Location of the Cannulae Implantation Site
represents cross sections of the rat brain showing the placement of the injection cannulae tips based on the microscopic analysis of cresyl violet-stained sections. show the placement of the injection cannula tips for the mPOA group providing repeated measure testing at both early (PPD7-8) and late postpartum (PPD13-14) stages (n=8). shows the location of the mPOA infusion sites for the group of postpartum females tested only during PPD13-14 (n=9). Histological analysis indicated that both mPOA groups had infusion sites distributed in a similar pattern, with most of the bilateral infusion sites located between -0.26 and -0.88 mm from bregma.
Figure 1 Schematic representation, based on the microscopic analysis of cresyl violet-stained sections, of infusion sites for female rats tested for maternal behavior following infusion treatments. (A) mPOA cannulated postpartum females tested at both early and (more ...)
Postpartum females included in the dorsal control group (n=7) had cannulae bilaterally placed dorsal to the anterior commissure, ranging between -0.26 and -0.40mm from bregma (). Nine additional postpartum females had infusion sites that bilaterally targeted control structures outside of the mPOA. One female had an asymmetrical placement that resulted in a unilateral infusion into the lateral mPOA and a unilateral infusion into the third ventricle, 3 females had their cannulae lateral to the mPOA into the LPOA, 2 females had their cannulae rostral to the mPOA into the diagonal band of Broca; and 3 females had their cannulae caudal to the mPOA into the anterior hypothalamus.
Transient mPOA inactivation reversibly inhibited early postpartum maternal behavior
Early postpartum females receiving bupivacaine infusion into the mPOA exhibited severe deficits in their maternal behavior. As shown in , bupivacaine-treated postpartum females exhibited significantly fewer retrievals (T=0.0, N=8, P=0.012, Wilcoxon’s matched pairs signed-ranks test) and mouthings (T=0.0, P=0.012) than after receiving saline infusions into mPOA (). In fact, during mPOA inactivation, none of the 8 postpartum females completed retrieving and grouping the pups into the nest; 4 of 8 did not retrieve any pups, and the remaining 4 retrieved ≤3 pups of their 8-pup litter. Moreover, the number of corporal (T=0.0, P=0.012) and anogenital lickings (T=0.0, P=0.012) was also dramatically reduced, and nest building was virtually absent (T=0.0, P=0.02). It is important to mention that the inserted cage barriers delayed, but did not prevent the pups from crawling into the nest area and grouping together in a huddle. Thus, even though none of the bupivacaine-treated postpartum females completed retrieving and grouping of the pups, their litters did group themselves into the nest within 594.5 ± 118.38 s, where they generally stayed together until the end of the behavioral test. Although most mPOA-inactivated females did not group their pups into the nest, they did approach and investigate them, and eventually hovered over the pups and nursed them (). In fact, the percentage of females that did hover over and nurse their litters was not significantly different after bupivacaine and vehicle infusions (6/8 and 8/8, respectively; Fisher exact probability test P=ns). However, mPOA-inactivated females spent significantly less time in contact with pups (T=0.0, P=0.012), due to an increased latency for dams to begin hovering over the pups (T=0.0, P=0.012; see ).
Figure 2 Effect of transient inactivation of the mPOA on median ± SIQR number of active maternal responses over a 30 min-behavioral test. Female rats were tested on early PPD 7 and 8 and then again on late PPD13 and 14. Data shown is for the behavioral (more ...)
Latencies and durations of maternal behaviors
Importantly, infusion of saline vehicle into mPOA had no significant effect on any maternal component measured ( and ). Thus, early postpartum females receiving vehicle infusion into the mPOA exhibited full maternal behavior that was not different from the unoperated control group (Mann-Whitney U test, all Ps=ns).
Transient mPOA inactivation reversibly increased late postpartum maternal behavior
Inactivation at the very same mPOA location had a completely different effect during late postpartum, causing a significant increase in maternal behavior compared to that seen after vehicle infusion (). Thus, bupivacaine-treated postpartum females that exhibited impaired maternal behavior during early postpartum showed notably increased maternal behavior when infused with bupivacaine in late postpartum ( and ). Importantly, the additional mPOA group tested only in late postpartum similarly showed increased maternal behavior toward pups following bupivacaine compared to saline infusions ( and ), in a manner that did not differ from the group that received repeated bupivacaine infusions. (Repeated mPOA inactivation vs. Single mPOA inactivation, all Ps=ns, Mann-Whitney U test). The great majority of bupivacaine-treated late postpartum females completed retrievals and grouped all pups into the nest (). Of the 17 late postpartum females (both mPOA groups), 16 retrieved all pups and the remaining subject retrieved 3 of the 8-pup litter after infusion of bupivacaine, while after saline infusion only 3 of these same 17 females grouped all pups, 9 retrieved ≤4 pups and the remaining 5 did not retrieve any pups at all. Concerning those 14 vehicle-treated females that did not complete retrieving, their pups grouped together into the nest by themselves with a median latency of 283.0 ± 81.0 s. In marked contrast, following bupivacaine infusion into the mPOA these same late postpartum females retrieved and grouped their litter into the nest, and did so significantly faster. Once the pups were grouped into the nest, they usually stayed together in a close group. Furthermore, it was observed that 13-14 day-old pups sometimes struggled when picked up by their mother, but this behavior from the pups neither prevented bupivacaine-treated late postpartum females to complete retrievals, nor increased the number of retrievals per pup needed by the mothers to group the entire litter into the nest (Figures and , ). This highlights the robust activational effect on the maternal behavior of late postpartum females following bupivacaine infusion into the mPOA. The numbers of mouthings (T=0.0, N=17, P=0.001, Wilcoxon’s matched pairs signed-ranks test), corporal (T=0.0, P=0.0003) and anogenital licking (T=0.0, P=0.0003), and nest building (T=6.0, P=0.001) behaviors also increased significantly when females received bupivacaine versus when these same females received a saline infusion into the mPOA (Figures and ). Further, late postpartum females spent more time in contact with pups after mPOA infusion of bupivacaine than saline vehicle (T=15.0, P=0.004).
Figure 3 Replication of late postpartum group: Effect of transient inactivation of the mPOA on median ± SIQR number of active maternal responses over a 30 min-behavioral test. An additional independent group of mPOA cannulated female rats was tested only (more ...)
Importantly, no significant differences were found between behavioral control and either vehicle-treated late postpartum female group, in any maternal behavior measured (Mann-Whitney U tests: all Ps=ns; Figures and , ).
Demonstration of regional specificity
Females with infusion sites that bilaterally targeted control structures outside of the mPOA did not show any change in maternal behavior after bupivacaine or saline infusions (dorsal, n=7; rostral, n=2; lateral n=3; and caudal to the mPOA, n=3). In particular, neither bupivacaine nor saline infusion into adjacent dorsal sites to the mPOA had any effect on the expression of early and late maternal behavior (Wilcoxon’s matched pairs signed-ranks tests: N=7, all Ps=ns; and ). Moreover, the maternal behavior of these anatomical control females was not different from that of behavioral control females (Mann-Whitney U tests: all Ps=ns; and ).
Figure 4 Effect of transient inactivation of dorsal sites to the mPOA on median ± SIQR number of active maternal responses over a 30 min-behavioral test. No significant differences in early and/or late postpartum maternal behavior were found in females (more ...) Locomotor activity
Home cage activity, including general exploration and self grooming did not differ both within and between groups (all Ps=ns, data not shown). Moreover, mPOA inactivation did not alter the locomotor activity of postpartum females relative to vehicle treatment, measured in the automated apparatus (time by treatment interaction: F (11, 77) = 0.2684, P=ns; ).
Figure 5 A. Locomotor activity (mean ± SEM) graphed in 5-min intervals over a 60min-test session for postpartum females with bilateral infusions of either 2% bupivacaine or vehicle into the mPOA. B. Schematic representation, based on the microscopic analysis (more ...)
In summary, inactivation of dorsal, lateral, anterior or posterior structures to the mPOA was ineffective in affecting maternal behavior, thereby confirming spatial specificity. Saline injections within mPOA had no effect indicating that neuronal inactivation, not mechanical or osmotic effects of drug injection, affected expression of maternal behavior in postpartum females. Moreover, that fact that saline infusions did not produce any behavioral effects even when preceded by one or two bupivacaine injections - while bupivacaine injections continued to have behavioral effects — shows that the target tissue remains stable over multiple injections. Unilateral mPOA inactivation was also without behavioral effect. Thus, the above described changes in maternal behavior produced by bupivacaine infusion into the mPOA are mainly due to effects within and not beyond mPOA.