self-administration sessions are far greater than the number during cr tests with a vehicle pretreatment. this is a consequence of i) lever presses during cr tests are not reinforced with meth and ii) self-administration sessions are 3 hrs long whereas cr tests are shown in15min intervals. sdz, the 5-ht2c antagonist, had no effect on number of active or inactive lever pressing at any dose tested. two-way rmanova comparing active lever pressing revealed no significant treatment effect  =  ), a significant time effect  =  ), and no treatment x time interaction  =  ). in contrast, administration of the 5-ht2c inverse agonist sb attenuated active lever pressing in the first 15 min at all doses tested resulting in an approximately 25 %, 50 %, and 95 % reduction in active lever pressing by,, and  mg/kg, respectively. two-way rmanova revealed a significant effect of treatment  =  ) and time  =  ), and a treatment x time interaction  =  ). inactive lever pressing was attenuated by approximately and 100 % for,, and  mg/kg sb respectively, in the first 15 min of testing. while the percentage change in inactive lever pressing appears dramatic, this only corresponds to approximately and fewer inactive lever presses by,, and  mg/kg, respectively. similar to the effects seen with sdz,  mg/kg of sb had no effect on active or inactive lever pressing when administered alone. pretreatment with  mg/kg sb 15 min prior to administration of or  mg/kg of sb however, did not attenuate the sb206-induced suppression of active and inactive lever pressing. two-way rmanova revealed a significant treatment effect  =  ), time effect  =  ), and treatment x time interaction  =  ) comparing the effects of  mg/kg sb and  mg/kg sb as well as a significant treatment effect  =  ), time effect  =  ), and treatment x time interaction  =  ) comparing the effects of  mg/kg sb and  mg/kg sb our antagonist data are consistent with the inability of 5-ht2c antagonism to alter cue-induced reinstatement of cocaine-seeking. however, because the sb did not prevent sb206-induced decreases in lever pressing, it is unclear whether sb effects are 5-ht2c dependent. normal exploratory and meth-evoked motor activity to assess the ability for the 5-ht2c ligands to impact non-operant-related motor activity, sdz and sb were tested for motor effects in the absence and presence of meth. in the absence of meth, neither the sdz nor sb altered motor activity. sdz had no effect on meth-evoked motor activity,. likewise, the lower dose of  mg/kg sb which was sufficient to attenuate meth-seeking, had no effect on meth-evoked motor activity. the higher dose only significantly attenuated meth-evoked vertical activity. rats that had undergone repeated cue reactivity testing were withdrawn from methamphetamine and assessed for motor effects after pretreatment with  mg/kg sdz ser,  mg/kg sb,  mg/kg sb and respective vehicles. serotonin2c receptor antagonism and inverse agonism had no effect on motor activity. rats that had undergone repeated cue reactivity testing were withdrawn from methamphetamine and assessed for methamphetamine-evoked motor effects after a 30 min pretreatment with  mg/kg sdz ser,  mg/kg sb,  mg/kg sb and respective vehicles. serotonin2c receptor antagonism had no effect on methamphetamine-evoked motor activity whereas inverse agonism with  mg/kg sb significantly attenuated vertical activity. discussion sb attenuates methamphetamine-seeking results from the current study are the first to investigate the role of 5-ht2c receptor activity in meth-mediated behaviors and the first to test sb in a rodent model of addiction. we revealed that the putative inverse agonist robustly and dose-dependently attenuated meth-seeking without impacting exploratory motor activity at  mg/kg and  mg/kg. the 5-ht2c antagonists had no effect on either meth-seeking or meth-induced motor activity. these results suggest that our prior work with mirtazapine may reflect the ability of mirtazapine to act as an inverse agonist, not as a 5-ht2c antagonist. the finding that the antagonist sb did not attenuate the effects of the inverse agonist sb was unexpected, and raised the question regarding the receptor selectivity of sb sb has high affinity for both the 5-ht2c and 5-ht2b receptors with fold or greater selectivity over other receptor targets including 5-ht2a receptors. in the current study, sb may antagonize 5-ht2b receptors, which are known to regulate behavioral and da-enhancing effects of amphetamine. however, the increase in accumbal da seen with  mg/kg sb is not altered with coadministration of the 5-ht2b antagonist ly indicating that  mg/kg sb does not substantially antagonize 5-ht2b receptors. antagonism of 5-ht2a receptors are a third 5-ht receptor that may be contributing to our findings. 5-ht2a antagonism with m attenuates cue-induced reinstatement of cocaine-seeking and meth-seeking. as mentioned above, sb demonstrates fold selectivity over the 5-ht2a receptor. 5-ht2a antagonism by sb particularly at  mg/kg, is plausible but unlikely at and  mg/kg, for amphetamine-evoked da concentrations in the nucleus accumbens and striatum are attenuated by 5-ht2a antagonism but  mg/kg sb has no effect suggesting that at this dose, sb does not act on 5-ht2a receptors. additionally,  mg/kg sb and 5-ht2a antagonism with sr 46349b have oppositional effects demonstrated by striatal 11 c]raclopride binding lending further evidence to the selectivity of sb for 5-ht2c over 5-ht2a receptors. taken together, the available in vivo data suggests that sb does not antagonize 5-ht2a receptors at  mg/kg. sb may also act as a positive allosteric modulator of the α nicotinic acetylcholine receptor with an ec of  μm for potentiation of responses evoked in gh4c cells by ec nicotine. dunlop et al., find that and 30 mg/kg sb reverses mk-801-induced deficits in prepulse inhibition, a classical model of schizophrenia, but only confirm actions as a positive allosteric modulator using a nicotinic antagonist against  mg/kg sb it is unclear whether lower doses, such as and  mg/kg sb would also act as a positive allosteric modulator at α nicotinic receptors. consistent with our findings, antagonism of α nicotinic receptors in the ventral tegmental area attenuates the reward-facilitating effect of cocaine whereas nicotine administration attenuates reinstatement of meth-seeking. the role of α nicotinic receptors and in particular, positive allosteric modulation of this receptor, in meth abuse is understudied. future investigation are needed to determine if this receptor is engaged by sb in the dose range tested before actions at the α receptor can be ruled out. debating receptor specificity based on our findings is difficult; however, it is important to note that a prior report showed that  mg/kg sb attenuates  mg/kg sb206-induced increases in striatal and accumbal da. reasons why spampinato and colleagues were able to antagonize sb effects and we were not may relate to the treatment history of rodent subjects. in the aforementioned neurochemical study, subjects were stimulant naïve, halothane anesthetized rats. in our study, rats had an extensive history of meth self-administration. this chronic meth history may have modified the 5-ht2c system, for we have observed a functionally upregulated response to systemic 5-ht2a/c receptor agonism in the ventral pallidum of rats chronically administered meth. repeated meth administration may result in changes in mrna editing profiles. proof of concept for this hypothesis is seen with studies on stress, antidepressant, and antipsychotic administration where mrna editing of the 5-ht2c receptor is altered. in addition to changing constitutive activity, mrna editing of the 5-ht2c receptor also alters the binding affinity of ligands whereby increased editing most often results in decreased agonist affinity for the receptor with enhanced or no effect on antagonists and inverse agonist binding affinity. it is unknown how editing patterns affect the affinities of sb and sb thus, the inability of sb to augment the effects of sb on cr may reflect changes in affinity for constitutively active isoforms and/or in the allosteric constant. unfortunately ex vivo binding studies, which would greatly facilitate our studies, are hampered by a lack of available tritiated ligands with 5-ht2c specificity. while future studies are needed to explore the mechanism of action for sb and determine receptor specificity in vivo, we speculate that despite a lack of antagonism by sb 5-ht2c receptors may still be involved. moreover, continued study is necessary to determine the effects of 5-ht2c receptor ligands on neuron function; to provide further insights our laboratory is conducting patch clamp studies in ex vivo slice preparations to determine the effects of 5-ht2c ligands, including sb on neurophysiology. sb but not sdz ser attenuates methamphetamine-evoked motor activity acute antagonism of 5-ht2c receptors enhances the motor effects of acute cocaine administration; however, this enhancement is lost in rats that are motorically sensitized by repeated injections of cocaine. this is comparable to our current findings wherein rats with a history of meth self-administration, 5-ht2c antagonism did not alter meth-evoked motor activity in rats. a single study testing sb on cocaine-evoked motor activity finds an attenuation of cocaine-evoked motor activity by  mg/kg sb but a potentiation with  mg/kg; however, this was an acute study with a single injection of cocaine making it difficult to compare with the current investigations using meth self-administration. in rats with a history of meth self-administration, only meth-evoked vertical activity was attenuated by  mg/kg sb both our findings and are in agreement in that sb does not alter normal exploratory motor behavior suggesting that sb is not sedating; differences in our study and regarding the ability to augment psychostimulant-evoked motor activity may reflect behavioral differences consequent to meth-induced plasticity. this hypothesis is supported by and our current findings wherein 5-ht2c antagonism fails to augment cocaine-evoked and meth-evoked motor activity in rats with a history of repeated cocaine or meth administration. sb decreases inactive lever pressing antagonists had no effect, whereas sb attenuated inactive lever pressing. mirtazapine, also an inverse agonist at the 5-ht2c receptor, similarly attenuates inactive lever pressing in a cue-induced reinstatement paradigm without effecting non-operant related motor activity in a small animal activity chamber in rats with a history of meth self-administration or rotorod performance in naïve rats. moreover, in naïve rats, administration of and 4 mg/kg sb has no effect on motor activity. a similar pattern is found for gabab receptor activation. the gabab agonist decreases inactive lever pressing in an operant task, yet has no effect on performance in an intracranial self-stimulation procedure. in summary, as sb had no effect on motor activity in in rats with a meth history or in naïve rats, and that mirtazapine, also an inverse agonist at 5-ht2c receptors, does not impair motor activity in naïve rats, we considered that decreased lever pressing of both active and inactive levers seen in the current study was not a consequence of decreased motor activity. accordingly, we propose that sb blunted the salience of the levers, both active and inactive, rather than impairing the ability of the rats to perform the operant task. this interpretation is supported by our prior demonstration that significance of meth-associated context cues are reduced by mirtazapine. additional studies aimed at exploring the effect of sb on non-drug reinforced behaviors may advance this hypothesis; however, as 5-ht2c receptors regulate food intake as well as intracranial self-stimulation behavior, such evaluations may challenging to conduct and difficult to interpret.  the presented findings are the first to explore the effects of 5-ht2c antagonists and putative inverse agonist, sb on meth-seeking and meth-induced motor activity. serotonin2c antagonism had no effect on meth-seeking or motor activity; in contrast, sb attenuated meth-seeking. this effect was selective, as sb agonist did not alter exploratory behavior, and only the higher dose reduced one motor parameter evoked by meth. there is a growing interest in assessing the role of constitutively active 5-ht2c receptors in vivo. it is currently unclear whether sb elicits the observed effects via 5-ht2c receptor inverse agonism; nonetheless, based on the dramatic reduction in seeking behavior, further investigation of this compound is merited as well as continued study of 5-ht2c regulation of psychostimulant-mediated behaviors. abbreviations meth, methamphetamine; da, dopamine; sdz, sdz ser 082; sb sb 206553; sb sb 242084; 5-ht, serotonin; cr, cue reactivity. competing interests the authors have no competing interests or financial disclosures to report. authors’ contributions experiments and data analysis were conducted by smg. experimental design and manuscript writing were done by smg and tcn. all authors read and approved the final manuscript. 
mirtazapine historically has been considered to be an antagonist at 5-ht2c receptors, but more recently shown to exhibit inverse agonism at constitutively active 5-ht2c receptors. to help distinguish the roles for antagonism vs. inverse agonism, here we explored the ability of a more selective 5-ht2c inverse agonist, sb to attenuate meth-seeking behavior, and compared its effects to those obtained with 5-ht2c antagonists, sdz ser and sb to do so, rats were trained to self-administer meth and tested for seeking-like behavior in cue reactivity sessions consisting of contingently presenting meth-associated cues without meth reinforcement. we consider 5-ht2c receptors as viable therapeutic targets. the lack of effect by 5-ht2c antagonists suggests that meth-seeking and meth-evoked motor activity are independent of endogenous 5-ht acting at 5-ht2c receptors. while sb dramatically impacted meth-evoked behaviors it is unclear whether the observed effects were 5-ht2c receptor mediated. we also explored motor function to determine the influence of sb and sdz ser on motor activity in the presence and absence of meth. motor function was largely unaltered by the 5-ht2c ligands; however, sb at the highest dose tested, attenuated meth-induced rearing behavior. we recently revealed that the atypical antidepressant, mirtazapine, attenuates meth-seeking in a rodent model of human substance abuse. in contrast, the antagonists, sdz ser and sb had no effect on cue reactivity.
 methamphetamine dependence presents a substantial socioeconomic burden. despite the need, there is no fda-approved pharmacotherapy for psychostimulant dependence. we consider 5-ht2c receptors as viable therapeutic targets. we recently revealed that the atypical antidepressant, mirtazapine, attenuates meth-seeking in a rodent model of human substance abuse. mirtazapine historically has been considered to be an antagonist at 5-ht2c receptors, but more recently shown to exhibit inverse agonism at constitutively active 5-ht2c receptors. to help distinguish the roles for antagonism vs. inverse agonism, here we explored the ability of a more selective 5-ht2c inverse agonist, sb to attenuate meth-seeking behavior, and compared its effects to those obtained with 5-ht2c antagonists, sdz ser and sb to do so, rats were trained to self-administer meth and tested for seeking-like behavior in cue reactivity sessions consisting of contingently presenting meth-associated cues without meth reinforcement. we also explored motor function to determine the influence of sb and sdz ser on motor activity in the presence and absence of meth.  like mirtazapine, pretreatment with sb, attenuated meth-seeking. in contrast, the antagonists, sdz ser and sb had no effect on cue reactivity. sb failed to attenuate the effects of and 10 mg/kg sb on cr. motor function was largely unaltered by the 5-ht2c ligands; however, sb at the highest dose tested, attenuated meth-induced rearing behavior.  the lack of effect by 5-ht2c antagonists suggests that meth-seeking and meth-evoked motor activity are independent of endogenous 5-ht acting at 5-ht2c receptors. while sb dramatically impacted meth-evoked behaviors it is unclear whether the observed effects were 5-ht2c receptor mediated. thus, sb deserves further attention in the study of psychostimulant abuse disorders. methamphetamineserotoninself-administrationmotorinverse agonistseeking 