Epigenetic processes associated with neonatal spinal transection.

In early development, the spinal cord in healthy or disease states displays remarkable activity-dependent changes in plasticity, which may be in part due to the increased activity of brain derived neurotrophic factor (BDNF). Indeed, BDNF delivery has been efficacious in partially ameliorating many of the neurobiological and behavioral consequences of spinal cord injury (SCI), making elucidating the role of BDNF in the normative developing and injured spinal cord a critical research focus. Recent work in our laboratory provided evidence for aberrant global and locus-specific epigenetic changes in methylation of the Bdnf gene as a consequence of SCI. In the present study, animals underwent thoracic lesions on P1, with cervical and lumbar tissue being later collected on P7, P14, and P21. Levels of Bdnf expression and methylation (exon IX and exon IV), in addition to global methylation levels were quantified at each timepoint. Results indicated locus-specific reductions of Bdnf expression that was accompanied by a parallel increase in methylation caudal to the injury site, with animals displaying increased Bdnf expression at the P14 timepoint. Together, these findings suggest that epigenetic activity of the Bdnf gene may act as biomarker in the etiology and intervention effort efficacy following SCI.

Acute intrathecal administration of quipazine elicits air-stepping behavior.

Serotonin plays a pivotal role in the initiation and modulation of locomotor behavior in the intact animal, as well as following spinal cord injury. Quipazine, a serotonin 2 receptor agonist, has been used successfully to initiate and restore motor behavior in rodents. Although evidence suggests that the effects of quipazine are spinally mediated, it is unclear whether intrathecal (IT) quipazine administration alone is enough to activate locomotor-like activity or whether additional stimulation is needed. Thus, the current study examined the effects of IT administration of quipazine in postnatal day 1 rats in two separate experiments. In experiment 1, quipazine (0.1, 0.3, or 1.0 mg/kg) was dissolved in saline and administered via IT injection to the thoracolumbar cord. There was no significant effect of drug on hindlimb alternating stepping. In experiment 2, quipazine (0.3 or 1.0 mg/kg) was dissolved in a polysorbate 80-saline solution (Tween 80) and administered via IT injection. Polysorbate 80 was used to disrupt the blood-brain barrier to facilitate absorption of quipazine. The injection was followed by tail pinch 5 minutes post-injection. A significant increase in the percentage of hindlimb alternating steps was found in subjects treated with 0.3 mg/kg quipazine, suggesting that IT quipazine when combined with sensory stimulation to the spinal cord, facilitates locomotor-like behavior. These findings indicate that dissolving the drug in polysorbate 80 rather than saline may heighten the effects of IT quipazine. Collectively, this study provides clarification on the role of quipazine in evoking spinally-mediated locomotor behavior.

Neonatal Spinal Cord Transection Decreases Hindlimb Weight-Bearing and Affects Formation of Achilles and Tail Tendons.

Mechanical loading may be required for proper tendon formation. However, it is not well understood how tendon formation is impacted by the development of weight-bearing locomotor activity in the neonate. This study assessed tendon mechanical properties, and concomitant changes in weight-bearing locomotion, in neonatal rats subjected to a low thoracic spinal cord transection or a sham surgery at postnatal day (P)1. On P10, spontaneous locomotion was evaluated in spinal cord transected and sham controls to determine impacts on weight-bearing hindlimb movement. The mechanical properties of P10 Achilles tendons (ATs), as representative energy-storing, weight-bearing tendons, and tail tendons (TTs), as representative positional, non-weight-bearing tendons were evaluated. Non- and partial weight-bearing hindlimb activity decreased in spinal cord transected rats compared to sham controls. No spinal cord transected rats showed full weight-bearing locomotion. ATs from spinal cord transected rats had increased elastic modulus, while cross-sectional area trended lower compared to sham rats. TTs from spinal cord transected rats had higher stiffness and cross-sectional area. Collagen structure of ATs and TTs did not appear impacted by surgery condition, and no significant differences were detected in the collagen crimp pattern. Our findings suggest that mechanical loading from weight-bearing locomotor activity during development regulates neonatal AT lateral expansion and maintains tendon compliance, and that TTs may be differentially regulated. The onset and gradual increase of weight-bearing movement in the neonate may provide the mechanical loading needed to direct functional postnatal tendon formation.

Pericytes improve locomotor recovery after spinal cord injury in male and female neonatal rats.

OBJECTIVE: It is not known how activation of the hypoxia-inducible factor (HIF) pathway in pericytes, cells of the microvascular wall, influences new capillary growth. We tested the hypothesis that HIF-activated pericytes promote angiogenesis in a neonatal model of spinal cord injury (SCI). METHODS: Human placental pericytes stimulated with cobalt chloride and naïve pericytes were injected into the site of a thoracic hemi-section of the spinal cord in rat pups on postnatal day three (P3). Hindlimb motor recovery and Doppler blood flow perfusion at the site of transection were measured on P10. Immunohistochemistry was used to visualize vessel and neurofilament density for quantification. RESULTS: Injection of HIF-activated pericytes resulted in greater vascular density in males but did not result in improved motor function for males or females. Injection of non-HIF-activated pericytes resulted improved motor function recovery in both sexes (males, 2.722 ± 0.31-fold score improvement; females, 3.824 ± 0.58-fold score improvement, P < .05) but produced no significant changes in vessel density. CONCLUSIONS: HIF-activated pericytes promote vascular density in males post-SCI. Acute delivery of non-HIF-activated pericytes at the site of injury can improve motor recovery post-SCI.

Histamine modulates spinal motoneurons and locomotor circuits.

Spinal motoneurons and locomotor networks are regulated by monoamines, among which, the contribution of histamine has yet to be fully addressed. The present study investigates histaminergic regulation of spinal activity, combining intra- and extracellular electrophysiological recordings from neonatal rat spinal cord in vitro preparations. Histamine dose-dependently and reversibly generated motoneuron depolarization and action potential firing. Histamine (20 µM) halved the area of dorsal root reflexes and always depolarized motoneurons. The majority of cells showed a transitory repolarization, while 37% showed a sustained depolarization maintained with intense firing. Extracellularly, histamine depolarized ventral roots (VRs), regardless of blockage of ionotropic glutamate receptors. Initial, transient glutamate-mediated bursting was synchronous among VRs, with some bouts of locomotor activity in a subgroup of preparations. After washout, the amplitude of spontaneous tonic discharges increased. No desensitization or tachyphylaxis appeared after long perfusion or serial applications of histamine. On the other hand, histamine induced single motoneuron and VR depolarization, even in the presence of tetrodotoxin (TTX). During chemically induced fictive locomotion (FL), histamine depolarized VRs. Histamine dose-dependently increased rhythm periodicity and reduced cycle amplitude until near suppression. This study demonstrates that histamine induces direct motoneuron membrane depolarization and modulation of locomotor output, indicating new potential targets for locomotor neurorehabilitation.

Responsiveness of rat fetuses to sibling motor activity: Communication in utero?

Previous research has revealed that fetuses detect and respond to extrauterine stimuli such as maternal movement and speech, but little attention has been cast on how fetuses may directly influence and respond to each other in the womb. This study investigated whether motor activity of E20 rat fetuses influenced the behavior of siblings in utero. Three experiments showed that; (a) contiguous siblings expressed a higher frequency of synchronized movement than noncontiguous siblings; (b) fetuses that lay between two siblings immobilized with curare showed less movement relative to fetuses between saline or uninjected controls; and (c) fetuses between two siblings behaviorally activated by the opioid agonist U50,488 also showed less activity and specific behavioral changes compared to controls. Our findings suggest that rat fetuses are directly impacted by sibling motor activity, and thus that a rudimentary form of communication between siblings may influence the development of fetuses in utero.

Maternal behavior influences development of a reflexive action pattern in the newborn rat.

This study examined the effect of maternal behavior on the expression and postnatal development of a reflexive behavior in rat pups. In neonatal rats, the leg extension response (LER) is a bilateral hyperextension of the hindlimbs in response to maternal anogenital licking (AGL). Past research has found that intranasal application of zinc sulfate (ZnSO4 ) to the dam induces hyponosmia, thereby reducing the incidence of AGL. In this study, pregnant dams received intranasal application of air (control), distilled water (control), or ZnSO4 on the day before birth and every other day thereafter until postnatal day 9 (P9). The LER was experimentally evoked in pups, using a vibrotactile device, at P1, P5, or P10. Pups born to ZnSO4 -treated dams showed significantly shorter bilateral LER durations and significantly smaller ankle angles than pups born to control dams. Reduction of overall maternal AGL approached significance, and afternoon AGL was significantly reduced. These data suggest that maternal behavior influenced development of the LER in rat pups, demonstrating the influence of maternal care on behavioral development during the perinatal period.

Developmental plasticity of coordinated action patterns in the perinatal rat.

Some of the most simple, stereotyped, reflexive, and spinal-mediated motor behaviors expressed by animals display a level of flexibility and plasticity that is not always recognized. We discuss several examples of how coordinated action patterns have been shown to be flexible and adaptive in response to sensory feedback. We focus on interlimb and intralimb coordination during the expression of two action patterns (stepping and the leg extension response) in newborn rats, as well as interlimb motor learning. We also discuss the idea that the spinal cord is a major site for supporting plasticity in the developing motor system. An implication of this research is that normally occurring sensory stimulation during the perinatal period influences the typical development and expression of action patterns, and that exploiting the developmental plasticity of the motor system may lead to improved strategies for promoting recovery of function in human infants with motor disorders.

Effectiveness of topical anesthetics on reducing tactile sensitivity in the paws of newborn rats.

The aim of this study was to evaluate the effectiveness of three local, topical anesthetics on touch response thresholds of the paws of 1-day-old rats. Touch response thresholds were measured using Semmes Weinstein monofilaments after treatment of the paws with EMLA (2.5% lidocaine and 2.5% prilocaine), alcaine (.5% proparacaine), triocaine (20% benzocaine, 6% lidocaine, and 4% tetracaine), or petroleum jelly (treatment control). Touch thresholds significantly increased after treatment with EMLA 18% of the time, and there was no evidence of a systemic effect. Touch thresholds were not significantly altered after treatment with alcaine, triocaine, or petroleum jelly. Therefore, EMLA appears to be a slightly effective topical anesthetic for reducing tactile sensitivity in newborn rats.

Sensorimotor training during expression of the leg extension response (LER) in 1-day-old rats.

In newborn rats, the leg extension response (LER) is a coordinated hyperextension of the hindlimbs that is shown in response to anogenital stimulation. Here we examined the influence of sensorimotor training on LER expression in postnatal day 1 rats. In Experiment 1, we examined if proprioceptive feedback facilitates LER expression. We did this by repeatedly stimulating the pup's anogenital region with a vibrotactile device, to experimentally evoke the LER, thus increasing LER-relevant hindlimb proprioceptive feedback during training. In trained subjects, the LER was evoked every 4 min for 15 trials, followed by a final LER test. Results indicated that proprioceptive feedback on its own did not alter later expression of the LER. In Experiment 2, we examined the effect of both proprioceptive and cutaneous feedback on LER expression, through the use of a range of motion (ROM) restriction during training. During ROM restriction, a Plexiglas plate was placed beneath the pup at 50% of limb length. After the 15th training trial, a final LER test occurred with no ROM restriction in place. Compared to controls, pups that experienced ROM restriction exhibited a significantly shorter LER duration, and smaller hip and ankle angles during the LER test (indicating greater limb flexion). Together these findings show that concurrent proprioceptive and cutaneous feedback, but not proprioceptive feedback alone, has persistent effects on expression of this newborn action pattern.

Visualization of rat tendon in three dimensions using micro-Computed Tomography.

Micro-computed tomography (CT) is an X-ray-based imaging modality that produces three-dimensional (3D), high-resolution images of whole-mount tissues, but is typically limited to dense tissues, such as bone. The X-rays readily pass-through tendons, rendering them transparent. Contrast-enhancing chemical stains have been explored, but their use to improve contrast in different tendon types and across developmental stages for micro-CT imaging has not been systematically evaluated. Therefore, we investigated how phosphotungstic acid (PTA) staining and tissue hydration impacts tendon contrast for micro-CT imaging. We showed that PTA staining increased X-ray absorption of tendon to enhance tissue contrast and obtain 3D micro-CT images of immature (postnatal day 21) and sexually mature (postnatal day 50) rat tendons within the tail and hindlimb. Further, we demonstrated that tissue hydration state following PTA staining significantly impacts soft tissue contrast. Using this method, we also found that tail tendon fascicles appear to cross between fascicle bundles. Ultimately, contrast-enhanced 3D micro-CT imaging will lead to better understanding of tendon structure, and relationships between the bone and soft tissues.•Simple tissue fixation and staining technique enhances soft tissue contrast for tendon visualization using micro-CT.•3D tendon visualization in situ advances understanding of musculoskeletal tissue structure and organization.

Sensory feedback alters spontaneous limb movements in newborn rats: effects of unilateral forelimb weighting.

Perinatal mammals show spontaneous movements that often appear random and uncoordinated. Here, we examined if spontaneous limb movements are responsive to a proprioceptive manipulation by applying a weight unilaterally to a forelimb of postnatal day 0 (P0; day of birth) and P1 rats. Weights were calibrated to approximate 0%, 25%, 50%, or 100% of the average mass of a forelimb, and were attached at the wrist. P0 and P1 pups showed different levels of activity during the period of limb weighting, in response to weight removal, and during the period after weighting. Pups exposed to 50% and 100% weights showed proportionately more activity in the nonweighted forelimb during the period of weighting, suggesting a threshold for evoking proprioceptive changes. Findings suggest that newborn rats use movement-related feedback to modulate spontaneous motor activity, and corroborate studies of human infants that have suggested a role for proprioception during early motor development.

A preliminary investigation of high retinoic acid exposure during fetal development on behavioral competency and litter characteristics in newborn rats.

Myelomeningocele (MMC) is the most common and severe type of spina bifida in which the developing spine and neural tube fail to close during prenatal development. This typically results in a small portion of the lower spinal cord and meninges protruding from the back of the individual, accompanied by severe motor and sensory deficits. In rats, retinoic acid (RA) exposure in high doses during fetal development has been shown to induce morphologic and clinical symptoms similar to humans with MMC. The aim of the current study was to examine litter characteristics and sensorimotor function in MMC-affected rat pups. Pregnant rats were gavage-fed 2 ml olive oil or all-trans RA (40, 45, 50 mg/kg) on gestational day 11. Pups underwent behavioral testing on postnatal day 2. Litter characteristics and newborn sensorimotor function varied across RA doses. Pups prenatally exposed to 45 and 50 mg/kg RA weighed significantly less than olive oil and 40 mg/kg RA pups. Litters exposed to 45 mg/kg RA suffered significantly higher mortality rates compared to other groups. Additionally, bladder function was significantly impaired in pups exposed to 40 mg/kg RA. Sensorimotor function findings demonstrated that for most behavioral assessments there was not a significant difference between control and RA-exposed subjects. However, pups treated with 40 mg/kg RA showed increased facial wiping, suggesting a hyper-responsiveness to sensory stimuli. Overall, the findings of the current study provide evidence for a model to examine litter characteristics and behavioral effects as well as morphology.

Low-cost, open-source, variable speed and incline treadmill for studying impacts of neonatal locomotion.

There is a need for a small-scale, laboratory treadmill to investigate impacts of neonatal locomotion on neuromuscular and musculoskeletal development in small animal models. Adult mice and rats are routinely assessed using commercially available treadmills, but these treadmills can be relatively expensive and they may lack features needed to evaluate developing animals. Therefore, to overcome these limitations, a new treadmill was designed, built and calibrated. This open-source treadmill was designed specifically for neonatal and postnatal mice and rats, and it fits within a neonatal incubator. By using predominantly off-the-shelf and 3D printed components, and a microcontroller, this treadmill was low cost and easy to reproduce. The design also included variable incline, and a transparent belt and enclosures for video and gait analysis. A touchscreen interface provided user-friendly control over belt speed and run time. Moreover, validation experiments showed high accuracy in belt speed control, allowing for tightly regulated experimental conditions. Overall, this new low-cost, open-source, variable speed and incline treadmill can be used to advance understanding of neonatal locomotion, and neuromuscular and musculoskeletal development.

A Cohort Study of Relations Among Caregiver-Infant Co-Occupation and Reciprocity.

Co-occupation is the mutual engagement of two people in a shared occupation. Recent research has investigated co-occupational activities during sensitive periods to inform clinical practice. However, there remains a dearth of applied research to bridge gaps between research and practice within salient co-occupational relationships between caregivers and infants. The study applied co-occupational constructs of physicality, emotionality, and intentionality within caregiver-infant dyads across infancy. These constructs were examined in relation to caregiver-infant reciprocity in other domains (i.e., language, motor, and affective) to determine the overlapping features of reciprocal co-occupation with established aspects of reciprocity. Results suggest that as infants transitioned into toddlerhood and became more mobile and intentional in behavior, there were observable changes in caregiver-infant reciprocity. Caregiver utterances, affect, touch, and co-occupation were significantly related within and across time, highlighting the need for more studies to disentangle these relations in reference to infant development.

Locomotion and posture development in immature male and female rats (Rattus norvegicus): Comparison of sensory-enriched versus sensory-deprived testing environments.

The aim of the current study was to provide normative data on spontaneous locomotion and posture behavior in developing rats (Rattus norvegicus), during the first 2 postnatal weeks. Male and female rat pups were tested daily from P1 (postnatal day 1; ∼24 hr after birth) to P15 in a sensory-enriched or sensory-deprived testing environment, which was enclosed in a temperature-controlled incubator. Pups in the sensory-deprived condition were tested individually and placed in a square, Plexiglas box (open-field) for a 20-min test period. Pups in the sensory-enriched condition were placed in the same box with the siblings and bedding from the home cage to provide sensory stimulation that mimicked the home nest. Subjects in this condition were tested two at a time, with an additional two siblings (2 males and 2 females total in box). It was hypothesized that pups in the sensory-enriched testing condition would demonstrate more mature patterns of behavior, given the presence of behavior-activating sensory stimuli in the box. It was found that rat pups exhibited spontaneous pivoting and crawling as early as P1, regardless of sensory stimulation present in the testing environment. These behaviors were shown at least 1 to 3 days earlier than reported in prior studies. Quadrupedal walking occurred as early as P4 but was not reliably expressed until P10/11. These findings suggest that controlling temperature during testing influences the typical age of first occurrence of these behaviors. Finally, there were no sex differences in the duration of locomotion and posture behaviors. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

DNA methylation and behavioral changes induced by neonatal spinal transection.

Although the importance of epigenetic mechanisms in behavioral development has been gaining attention in recent years, research has largely focused on the brain. To our knowledge, no studies to date have investigated epigenetic changes in the developing spinal cord to determine the dynamic manner in which the spinal epigenome may respond to environmental input during behavioral development. Animal studies demonstrate that spinal cord plasticity is heightened during early development, is somewhat preserved following neonatal transection, and that spinal injured animals are responsive to sensory feedback. Because epigenetic alterations have been implicated in brain plasticity and are highly responsive to experience, these alterations are promising candidates for molecular substrates of spinal plasticity as well. Thus, the current study investigated behavioral changes in the development of weight-bearing locomotion and epigenetic modifications in the spinal cord of infant rats following a neonatal low-thoracic spinal transection or sham surgery on postnatal day (P)1. Specifically, global levels of methylation and methylation status of the brain-derived neurotrophic factor (Bdnf) gene, a neurotrophin heavily involved in both CNS and behavioral plasticity, particularly in development, were examined in lumbar tissue harvested on P10 from sham and spinal-transected subjects. Behavioral results demonstrate that compared to shams, spinal-transected subjects exhibit significantly reduced partial-weight bearing hindlimb activity. Molecular data demonstrate group differences in global lumbar methylation levels as well as exon-specific group differences in Bdnf methylation. This study represents an initial step toward understanding the relationship between epigenetic mechanisms and plasticity associated with spinal cord and locomotor development.

Onset of neonatal locomotor behavior and the mechanical development of Achilles and tail tendons.

Tendon tissue engineering approaches are challenged by a limited understanding of the role mechanical loading plays in normal tendon development. We propose that the increased loading that developing postnatal tendons experience with the onset of locomotor behavior impacts tendon formation. The objective of this study was to assess the onset of spontaneous weight-bearing locomotion in postnatal day (P) 1, 5, and 10 rats, and characterize the relationship between locomotion and the mechanical development of weight-bearing and non-weight-bearing tendons. Movement was video recorded and scored to determine non-weight-bearing, partial weight-bearing, and full weight-bearing locomotor behavior at P1, P5, and P10. Achilles tendons, as weight-bearing tendons, and tail tendons, as non-weight-bearing tendons, were mechanically evaluated. We observed a significant increase in locomotor behavior in P10 rats, compared to P1 and P5. We also found corresponding significant differences in the maximum force, stiffness, displacement at maximum force, and cross-sectional area in Achilles tendons, as a function of postnatal age. However, the maximum stress, strain at maximum stress, and elastic modulus remained constant. Tail tendons of P10 rats had significantly higher maximum force, maximum stress, elastic modulus, and stiffness compared to P5. Our results suggest that the onset of locomotor behavior may be providing the mechanical cues regulating postnatal tendon growth, and their mechanical development may proceed differently in weight-bearing and non-weight-bearing tendons. Further analysis of how this loading affects developing tendons in vivo may inform future engineering approaches aiming to apply such mechanical cues to regulate engineered tendon formation in vitro.

The Spinal Cord, Not to Be Forgotten: the Final Common Path for Development, Training and Recovery of Motor Function.

Research on learning, memory, and neural plasticity has long focused on the brain. However, the spinal cord also exhibits these phenomena to a remarkable degree. Following a spinal cord injury, the isolated spinal cord in vivo can adapt to the environment and benefit from training. The amount of plasticity or recovery of function following a spinal injury often depends on the age at which the injury occurs. In this overview, we discuss learning in the spinal cord, including associative conditioning, neural mechanisms, development, and applications to clinical populations. We take an integrated approach to the spinal cord, one that combines basic and experimental information about experience-dependent learning in animal models to clinical treatment of spinal cord injuries in humans. From such an approach, an important goal is to better inform therapeutic treatments for individuals with spinal cord injuries, as well as develop a more accurate and complete account of spinal cord and behavioral functioning.