Virgin females will strongly prefer a pup-associated chamber after being exposed to pups for prolonged periods of time (24 h/day for 21 days), rivaling the robust pup-associated chamber preference seen in parturient females during the early postpartum period, when maternal motivation is strongest [9
]. Fewer virgin females (33%) exposed to pups for relatively brief periods of time (1 h/day for 7 days) preferred the pup-associated chamber, compared to virgin females given prolonged pup-exposure (42%) and, particularly, early postpartum females (50%), revealing a subtle stepwise relationship between length of pup-exposure and strength of maternal motivation. No females lacking full exposure to pups and receiving only distal (auditory and olfactory) pup-cues preferred the pup-associated chamber. This work reveals, for the first time, that even brief exposure to pups is sufficient to promote maternal motivation in a strong subpopulation of virgin females and that this maternal motivation can be enhanced to postpartum levels by increasing the length of exposure to pups.
Importantly, our prolonged pup-exposure regimen is the closest published replication of the natural behavioral patterns of the parturient female during the first eight days of the postpartum period, when the strength of her maternal motivation peaks [9
]. First, the maternal female expresses vigorous, consistent maternal behaviors toward her pups during the early postpartum period [1
]. In the present study, virgin females given prolonged pup-exposure expressed maternal behavior that improved significantly over time and was maximally established by CPP testing. That nearly half of these virgin females preferred the pup-associated chamber at CPP test suggests that maximizing females’ maternal responsivity may increase her attribution of incentive value to pups.
Second, the parturient female spends the majority of her time with her pups during the early postpartum period [2
]. In the present study, females were only deprived of pups for relatively brief, biologically relevant periods (2 h) prior to pup-conditioning [10
]. Our data demonstrates, for the first time, that dramatic pup-deprivation is not necessary for virgin females to strongly prefer a pup-associated chamber [17
]. As even parturient females will not prefer a pup-associated chamber following brief pup-deprivations in late postpartum, when increasing amounts of time are spent away from pups and maternal care declines [24
], maternal motivation in virgin females can actually surpass that of the naturally parturient female in late postpartum.
Together, our data demonstrate that, by mimicking the natural behavioral patterns seen in early postpartum, virgin females can express strong maternal motivation that rivals that of the parturient female. We conclude that maternal motivation is not established exclusively by the complex neuroendocrine states of gestation, parturition, and postpartum, or by the physiological, behavioral, and polysensory experiences of parity.
Additionally, many virgin females given brief pup-exposure, which never expressed fully maternal behavior, expressed strong preference for the pup-associated chamber. These non-maternal females expressed a markedly reduced locomotor response during the first pup-conditioning session compared to the fully maternal females given prolonged pup-exposure and expressed mostly freezing and/or avoidant behavior toward pups. By the last conditioning session, brief pup-exposure females were increasingly tolerant of pup-initiated physical interaction and no longer expressed freezing/avoidant behaviors, and locomotor response to pups matched that of prolonged pup-exposure females. We posit that the emergence of these inconsistent, maternal-like behaviors represents a transitional state of maternal responsivity during which pups shift from an aversive to a neutral or slightly attractive stimulus to the female. Females given brief pup-exposure did not express locomotor habituation across pup-conditioning sessions, as did prolonged pup-exposure females, indicating that the females’ perception of the pup stimulus differed dramatically across these four exposures [38
]. We posit that the initial tolerance for pup-initiated contact (e.g. rooting) represents an emerging transition in the motivational state of the female that is potentiated by the changing incentive-motivational status of pups and ultimately expressed as a drive to initiate contact and interact with pups.
Notably, the motivational transition observed in virgin females given brief pup-exposure was not
observed in females given only distal exposure to a limited range of pup-related stimuli. While both groups of females were completely non-maternal, the olfactory and auditory cues provided by distal pup-exposure were insufficient to initiate a similar shift in the incentive-motivational value of pups to the distally exposed female. We posit that the limited time spent in the pup-associated chamber by these distally exposed females reflects their sub-threshold attraction to the novel odors and vocalizations of pups enclosed within the mesh bag [39
Together, these findings suggest that pup-associated chamber preference in the virgin female requires a basal level of physical interaction between the female and pups, which distal pup-exposure prevents. Pups confined inside a mesh bag were prevented from initiating physical contact with these non-maternal virgin females, e.g. rooting at females’ ventrum and nipples, whereas pup-initiated contact occurred in all other groups of females, even those given brief pup-exposure, with contact reciprocated and supplemented by maternally responsive females. Prior work by Fleming and colleagues [7
] suggests that maternal motivation is contingent upon direct female-pup interactions: bar-pressing for access to pups declines dramatically if pups cannot be retrieved upon delivery [7
] and postpartum females will not prefer a pup-associated chamber if prevented from interacting with pups in that chamber [25
]. Our data importantly extend these findings by revealing that physical interactions can be initiated by the maternally responsive female (i.e. during prolonged pup-exposure) or by pups (i.e. during brief pup-exposure) and must include a complete range of polysensory input in order to promote maternal motivation in both virgin and parturient females.
It is also important to note that the expression maternal behavior, an important correlate to our appetitive-motivational CPP measures, remained remarkably consistent between virgin females preferring the pup-associated or empty chamber. At no point did any female’s maternal behavior in the homecage or behavioral patterns recorded during pup-conditioning predict or correlate with her preference for the pup-associated or empty chamber; this dissociation was evident across our novel, prolonged pup-exposure regimen, including the 15 days prior to CPP, the six days of CPP (), and during each pup-conditioning session (data not shown; see for exemplar). Strongly motivated, ‘active’ maternal behaviors (retrieval and nest-building) [12
], which were expressed consistently after prolonged pup-exposure and were emerging after brief pup-exposure, did not predict any females’ preference for the pup-associated chamber. Thus, even well-established, motivated caretaking behaviors do not predict a virgin females’ maternal motivation and that behavior and motivation remain distinguishable components of the maternal state, in accord with others’ work [7
As females are the primary providers of parental care in rodents and most other small mammals, many factors may mediate the shift of the incentive-motivational value of pups from aversive to attractive, initiate the emergence of a drive to interact with pups in the non-maternal female, and promote the expression of a range of maternal behaviors that further strengthen maternal motivation in the female [1
]. These factors include neurobiological, environmental, and experiential factors, which act in concert to regulate maternal responsivity in the female rat. The present work uniquely addresses how two of these critical factors, postpartum status and exposure to pups, affect maternal motivation in the female rat, a model that is uniquely evolutionarily primed to respond to changes in the incentive-motivational value of pups.
It has been proposed that females’ motivated behavior may be influenced by dynamic differences in levels of gonadal hormones and lactogenic peptides in cycling and postpartum females. While preference for a pup-associated chamber is strongest in gonadally intact (versus ovariectomized) females [17
], this preference is likely attributable to hormonally facilitated female-pup interactions [1
]. In the present study, pup-exposed virgin and postpartum females expressed comparable preference for the pup-associated chamber, despite dramatic neuroendocrine differences [41
]. Furthermore, CPP is relatively insensitive to differences across the estrus cycle [30
], suggesting that any perseverative changes in gonadal hormone levels induced by pups [41
] do not influence CPP, either. Mere exposure to pups can up-regulate long-form prolactin receptor mRNA in both virgin and postpartum females [46
]; circulating prolactin levels are substantially higher in females expressing pup-induced maternal behavior than in non-maternal females exposed briefly to pups [41
]. Prolactin has been proposed as a likely factor promoting maternal motivation [2
], and it is possible that pup-exposed virgin females that prefer the pup-associated chamber express higher levels of centrally acting prolactin.
The mesolimbic dopamine (DA) system also contributes importantly to the expression of highly motivated maternal behaviors [6
] and, as emerging evidence suggests, the attribution of incentive-motivational value to pups in the parturient female [6
]. Dopamine release into the nucleus accumbens increases markedly during bouts of licking/grooming [57
], while blocking dopamine transmission impairs both licking and motivated pup retrieval [2
]. Recent work indicates that dopamine antagonism blocks the acquisition of pup-associated chamber preference in postpartum females [17
], whereas stimulation of D1 receptors can promote it [2
]. Thus, we posit that individual differences in D1-receptor responsivity may modulate the incentive value attributed to pups by the maternally responsive female. This hypothesis was supported by a recent study [33
] revealing that accumbal dopamine is chronically elevated in maternally responsive virgin females; we posit that this persistent increase in mesolimbic dopamine may contribute to strong maternal motivation in our virgin females given prolonged pup-exposure. Also, as reunion with pups transiently increases accumbal dopamine in the maternal female [57
], we further posit that such increases in dopamine may also enhance the incentive-motivational value of pups during each pup-conditioning session, facilitating the shift in incentive-motivational value of pups in our virgin females given brief pup-exposure.
It is possible that pup-induced elevations in dopaminergic transmission may have enhanced associative learning in all pup-exposed females [59
], thereby preventing most females given only distal pup-exposure from strongly preferring a conditioning chamber. However, pup-associated stimuli (e.g. odors, vocalizations) and novel stimuli, such as the mesh bag containing pups, elicit arousal in females and depends upon mesolimbic dopamine activity [60
]. Thus, whether in the form of vigorous caretaking behaviors (maternal females) or as mild anxiety (non-maternal females) [1
], we believe that attention and learning of conditioned associations (e.g. CPP) was similarly enhanced in all females in this study [62
Ultimately, the present study reveals, for the first time, striking levels of maternal motivation in the virgin female rat. This motivation is contingent upon physical interactions between the female and pups, including full exposure to a broad spectrum of polysensory stimuli associated with pups. Furthermore, maternal motivation can be enhanced by systematically extending the length of time that females are exposed to pups. Early work revealed that consistent, repeated exposures to a stimulus can increase the attractiveness of that stimulus [63
]; we posit that the systematic increases in maternal motivation seen in the present study may be attributable to the systematic increases in pup-exposure length and, as a consequence, the quality of maternal behavior. This critical shift in incentive-motivational value of pups from aversive to attractive may be attributable to endocrinological, neurochemical and behavioral changes emerging during initial pup-exposures. Importantly, this motivational shift contributes critically to pups’ survival and health by promoting motivated caregiving and, as revealed in the present study, can occur equally in both virgin and parturient female rats.