Neonatal paw pricking alters adolescent behavior in a sex-dependent manner and sucrose partially remediates the effects

Abstract

Neonatal exposure to noxious stimuli such as repeated heel lances can cause behavior changes. In the NICU sucrose given prior to procedures attenuates the immediate behavioral response to noxious stimuli but may not ameliorate the long-term consequences, and treatment with 24% sucrose can brain structure and behavior in adult rodents. We used a rat model to determine whether paw pricks during the neonatal period alter social interaction and/or paw withdrawal thresholds (PWT) in adolescence, and if 7% sucrose mitigates these effects. One male and one female pup per litter was assigned to each of six experimental groups (no paw prick (control), 1 paw prick (1PP), or 2PP, ± sucrose). Hind paws were pricked once or twice each day between postnatal day (P)3 and P10. Social behavior and PWT were tested in adolescence using the modified social interaction test and von Frey filaments, respectively. Social behavior was altered in the 2PP group; total time interacting was lower in 2PP rats, primarily due to less time sniffing a play partner. Sucrose did not mitigate effects of paw prick but trended to alter social behaviors in males; it decreased time in contact but increased social motivation (movement toward a play partner). PWTs were higher in 2PP animals, this was not altered by sucrose. Thus, rat pups exposed to paw pricks in the neonatal period have some altered behaviors in adolescence. The nature of the behavioral changes is sex-dependent, but sucrose did not mitigate these changes.

INTRODUCTION

It has long been known that early life experiences can affect brain development and lead to changes in structure and function (Krech et al., 1960; Wiesel and Hubel, 1963), and that these changes may be sex-dependent (Camp et al., 1984). Experiences that can alter brain development include exposure to nociceptive, presumably painful, stimuli (Bell, 2018). Infants in the neonatal intensive care unit (NICU) endure frequent invasive procedures, the most common being heel lance for blood draws (Barker and Rutter, 1995; Bueno et al., 2023; Yamada et al., 2023), i.e., a rapid pinprick to the heel to collect blood. Frequent heel lances can cause repetitive pain/discomfort and/or tissue injury. The latter may lead to prolonged local inflammation, but both may affect development of neural pathways and alter behavior later in life (Cook et al., 2023; Grunau, 2013; Walker, 2019).

Pain is a complex phenomenon with multiple aspects that involve many different parts of the brain (e.g., (Xie et al., 2009). In neonates, pain modulation is immature. Pain pathways have immature afferent inputs and integrated cortical connections, as well as immature inhibitory connections (Fitzgerald and Beggs, 2001). Exposing neonates to pain during this critical time may alter the normal development of pain pathways and behavior (Anand et al., 1999; Fitzgerald and Beggs, 2001). Such changes have been reported in somatosensory and pain processing systems, including medial prefrontal cortex, and include changes in regional volumes as well as connectivity among regions (Chang et al., 2022; Cook et al., 2023; Huang et al., 2019; Schwaller and Fitzgerald, 2014). Nociceptive stimuli also trigger a spinal reflex, which causes rapid withdrawal from the stimulus.

Human studies suggest that pain exposure during the neonatal period, a critical period of plasticity, leads to a long-term alteration in pain sensitivity as well as in cognitive and motor outcomes. Long-term alterations in pain responsivity, such as lower pain thresholds to heat and higher somatization scores in ex-NICU school-aged children have been described (Grunau et al., 1994; Hermann et al., 2006), and pain in the neonatal period has been associated with worsening internalizing behaviors as toddlers (Vinall et al., 2013).

Effects of neonatal exposure to noxious (presumably painful) stimuli have also been studied in animal models. The long-term effects of paw prick on brain structure, basal pain sensitivity, and social and emotional behavior have been reported in rodents. Neonatal rodents given multiple daily paw pricks demonstrated alterations in brain white and grey matter volumes (Tremblay et al., 2017), as well as changes in anxiety-like behaviors, social discrimination, memory, and alcohol preference in adulthood (Anand et al., 1999; Page et al., 2005). Male, but not female, rats that underwent plantar incision showed hypersensitivity to heat, cold, and to mechanical stimulation (i.e., the von Frey filament test) as well as abnormalities in social behavior in adulthood (Burke and Trang, 2017). Both male and female rats given multiple paw pricks showed hypersensitivity to mechanical stimulation in the neonatal period (van den Hoogen et al., 2019, 2018). Interestingly, paw pricked animals exhibit hyposensitivity to mechanical stimulation in adulthood, an extended time of hypersensitivity following re-injury during adulthood, and enhanced electrophysiological responses of dorsal horn neurons indicating long-term effects of the neonatal insult (Knaepen et al., 2013; van den Hoogen et al., 2020, 2018).

In the NICU, 2–21% of infants receive analgesia during invasive procedures (Carbajal et al., 2008; Simons et al., 2003). Sucrose, commonly used in the NICU as an analgesic, has been shown to immediately alleviate pain-like behaviors when given during certain procedures such as heel lance (Stevens et al., 2016). In male rats, the efficacy of sucrose (7.5% w/v) is age-dependent, with maximal effect from postnatal day (P)3 until P10 (Anseloni et al., 2002). Higher concentrations of sucrose (24% w/v) protect against the sensitivity to mechanical and/or thermal stimuli in adulthood following piercing of the paw (inserting a needle through the paw) in the neonatal period (Nuseir et al., 2017), but may be harmful. Multiple brain regions were reported to be smaller in mice given repeated treatment with 24% sucrose (Tremblay et al., 2017). And it was recently reported that the high concentration of sucrose was not effective at ameliorating effects of four paw piercings per day for 14 days on sensitivity to thermal stimuli in mid- or late-adolescence, or on motor behaviors and a learning and memory task in late adolescence, and in fact sucrose-treated rats often showed worse performance on the tasks suggesting some long-term harm (Nuseir et al., 2024). At this time, it is not clear if the harmful effects of sucrose are concentration dependent.

There are clear effects of neonatal exposure to noxious stimuli on late-adolescent and adult behaviors, and inconsistent evidence on whether sucrose is effective in mitigating outcomes or not, but the early adolescent period has not been well-studied. Both social behaviors and response to the von Frey filament test differ between adolescence and adulthood. Adolescent rats typically exhibit more play fighting than adults whereas adults show more social investigation (sniffing) behavior (Mooney and Varlinskaya, 2011a; Vanderschuren et al., 1997; Varlinskaya and Spear, 2008, 2002); and paw withdrawal thresholds increase across the juvenile period (postnatal day (P)7 – P21; (Ling et al., 2023)) but are reported to be lower in adolescent or young adults compared with older animals (Muralidharan et al., 2020; Nuseir et al., 2024). There are also sex differences reported for social behaviors (e.g., (Auger and Olesen, 2009; Mooney and Varlinskaya, 2011a) and for the von Frey test (e.g., (Tsao et al., 2023)). Because the behavioral profiles may differ between sexes and across age, it is possible that neonatal insult has different effects on adolescent behavior than those reported by others for neonatal or adult animals.

In this study, we test the hypothesis that repeated neonatal exposure to a mild paw pricking insult alters social behavior and paw withdrawal thresholds in early adolescence and examined the effectiveness of low concentration (7.5%) sucrose in improving behavioral outcomes and paw withdrawal thresholds. Because the somatosensory and limbic systems are implicated in pain and both are important for social behavior, we also tested rats on a gap crossing test that uses the somatosensory system (Lee et al., 2009) but does not use the limbic system.

METHODS

Animals and experimental design

Timed Pregnant Long-Evans rats (Envigo, Frederick MD) arrived on gestational day 14–18. Following birth litters were culled to 10 with equal numbers of males and females as best as possible. Two males and two females per litter was assigned to each of three exposure groups: a single paw prick each day given in the same hind paw (left or right) at the same time each day (0900; 1PP), two paw pricks each day in the right hind paw ~four h apart (0900 & 1300; 2PP), control animals (CON) that were handled once per day. All treatments occurred from postnatal day (P) 3 through P10, inclusive. From the two rats per sex per group, one was untreated and the other was given sucrose (see below). Thus, all animals of one sex in an experimental group came from a different litter and each litter was represented in more than one experimental group. A total of 17 litters was used for this experiment and the number of animals per group is shown in Table 1.

TABLE 1:

Number of rats per group

GROUP	MALES	FEMALES
CON	11	9
CON-Suc	12	11
1PP	6	7
1PP-Suc	7	6
2PP	7	7
2PP-Suc	7	6

1PP – one daily paw prick; 2PP – two daily paw pricks; CON – control; Suc – sucrose

Animals were housed in the AAALAC-accredited animal facility at the University of Maryland, Baltimore. The facility is temperature- (22°C) and humidity-controlled (40–45%) and is kept on a 12/12-hr light/dark cycle (lights on at 0700). All procedures were performed in accordance with the approval of the Institutional Animal Care and Use Committee (IACUC) at the University of Maryland, Baltimore and were within the guidelines for animal care established by the National Institutes of Health.

On postnatal day (P)3 pups were tattooed on one paw for identification using AIMS ATS-3 system (Animal Identification and Marketing Systems, Inc., Hornell NY). If the subject was not assigned to a paw prick group, a front paw was tattooed and numbing cream (7.5% benzocaine, source: Orajel^®, Church and Dwight, Inc.) was applied prior to tattoo. Animals in paw pricked groups were tattooed on a hind paw with no numbing cream. Because the needle was inserted 1 mm, as for subsequent paw prick experiences, the tattoo was considered equivalent to a paw prick for P3. Pups were separated from the dam during paw pricking but were kept with littermates in a cage on a heat pad. The dam was returned to the cage once exposure / treatment was complete for the litter. Rats were weaned on P21 and housed in groups of two or three same sex littermates. Body weight was measured for all animals on P3, P10, and P28.

Exposure

The dam was removed from the cage prior to beginning exposure / treatment of the litter and returned once all pups had been treated. Paw pricked (PP) animals had a 27-gauge needle inserted to a depth of 1 mm into the same hind paw either once (1PP) or twice (2PP; ~4h apart) per day between P3 and P10, inclusive, with the tattoo experience being the first paw prick for all rats. If bleeding did not stop from the pressure of walking, pressure from cotton swab was applied before returning pups to the dam. Paws were observed daily for signs of inflammation.

Handled animals were picked up, briefly inverted supine to identify the tattoo, and placed back on their paws.

For animals given sucrose, 10 μl of a sucrose solution (7.5% w/v in water) was administered orally during handling or immediately prior to each paw prick between P3 and P10 (as described (Anseloni et al., 2002)).

Behavior

Animals underwent behavior testing as follows: modified social interaction test on P28 or P29, gap crossing test on P31 or P32, von Frey test between P33 and P35. Rats always had at least 24 h between tests.

Modified social interaction test

Animals were tested for social behavior during early adolescence (P28 or P29) using the modified social interaction test (Mooney and Varlinskaya, 2011), using a Plexiglass box, 30 cm long × 24 cm wide × 20 cm high (with a clear partition in the middle containing a semicircular hole (7 cm wide × 5 cm high) that allowed animals to cross between compartments. Tests were videotaped under dim white lighting (i.e., by lamps rather than overhead lights) for later scoring.

Experimental animals were marked with Sharpie^™ for identification, isolated for 20 minutes, then habituated alone to the interaction chamber for 10 minutes. Distance traveled in the box during the 10-minute habituation was recorded using the AnyMaze^™ system. After 10 min, an unmarked, non-habituated play partner was introduced to the chamber and the test was videotaped for another 10 minutes. Play partners were the unmanipulated offspring from another litter and were matched for age, sex, and weight (± 10 g).

Videotapes were manually scored for time the experimental animal spent in four individual behaviors by an observer blinded to experimental group. A subset of tapes was scored by a second observer to insure reproducibility. Outcome measures were time spent in four social behaviors: sniffing (sniffing in close proximity and active exploration of play partner), chasing (experimental animal actively pursues play partner), play fighting (experimental animal pins play partner or attacks nape of neck), and contact (bodies are in contact aside from sniffing or play fighting). Time play fighting and chasing are considered “active interaction” and sniffing and contact are combined into “passive interaction”. Total time in the four individual behaviors was also added and reported as total social interaction time, and each of the four behaviors is also presented as percent of time interacting.

Social motivation

Social motivation was calculated as the number of crossovers (movement through the partition from one side of the box to the other) to the partner – the number of crossovers away from the partner)/(the total number of crosses) × 100 (e.g., (Mooney and Varlinskaya, 2011a; Varlinskaya and Spear, 2008).

Gap crossing

Animals underwent the gap crossing test (Lee et al., 2009) on P31 or P32 as done previously (Waddell et al., 2016; Wellmann et al., 2015; Wellmann and Mooney, 2015) to test function of the somatosensory system (Lee et al., 2009). Briefly, rats were placed in a brightly lit (overhead room white lights on) white box on a platform 60 cm long × 15 cm wide and ~30 cm above the floor. Opposite the platform end was a solid, dark box (Supplementary Figure 1). Animals had two trials (up to 120 s each) to cross the distance (gap) between the end of the platform and the box. With each successful trial the distance between the platform and box was increased 1 cm. The longest distance successfully crossed was recorded.

von Frey filament test

Rats underwent the von Frey filament test of mechanical sensitivity beginning on P33 or P34. Animals were habituated to sitting on a mesh platform in a transparent testing chamber (20 cm long × 20 cm wide × 14 cm high) for 30 minutes on the day prior to testing. On the day of testing, 24h later on P34 or P35, von Frey filaments (Ugo Basile, Camerio, Italy) were inserted through the mesh and applied to a hind paw until a paw withdrawal response was elicited. Paw withdrawal response was defined as abrupt lifting or licking of the paw.

von Frey filaments bend with a known force (0.6 – 26 g) when applied to a paw. Starting with the 2g filament, 20 trials were performed on the pricked paw. A filament of known force was manually applied to the middle of the plantar surface of the paw and pressure was applied such that the filament bent. The up-down method dictates what the next filament in the test will be; a lack of response (no paw withdrawal) to the chosen filament is followed by a filament that requires more force to bend whereas a positive response is followed by a filament that requires less force to bend. The Dixon formula incorporating all 20 trials was then used to calculate the overall final force (Dixon, 1980).

$$\frac{\sum }{N}+\frac{d}{N}(A+C)$$

Where ∑ is the sum of all force values, N is the number of trials, d is log(force_max – force_min). C is number of consecutive trials with no response. To determine A and C, calculate the sum of trials without a response – sum of trials with a response, then use Table 2 in (Dixon, 1965) to determine the paw withdrawal threshold (PWT). An example is shown in Supplementary Figure 2.

Statistics

Data were used to generate means and standard deviations (SD) for each group. Repeated measures analysis of variance (RM ANOVA) was used to analyze body weights. Behavior data were initially analyzed using three-way ANOVA with sex, exposure (±paw prick), and treatment (±sucrose) as the factors. For most outcomes, ANOVA identified that sex showed significance or trend to interact with other factors, therefore data were also analyzed separately for each sex using two-way ANOVA and reported herein. Post-hoc analyses were done using the Bonferroni test. Alpha was set at ≤0.05, and a trend was identified if 0.05<p≤0.10. Grubb’s test was used to detect outliers and, where identified, ANVOAs were run without the outlier as noted in the Results. Sigma Plot (Systat Software Inc., San Jose CA) and SPSS software (IBM, Armonk, NY) were used for analyses.

RESULTS

Data

Grubb’s test identified outliers for most of the dependent measures as noted below, the exceptions being body weights, gap crossing, and social motivation.

Body weights

For both males and females, RM ANOVA identified the expected increase in body weight with age (F_2,44=1565.741, p<0.001, and F_2,44=5671.599, p<0.001, respectively; Figure 1). All groups gain weight across all ages (all p’s <0.001). In males, there was also a between-subjects effect of exposure (F_2,45=3.469, p=0.040); there was a trend for 2PP males to be heavier than CON (p=0.083) or 1PP (p=0.074); this was significant at P10 (p=0.012) (Supplemental Figure 3). Treatment with sucrose did not affect body weights.

Figure 1.

Body weights. Body weights were taken at the beginning and end of the exposure period, on postnatal day (P)3 and P10, and at the beginning of behavior testing on P28. Both males (TOP) and females (BOTTOM) showed significant increases in body weight across age. In males, the animals exposed to two daily paw pricks were heavier at P10 than control animals or those that received one paw prick (also see Supplementary Figure 3). Treatment with sucrose did not affect body weight at any age. Data are shown as mean and standard deviation, dots show individual data points. N = 6 – 12 per group (see Table 1). CON, control; Suc, treated with sucrose; 1PP one paw prick per day; 2PP, two paw pricks per day. ^& significant effect of age. * significant difference between groups at P10 as described in text.

Locomotor Activity

Distance traveled during the 10-minute habituation in the social behavior box was recorded. One female in the 1PP group (without sucrose) was identified as an outlier and removed from analysis. There were no effects of exposure or treatment on this measure (Figure 2).

Figure 2.

Locomotor Activity. The distance traveled by the experimental animal during a 10 min habituation to the social interaction box was recorded. No effects of exposure or treatment were identified. Data are shown as mean and standard deviation, dots show individual data points. N = 6 – 12 per group (see Table 1). CON, control; Suc, treated with sucrose; 1PP one paw prick per day; 2PP, two paw pricks per day.

Modified Social Interaction Test

For the social interaction test, one female (1PP no sucrose) was identified as an outlier for both total time interacting and time sniffing, one female (2PP no sucrose) was an outlier for time spent in contact, one female (CON no sucrose) was an outlier for time spent chasing, and one female (CON + sucrose) was an outlier for time play fighting.

Males showed a trend for an effect of exposure on total time spent interacting (F_2,45=2.615, p=0.084; Figure 3A), where 2PP rats spent less time interacting than CON (p=0.093). After separating the social interaction into individual components, the two-way ANOVA identified a significant effect of exposure on time spent sniffing (F_2,45=4.845, p=0.012; Figure 3C): this was significantly lower in 2PP males than 1PP (p=0.028) or CON (p=0.027). There was a trend for an effect of treatment on time spent in contact (F_1,45=3.682, p=0.061; Figure 3E) where animals given sucrose spend less time in contact than untreated rats. There were no effects of exposure or treatment on time spent chasing or play fighting (Figure 3G & 3I). Graphs showing data collapsed across treatment groups are included as Supplementary Figure 4, a graph showing time in contact collapsed across exposure is included as Supplementary Figure 5.

Figure 3.

Modified social interaction test. Total time spent interacting (A, B) and time in each of four social behaviors is shown (C – J). In males (LEFT) exposure to two daily paw pricks (2PP) decreased total time interacting (A) and time spent sniffing (C), and treatment with sucrose showed a trend to decrease time in contact (E). In females (RIGHT), total time interacting (B), time sniffing (D), and time chasing (H) were all lower in 2PP rats. Data are shown as mean and standard deviation, dots show individual data points. Graphs showing data collapsed across groups are shown in Supplementary Figure 4. N = 6 – 12 per group (see Table 1). CON, control; Suc, treated with sucrose; 1PP one paw prick per day; 2PP, two paw pricks per day. * significantly different to CON, (*) a trend to be different to CON, # significantly different to 1PP, (@) trend for effect of treatment.

In females, the total time spent interacting showed a significant effect of exposure (F_2,40=4.200, p=0.022), where 2PP rats interacted less than 1PP (p=0.035) and showed a trend to interact less than CON (p=0.065; Figure 3B). Treatment with sucrose did not show a main effect or an interaction. Individual components were also altered. Exposure significantly affected time spent sniffing (F_2,40=7.915, p=0.001) and chasing (F_2,40=5.339, p=0.009). The time spent sniffing was significantly lower in 2PP females than CON (p=0.011) or 1PP (p=0.002; Figure 3D). 2PP females spent significantly less time chasing than 1PP (p=0.007; Figure 3H). Time spent in contact or play fighting was not significantly different among the groups (Figures 3F & 3J). There were no significant main effects of treatment or interactions between exposure and treatment. Graphs showing data collapsed across treatment groups are included as Supplementary Figure 4.

Because total time interacting was different among groups, percent time spent in each of the behaviors was also examined (Figure 4). Main effects of exposure were identified in males for sniffing (F_2,45=7.268, p=0.002) and play fighting (F_2,45=6.213, p=0.004) and in females for sniffing (F_2,37=7.481, p=0.002) and contact (F_2,37=4.820, p=0.014). Treatment with sucrose did not affect percent time in individual behaviors.

Figure 4.

Percent time in social behaviors. The percent time spent in each behavior was calculated. In males (LEFT), percent time sniffing (dark blue) was lower and percent time play fighting (light green) was higher in the 2PP group compared with CON or 1PP. In females, percent time sniffing (dark blue) was lower and percent time in contact (light blue) was higher in 2PP females than CON or 1PP. Green colors show “active” behaviors as described in the text; percent time in these is higher in 2PP males than CON or 1PP males. No differences were seen in females. N = 6 – 11 per group (see Table 1). CON, control; Suc, treated with sucrose; 1PP one paw prick per day; 2PP, two paw pricks per day. * significantly different to CON, # significantly different to 1PP.

CON males spent around 65% of their time sniffing the play partner, 12.5% in contact, ~7% chasing, and the final 15% play fighting (Figure 4). 1PP males spent were not significantly different to CON. In contrast, 2PP males spent significantly less time sniffing (53%) than CON (p=0.026) or 1PP (p=0.002) and more time play fighting (265%) than CON (p=0.031) or 1PP rats (p=0.005).

CON females also spent most of their time (~66%) sniffing the play partner, 12% time in contact, 8% chasing, and 14% play fighting (Figure 4). 1PP females were not different to CON, whereas 2PP females spent significantly less time sniffing (54%) than CON (p=0.033) or 1PP (p=0.002) and more time in contact (18%) than 1PP (p=0.011).

When time play fighting and chasing are combined into “active interaction” and sniffing and contact are combined into “passive interaction”, these are different by exposure group in males (F 2,45=6.551, p=0.003), but not females. 2PP males spent approximately 33% of their interaction time in active behaviors, significantly more than CON (22%, p=0.037) or 1PP rats (16%, p=0.003).

Social Motivation

In males, social motivation was unaffected by exposure but showed a trend to differ by treatment (F_1,45=3.744, p=0.059); rats that received sucrose showed more social motivation than those that were untreated (Figure 5), i.e., they were more likely to cross through the partition from one side of box to the other towards the play partner. In females, neither exposure nor treatment affected social motivation (Figure 5). A graph showing male data collapsed across exposure groups is included as Supplementary Figure 5.

Figure 5.

Social motivation. Social motivation is calculated from the number of crossings towards and away from the play partner as described in the text. Males showed a trend for a higher index in rats treated with sucrose compared with untreated (Data are shown collapsed across treatment group in Supplementary Figure 5). No differences were found among the female groups. Data are shown as mean and standard deviation, dots show individual data points. N = 6 – 12 per group (see Table 1). CON, control; Suc, treated with sucrose; 1PP one paw prick per day; 2PP, two paw pricks per day. (@) trend for effect of treatment.

Gap Crossing

There were no effects of exposure, treatment, or interactions on the gap crossing test (Figure 6).

Figure 6.

Gap crossing. The longest distance crossed between the end of a platform and a dark box was recorded. No effects of exposure or treatment were identified. Data are shown as mean and standard deviation, dots show individual data points. N = 6 – 12 per group (see Table 1). CON, control; Suc, treated with sucrose; 1PP one paw prick per day; 2PP, two paw pricks per day.

von Frey Test

In males, there was a significant effect of exposure on PWTs (F_2,45=6.844, p=0.003); these were significantly higher in 2PP than CON (p=0.002) and showed a trend to be higher in 2PP than 1PP (p=0.061; Figure 7).

Figure 7.

Paw withdrawal thresholds. The von Frey filament test was used to determine paw withdrawal thresholds (PWTs). PWTs were higher in males exposed to 2 paw pricks per day (2PP) compared to control (CON) and trended to be higher than in animals exposed to 1 paw prick per day (1PP). In females, PWTs were higher in 2PP compared with CON and 1PP. Data are shown as mean and standard deviation, dots show individual data points. N = 6 – 12 per group (see Table 1). CON, control; Suc, treated with sucrose; 1PP one paw prick per day; 2PP, two paw pricks per day. * significantly different to CON, # significantly different to 1PP, (#) a trend to be different to 1PP.

A similar outcome was seen in females. There was a significant effect of exposure on PWTs (F_2,40=9.174, p<0.001); these were higher for 2PP animals than CON (p=0.001) or 1PP (p=0.002; Figure 7).

Treatment with sucrose did not mitigate the effect of exposure in males or females. Graphs showing data collapsed across treatment groups are included as Supplementary Figure 6.

DISCUSSION

This study examined whether sucrose administration (“treatment”) protected against the effects of paw pricking in the neonatal period (“exposure”) on adolescent behaviors; it is assumed that the exposure caused transient pain somewhat akin to a heel stick in humans and that one or two paw pricks per day is likely to be a mild insult given that infants in the NICU experience as many as 8 – 15 heel sticks per day (Britto et al., 2014). Overall, we found that exposure to two daily paw pricks between P3 and P10 affected social behavior and altered paw withdrawal thresholds, consistent with findings reported by others (Anand et al., 1999; Page et al., 2005; Ranger et al., 2019), whereas a single daily paw prick showed little effect. Treatment with sucrose showed subtle effects on social behaviors in males but did not mitigate effects of paw prick on any of the behaviors examined in males or females.

Because animals in the 2PP group spent less time interacting in the social interaction test, we examined the architecture of their behavior by assessing percent time in each of the four categories of social behavior. For both males and females, most (60–70%) of the social behavior was sniffing of the play partner (also termed social investigation by others, e.g., (Varlinskaya and Spear, 2002) for CON and 1PP rats. This decreased to ~50% of time in the 2PP rats, both male and female. In males, there was an increase in percent time play fighting which in turn increased time in active forms of social interaction (sniffing + play fighting). In females, the decreased present time sniffing was primarily compensated by time in passive contact, although a non-significant increase in time spent play fighting meant that percent time in active forms of interaction did not significantly differ among the groups. Together, these findings suggest that 2PP (but not 1PP) changed how the animals interacted with a play partner. Given that all groups showed similar levels of activity during their time exploring the box prior to introduction of the play partner, it seems unlikely that the differences in the type of interaction between the groups is simply due to different activity levels. Impairment in social interactions, particularly in young animals, is linked to lasting effects on cognitive behaviors, in part by decreasing inhibitory circuits in the prefrontal cortex (Bijlsma et al., 2023, 2022). As such, the disrupted social behavior architecture in adolescence seen here may indicate animals at risk for other behavioral changes in later life.

The neonatal manipulations applied in this study altered mechanical sensitivity; animals given two daily paw pricks in the neonatal period required more force to elicit a response to the von Frey filaments in adolescence. This higher force suggests lower sensitivity to the mechanical stimulation of the von Frey filaments and is similar to a recent study that reported adult hyposensitivity after multiple neonatal paw pricks (van den Hoogen et al., 2020). And the lack of effect of one daily paw prick on PWTs in adolescence is somewhat similar to (Knaepen et al., 2013) where four paw pricks were given each day, but only one per day in each paw. In contrast, another study reports increased sensitivity; chronic paw pricking in neonatal rats (once per day for 7 days) resulted in lower PWTs in the von Frey test in adult males, but not females (Page et al., 2013). The lower sensitivity in the paw that received two insults per day could be caused by local damage to sensory receptors in the paw.

Previous studies in rodents assessed the effects of repeated paw pricks and sucrose administration in the neonatal period on adult brain and behavior (Nuseir et al., 2024; Ranger et al., 2019; Tremblay et al., 2017). Repeated sucrose administration was associated with smaller brain volumes in adult mice, and animals treated with sucrose prior to paw pricking had the smallest brain volumes, notably in hippocampus and cerebellum (Tremblay et al., 2017). Behaviorally, paw pricked mice showed deficits in short-term memory, and this was improved in the animals that also received sucrose, however, control mice that received 24% sucrose showed similar performance to the paw pricked mice suggesting some harm. A recent study in rats concurs, reporting that repeated administration of 24% sucrose not only failed to protect against behavioral changes caused by repeated neonatal paw piercing but also altered behavior in control animals (Nuseir et al., 2024). Together, these findings suggest that repeated sucrose treatment may be harmful. Our results do not necessarily agree, however, we used a milder insult (one or two paw pricks per day) and gave a much lower concentration of sucrose (7.5% as used by others (Anseloni et al., 2002) vs 24%). Given the widespread use of sucrose in the NICU, further investigation is warranted with particular attention being paid to concentration and amount.

Limitations of this study include that we used timed pregnant rats which may have caused some stress in early gestation, we only tested a single concentration of sucrose, and we only tested animal behavior in early adolescence. Additionally, we only scored social behavior for the experimental rat, whereas the test requires interaction with a conspecific. It is standard practice that only the experimental animal is scored in this specific test (e.g. ((Mooney and Varlinskaya, 2011a; Varlinskaya et al., 2020; Varlinskaya and Spear, 2008). That said, in a previous study (Waddell et al., 2016) we did score the play fighting behavior of both rats because the measured ultrasonic vocalizations were from both animals; in that study, the pattern of interaction was the same for both experimental animal and the play partner.

CONCLUSION

Experience of a chronic mild insult in the neonatal period alters paw withdrawal thresholds and some aspects of social behavior in insult- and sex-dependent ways. Oral sucrose did not mitigate most of the outcomes measured, however, unlike other studies it had only subtle effects on behavior, and these were only seen in males. Further research is still needed to ascertain the long-term benefits and risks of pretreated paw pricks and the need for consistent use of analgesia for paw prick in the neonatal period.

HIGHLIGHTS.

• Two daily paw pricks, but not one, alters social behavior and increases mechanical sensitivity in adolescent rats, and sucrose does not improve these outcomes.

• Paw prick exposure alters the architecture of social behavior in sex-dependent manner.

• Changes in social behavior occured in the absence of changes in motor behaviors.