Components of the Transport Response: postural regulation
Pups carried by an experimenter to mimic maternal oral carrying showed a characteristic compact posture, with flexion of the extremities as they grew (Figure 1A,B). While the hindlimbs of the postnatal day (PND) 10 pups were relaxed, those of a PND 14 pup carried by an experimenter were ventroflexed from the level of the pelvis (Figure 1B). To quantify the degree of body compaction and pliability during the Transport Response, we compared the posture during the Transport Response with the posture of the totally atonic condition during picking-up, by general anesthesia of the same age of pups. The lower back was significantly more curved in the normal PND 14 pups than that of the PND 14 anesthetized pups (t(20.651) = 9.7963, p < 0.01, Figure 1D). Consequently, the entire body of a normal PND 14 pup maintained its compact position during the Transport Response, as measured by the nose to the toe length during the Transport Response compared with the nose-toe length under general anesthesia (t(21.957) = –5.7821, p < 0.01, Figure 1E). The muscular tone required to maintain this compact posture was apparent when the posture of a normal PND 14 pup (Figure 1B) was compared with that of a pup of the same age under general anesthesia (Figure 1C).
When the carried pups were placed back on a tube in a supine position, the backs of the pups conformed to the curvature of the tube without resistance; this was similar to the behavior of the anesthetized pups (t(21.784) = –1.9323, p = 0.066, Figure 1F, G; note that the hindlimbs of the left pup are maintained in a ventroflexed position in Figure 1G). These data indicated that despite the postural maintenance, the trunk was not all rigid during the Transport Response. In particular, the neck and the upper-body trunk were flexible and pliable. Moreover, the eyes of the PND 16 pups became progressively narrowed after manual carrying (Freidman test: Chi-square = 17.4286, df = 2, p < 0.01, Figure 1H). This data was consistent with the previous anecdotal observations that the transported pups often kept their eyes close (see pictures in [2, 5]).
Components of the Transport Response: apparent analgesia
Another possible feature of the Transport Response was the behavioral insensitivity to pain, as suggested through our daily handling of the mouse pups for procedures such as biopsy for DNA genotyping. To directly measure the pups’ apparent pain threshold, the tails of the pups were pinched by a clip with a known pinching force under three different conditions: manual carrying (Carrying), gentle touching between the experimenter’s fingers on a paper towel (Touching; see Manual touching in Additional file 1 as an actual maneuver) or under undisturbed conditions (UD; Figure 2A).
Additional file 1:A movie of an actual Touching maneuver and a pup’s response for the tail pinch during Touching.(MOV 2 MB)
Pinching the tail using an artery clip with a 160 g pinching force elicited nociceptive responses in most of the pups in the UD and Touching groups. The pups responded to the pinch by squealing, rushing forward, or by turning back toward the tail and biting the clip (Figure 2A; Manual touching in Additional file 1). In contrast, only 5 of the 17 pups (29.4%) that were manually carried showed nociceptive responses. The other 10 pups (58.8%) exhibited no postural changes (Manual carrying in Additional file 2), and the remaining 2 pups (11.8%) further flexed their hind limbs (p < 0.01, Fisher’s exact test, Figure 2A).
Additional file 2:A movie of an actual Carrying maneuver and a pup’s response for the tail pinch during Carrying.(MOV 2 MB)
This apparent analgesic state during the Transport Response demonstrated a ceiling effect, as the nociceptive response to the tail pinch with a clip of 200 g pinching force did not differ between the groups (p = 1 in UD vs. Touching, p = 0.074 in Touching vs. Carrying, p = 0.3 in UD vs. Carrying, Fisher’s exact test, p- value adjustment by Holm’s method, Figure 2B), suggesting an increase in the pain threshold.
To determine whether opioid signaling was involved in the apparent analgesia during the Transport Response, we first examined pups from the μ-opioid receptor knockout (Oprm–/–) mouse line for their responses to manual carrying and the tail clip. Oprm–/– pups developed with no gross differences from the other genotypes in terms of appearance and weight gain (F(2, 15.2) = 0.67, p = 0.52 at PND 10, F(2, 17.15) = 1.88, p = 0.18 at PND 13). Mutant pups at PND 13 showed a normal Transport Response, including inhibition of voluntary movement (F(2, 41.71) = 0.64, p = 0.53, Figure 2C) and compact postural adaptation (Figure 2D) when compared with their wild-type littermates. We carried pups of each genotype to induce the Transport Response and then pinched their tails with a clip of 160 g pinching force. There were no significant differences in nociceptive response types between the genotypes (No response: 47.06% of Oprm–/–, p = 0.31 in Oprm+/+ vs. Oprm+/–, p = 0.10 in Oprm+/– vs. Oprm–/–, p = 0.89 in Oprm+/+ vs. Oprm–/–, Fisher’s exact test, p- value adjustment by Holm’s method, Figure 2E), suggesting that the apparent analgesic effect during the Transport Response persisted under the lack of the μ-opioid receptor. To further confirm the above finding, we also utilized the opioid receptor antagonist naloxone (Nx) [7]. The nociceptive responses to the tail pinch during the Transport Response were not significantly different between the C57BL/6 PND 13 pups injected with either Nx or saline (Sal) (p = 1 in no injection (NI) vs. Sal, p = 0.24 in Sal vs. Nx, p = 0.24 in NI vs. Nx, Fisher’s exact test, p- value adjustment by Holm’s method, Figure 2E). These results suggested that the expression of the nociceptive response is suppressed during the Transport Response via a non-opioidergic mechanism.
Ontogeny of the calming response
To address whether the Transport Response is a filial-specific response, the ontogeny of the various components of the Transport Response were examined in detail using mouse pups. First, we compared the inter-beat interval (IBI in Figure 3A, the inverse of heart rate) during two different conditions using pups aged from PND 4 to PND 14. One condition was “Carrying”, during which the pups were gently held between the tips of the experimenter’s first two fingers and picked up in the air. The other condition was “Holding”, in which the pups were only held by the experimenter’s fingers. The amount of difference (%) in the inter-beat interval during the two conditions (Carrying minus Holding) showed no significant difference from PND 4 to PND 8 (p = 0.65, Figure 3A). From PND 9 onward, the inter-beat interval increased rapidly at the start of Carrying, and its difference between Carrying and Holding became evident (F(5, 104) = 42.1, p < 0.001; Figure 3A). Next, we investigated the ontogeny of ultrasonic vocalization (USV) emissions during the Transport Response. The number of USV emissions was significantly lower in the Carrying condition from PND 4 to PND 9 compared with the Holding or UD (F(5, 298) = 4.65, p < 0.001; Figure 3B).
We also examined the ontogeny of the immobilization response (Figure 3C). Of the total 547 mice, 271 male and 276 female pups between PND 0–20 were manually carried and held still in the air by the experimenter’s fingers for 15 s or until the pup started exhibiting anti-gravitational voluntary movements. At the end of the first postnatal week, cessation of the initial immobilization was almost always followed by struggling (rapid turning and shaking of the limbs and tail). Moreover, the struggling pups never returned to the immobilized state again. The mean time period of immobility was approximately 8–9 s during the first postnatal week. In the first few days after birth, the pups did not clearly inhibit their voluntary movement during manual carrying and would often start to move their extremities choppily. During the second postnatal week, the pups were immobile for longer periods of time. This immobilization effect peaked at PND 10, was gradually reduced after PND 15 and diminished by PND 20; the immobilization effect was not observed afterward (PND 35, 56 in Figure 3C). In C57BL/6 laboratory mice, this immobilization response could not be extended or evoked by continuous daily handling by the experimenter (t(20.338) = 0.1365, p = 0.89, Figure 1D), although in adult rats such daily handling by experimenters could induce an immobilization response [5]. These data indicate that each component of the Transport Response, namely the cardiac deceleration, reduction of ultrasonic vocalization and immobility response, had a clear and separate time window within the preweaning period of mouse pups, and that did not observe in the adulthood.
Ontogeny of postural regulation
Next, we examined the ontogeny of the characteristic postural regulation described above, focusing on the hindlimb, forelimb and tail. Most of the pups immobilized with a symmetrical hindlimb posture; an asymmetrical posture was observed in only 8% of the 517 pups (data not shown). The symmetrical hindlimb postures were classified into three categories (Figure 4A): extension (magenta triangles), half flexion (green squares), and full flexion (blue filled squares). During the first postnatal week, most of the pups maintained the extended hindlimb posture. During PND 8–13, the pups would halfway flex their hindlimbs; alternatively, they would first fully flex their hindlimbs but then gradually let them down and extend them during the immobilization period. From PND 14 onward, most of the pups maintained their hindlimbs in the fully flexed position during the immobilization period. These observations suggested that the postnatal period could be roughly subdivided into three groups according to the hindlimb posture, the first postnatal week, PND 8–13 and PND 14 onward. To confirm this, we compared the hindlimb postural types at PND 6, 10 and 14 (Figure 4B). There were significant differences in the composition ration of postural types among the three PNDs (p < 0.001, Fisher’s exact test, p- value adjustment by Holm’s method, Figure 4B). The major type of the hindlimb posture was the extended posture at PND 6, the half flextion at PND 10 and the full flexion at PND 14.
The forelimb posture during immobilization was analyzed by classifying it into three categories (Figure 4C): symmetric extension, symmetric flexion and rare asymmetric positioning (data not shown). The developmental course of forelimb flexion was essentially similar to that of hindlimb flexion; the pups extended their forelimbs until PND 7 and then gradually maintained their forelimbs fully flexed throughout the immobilization period.
The tail posture was classified into two positions during postnatal development (Figure 4D). First, the tail was extended forward at PND 0 and PND 1. Next, the tail was extended downward from PND 2 onward.
Maternal retrieval and the concomitant pups’ responses
To examine the developmental course of mother-pup interactions in a more naturalistic setting, we used an experimental setup designated as the “maternal rescue of pups in a cup” [6] (Figure 5A). All of the pups were successfully rescued by their mothers until the pups were PND 16 (Figure 5B). As the pups grew older, they maintained their limbs in a more flexed position, as shown in Figure 4 and the right panel of Figure 5A. From PND 17 onward, the pups were able to climb up and get out of the cup independently (Figure 5B). The maternal attempts to orally retrieve the pup, support the pup’s escape by pulling the pup over the edge of the cup from the outside, or stay attentively near the pup until the pup got out remained until PND 19 or PND 20. This was the time period when almost all of the pups could independently get out of the cup within a few minutes. In this assay, no pup was left inside of the cup for more than 9 min after the start of the test session. These observations suggested that in this experimental setup, the development of the pups’ motor ability was a major determinant in diminishing the maternal retrieval rate. As shown in Figure 4, the attenuation of the immobilization response progressed in parallel with the pups’ increased motor ability. The expression of the Transport Response in the mouse pups coincided well with the pup’s development when they needed maternal rescue for their transport.