Females were randomly assigned to one of the following groups: (1) females stereotaxically implanted with bilateral guide cannulae aimed at the mPOA, or (2) at locations aimed immediately outside the mPOA: (a) dorsal control and (b) lateral, rostral, or caudal control groups, and (3) females not subjected to stereotaxic surgery (behavioral control group).

All testing was conducted during the light phase of the light/dark cycle. Following infusions of either bupivacaine or saline, postpartum females were returned to their home cage. Five minutes after, their eight pups were scattered in the home cage opposite to the female’s nest. The number of the following maternal behavioral components was continuously recorded for 30 min: retrievals of the pups into the nest, mouthings (oral repositioning of the pups into the nest), sniffings, full body and anogenital lickings, and nest building. In addition, the total duration of huddling behaviors, including lying in contact with pups and hovering over the pups in the nest while actively performing other behaviors (i.e. licking of pups or self grooming), and the nursing posture kyphosis, a quiescent upright crouching over pups [99] were recorded. Total time in contact with pups was the summed durations of huddling plus nursing behaviors. Also, the latency (i.e. time from the introduction of the pups to the first behavioral response by the female) to begin retrieving pups, to reunion of the entire litter into the nest as well as to begin hovering over and nursing was registered. Only those postpartum females that retrieved each of their eight pups into the nest were considered to have grouped the litter. The latency to begin hovering over or nursing the pups was the first occurrence of a bout of each behavior ≥2 min in duration. A latency of 1800 s was given for any category of behavior that was not initiated (or completed, i.e. reunion of the litter) within the 30-min observation period. Other behaviors recorded included general exploration (line crosses and rearings), self-grooming and eating/drinking.

Early postpartum females receiving bupivacaine infusion into the mPOA exhibited severe deficits in their maternal behavior. As shown in Figure 2, 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 (Figure 2). 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 (Table). 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 Table).

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 (Figure 2). 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 (Figure 2 and Table). Importantly, the additional mPOA group tested only in late postpartum similarly showed increased maternal behavior toward pups following bupivacaine compared to saline infusions (Figure 3 and Table), 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 (Table). 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 ​(Figures22 and ​and3,3, Table). 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 ​(Figures22 and ​and3).3). 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).

Remarkably, all bupivacaine-treated late postpartum females retrieved and grouped pups into the nest, and engaged in more licking and nest building relative to controls during the 30-min maternal behavior test. The present results provide strong evidence for the facilitatory effect of mPOA inactivation on late postpartum maternal behavior, as both repeated and single mPOA inactivation groups showed a notable increase in maternal behavior. To our knowledge, this is the first study to demonstrate that the mPOA is differentially engaged during early vs late postpartum in the regulation of maternal responsiveness. For instance, inactivation of the mPOA did not inhibit maternal behavior in late postpartum, suggesting that with the progression of the postpartum period, maternal responsiveness is progressively less dependent on mPOA activity. Not only do the present results demonstrate that mPOA is not necessary for the expression of maternal behavior in late postpartum, the finding that mPOA inactivation facilitated late postpartum maternal behavior suggests that the regulation of maternal responsiveness is more at this point distributed in the maternal circuitry such that additional network components are recruited with maternal experience. An important implication of this is that mPOA may play an active role in suppressing the ability of other brain structures to inappropriately influence maternal behavior in late postpartum.

Bupivacaine inactivation has been successfully used in behavioral studies to induce effective temporary neural inhibition of discrete brain areas [8, 16, 21, 37, 85, 108]. The effective spread of neural inactivation (i.e. the distance over which there is a physiological effect) of the structurally similar amide-linked anesthetic lidocaine has been well characterized both in cortical and subcortical structures using electrophysiological and other indices of neural activity [5, 50, 82, 90, 91, 102]. Accordingly, the 1.0μL infusion volume used in the present experiments likely produced a spherical area of functional inactivation with an approximate radius of 500-620 μm from the tip of the injector [5, 102], which approximately corresponds to the volume of the mPOA. Given (1) the central location of infusion sites within the mPOA, (2) the limited diffusion of bupivacaine, and (3) the robust effect of bupivacaine on maternal behavior with little intergroup variability, it is highly likely that the vast majority of the physiological effects of bupivacaine injections in the majority of our females remained primarily within, but not beyond the mPOA. Nevertheless, considering the distribution of the mPOA infusion sites in the rostro-caudal axis, in some limited cases, bupivacaine might have diffused rostrally to the caudal aspect of the diagonal band of Broca, and in others, caudally to the most rostral parts of the anterior hypothalamus and anterior paraventricular nucleus of the hypothalamus. However, bupivacaine infusions into the above-mentioned sites, at both early and late postpartum stages, had no effect on the maternal behavior of postpartum females (anatomical controls). In agreement with these findings, previous studies have shown that discrete permanent bilateral lesions of either the anterior hypothalamic area [26], the paraventricular nucleus of the hypothalamus [64], or the ventromedial hypothalamus [30] of early postpartum females did not affect the expression of maternal behavior.