Location of the Cannulae Implantation Site
, , and represent schematic cross sections of the rat brain showing the placement of the injection cannulae tips based on the microscopic analysis of cresyl violet-stained sections. shows the placement of the injection cannula tips for the mPOA (nBUP=10 and nVEH=9) groups tested for pup-CPP, and shows those for the groups of postpartum females tested for cocaine-CPP (n=12). shows the location of the mPOA infusion sites for the postpartum groups tested in the concurrent pup/cocaine choice CPP procedure (nBUP=9 and nVEH=8). Histological analysis demonstrated that all mPOA groups had sites distributed in a similar overall pattern, with most of the bilateral injections sites located between −0.26 and −0.88 mm from bregma.
Figure 2 Effect of transient inactivation of the mPOA on expression of pup-induced CPP. (A) Schematic representation, based on the microscopic analysis of cresyl violet-stained sections, of mPOA and dorsal control sites for all rats receiving infusion treatments. (more ...)
Figure 3 Effect of transient inactivation of the mPOA on expression of cocaine-induced CPP. (A) Schematic representation, based on the microscopic analysis of cresyl violet-stained sections, of injection sites for all rats receiving infusion treatments. (B) Cocaine-associated (more ...)
Figure 4 Effect of transient mPOA inactivation on motivated choice of pup- versus cocaine-associated environments in a concurrent pup/cocaine-CPP. (A) Schematic representation, based on the microscopic analysis of cresyl violet-stained sections, of injection sites (more ...)
Experiment 1 - Effect of Transient mPOA Inactivation on Expression of Pup-induced CPP
In the preconditioning test, the majority of postpartum females showed no preference for either side compartments, and, on average, spent similar amounts of time in each chamber of the CPP apparatus. Importantly, unconditioned chamber preferences [χ2=5.137, df=6, P=ns] and times were similar and did not differ among groups [chamber × group interaction: F(3,32)=0.1649, P=ns], indicating that differences in baseline responding cannot account for any selective effect of the bupivacaine-induced changes in conditioned preferences.
After conditioning, non-surgical control females exhibited a robust CPP for the pup-associated environment as demonstrated by comparison analysis of pre- and postconditioning environment preferences [χ2 (3, N=10)=40.533, P<0.05]. Similarly, postpartum females receiving infusion of saline vehicle into the mPOA prior to CPP testing exhibited substantial conditioned preference for the pup-associated environment [preconditioning versus postconditioning environment preferences: χ2 (3, N=9)=40.083, P<0.05]. Thus, the majority of females receiving vehicle infusion into the mPOA preferred the environment associated with the pups (78%), and this proportion was not different from the non-surgical control group (70%) (Fisher’s Exact Test, P=ns), demonstrating that surgery, handling and intracranial drug administration procedures neither affected the acquisition nor the expression of pup-CPP of postpartum females (). Furthermore, the time spent in the pup-associated environment significantly increased in both the non-surgical control group and in the group receiving microinfusion of saline vehicle into the mPOA following conditioning [session × group interaction: F(3,32)=4.36, P<0.05; chamber × session interaction: F(1,32)=35.4, P<0.001; chamber × group × session interaction: F(3,32)=5.7, P<0.01]. In addition, both groups similarly spent more test time in the pup-associated environment than in the alternative one associated with no unconditioned stimuli (Tukey’s HSD test: both Ps<0.05; ). Thus, both environment preferences and times changed significantly as a function of conditioning, indicating a robust CPP associated with pups (see ).
In contrast, intra-mPOA infusions of bupivacaine prior to CPP testing completely blocked the expression of the conditioned preferences for the pup-associated environment. Thus, none of the mPOA bupivacaine-treated females preferred the environment associated with pups (0%), whereas the majority of control females (both groups) preferred the pup-associated option (70–80%, Fisher’s Exact Test, both Ps<0.05; ), indicating that neuronal inactivation, not the mechanical or osmotic effects of drug infusion, affected expression of pup-CPP in postpartum females. Furthermore, the time spent in the pup-associated environment was significantly reduced in mPOA-inactivated females compared to notable time spent in the pup-associated environment by both control groups (Tukey’s HSD test: both Ps<0.001; ).
Proof of Anatomical Specificity
Regional specificity limited to mPOA of the CPP effects was demonstrated since inactivation of anatomical sites adjacent to the mPOA had no effect on CPP for pup-associated environments. shows the implant locations for the group of postpartum females tested for pup-associated CPP following infusion of bupivacaine into adjacent dorsal sites to the mPOA (n=7). In addition to this deliberately chosen dorsal control site, as a result of the normal variation in placements found with stereotaxic surgery targeting a small brain region, nine additional postpartum females treated with bupivacaine before CPP testing had infusion sites that bilaterally targeted control structures immediately outside the mPOA. Three females had an asymmetrical placement that resulted in a unilateral infusion into the lateral mPOA and a unilateral infusion into the third ventricle, 2 females had their cannulae rostral to the mPOA into the diagonal band of Broca; and 4 females had their cannulae caudal to the mPOA into the anterior hypothalamus.
Most of theses control females receiving pre-testing bupivacaine infusion into dorsal sites preferred the environment associated with pups (71%). Thus, the conditioned environment preferences and times of dorsal control females were not different from those of non-surgical and vehicle-treated control females [χ2=1.778, df=6, P=ns; session × chamber × group interaction: F(2, 23)=0.001, P=ns; ]. Similarly, of those postpartum females with cannulae sites that bilaterally targeted control structures either rostral or caudal to the mPOA, 78% showed robust pup-associated CPP after bupivacaine infusions (caudal to the mPOA, 3 of 4; rostral, 2 of 2; unilateral 2 of 3 preferred the environment associated with pups). Thus, the disruptive effect on pup-associated CPP responses produced by pre-testing bupivacaine infusion into the mPOA appear unlikely to have been mediated by diffusion to these other dorsal, anterior, or posterior structures.
Experiment 2 - Effect of Transient mPOA Inactivation on Expression of Cocaine-induced CPP
Prior to conditioning, unconditioned chamber preferences [χ2=3.152, df=3, P=ns] and times [chamber × group interaction: F(1,22)=0.078, P=ns] were similar and did not differ among groups. Following conditioning, a significant CPP to 5.0 mg/kg IP cocaine occurred in both the non-surgical and the vehicle-treated control groups, with chamber preferences [preconditioning versus postconditioning environment preferences: χ2 (3, N=12)=8.643, P<0.05 and χ2 (3, N=12)=8.637, P<0.05, respectively] and times [session × chamber interaction: F(1,22)=57.2, P<0.01] differing statistically between the pre- and postconditioning sessions (see ).
There was no significant difference in chamber preference [χ2=, df=3, P=ns] nor times [session × chamber × group interaction: F(1,22)=0.61, P=ns] between the non-surgical control and saline microinjection groups during the CPP test ().
Transient mPOA inactivation had no impact on the motivational responsivity of postpartum females to cocaine-related stimuli (). Thus, intra-mPOA bupivacaine infusion prior to CPP testing affected neither the number of early postpartum females preferring the cocaine-associated environment (Fisher’s Exact Test, both Ps=ns), nor the time spent by the group in the cocaine-associated environment (Tukey’s HSD test, both Ps=ns) compared to the non-surgical control group. Consequently, postconditioning environment preferences did not differ within and between groups, with the vast majority of postpartum females, regardless of treatment, preferring the cocaine-associated environment. All groups also similarly spent more time in the cocaine-associated environment than in the saline-associated environment (Tukey’s HSD test, all Ps=ns; ).
Experiment 3 - Effect of transient mPOA inactivation on motivated choice of pup- versus cocaine-associated environments in a concurrent pup/cocaine choice CPP
Prior to conditioning (at preconditioning test) the majority of postpartum females showed no preference for either side compartment, and, on average, spent similar amounts of time in each chamber of the CPP apparatus. Furthermore, there were no differences in the unconditioned chamber preferences between groups [χ2=1.893, df=6 P=ns], nor were there significant group [F(2,24)=0.047, P=ns], chamber [F(1,24)=0.0001, P=ns] and group × chamber interaction [F(2,24)=0.055, P=ns] effects. A robust CPP effect was demonstrated, as environment preferences [behavioral control group: χ2(3, N=10)=22.67, P<0.05 and vehicle-treated group: χ2(3, N=8)=11.2, P<0.05] and times changed significantly between the pre- and postconditioning sessions (see ).
After conditioning, most females from the two control groups developed a preference for environments associated with one of the two unconditioned stimuli, either cocaine or pups (). There was no significant difference in environment preferences between the non-surgical and saline microinjection control groups [χ2=0.064, df=3, P=ns; ]. Thus, 40% of the non-surgical control females showed a preference for the cocaine-associated environment, and 40% preferred the pup-associated environment. Similarly, the vehicle-treated group showed a bimodal distribution in which postpartum females preferred either the pup- or the cocaine-associated environment after conditioning.
In contrast, intra-mPOA infusions of bupivacaine just prior to conditioned place preference testing selectively and entirely blocked the conditioned preference of postpartum females for the pup-associated environment (). Furthermore, a substantial and statistically significant majority of the postpartum females infused with bupivacaine into the mPOA preferred the cocaine-associated environment (78%). Thus, the majority of early postpartum females preferred the cocaine- over the pup-associated environment following pre-testing bupivacaine infusion into the mPOA, highly contrasting the bimodal distribution of conditioned preferences of both control groups for pup- and cocaine-associated environments ().
As shown in , the analysis of environment times during the CPP test revealed a significant chamber × group interaction effect [F(2,24)=3.74, P<0.05], with overall time spent in the cocaine-associated environment significantly longer in the bupivacaine-treated group than in either non-surgical or vehicle-treated control groups (Tukey’s HSD test, both Ps<0.05). Pup- and cocaine-associated environment times during the expression test were similar and did not differ between the two control groups (Tukey’s HSD test, all Ps=ns). However, the time spent in the cocaine-associated environment by bupivacaine-treated females was significantly longer than the time spent in the pup-associated environment (Tukey’s HSD test, P<0.05; ).
Five additional postpartum females treated with bupivacaine before testing had infusion sites that bilaterally targeted structures immediately rostral or caudal to the mPOA. Of these females, 3/5 preferred the pup-associated environment and 2/5 the preferred the cocaine-associated environment, resulting in a postconditioning environmental preference pattern similar to that of the control groups. This further supports the anatomical specificity of the data, showing that bilateral infusions of bupivacaine into the mPOA are needed to block the conditioned preferences of postpartum females for the pup-associated environment.
It is important to note that the CPP responses of mPOA bupivacaine-treated females for cocaine-associated environments in the concurrent pup/cocaine choice procedure are comparable in magnitude to those of postpartum females presented with a CPP choice between an environment associated with 1.0mg/kg IP cocaine and one associated with saline (78% and 75%, respectively; and ). This together with the fact that transient inactivation of the mPOA completely and selectively blocked the conditioned preference of postpartum females for the pup-associated environment, regardless of which alternative option was presented (Experiments 1 and 3), suggest that the shift in the choice preference toward the cocaine-associated environments induced by mPOA inactivation in the concurrent choice procedure is most likely due to the result of a reduction in pup-associated environment preferences.
Transient inactivation of mPOA did not affect locomotor activity during CPP test
Locomotor activity during the expression test did not differ between the groups, suggesting that exploratory behaviors remained intact during CPP testing. Across mPOA-cannulated groups, locomotor scores during the 60-min CPP test were similar regardless of whether females received bupivacaine or saline treatment, and were not different from non-surgical controls [treatment × group interaction: Experiment 1: F(2,24)=0.06, P=ns; Experiment 2: F(1,22)=0.0137, P=ns; Experiment 3: F(2,26)=1.17, P=ns].