  the conditioned place preference procedure in rodents is used to study the conditioned - rewarding properties of drugs of abuse ( e.g. , cocaine ) that can contribute to substance abuse and dependence in humans [ 1 , 2 ] . during the development of conditioned place preference ,
cocaine injection is paired with a distinct context , and vehicle is paired with a different context .
the expression of place preference is examined in a cocaine - free state when the rodent is given free choice access to both contexts .
conditioned place preference ( cpp ) is defined as an increase in the amount of time spent on the drug paired side ( dps , e.g. , cocaine context ) , relative to the amount of time spent on this same side during a preconditioning trial ( cpp score = dpspostest   dpspretest ) .
the development and expression of cocaine - induced conditioned place preference have most frequently been attributed to the drug 's efficacy to regulate dopamine signaling in the brain ; however , other neural circuits  such as those associated with memory , motor control , and anxiety  play an important role in this effect [ 35 ] .  
sigma receptors contribute to cocaine 's behavioral effects in rodents , including the development and expression of cpp [ 68 ] .
overall , sigma receptor antagonists attenuated or blocked cocaine 's conditioned - rewarding properties [ 912 ] .
for example , coadministration of bd-1047 with cocaine prevented the development of cpp in mice . in a separate study , after the development of cocaine cpp , bd-1047 blocked the expression of cocaine 's conditioned - rewarding properties in rats .
the sigma ligand alone did not have conditioned - rewarding or conditioned - aversive properties , as there were no differences in cpp induced between rats that administered only bd-1047 and those that administered saline during the conditioning phase .
recently , the n - phenylpropyl - n-substituted piperazine sigma receptor ligand sa-4503 ( 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine ) blocked the development of cocaine cpp in rats .
sa-4503 is regarded as a sigma receptor agonist ; however , it also blocked cocaine - induced hyperactivity in mice , an antagonist effect observed with other ligands in its chemical class .
these seemingly contradictory results suggest a mixed agonist / antagonist profile of n - substituted piperazine ligands .  
yz-185 ( n - phenylpropyl - n-(3-methoxyphenethyl)piperazine ) , a structural congener to sa4503 in that it only lacks the 4-methoxy group on the phenethyl moiety , binds with high affinity to 1 and 2 sigma receptors ( ki values  1.4 and 10.2  nm , resp . ) and blocks convulsions and death induced by a lethal ( ~198  mol / kg ) cocaine dose in mice .
recently , our laboratory observed that a high yz-185 dose ( 31.6  mol / kg ) attenuated cocaine - induced hyperactivity ( 66  mol / kg ) , while a low yz-185 dose ( 0.1  mol / kg ) enhanced cocaine - induced hyperactivity .
it is possible that yz-185 functions as a sigma receptor antagonist at the higher dose and an agonist at the lower dose ; demonstrating the characteristic mixed agonist / antagonist profile observed in the related compound sa-4503 .
the goal of the present study was to continue investigating the behavioral properties of yz-185 .
the intrinsic conditioned - rewarding properties of yz-185 and its effect on the development and expression of cocaine - induced conditioned place preference were determined . to further characterize yz-185
, we also evaluated its effect on anxiety - related behavior with the elevated zero maze procedure [ 1719 ] and coordinated motor skill behavior using the rotarod [ 20 , 21 ] .
cocaine hydrochloride was purchased from sigma - aldrich ( st . louis , mo , usa ) .
drug doses refer to the free base weight and are presented in mol units to allow for comparison to other sigma ligands .  
male cd-1 mice ( charles river , 2022  g at arrival ) were housed , 4 or 5 mice per cage with standard rodent chow and water available ad libitum .
the colony was maintained under a 12  hr/12  hr light / dark cycle , and all experiments were conducted during the light phase of the cycle .
all procedures were approved by the institutional animal care and use committee of the university of missouri .   the dose - response for cocaine to induce conditioned place preference was determined .
place conditioning experiments were performed in acrylic shuttle boxes ( 48  20  20  cm ) . for conditioning sessions
, the boxes were divided into two equal - sized compartments ( 20  20  20  cm ) by partitions .
one side had vertical black / white stripes on the four walls , and the floor was a metal mesh .
the other side had horizontal stripes on the walls and a metal rod floor . for pre- and posttest sessions
sides were separated by a center area ( 8  20  20  cm ) with black walls and a smooth black acrylic floor .
an incandescent lamp was placed approximately 105  cm above the box floor and was the only room lighting . to mask sounds from outside the room ,
a video camera above the maze was connected to a computer , running any - maze software ( version 4.81 , stoelting co. , wood dale , il , usa ) , which measured time spent on each side and distance traveled during both pre- and postconditioned test of preference sessions .
side determination was set at a criterion of 75% of body area existing on that side , calculated in real time by the video tracking software .    during the preconditioning test session ( day 1 )
, mice were given free access to either side of the box for 20 minutes .
initial preference  defined as the side in which the mouse spent the most time  was determined .
mice were then assigned a dps in a counter - balanced fashion such that half of the mice had drug paired with their initially preferred side , while the other half received drug on their nonpreferred side .
conditioning assignments followed a counter - balanced fashion over the following six consecutive days , such that half of the mice were given cocaine ( 16 , 33 or 66  mol / kg ( 5 , 10 , or 20  mg / kg ) ) or saline ( control ) injection and confined to their dps for 20 minutes on days 2 , 4 , and 6 .
the remaining half of mice were given cocaine or saline injection and confined to their dps for 20 minutes on days 3 , 5 , and 7 . during non - dps conditioning days , all mice received saline injection and were confined to their non - dps for 20 minutes .
a post - conditioning test following the same procedure as the pre - conditioning test was performed on the day after the last conditioning session ( day 8) .  
the dependent variable was a conditioned place preference score ( cpp score ) defined as the change in the amount of time ( in seconds ) a mouse spent on the dps from pretest to posttest ( i.e. , cpp score = dpspostest   dpspretest ) .
data were analyzed via analysis of variance ( anova ) with cocaine dose as a between - groups factor .
newman - keuls post hoc tests were followed when appropriate ( p < 0.05 ) .    the ability of yz-185 to induce place conditioning was determined via the procedures described .
however , on three conditioning days , mice ( n = 712 mice / group ) received yz-185 ( 0.1 , 3.16 , or 31.6  mol / kg ( 0.042 , 1.31 , or 13.1  mg / kg ) ) or saline ( 0  mol / kg yz-185 ) instead of cocaine .
conditioning scores were analyzed via anova with yz-185 dose as a between - groups factor .  
the effect of yz-185 on the development of cocaine - induced place preference also was determined via procedures described .
however , on three dps conditioning days , mice ( n = 812 mice / group ) were pretreated with yz-185 ( 0.1 , 3.16 , or 31.6  mol / kg ) or saline ( 0  mol / kg yz-185 ) 15 minutes prior to cocaine ( 66  mol / kg ) or saline administration . on the three non - dps conditioning days , all mice received two saline injections .
conditioning scores were analyzed via anova with yz-185 dose as a between - group factor .  
the effect of yz-185 on the expression of cocaine - induced place preference was also determined .
procedures for inducing cocaine ( 66  mol / kg ) place preference were performed as described . however , on the posttest , mice ( n = 513 mice / group ) were pretreated with yz-185 ( 0.1 , 3.16 , or 31.6  mol / kg ) or saline ( 0  mol / kg yz-185 ) 15 minutes prior to being given free access during the post - conditioning test .
conditioning scores were analyzed via anova with yz-185 dose as a between - group factor .   to examine effects of yz-185 on anxiety - related behaviors ,
additional groups of mice ( n = 10 - 11 mice / group ) were randomly assigned to receive yz-185 ( 0.1 , 3.16 , and 31.6  mol / kg ) or saline ( 0  mol / kg yz-185 ) injection .
30 minutes following treatment , mice were placed on an elevated zero maze ( bond life sciences center , columbia , mo , usa ) for 5 minutes .
the maze was constructed of grey acrylic in a circular track 10  cm wide , 105  cm in diameter and elevated 72  cm from the floor .
two arms were open , and two arms were bounded by grey acrylic walls 28  cm in height .
an incandescent lamp was placed approximately 2  m above the maze and was the only room lighting .
a video camera above the maze recorded the location of the mice , and scoring was later completed by an observer blind to the treatment condition .
time in the open arm and the number of entries into the open arm were determined .
these two variables were analyzed via separate anova with yz-185 dose as a between - groups factor . to examine possible effects of yz-185 on coordinated motor skill behavior
, mice were placed on a rotarod apparatus ( env-577 m , med associates , georgia , vt , usa ) consisting of a horizontal rod ( 3.2  cm diameter ) 16.5  cm above the floor of the apparatus .
on three consecutive days ( days 13 ) , mice ( n = 10 - 11 mice / group ) were placed on the rod , and its rotation increased at a consistent rate ( 440  cm / min ) across a 5-minute session .
the duration of time each mouse remained on the rod was recorded . on the following day ( day 4 ) , mice were randomly assigned to receive yz-185 ( 0.1 , 3.16 , and 31.6  mol / kg ) or saline ( 0  mol / kg yz-185 ) injection and after 30 minutes were placed on the rod .
data from days 13 were analyzed via anova with day as a within - subjects factor .
the data from day 4 were analyzed via anova with yz-185 dose as a between - groups factor .
  in the experiment to determine cocaine 's efficacy to produce conditioned place preference , analysis of conditioning scores revealed a significant main effect of cocaine dose ( f(3,68 ) = 11.80 ; p < 0.001 ) .
post hoc tests revealed that cpp scores were greater for mice that received 16  mol / kg ( mean = 135.4  s ; sem = 48.2  s ) , 33  mol / kg ( mean = 123.4  s ; sem = 52.2  s ) , or 66  mol / kg ( mean = 148.5  s ; sem = 20.1  s ) cocaine than for mice that received only saline ( mean = 49.1  s ; sem = 24.7  s ) . there were no significant differences between the three groups of mice that administered cocaine . thus , cocaine produced conditioned place preference , although the effect was not dose dependent .   the efficacy of yz-185 to produce a conditioned place preference or aversion was determined , and the data are presented in figure 1(a ) .
there were no significant differences ( f(3,34 ) = 0.77 ; p = 0.52 ) between the groups of mice that administered yz-185 ( 0.131.6  mol / kg ) or saline ( 0  mol / kg yz-185 ) , indicating that yz-185 did not have conditioned - rewarding or conditioned - aversive properties . 

figure 1(b ) depicts cpp scores for mice that received concurrent yz-185 and cocaine administration during conditioning to investigate the development of cocaine cpp .
a significant main effect of yz-185 dose was revealed ( f(3,36 ) = 6.32 ; p < 0.01 ) .
post hoc analyses revealed that cpp scores for mice that received 31.6  mol / kg yz-185 and cocaine were less than scores for mice that received saline and cocaine .
there were no significant differences in cpp scores between mice that received 0.1  mol / kg or 3.16  mol / kg yz-185 and cocaine and mice that received saline and cocaine .
thus , only the high yz-185 dose blocked the development of cpp to cocaine . 

figure 1(c ) depicts conditioning scores for mice that received yz-185 only on the posttest to investigate the expression of cocaine cpp .
there were no significant differences ( f(3,36 ) = 0.15 ; p = 0.93 ) between groups , indicating that the sigma receptor ligand did not affect the expression of cocaine cpp .  
the effect of yz-185 on time spent in the open quadrants of the zero maze and the number of quadrant entries were analyzed and are presented in figures 2(a ) and 2(b ) , respectively .
the main effect of yz-185 dose was not significant for either dependent variable ( f(3,42 ) = 0.71 ; p = 0.56 , and f(3,42 ) = 1.15 ; p = 0.34 , resp . ) .
thus , yz-185 did not affect anxiety - like behavior as assessed by the zero maze . 

there was a significant increase in time on the rod on days 2 and 3 , relative to day 1 ( f(2,82 ) = 19.24 ; p < 0.001 ) , indicating the development of motor skill learning .
however , the main effect of yz-185 dose from day 4 , when mice received yz-185 treatment before rod placement , was not significant ( f(3,42 ) = 0.50 ; p = 0.68 ) .
these data indicate that yz-185 did not alter the expression of previously learned motor skill behavior .
in our previous study , 66  mol / kg cocaine produced hyperactivity that was altered by the n - phenylpropyl - n-substituted piperazine sigma ligands tested , including yz-185 .
in fact , cocaine - induced hyperactivity was suppressed by 31.6  mol / kg yz-185 . presently , this same cocaine dose induced significant cpp that was sensitive to yz-185 administration .
pretreatment with 31.6  mol / kg yz-185 during conditioning blocked cocaine - induced cpp . in previous cpp studies , other sigma receptor antagonists ( e.g. , bd-1047 and cm-156 ) also diminished the development of cocaine 's conditioned - rewarding properties [ 10 , 12 ] .
furthermore , matsumoto and colleagues   reported that this yz-185 dose range markedly decreased cocaine - induced seizures in mice .
this pattern suggests yz-185 functions as a sigma receptor antagonist , at least at higher doses .
relatedly , mori and colleagues   recently reported that the putative sigma receptor agonist sa-4503 , an n - substituted piperazine similar to yz-185 , prevented the development of cpp to cocaine in rats .
though initially characterized as a sigma receptor agonist , sa-4503 could be a mixed agonist / antagonist . like sa-4503 , yz-185 has also been shown to display agonist properties .
presently , 0.1  mol / kg yz-185 did not significantly affect the development of cpp to cocaine , but showed a trend toward enhancement .
yz-185 too may have a mixed agonist / antagonist profile that is dose dependent and selective for specific aspects of rodent behavior .
neither 0.1 nor 31.6  mol / kg yz-185 induced cpp or altered behavior in the zero maze or rotarod , further indicating a selective behavioral profile .
the development of cpp reflects the association of the unconditioned stimulus ( us , cocaine ) with the conditioned stimulus ( cs , the context ) [ 2 , 22 ] .
sigma receptors may contribute specifically to the interoceptive properties of the us and/or the cs [ 23 , 24 ] or more globally interfere with the cs - us association .
previous studies where yz-185 diminished cocaine 's effects on locomotor activity   and convulsions   suggest that yz-185 diminished cocaine - induced cpp by attenuating the us properties of cocaine ( i.e. , it diminishes cocaine 's efficacy ) .
however , future studies on yz-185 and other sigma receptor ligands on classical conditioning are warranted , considering the prophylactic potential of sigma ligands in models of cognitive diseases . in the absence of cocaine and across the dose range examined , yz-185 did not induce cpp , indicating that yz-185 does not have conditioned - rewarding properties like cocaine or other abused drugs . additionally , the finding indicates that yz-185 does not have conditioned aversive properties , indicated by reduced time spent on dps , and thus did not block the development of cpp to cocaine by inducing an aversive state ( e.g. , malaise or anxiety ) . while yz-185 blocked the development of cocaine cpp , it was ineffective at blocking the expression of the cpp after cocaine 's conditioned - rewarding properties were established .
systematic evaluation of sigma receptor ligands on the development versus expression of cocaine place preference has not been reported , although bd-1047 blocked the development in mice   and the expression in rats .
it is possible that yz-185 could diminish the expression of cpp established given fewer cs - us pairings ( e.g. , a single conditioning trial rather than three trials ) or given a lower cocaine dose .
the regimen used to establish cocaine - induced cpp in the present study was selected based on the cocaine cpp literature , and the 66  mol / kg cocaine dose was susceptible to n - substituted piperazine treatment in another behavioral measure ( i.e. , cocaine - induced hyperactivity ) .
yz-185 was examined in the zero maze and rotarod assays to further characterize its pharmacological profile and understand potential behavioral mechanisms to alter cocaine 's effects .
the elevated zero maze is commonly used to measure a drug 's anxiolytic / anxiogenic properties [ 17 , 24 ] .
some sigma receptor ligands diminish anxiety in rodents [ 28 , 29 ] , and these receptors are a clinical target for the development of anxiolytics .
yz-185 did not induce or minimize anxiety - like behaviors in mice , relative to mice treated with saline vehicle suggesting that this ligand lacks efficacy to regulate stress and anxiety .
the rotarod test is used to evaluate motor skill learning / memory in rodents and is sensitive to treatment with neurotoxins , including those targeting dopamine pathways in brain [ 20 , 21 ] .
yz-185 did not alter performance on the rotarod , relative to mice treated with vehicle , consistent with previous work with bd-1047 .
this suggests that yz-185 's effects on cocaine - induced hyperactivity and cpp are not related to impairments in motor control .   the n - phenylpropyl - n-substituted piperazine sigma receptor ligand yz-185 binds with high affinity ( ki values  110  nm ) to sigma receptors in brain   and blocks the development of cocaine - conditioned place preference at a high ( 31.6  mol / kg ) dose .
these results suggest that yz-185 is a sigma receptor antagonist , at least at this dose .
the present results also extend previous research suggesting a role for sigma receptors in the development of cocaine 's conditioned - rewarding properties [ 912 ] that can contribute to substance abuse and dependence in humans .
 sigma receptor antagonists diminish the effects of cocaine in behavioral assays , including conditioned place preference . 
 previous locomotor activity experiments in mice determined that the sigma receptor ligand yz-185 ( n - phenylpropyl - n-(3-methoxyphenethyl)piperazine ) enhanced cocaine - induced hyperactivity at a lower ( 0.1  mol / kg ) dose and dose - dependently attenuated cocaine - induced hyperactivity at higher ( 3.1631.6  mol / kg ) doses . 
 the present study investigated the effect of yz-185 on cocaine 's conditioned - rewarding properties in mice . 
 yz-185 ( 0.1 , 0.316 , 3.16 , and 31.6  mol / kg ) did not have intrinsic activity to produce conditioned place preference or aversion . 
 a higher ( 31.6  mol / kg ) yz-185 dose , but not lower ( 0.13.16  mol / kg ) yz-185 doses , prevented the development of place preference to cocaine ( 66  mol / kg ) . 
 yz-185 did not alter the expression of cocaine place preference . 
 to further characterize yz-185 's behavioral profile , its effects in the elevated zero maze and rotarod procedures were also determined ; yz-185 produced no significant change from baseline in either assay , indicating that the sigma receptors probed by yz-185 do not regulate anxiety - like or coordinated motor skill behaviors . 
 overall , these results suggest that yz-185 is a sigma receptor antagonist at the 31.6  mol / kg dose and demonstrate that sigma receptors can mediate the development of the conditioned - rewarding properties of cocaine . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions 