Early adversity alters neurodevelopmental milestones in male and female rat pups and motor cortex morphology and gait in juveniles.

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Learn more: PMC Disclaimer | PMC Copyright Notice Neurobiol Stress . 2026 Apr 3;42:100814. doi: 10.1016/j.ynstr.2026.100814 Search in PMC Search in PubMed View in NLM Catalog Add to search Early adversity alters neurodevelopmental milestones in male and female rat pups and motor cortex morphology and gait in juveniles Annie Phan Annie Phan a Integrated Program in Neuroscience, McGill University, Montreal, Quebec, H4H 1R3, Canada b Douglas Institute in Mental Health, Montreal, Quebec, H4H 1R3, Canada Find articles by Annie Phan a, b, 1 , Michael Ru Michael Ru b Douglas Institute in Mental Health, Montreal, Quebec, H4H 1R3, Canada Find articles by Michael Ru b, 1 , Brianna Latremouille Brianna Latremouille b Douglas Institute in Mental Health, Montreal, Quebec, H4H 1R3, Canada Find articles by Brianna Latremouille b , Hong Long Hong Long b Douglas Institute in Mental Health, Montreal, Quebec, H4H 1R3, Canada Find articles by Hong Long b , Claire-Dominique Walker Claire-Dominique Walker b Douglas Institute in Mental Health, Montreal, Quebec, H4H 1R3, Canada c Dept of Psychiatry, McGill University, Montreal, Quebec, H4H 1R3, Canada Find articles by Claire-Dominique Walker b, c, ⁎ Author information Article notes Copyright and License information a Integrated Program in Neuroscience, McGill University, Montreal, Quebec, H4H 1R3, Canada b Douglas Institute in Mental Health, Montreal, Quebec, H4H 1R3, Canada c Dept of Psychiatry, McGill University, Montreal, Quebec, H4H 1R3, Canada ⁎ Corresponding author. Dept of Psychiatry & Dept of Anatomy and Cell Biology McGill University Douglas Institute Research Center Montreal, QC, H4H 1R3, Canada. [email protected] 1 Contributed equally to the work. Received 2025 Nov 24; Revised 2026 Mar 19; Accepted 2026 Apr 2; Collection date 2026 Jun. © 2026 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). PMC Copyright notice PMCID: PMC13091161  PMID: 42005377 Abstract Early life stress (ELS) and adversity influences neurodevelopmental trajectories and affects maturation of brain circuits, network connectivity and epigenetic regulation, leading to increased vulnerability to adult psychopathology. ELS timing targets sensitive periods for neural plasticity, which are identified by the ability to reach physical and sensorimotor developmental milestones. In particular, motor development, being highly dependent on coordinated activity across several brain regions, is especially susceptible to environmental challenges, although detailed modifications are unclear. Here we used the limited bedding (LB) paradigm in rats to examine the effects of ELS longitudinally on physical and sensorimotor milestones as well as morphological changes in the motor cortex (M1) and motor behavior in male and female juveniles. Neonates from the LB litters displayed delayed eye opening and reduced weight gain compared to NB pups. Reaching sensory milestones such as cliff avoidance, ear twitch and auditory startle responses was delayed by LB exposure, similarly to surface righting reflex, grasping reflex and orienting behavior that were significantly impaired in both male and female LB pups. In juvenile LB rats, locomotion in the openfield was reduced and hind limb gait was impaired. There were no bedding or sex effects on skilled walking. Morphological analysis of Layer V motor cortex neurons (M1) in juveniles revealed that LB reduced dendritic length and increased spine density, but in females only. These data show that LB exposure during the neonatal period delays the reaching of multiple developmental milestones in several domains associated with the maturation of motor function and coordination. Keywords: Neonatal rat, Limited bedding, Early adversity, Developmental milestones, Gait, Primary motor cortex Highlights • Exposure to early limited bedding (LB) conditions delays the reaching of several physical, sensory and reflex milestones equally in both male and female neonatal rats. • Motor development, orienting and gait are significantly affected by early adversity in juvenile LB offspring in association with morphological changes in M1 motor cortex neurons. • Impaired sensorimotor development induced by exposure to early adversity might represent a sensitive marker of neurodevelopmental consequences of stress in juvenile rats. 1. Introduction Exposure to early life stress (ELS) is recognized to cause alterations in neurobiological, emotional and cognitive processes both in the short- and long-term in humans and rodent models ( Smith and Pollak, 2020 ). In humans, ELS increases the risk for psychopathology including major depression and anxiety from early adolescence ( Burkholder et al., 2016 ; Lemoult et al., 2020 ) into adulthood ( VanTieghem and Tottenham, 2018 ; Spadoni et al., 2022 ). ELS modifies brain structure and function ( Krugers and Joëls, 2014 ; Teicher et al., 2016 ) as well as behavioral regulation at a time of development that is characterized by heightened neural plasticity, increased proliferation, synaptogenesis, and myelination ( Nelson and Gabard-Durnam, 2020 ), all processes that are critically influenced by environmental conditions. For instance, negative early life experiences including poor quality or decreased quantity of care, emotional or physical abuse, and reduced access to resources have been associated with altered brain maturation ( Honeycutt et al., 2020 ), delayed sexual maturation ( Manzano et al., 2019 ), and impairment in the stress response ( Van Bodegom et al., 2017 ) and emotional circuitry ( Opendak et al., 2020 ; Guadagno et al., 2021 ). Early life stress in humans can take different forms, such as parental loss, abuse, or poverty, which can lead to different outcomes ( Juen et al., 2024 ). To better understand the effects of ELS on brain networks development and their associations with behavioral outcomes, animal models have been developed, including maternal separation, scarcity-adversity and limited bedding and nesting (LBN) models ( Walker et al., 2017 ) to allow for precise control of genetic and environmental factors and testing of mechanistic hypotheses ( Takahashi and Shelton, 2025 ). The maternal separation model remains popular in the exploration of the effects of restricting maternal care on later life outcomes ( Gildawie et al., 2020 ). However, this paradigm can produce inconsistent and contradictory results due to considerable heterogeneity between maternal separation studies and variation in maternal compensatory behavior upon reunion with her pups ( Wang et al., 2020 ). The limited bedding (LB) paradigm and scarcity-adversity variant have been used as a naturalistic animal model to simulate a lack of resources and impoverished environment affecting developmental outcomes of the offspring ( Ivy et al., 2008 ; Walker et al., 2017 ; Deckers et al., 2024 ; Grillo Balboa et al., 2025 ). In particular, the impoverished cage environment, where the dam is unable to create a satisfactory nest, usually leads to fragmented and unpredictable maternal care ( Gallo et al., 2019 ; Glynn and Baram, 2019 ; Porras et al., 2025 ). Important changes in early environmental and social (maternal) factors may also considerably affect the achievement of developmental physical, sensory and motor reflex milestones, a hallmark of general brain maturation processes. In children suffering from neurodevelopmental disorders such as autism spectrum disorder (ASD) or intellectual disability, delays in motor, social and sensory processes are observed in the first few years of life such that they miss key developmental milestones ( Meredith, 2015 ). In rodents, delays in reaching specific developmental milestones have been documented after postnatal maternal separation ( Demaestri et al., 2020 ), scarcity-adversity ( Lauraine et al., 2024 ) and limited bedding ( Demaestri et al., 2020 , Pardo et al., 2023 ) models of ELS as well as gestational pharmacological models using valproic acid administration ( Ruhela et al., 2019 ) or methadone ( Grecco et al., 2021 ). Several of these studies have used common developmental milestones such as eye opening, surface righting reflex, grasp reflex to assess development at specific times during the postnatal period. However, this snap shot of developmental processes lacks the “visualization” of the precise timeline of reaching the specific milestones in an entire population of animals from different early conditions. Interestingly, while sexual differences exist in the long-term consequences of ELS (Guadagno et al. 2017; Goodwill et al., 2019 ; White et al., 2020 ), potentially due to differences in hormonal regulation and brain maturation rates ( Bath et al., 2016 ), delays in reaching neurodevelopmental milestones after ELS appear to be similar between male and female rodents ( Demaestri et al., 2020 ; Pardo et al., 2023 ; Lauraine et al., 2024 ). However, because milestones measures have been discrete and not continuous, it is still unclear whether the reaching of specific neurodevelopmental milestones is differentially modified in male and female offspring and whether specific domains are preferentially affected. Among the categories of neurodevelopmental milestones routinely examined after ELS, changes in motor reflexes and function appear to be predominant. For instance, the surface righting reflex appears within the first week of life and constitutes a complex motor function mediated by vestibular pathways, spinal interneurons and motor neurons ( Altman and Sudarshan ). In contrast, grasp reflexes may be more reliant on the maturation of the corticospinal tract and sensory afferents to the spinal cord than on motor cortex neurons as maturation process of motor control after birth involves the disappearance of primitive reflexes ( Laliberte et al., 2022 ). In the context of ELS, previous studies have found that LB or maternal separation had a significant effect on the development of reflexes in both rats ( Lauraine et al., 2024 ) and mice ( Demaestri et al., 2020 ). A delayed arrival of theses milestones after LB or maternal separation was associated with a higher expression of the immature protein doublecortin (dcx) mRNA in the motor cortex of neonatal mice ( Demaestri et al., 2020 ), suggesting maturational delays in these neurons. Motor cortex layer V pyramidal neurons (LVPNs) regulate the voluntary control of motor output and fine motor coordination in adults and the morphology of LVPNs is a strong indicator of their functional capabilities ( Sakai, 2020 ). During development, changes in neural architecture are essential for optimized function to create more robust and efficient neural communication network. Pruning is when underutilized synapses are eliminated ( Sakai, 2020 ), and when this process is impaired, for instance after ELS, it could lay the foundation for variable deficits in motor coordination such as gait and skilled walking. In these studies, we used a continuous longitudinal assessment of multiple neurodevelopmental milestones across the first 2 weeks of age between individual control and LB-exposed male and female rat pups to identify the differential timing of milestone achievement maturation and the potential sex-related differences in attaining these milestones. Since behavioral analyses of potential motor deficits can also provide valuable macroscopic insight into the impact of ELS on motor development, we examined locomotor activity in the openfield, gait as an essential component of motor coordination and skilled walking in juvenile rats. Finally, in order to determine whether behavioral changes were associated with impaired motor neuron properties, we examined morphological changes induced by LB in LVPN of the primary motor cortex in juveniles. Our results demonstrate that exposure to LB conditions delays the reaching of several physical, sensory and reflex milestones equally in both male and female neonatal rats and affects orienting and gait in juveniles together with increased spine density in M1 LVPV neurons, particularly in female juveniles. Thus, impaired sensorimotor development induced by exposure to early adversity might represent a sensitive marker of neurodevelopmental consequences of stress in neonatal and juvenile rats. 2. Methods 2.1. Animals Pregnant Sprague-Dawley female rats (C.River, Kingston colony, USA) were received in our animal facility on gestation day 14 and maintained under controlled conditions of temperature (22-24 °C) and humidity (70-80%). Animals were kept on a 12h:12h light-dark schedule (lights on at 08:00h) and fed rat chow and water ad libitum . The day of birth was considered PND0 and litters were culled to 10 pups per mother on PND1 with an equal sex ratio if possible. Six experimental cohorts were run for milestones at 3-4 weeks interval for a total of 20 litters and 86 male and 66 female pups. Separate cohorts of animals were used for gait analysis (4 litters, 24 male and 14 female pups) and determination of skilled walking (8 litters, 46 male and 33 female pups). All experimental procedures were approved by the University Animal Care Committee at McGill University in accordance with the guidelines of the Canadian Council on Animal Care. 2.2. Limited bedding conditions as an early life stress The LB paradigm ( Walker et al., 2017 ) was used between PND1-9 according to a protocol adapted from Baram and colleagues ( Molet et al., 2014 ) with cage changes on PND4 and PND10. On PND1, mothers and their litters were randomly assigned to the limited bedding (LB) or normal bedding (NB) condition. LB mothers and their litters were placed on a wire mesh platform 2.5 cm above the cage floor. Approximately 1.5 cm of bedding was added below the platform to cover the cage floor. The dams were given one-half of one paper towel for nesting material. The NB cages received a 2.5 cm layer of woodchips and one-half of one paper towel. On PND10, all LB mothers/litters were returned to NB conditions. All litters were kept with their biological mother for the duration of the experiments. The weights of all pups were recorded daily while tested for developmental milestones. After PND15, litters were left undisturbed unless for cage changes until weaning on PND21, behavioral testing between PND25-28 and tissue collection on PND28. 2.3. Maternal behavior recording Maternal behavior was video recorded for 24hrs between PND5-6 across all scored experimental cohorts (10 NB and 10 LB litters). The following maternal behaviors were recorded: active/passive nursing, pup grooming, pup retrieving, self-grooming, sleeping, eating, drinking and wandering. Video recordings started shortly after daily manipulation for milestone testing. Behavior was scored every minute during four 1-hr sessions (2 during the light and 2 during the dark phase) as previously described {McLaughlin, 2016 #60}. The fragmentation of overall behavior, i.e., the degree to which maternal behaviour occurs in many short bouts ( Baram et al., 2012 ), during each observation period was determined using a behavioral consistency score in which a score of “1” was given when behavior changed from one epoch (minute) to the next, and “0” when there was no change in the type of behavior exhibited ( Ivy et al., 2008 ). 2.4. Testing for developmental milestones Rat pups (4 males and 4 females/litter) were randomly assigned to evaluation of milestones between PND2-15 and handled daily in specific tests. Pups from each litter were individually identified by india ink tatoo in either the forepaw or hindpaw. Two pups from each litter were not tested in any of the developmental milestones tests and served as controls for the handling associated with testing. Testing occurred in the morning between 9 and 10:30AM. When tested, the whole litter was removed from the mother and kept on a warming pad in the testing room adjacent to the housing room. Daily separation from the mother averaged 13.7 ± 0.37 min (Mean ±SEM, n = 108). We tested a total of 152 pups from 20 litters that were subdivided in either NB or LB conditions. Physical, sensory, and motor developmental characteristics were tested according to Heyser (2004) with recording of the day of achievement of each developmental milestone. The day of achievement is defined as the day at which the pup successfully performed the task for 2 consecutive days. On PND 1, pups were weighed, sexed, and identified by tattoos using india ink injected in the forepaw and/or hindpaw. Pups were weighed and tested daily between PND2 and 15-16 between 9 and 10AM. Testing for each day varied, as indicated in Fig. 1 . Fig. 1. Open in a new tab A: Diagram of experimental procedures illustrating milestone testing between PND1-15 in rat pups from normal bedding (NB) or limited bedding (LB) litters. LB was applied between PND1-10 and on PND10, all litters were returned to NB conditions. Behavioral testing (openfield, gait and skilled walking on the ladder rung test) was performed between PND26-29. Brain collection for motor neuron morphology by Golgi analyses was performed on PND29-30. B: Timeline of testing for the different milestones as a function of pup's age. Each modality (physical, sensory or reflex) is represented by a different color. Four males and four females per litter were individually tested for these milestones at each age between PND1-2 and PND15-16. 2.5. Physical milestones For physical development, the anogenital distance, nose to anus distance, lower incisor eruption, fur development, eye opening, pinnae detachment, and auditory canal opening were recorded. Anogenital distance was measured on PND1 and nose to anus distance, an index of pup growth, was measured on PND15 with a precision ruler. To probe for incisor eruption, forceps were used to inspect the pup's mouth and the age when the lower teeth broke the surface of the gum was recorded. Fur development was recorded at the age at which dorsal and ventral pigment and fur appeared. For eye opening, we attributed the milestone achievement when both eyes were open. The age at which both pinnae were detached completely from the cranium was recorded. Using forceps for better view of the auditory canal, the day at which the auditory canal was open and visible was recorded. For this variable, we attributed the milestone achievement when both auditory canals were open. 2.6. Sensory and reflex development milestones For sensory development, negative geotaxis, cliff avoidance, vibrissae placing response, ear twitch, and auditory startle response were examined. Reflexes such as surface righting reflex, grasping reflex, vertical screen test, and bar holding were recorded. For the negative geotaxis test, the pup is placed head down on an inclined board (30°) of 30 cm by 30 cm, covered with 16-mesh wire screen. The test begins with the hind limbs of the pup in the middle of the board. If the pup falls immediately (within 10 s), the test is repeated. Achieving the task requires that the pup turn around (180°) and climb up the board with their forelimb within 30 s. To test cliff aversion, the pup is first positioned at the edge of a stand (height 30 cm) with the digits of the forepaws and the snout hanging over the ledge. The time in seconds for the pup to turn and begin to crawl away from the edge is measured. If the pup falls immediately (within 10 s), the test is repeated. The pup must perform this task in less than 30 s and for two consecutive days. To test vibrissae placing response, the pup is held around the trunk and suspended in the air. A thin metal rod is used to make contact with the vibrissa. The reflex is observed when the pup raises its head and extends its forelimb to grasp the object upon contact. To test the ear twitch reflex, a cotton tip of an applicator is used with the ends pulled out and twisted to form a fine cotton filament. The pup is placed on a flat surface, and the filament is gently brushed against the tip of the ear. The reflex is observed when the pup flattens the ear against the side of the head. To test for auditory startle response, the pup is first brought to a different room, isolated from the rest of the litter. Then, a clicker is employed at a distance of 25 to 30 cm above the pup to test for the startle reflex. The reflex is present if the clicker elicits a slight jerk, kicking, and/or squirming reaction. For reflex development, surface righting reflex, grasping reflex, vertical screen test, and bar holding were tested. For surface righting reflex, the pup is placed on its back. The time to turn over and reach the position in which all four paws make contact with the surface is recorded. The pup must successfully complete this task within 1 s for 2 consecutive days. For grasping reflex, the pup is gently held around its trunk, and a thin metal rod is used to touch the forelimbs of the pup. The day at which the pup grasps the rod upon contact is recorded. To perform the vertical screen test, the pup is placed on a screen (50 cm × 22 cm, grid openings of 15 mm) in the horizontal position. Within the first 2 s of the test, the screen is turned to the vertical position, and the rat is observed for 5 s in this vertical position. To perform this task successfully, the pup must grip to the screen for the full 5 s. To set up the bar holding test, a cage with a thin metal bar hanging across is employed. The pup is positioned close to the metal bar, and the test begins when the pup holds the bar with its front paws, and the animal is released to hang only by its front paws. The pup must hold on to the bar for at least 10 s on two consecutive days. 2.7. Orienting motor behavior and locomotion in preweaning and juvenile rats To test for the extinguishing of the pivoting motor behavior, the pup (PND10-15) is placed on a plastic sheet in the center of a circle (13 cm in diameter). The length of time the pup takes to travel outside of the circle (with both forelimbs and hind limbs outside) is recorded. The pup must be able to travel beyond the boundary of the circle within 30 s for two consecutive days to reach criterion. General locomotor behavior was tested in an openfield arena (100x100 × 50cm) on PND26-27. Juvenile (PND26) male and female rats were placed in the arena for 5 min and their movement in the arena was videotaped. The openfield was divided into 3 sections: a center square section (center, 50cmx50cm), a shadow section (shadow, 100cmx18cm) along one of the walls of the openfield (constituted by the natural wall shadow) and a peripheral section (periphery, 100 × 100 cm) that included space around the other 3 walls of the openfield that were more brightly lit than the shadow part. This subdivision between lit and shadow periphery was introduced in order to provide a gradient of environmental conditions. Duration spent and distance were recorded for all 3 sections of the openfield. 2.8. Gait analysis and skilled walking in juvenile offspring Gait dynamics were evaluated in NB and LB offspring on PND27 (4 litters, 38 pups) using the DigiGait (Mouse Specifics, Inc.) imaging system. The DigiGait apparatus consists of a transparent treadmill belt enclosed in a polycarbonate compartment and a camera located below the belt to capture digital images of paw placement at 125 frames/sec. Rats were placed on the treadmill and ran at a velocity of 12 cm/s. The area of the underside of each paw relative to the area of the treadmill belt at each frame was used for spatial and temporal measurements. Recordings were trimmed to approximately 4-6 s of consecutive walking and manual adjustments such as contrast adjustment or artifact removal were performed prior to software processing. Main gait indices such as stride length, stride time, brake and propulsion times were analyzed using DigiGaitAnalysis 15 software. 2.9. Skilled walking ladder rung test Adapted from Metz and Whishaw (2002) , the ladder rung test evaluates skilled fore- and hind limb stepping, placing, and coordination. The ladder rung apparatus was constructed with Plexiglass walls and removable metal rungs (3 mm diameter) that are spaced 1 cm apart. The entire apparatus was elevated 30 cm above the ground. A video camera (GoPro Hero7) was angled ventrally so that all paws could be recorded simultaneously while the rat moved along the ladder apparatus. This camera was mounted on a skater dolly, which allowed the experimenter to manually follow the movement of the subject across the ladder. The shutter speed was set at 1/500 s. Two different ladder rung arrangements were used for the trials, the first being a regularly spaced arrangement with all rungs available, and the second being an irregular, novel arrangement where there was a maximum of 3 cm between rungs. The starting point was blocked after the rats were placed on the ladder and a dark box was placed at the end of the ladder. Each rat (PND28-29) underwent five trials: one initial training session where rats were left on the apparatus (regular rung) for 2 min to familiarize themselves with the environment, two regular, and two irregularly spaced rung trials. The video recordings were analyzed frame-by-frame and steps were scored based on the 7-category scoring system developed by Metz and Whishaw (2002) where a total miss = 0, a deep slip = 1 and a correct placement = 6. Only consecutive steps of each limb were analyzed and thus, the last step before the stop and the first step after the stop were excluded. When different errors occurred at the same time, the lowest of the scores was recorded. 2.10. Morphological analyses of motor cortex (M1) A subset of animals tested for milestones (males and females, NB and LB) were anesthetized on PND29-30 and perfused with ice-cold 0.9% saline-heparin (5USP units/mL heparin) for 5 min. Following perfusion, brains were kept in Golgi-Cox solution (1.04% K 2 Cr 2 O 7 , 1.04% HgCl 2 , and 0.83% K 2 CrO 4 diluted in distilled water, all reagents from Fisher Scientific, Fair Lawn, NJ) for 14 days in the dark at room temperature before being transferred in 30% sucrose at 4 °C for 2-7 days ( Guadagno et al., 2018 ). Coronal sections (200 μm) were cut using a vibratome (Leica, Concord, ON) and immersed into a 6% sucrose solution. Sections were placed serially onto 2% gelatin-coated slides, dried and stained with 100% ammonium hydroxide followed by fixative (Carestream GBX fixer, diluted 1:1 in distilled water, Sigma-Aldrich, St Louis, MO). Sections were dehydrated in serial alcohol rinses, cleared in xylene and coverslipped. Golgi-Cox stained pyramidal neurons in the primary motor cortex region (M1, layer V) were selected for analysis on the basis of morphological criteria described elsewhere ( Wang et al., 2020 ). Neurons were manually traced by an experimenter blind to sex and bedding condition, with the Neurolucida software (MicroBrightField, Williston, VT) using a Zeiss Imager M1 microscope with a 100× objective and a Hamamatsu camera. Digital images were captured with 20x and 100× objectives. A total of 3-4 neurons were analyzed per animal (6-8 male and female pups/bedding group). Branched structure analyses were performed in Neuroexplorer (MicroBrightField, Williston, VT) on reconstructed neurons to determine total dendritic length, number of branch points (nodes) and number of dendritic spines. To calculate spine density (spines/μm), the total number of spines was divided by the total dendritic length for each neuron. Sholl analysis was performed using Neurolucida (MicroBrightField, Williston, VT) with a cut off distance set at 220 μm from the soma. 2.11. Statistical analyses Behavioral endpoints for daily milestones were first analyzed with the Chi square test as a preliminary analysis for identifying the significant milestone endpoints. Then, a stepwise multiple regression model was computed for each significant endpoint to find their significant predictors. The examined predictors were body weight on PND1, body weight on the day before emergence of the milestone endpoint, sex, and bedding condition. Time of achieving milestones were compared between sex and bedding groups using 2-way ANOVA with and Tukey HSD post-hoc tests when appropriate. Openfield and freezing behavior data as well as morphological data were analyzed using 2-way ANOVA and Tukey HSD post-hoc tests when appropriate. Data for maternal behavior were analyzed with a repeated measures ANOVA (across light phases) with bedding and light as factors. Statistical significance was set at p < 0.05. 3. Results 3.1. Maternal behavior We scored nursing time, pup grooming and self grooming time as well as fragmentation in NB and LB mothers between PND5-6. All variables, except pup grooming, differed significantly as a function of the light/dark phase of the cycle (p < 0.001) ( Supplemental Table 1 ). Nursing time, pup grooming and fragmentation were not altered significantly by bedding condition. Self grooming displayed a significant effect of bedding (F(1,19) = 16.01, p = 0.0008), light phase (F(1,19) = 26.6, p < 0.0001) and a significant light by bedding interaction (F(1,19) = 12.84, p = 0.002). NB mothers exhibited a higher level of self grooming during the dark phase of the cycle compared to LB mothers (p < 0.001) and compared to the light phase (p < 0.001). 3.2. Effect of limited bedding conditions (LB) on developmental milestones Physical milestones: Some, but not all physical developmental characteristics were affected by the early change in bedding condition in either males or female pups. As depicted in Fig. 2 A, the distribution of eye opening (χ 2 (2) = 7.37 p = 0.025) and auditory canal opening (χ 2 (2) = 23.27, p < 0.001) was significantly different between NB and LB pups. In contrast, fur development was not significantly affected by bedding condition (χ 2 (3) = 5.54, p = 0.137). Two-way ANOVA showed that there was no sex effect or sex by bedding interaction for either eye opening, auditory canal opening or fur development. Body weight increase as a function of age was not significantly different between NB and LB pups in either sex ( Fig. 2 B), although there was a trend for LB pups to show reduced body weight in both males and females as previously reported ( Shupe and Clinton, 2021 ). Anogenital distance on PND1, timing of incisor eruption (onset at PND5-6: NB = 8 pups, LB = 3 pups), and pinnae detachment (onset at PND9: NB = 3 pups, LB = 1 pup, peak: NB=PND10, LB=PND11) were not different between sex or bedding groups ( Table 1 ). There was no sex effect or sex by bedding interactions for these variables. There was no sex or bedding effect on nose to anus distance taken on PND15 and expressed as a function of body weight at this age (males: NB = 0.273 ± 0.0065; LB = 0.286 ± 0.0034; females: NB = 0.280 ± 0.007; LB = 0.287 ± 0.042). Fig. 2. Open in a new tab Top: Frequency histograms (number of pups reaching milestones) of eye opening and fur development as a function of age in NB and LB rat pups. The distribution of eye opening was significantly delayed in LB pups (n = 44, males: n = 22; females: n = 22) compared to NB pups (n = 61, males: n = 31, females: n = 30) (χ 2 (2) = 7.37, p = 0.025). Fur development was not significantly different between NB (n = 56; males: n = 28; females: n = 28) and LB (n = 46; males: n = 23, females: n = 23) pups. Bottom : Evolution of body weight in male (left) and female (right) from NB (n = 7 litters) and LB (n = 7 litters) pups. Body weight is represented as the average weight of males and females in each litter (n = 4 males and females/litter). Although not significant, body weight tended to be reduced by LB in both sexes. Values represent the Mean±SEM. Table 1. Chi-square analysis and multiple stepwise regression model for predictor identification in the physical, sensory and reflex milestones for NB and LB rat pups. Milestone Endpoint Chi analysis Regression Model Summary Significant predictors Yates Chi df Yates p Adjusted R Square F Change Sig F Change Predictors t p Physical Fur Development 5.536 3 0.1365 0.411 4.334 0.040 bwpnd6 −6.977 0.000 bedding 2.554 0.012 bwpnd1 2.082 0.040 Pinnae Detachment 0.1411 2 0.9319 Incisor Eruption 2.81 4 0.7285 Auditory Canal Opening 23.27 2 <0.0001 0.1723 27.69 <0.0001 bedding 5.262 <0.0001 Eye Opening 7.38 2 0.025 0.0508 5.51 0.0208 bedding 2.347 0.0208 Sensory Negative Geotaxis 5.34 10 0.8035 Cliff Avoidance 17.99 9 0.0353 0.006 1.661 0.020 bedding 1.289 0.020 Vibrissa Placing Response 1.93 5 0.8581 Ear Twitch 23.61 3 <0.0001 0.235 35.026 <0.0001 bedding 5.918 0.000 Auditory Startle Response 22.52 3 <0.0001 0.357 13.210 <0.0001 bwpnd10 −5.784 0.000 bedding 3.635 0.000 Reflex Surface Righting Reflex 48.15 9 <0.0001 0.349 60.469 0.000 bedding 7.776 0.000 Grasping 75.64 6 <0.0001 0.447 4.383 0.039 bedding 8.174 0.000 bwpnd2 −2.094 0.039 Level Screen 14.74 5 0.0115 0.123 6.255 0.014 bwpnd3 −2.724 0.008 bedding 2.501 0.014 Bar Holding 4.65 7 0.7031 Orienting 23.87 7 0.0006 0.091 12.152 0.001 bedding 3.486 0.001 Open in a new tab Sensory development milestones: We tested five sensory modalities across developmental age and three of the tests for sensory modalities yielded significant bedding effect ( Table 1 ).As depicted in the frequency histograms in Fig. 3 A–D), cliff avoidance (χ 2 (9) = 17.99, p = 0.0353), ear twitch (χ 2 (3) = 23.61, p < 0.0001) and auditory startle responses (χ 2 (3) = 22.52, p < 0.0001) were significantly delayed in LB compared to NB pups. In contrast, vibrissa placing responses (p = 0.858) or the age at which criterion was achieved for negative geotaxis (p = 0.804) was not significantly altered by bedding conditions. Regression analysis using bedding and body weight 1-2 days prior to the age of onset of criterion reaching ( Table 1 ) demonstrated that bedding was a significant predictor for ear twitch (p < 0.001) and the auditory startle response (p < 0.001). Changes in this last sensory modality was also significantly predicted by body weight on PND10, 2 days prior to the onset of criterion in both groups ( Fig. 3 D). Two-way ANOVA found no significant effects of sex or sex by bedding interactions for any of the sensory modalities tested. Fig. 3. Open in a new tab Frequency histograms (number of pups reaching milestones) of sensory ( A-D ) and reflex ( E-H ) development as a function of age in NB and LB rat pups between PND3-16. P value for Chi square analyses is displayed for each test. Reaching milestone criterion for cliff avoidance (A), ear twitch (B) and auditory startle responses (D) was all significantly delayed by LB conditions while negative geotaxis (C) was not significantly affected by bedding conditions. In the reflex category, surface righting (E) and grasping (F) were greatly delayed by LB conditions (p < 0.0001). Reaching milestones for vertical holding in the level screen test (G) and orienting in a straight line (H) was significantly delayed in LB compared to NB pups (p < 0.05-0.001). Values represent the Mean±SEM of n = 40-64 NB (males: n = 20-32; females: n = 20-32) and n = 32-54 (males: n = 16-27; females: n = 16-27) LB pups. Reflex development milestones : Four out of the five reflex modalities tested across development ( Fig. 3 E–H) exhibited significant effects of bedding, i.e. surface righting reflex (χ 2 (9) = 48.15, p < 0.0001), grasping reflex (χ 2 (6) = 75.64, p < 0.0001), level screen test (χ 2 (5) = 14.74, p = 0.0115) and gait pivoting or orienting (χ 2 (7) = 23.87, p < 0.001; see below). Bar holding was not affected by bedding conditions (p = 0.703) ( Table 1 ). Regression analysis using bedding and body weight 1-2 days prior to the age of onset of criterion reaching ( Table 1 ) demonstrated that bedding was a significant predictor for surface righting reflex (p < 0.001), grasping (p < 0.0001), and level screen test (p = 0.014). Changes in grasping reflex and level screen performance was also significantly predicted by body weight 1-2 days prior to the onset of criterion in both groups). Similarly to sensory milestones, two-way ANOVA found no significant effects of sex or sex by bedding interactions for any of the reflex modalities tested. 3.3. Effect of limited bedding conditions on orienting behavior and locomotion in the openfield As a first evidence of the disappearance of the typical pivoting motor behavior of neonates, we examined the onset of orienting in a straight line in pups from NB and LB mothers. In both male and female pups, LB significantly increased the time to reach the straight line milestone compared to the NB condition ( Fig. 4 A). In the LB condition, 37 pups (out of 56) had not reach the criterion after PND15 while only 16 (out of 56) NB pups did not reach criterion before or on PND15. Frequency histogram ( Fig. 3 ) and regression analysis confirmed the significant effect of bedding (χ 2 (7) = 23.87, p = 0.0006) ( Table 1 ). In order to determine if this difference was caused by significant changes in general motor behavior or by more subtle changes in gait, we analyzed behaviors in the openfield and examined gait and fine motor skills on the ladder rung test (see below) in juveniles (PND28-29). In all 3 compartments of the openfield (center, shadow and periphery), the distance travelled was reduced in LB juveniles compared to NB juveniles and in both sexes ( Fig. 4 A–C,E). In the periphery, there was a significant effect of bedding on distance travelled (F (1, 198) = 16.33, p < 0.001), but no sex or bedding × sex interaction. In the shadow, LB juveniles also displayed less locomotion than NB pups, with a significant effect of sex (F (1, 198) = 8.647, p = 0.0037), bedding (F (1, 198) = 5.315, p = 0.022), but no bedding × sex interaction. The significant bedding effect reported for the “safer” portion of the openfield was also observed in the center of the openfield, where LB juveniles displayed significantly less locomotion than NB juveniles (bedding effect: F (1, 198) = 5.402, p = 0.0211). Fig. 4. Open in a new tab Left: Distance (mm) (A,C,E) and Right: time (B,D,F) spent in the various compartment of the modified openfield test in juvenile (PND 26-27) NB (male: n = 49; female: n = 43) and LB (male: n = 58; female: n = 58) offspring. As indicated by the drawings, parameters were analyzed for the center (top panels), periphery (middle panels) and shadow (bottom panels) portions of the openfield. Significant main effects of bedding in the two-way ANOVA analysis are represented on the graphs with ∗ (p < 0.05) and ∗∗∗ (p < 0.0001). Main effects of sex and interactions between sex and bedding are not represented (see results section). Values represent the Mean±SEM of 43-58 animals/group. Early unfavorable (LB) bedding conditions significantly reduced time spent in the periphery (F (1, 198) = 6.060, p = 0.0147), but increased time spent in the shadow (F (1, 198) = 6.377, p = 0.0123)( Fig. 4 D–F). There were no significant changes in the time spent in the center of the openfield ( Fig. 4 B). In order to determine whether changes in openfield behavior were caused by changes in anxiety, we tested a subset of animals in the elevated plus maze (EPM) as juveniles (PND27-28, n = 9-12 juveniles/group). No significant bedding effect was observed in the EPM at this age and for both male and female juveniles (data not shown). 3.4. Changes in gait and fine motor skills in juveniles after early LB conditions Gait analysis in juveniles revealed that several characteristics of the gait were significantly impaired by the early changes in bedding conditions and more specifically for the hind limbs ( Fig. 5 ), while there were no significant changes for the front limbs. Two way ANOVA of stride time revealed that bedding had a significant effect to reduce stride time for both left (F(1,34) = 4.34, p = 0.0447) and right (F(1,34) = 4.51, p = 0.041) hind limbs, but there were no sex effects or bedding × sex interactions for this variable ( Fig. 5 A). Two way ANOVA analyses of stride length showed a significant effect of bedding for both the left (F(1,34) = 4.50, p = 0.041) and the right (F(1,34), p = 0.0309) hind limb. A significant effect of sex was found for the right hind limb (F(1,34) = 4.46, p = 0.042), but no bedding × sex interaction was found for either left or right hind limb ( Fig. 5 B). Propulsion time measures the time required for the foot to leave the floor and brake time, the time necessary to bring the foot on the floor. Two way ANOVA of propulsion time showed that bedding significantly reduced propulsion time for both left (F(1,34) = 9.50, p = 0.004) and right (F(1,34) = 6.42, p = 0.016) limbs, but there was no effect of sex or significant interaction between bedding and sex ( Fig. 5 C). In contrast, brake time was not affected by either sex or bedding (not shown). Fig. 5. Open in a new tab Analysis of gait in NB and LB male and female juveniles (PND27-28) according to the schema on top of figure. A full stride is illustrated and decomposed into propulsion, braking and swing time. Hind limb data are illustrated for NB and LB male and female juveniles including stride time ( A ), stride length ( B ) and propulsion time ( C ). Two way ANOVA of stride time and propulsion time showed a significant main effect of bedding (p < 0.05) for both left and right hind limb variables with values for the LB being inferior to those of NB offspring. There was no effect of sex or interaction for either parameter. Two-way ANOVA analyses of stride length showed a significant effect of bedding for both the left and the right (p < 0.05) hind limb. A significant effect of sex was found for the right hind limb (p = 0.042), but no bedding × sex interaction was found for either left or right hind limb. Brake time was not affected by either sex or bedding. Values represent the Mean±SEM of n = 11-13 male and n = 6-8 female juveniles. Because juvenile gait was significantly modified by early bedding conditions, we determined whether this had consequences for fine motor skills on the ladder rung with both regular and irregular spacing between the rungs. Consecutive steps along the rung were scored according to a 7-category scoring system developed by Metz and Whishaw (2002) where the lower the score, the less correct the run is. The percentage (%) of correct steps for forelimbs and hind limbs is indicated in Fig. 6 for both regular and irregular ladder rungs. In the regular rung, two way ANOVA indicated a significant sex effect (F(1,75) = 6.28; p = 0.0143) for the hind limb, but no bedding or sex × bedding interaction. LB females had a higher hind limb correct score than their male counterparts (p = 0.0108). Similarly, in the irregular rung, we found a significant sex effect (F(1,72) = 4.81; p = 0.031), but no bedding or sex × bedding interaction. Females in the NB group had a higher percentage of correct steps in the hind limb compared to NB males (p = 0.039). We also computed the percentage (%) of each error category by dividing the number of categorical errors by the total number of steps ( Supplemental Fig. 1 ). In the regular arrangement, LB females displayed reduced correct steps with their forelimbs (p = 0.035) and increased slight slips (p = 0.029) compared to their NB counterparts. Fig. 6. Open in a new tab Illustration of skilled walking test for male and female NB and LB juveniles (PND28-29) using both regular and irregular ladder rung settings as adapted from Metz and Whishaw (2002) . The ladder rung apparatus was elevated above ground allowing a mobile camera to record precise paw placement while the rat walked from one end to the other of the ladder. Percentage of correct placement of forelimb ( A,B ) and hind limb ( C,D ) is displayed for regular (left) and irregular (right) settings. In the regular rung, two way ANOVA indicated a significant sex effect (p = 0.0143) for the hind limb, but no bedding or sex × bedding interaction. LB females had a higher hind limb correct score than their male counterparts (p = 0.0108). In the irregular rung, there was a significant sex effect (p = 0.031), but no bedding or sex × bedding interaction. Females in the NB group had a higher percentage of correct steps in the hind limb compared to NB males (p = 0.039). Values represent the Mean±SEM of n = 21-25 male and n = 13-17 female juveniles. 3.5. Changes in juvenile M1 pyramidal neurons after exposure to LB We next examined whether changes in fine locomotion and gait induced by the early LB conditions were associated with lasting morphological changes in the M1 motor cortex pyramidal neurons of juveniles (PND29-30) and more specifically in the layer V that projects to brain stem and spinal cord areas to control movement. As indicated in Fig. 7 , in males (left panels), there was no significant effect of bedding on spine number or spine density, although LB significantly increased dendritic length compared to NB conditions (F(1,264) = 4.72; p = 0.031). In contrast, dendritic length was greatly reduced in LB females ( Fig. 7 , right panels) (F(1, 264) = 33.02; p < 0.001), leading to a significant increase in spine density in this group (F(1,264) = 52.53; p < 0.001). There were no significant differences due to the bedding conditions in the number ( Supplemental Fig. 2 ) and distribution of branch points as a function of the distance from soma (not shown) in either male or female juveniles. The branched analysis revealed a close to significant effect of bedding in spine density (F(1,24) = 3.616; p = 0.069) and an interaction between bedding and sex for dendritic length (F(1,24) = 4.157, p = 0.0526), where LB had an opposite effect in males and females ( Supplemental Fig. 2 ). Fig. 7. Open in a new tab Morphological analysis of motor cortex (M1) pyramidal neurons in the layer V of the cortex in juvenile NB and LB male and female (PND29-30) rats visualized by Golgi stain. A: Illustration of a M1 motor neuron analyzed (scale bar = 50 μm) and B : Changes in spine density between NB and LB male or female offspring (scale bar = 10 μm). Sholl analysis of number of spines ( C ), dendritic length ( D ) and spine density ( E ) are represented as a function of the distance from the soma in males NB and LB (left) and females NB and LB (right) juveniles. To calculate spine density (spines/μm), the total number of spines was divided by the total dendritic length for each neuron. Neurons were manually traced by an experimenter blind to sex and bedding condition, with the Neurolucida software (MicroBrightField, Williston, VT) using a cut off distance set at 220 μm from the soma. A total of 3-4 neurons were analyzed per animal (n = 6-8 pups of each sex/bedding group). Values represent the Mean±SEM. ∗, p < 0.05; ∗∗, p < 0.01 between NB and LB groups (main effect of bedding). 4. Discussion In this study, we used the limited bedding (LB) paradigm of early stress in rats to examine the effects of early neonatal adversity longitudinally on physical and sensorimotor milestones as well as morphological changes in the motor cortex (M1) and motor behavior in male and female juveniles. We found that LB exposure produced consistent delays in several physical, sensory and reflex milestones in neonates and reduced overall locomotion in the openfield in juveniles without a significant sex effect. Analysis of orienting behavior, an early indicator of locomotion in neonates also highlighted a neurodevelopmental delay in motor behavior, which was further documented with impaired gait characteristics in LB juveniles. These changes in locomotion after LB exposure were accompanied by sex-specific morphological alterations in spine density of L5 motor cortex (M1) neurons, suggesting that the ELS can potentially shape motor development through both behavioral and cellular pathways. A central objective of our study was to provide a longitudinal, individualized assessment of the timing to reach specific milestones in both NB and LB offspring and to test a wide range of modalities, physical, sensory and motor reflex across age. The LB paradigm is known to produce fragmentation of maternal behavior and can also lead to both hypervigilance and abuse depending on the type and timing of LB ( Ivy et al., 2008 ; Walker et al., 2017 ; Gallo et al., 2019 ; MacDowell Kaswan et al., 2025 ). In our study maternal behavior of LB females was not significantly modified compared to NB females on PND5-6, except for a modest increase in LB nursing time. However, the LB conditions had a significant effect on eye opening, acoustic canal opening and a trend towards reduced body weight as often documented in our studies (Guadagno et al., 2017) and those of others ( Eck et al., 2020 ). Delayed eye opening is consistently seen across mouse and rat studies with the limited bedding and the maternal separation paradigms ( Demaestri et al., 2020 , Pardo et al., 2023 ), but was not observed with the scarcity-adversity paradigm ( Lauraine et al., 2024 ). Eye opening was also delayed with ablation of the whiskers during the first week of life, suggesting that passive whisker touch and prevention of the transition to active sensing during this period can affect neurodevelopment ( Smirnov and Sitnikova, 2019 ). Although vision is not the primary sensory modality used by rodents and light can influence neurodevelopment before eye opening, a delay in eye opening could potentially delay visual cues-evoked stimulation, affecting the maturation of navigational systems and other systems that either depend on or are sensitive to these cues ( Shen and Xue, 2025 ). Similarly, a delay in auditory canal opening could be associated with later hearing development and changes in the threshold of auditory brainstem responses. Enhanced maternal care effects on these variables have been documented in Wistar rats after handling and cross-fostering, both leading to accelerated development of auditory brainstem responses ( Adise et al., 2014 ), in contrast to our results suggesting a potential delay in these responses. Interestingly, we found that LB pups also displayed a delay in acquisition of the acoustic startle response, a reflex response that is dependent on and correlates with the activity of several auditory brain stem regions ( Koch and Schnitzler, 1997 ). A similar pattern is observed in pups originating from dams fed a DHA-rich diet, which display both delayed acoustic startle responses and longer auditory brainstem conduction times ( Haubner et al., 2002 ). The timing of other physical milestones like fur development was not significantly affected by LB, although stepwise multiple regression analysis for this variable revealed that bedding, body weight on PND1 and on the day before the first animals from both groups reached the milestone (PND6 in this case) were significant predictors of the appearance of fur. This suggests that fur development might be correlated with body weight gain during the early postnatal period and that consequently, the effect of bedding on fur development could be conveyed through changes in body weight gain. In addition to LB-induced changes in physical milestones, we found that bedding conditions had a significant effect to delay the occurrence of sensory modalities and motor reflexes such as ear twitch, surface righting, grasping and level screen holding. Our results are consistent with other work using a number of different models of early adversity (perinatal and postnatal, social, nutritional or drug-induced) and showing that motor reflexes and coordination are compromised by early adversity ( Patin et al., 2004 ; Mesquita et al., 2007 ; Demaestri et al., 2020 ). For instance, neonatal hypoxic-ischemic injury delayed the onset of ear twitch and other neurological reflexes that were associated with early deficits in motor function ( Lubics et al., 2005 ) and maternal separation induced retardation in the acquisition of postural and motor reflexes like the surface righting reflex and negative geotaxis, but only in females ( Mesquita et al., 2007 ). In contrast to these studies, we did not observe sex differences in the reaching of any of the milestones tested. This was initially surprising as neurodevelopmental trajectories vary between sexes ( Davis and Pfaff, 2014 ) and our method of longitudinal assessment for reaching criterion in each category of milestones would increase the sensitivity of our measures. There is currently no consensus on sex-dependent effects of early adversity on neurodevelopmental milestones. Eye opening, a commonly used neurodevelopmental milestone was delayed in female LB mice, but not in males ( Pardo et al., 2023 ) while another report showed no significant sex effect on this variable ( Demaestri et al., 2020 ). In rats, latency to cliff avoidance after scarcity-adversity was impaired in females only ( Lauraine et al., 2024 ), but other studies, including our own, report no sex differences. A delay in acquisition of cliff avoidance was noted for LB compared to NB pups, but negative geotaxis was comparable between groups. Cliff avoidance evaluates the ability of neonates to avoid a dangerous situation and turn around to safety. This reflex response involves the timely integration between sensory inputs and locomotor output responses ( Altman and Sudarshan, ) thus if some of the components directing motor responses are compromised by early adversity, as demonstrated above for other motor reflex tasks, it would also naturally impair the ability of the neonates to avoid danger. Finally, orienting behavior and the ability to transition from mostly circular locomotor movements and crawling to a linear trajectory was delayed in LB offspring, consistent with detrimental effects of early adversity on motor control ( Torabi R et al., 2021 ). Another important aim of this study was to examine the impact of LB on fine motor control, limb coordination and morphology in the primary motor cortex (M1) once mature walking skills are acquired in juvenile rats ( Shriner et al., 2009 ). Previous studies found that either LB or maternal separation increased the expression of Doublecortin, a marker of immature neurons in PND8 mice, but morphological analyses of motor neurons were not performed after these adversity conditions ( Demaestri et al., 2020 ). Primary motor cortex (M1) is one of the cortical regions to mature early in neonatal life and it functions like a somatosensory area before developing its distinguished motor functions around adolescence (PND35). During neonatal life (PND8-12), a shift in M1 sensory responses occurs so that forelimb representation neurons lose their sensitivity to active sleep (REM) related twitches and become responsive to wake-related movements ( Dooley and Blumberg, 2018 ). This transition towards the sensory consequences of movement is observed just before the development of motor outflow. It is likely that processes leading to the maturation of M1 motor cortex neurons are exquisitely sensitive to early adverse environments as in both humans ( Pitcher JB et al., 2009 , Araújo, 2025 ) and rodents ( Mesquita et al., 2007 ; Farkas et al., 2009 ), impaired motor functions represent some of the early signs of neurodevelopmental delays. Accordingly, our results show that LB exposure leads to several sensorimotor deficits in neonatal life (surface righting reflex, grasping, level screen and orienting) as well as during pre-adolescence (locomotion, gait). Openfield testing revealed that both male and female LB offspring showed reduced locomotion and distance travelled in all parts of the openfield, including the “safety” zone of the shadow, where they spent significantly more time. These data point to an overall decreased motor function and a potential for increased anxiety-like behavior in LB juveniles, although we did not find significant differences between bedding conditions when testing a subset of animals in the elevated plus maze. This could be interpreted as either the number of animals tested in the subset cohort was insufficient to lead to significant differences or the context in which locomotion is assessed might be interpreted differently in juveniles compared to adults. To get a more comprehensive view of the fine motor components altered by LB exposure, we performed a gait analysis and subjected juveniles to a skilled walking test. Gait analysis revealed a hind limb-specific reduction in propulsion and stride with LB in both sexes. Presumably, the observed shorter but faster strides would facilitate less precise surface placement of the hind limbs before weight-bearing contact ( Clarke and Still, 1999 ). Exactly why the hind limbs are preferentially affected remains to be determined, but it could result from cell positioning defects in the spinal cord of LB rats ( Phelps et al., 2002 ). In contrast to the gait analysis, results of the skilled walking task showed no effect of bedding on either forelimb or hind limb correct placement but a sex effect in hind limb placement where females of either bedding group tend to display a greater percentage of correct placement compared to males. When the specific type of errors was computed for both regular and irregular rung arrangement ( Supplemental Fig. 1 ), LB females had less correct placement and more slight slip placement compared to NB females in the regular rung arrangement. These results were surprising as we expected that given the reduced locomotion and stride length characteristics of LB offspring, they would display more impairment compared to the NB controls in the irregular rung. It is possible that the lack of consistency between the behavioral tests used here reflects the intrinsic differences in what each test examines and that each test might recruit different circuits and adaptations. An important consideration is the age at which these experiments took place, PND28, reflective of the juvenile stage of neural development and the initial onset of adult skilled movement ( Shriner et al., 2009 ). Because neural networks have not fully consolidated, LB-induced changes to motor skills may become more apparent in adulthood ( Kokubo et al., 2018 ). Given the significant impact of early LB conditions in delaying achievement of specific sensorimotor milestones and overall locomotion, gait or skilled walking in juveniles, we next examined whether morphological characteristics of Layer V M1 motor neurons (L5) in NB and LB juveniles could associate with these changes. L5 projection neurons are essential for motor skill learning and project mostly to the brain stem, spinal cord and other cortical and subcortical structures ( Oswald et al., 2013 ; Kida et al., 2024 ). Structural modifications including increases in dendritic length and branching and increased synaptic plasticity are observed in these neurons following skilled motor training in adulthood ( Kolb et al., 2008 ; Kida et al., 2024 ). Our data in juvenile rats show a reduction of dendritic length of L5 neurons in LB females and a corresponding increase in spine density, which may indicate that LB causes mild dendritic atrophy and impairs the process of synaptic pruning by microglia and thus, the optimization of neural networks in these animals. Additional measures of processes related to synaptic pruning such as microglia number and reactivity, and pruning-related markers such as lysosomal marker CD68 for instance might help understand the LB-induced changes observed in females in our study. Spine density in males was not affected by LB, although dendritic length was slightly elevated. Although not measured in our study, further characterization of the type of spines affected by the LB procedure could provide valuable information on the stage of spines that might be preferentially affected by early adversity (mature vs. immature spines). Similarly to our data, juvenile motor neurons have reduced dendritic branches after neonatal food restriction ( Torrero et al., 2005 ) and neurons in the adolescent somatosensory cortex display a reduction in spine elimination after neonatal whisker trimming ( Zuo et al., 2005 ). Since we did not identify the phenotype of L5 neurons analyzed nor the type of spines affected, further studies are required to identify the projection subtype of L5 motor neurons affected by the LB paradigm and the long-term behavioral effects of these morphological changes. Generally, the study of refined motor functionality is complex due to the contribution of several neural networks including the somatosensory system in providing sensory input, the cerebellum in controlling balance and coordination, among other tasks. Our current data in juveniles reinforce the notion that impaired motor functions represent early signs of neurodevelopmental delays, but evaluation of the changes in motor behavior and function in adult LB offspring might provide valuable information on the type of changes and underlying mechanisms that are maintained or even exacerbated in adulthood. Our current research seeks to provide preliminary indications of whether there are indeed impacts from LB on a primary area of motor development. There are some limitations to our study. The first being that we tested all the pups daily for specific milestones and in doing so, separated the pups from their mother for a duration of approximatively 15 min (13.7 min), which corresponds to the “handling” paradigm defined by the late Dr Seymour Levine ( Erskine et al., 1975 ). It is possible therefore that maternal behavior was altered by this repeated short-term separation and that the effect of LB was attenuated by the “beneficial” effects of “handling” on neurodevelopment. A second limitation is related to the scoring of maternal behavior as we chose to measure maternal behavior between PND5-6 as in our previous studies. However, in these maternal cohorts that were handled daily, it is possible that significant effects on maternal “fragmentation” could have been observed in earlier time points (i.e. on PND2-3), before the establishment of a “pattern” of daily disturbances. In addition, we did not measure nest sorties and the duration of nesting bouts, which could represent other important direct measures of fragmented maternal behavior. A more complete and longitudinal assessment of maternal behavior as provided in other studies may refine the characterization of the changes induced by LB conditions. Finally, in the skilled walking test, we relied on manual scoring, which despite blind assessment, may introduce error through observer variability. In future work, computational approaches could be implemented through the open-source ALMA (Automated Limb Motion Analysis) toolbox, which can apply machine learning algorithms to facilitate the comprehensive analysis of locomotion. In summary, our study provides valuable insight into the effects of ELS induced through LB conditions on neurodevelopmental milestones and identified the timing of physical, sensory and motor modalities that are affected by this paradigm of early adversity. We also document the enduring effects of early adversity on motor development at the macroscopic and cellular levels and provide evidence for morphological changes in pyramidal neurons of the motor cortex after LB conditions. Whether these changes persist into adulthood is subject to future studies, but they could reflect the long-term consequences of ELS on microglia activation and other cellular mechanisms responsible for the functional regulation of motor circuits and behavior. Disclosures The authors have no financial interest in, or conflict of interest with the subject and materials discussed in the present manuscript. CRediT authorship contribution statement Annie Phan: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft. Michael Ru: Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – original draft. Brianna Latremouille: Investigation, Methodology. Hong Long: Investigation, Methodology. Claire-Dominique Walker: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing – review & editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors wish to thank Dr Joseph Rochford (Douglas Institute Research Center) for his help with the statistical analyses and the coordinator of the Molecular and Cellular Microscopy Platform at the Douglas Institute Research Center for help with the Golgi analyses. This study was supported by a grant from the Canadian Institutes for Health Research (CIHR), grant # PG407226 to CDW. Footnotes This article is part of a special issue entitled: Gig Levine Special Issue published in Neurobiology of Stress. Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.ynstr.2026.100814 . Appendix A. Supplementary data The following are the Supplementary data to this article. Multimedia component 1 mmc1.docx (20.9KB, docx) Supplemental Figure 1 Percent (%) distribution of foot placement scores in each error category in the skilled walking test (ladder rung) for male and female NB and LB juveniles (PND28-29) using both regular and irregular ladder rung settings as adapted from Metz and Whishaw (2002) . The percentage of error was computed by dividing the number of categorical errors by the total number of steps. Student-t-tests (GraphPad) were used to determine significant bedding effects for each category. Under the regular arrangement conditions, the effect of bedding significantly decreased the number of forelimb “correct” steps in LB females (p = 0.035), but increased the percentage of slight slips (p = 0.029) in the same group as assessed by multiple unpaired t-tests. No other significant changes due to bedding or sex were confirmed using two-way ANOVA. Values represent the Mean±SEM of n = 21-25 male and n = 13-17 female juveniles. ∗, p < 0.05 between NB and LB groups. mmc2.pptx (167.9KB, pptx) Supplemental Figure 2 Branched structure analysis of motor cortex (M1) pyramidal neurons in the layer V of the cortex in juvenile NB and LB male and female (PND29-30) rats visualized by Golgi stain. Analyses of pyramidal neurons were performed in Neuroexplorer (MicroBrightField, Williston, VT) on reconstructed neurons to determine number of dendritic spines ( A ), total dendritic length ( B ) and number of branch points or nodes ( C ). To calculate spine density ( D ) (spines/μm), the total number of spines was divided by the total dendritic length for each neuron. A close to significant bedding effect was observed for spine density (p = 0.069) and interaction between sex and bedding for dendritic length (p = 0.053). Values represent the Mean±SEM of 6-8 juveniles per group. 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Supplementary Materials Multimedia component 1 mmc1.docx (20.9KB, docx) Supplemental Figure 1 Percent (%) distribution of foot placement scores in each error category in the skilled walking test (ladder rung) for male and female NB and LB juveniles (PND28-29) using both regular and irregular ladder rung settings as adapted from Metz and Whishaw (2002) . The percentage of error was computed by dividing the number of categorical errors by the total number of steps. Student-t-tests (GraphPad) were used to determine significant bedding effects for each category. Under the regular arrangement conditions, the effect of bedding significantly decreased the number of forelimb “correct” steps in LB females (p = 0.035), but increased the percentage of slight slips (p = 0.029) in the same group as assessed by multiple unpaired t-tests. No other significant changes due to bedding or sex were confirmed using two-way ANOVA. Values represent the Mean±SEM of n = 21-25 male and n = 13-17 female juveniles. ∗, p < 0.05 between NB and LB groups. mmc2.pptx (167.9KB, pptx) Supplemental Figure 2 Branched structure analysis of motor cortex (M1) pyramidal neurons in the layer V of the cortex in juvenile NB and LB male and female (PND29-30) rats visualized by Golgi stain. Analyses of pyramidal neurons were performed in Neuroexplorer (MicroBrightField, Williston, VT) on reconstructed neurons to determine number of dendritic spines ( A ), total dendritic length ( B ) and number of branch points or nodes ( C ). To calculate spine density ( D ) (spines/μm), the total number of spines was divided by the total dendritic length for each neuron. A close to significant bedding effect was observed for spine density (p = 0.069) and interaction between sex and bedding for dendritic length (p = 0.053). Values represent the Mean±SEM of 6-8 juveniles per group. Sholl analysis of this same cohort of animals is displayed in Fig. 7 . mmc3.pptx (120.3KB, pptx) Data Availability Statement Data will be made available on request. 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