Early Postnatal Days Research Articles

SUN-218 The Role Of Fstl3 In Spermatogenesis: A Transcriptome Analysis

Spermatogenesis in mammals depends on the function of Sertoli cells (SC). The total number of SC present in the testes is determined in the pre-pubertal stages of development. Activin, a TGFβ family ligand, regulates SC proliferation in the early postnatal phase in mice. During this phase immature SC are unable to assemble tight-junctions which are required to form the blood-testis-barrier (BTB) and consequently do not support spermatogenesis. Overexpression of activin beyond pre-pubertal stages results in the disruption of spermatogenesis and results in infertility. Follistatin Like-3 (Fstl3) is a glycoprotein known to bind and inhibit activin. Contrary to overexpression of activin, however, global deletion of Fstl3 in mice results in increased numbers of SC and increased sperm production in older males. Based on this striking observation, in this study, using the global deleted Fstl3 mouse model, we sought to elucidate the function of Fstl3 in testicular development during the transition from pre-pubertal to post-pubertal stages. In testis sections from BrdU treated Fstl3 KO mice, we found that SC number per tubule is maintained from pre-pubertal to post-pubertal stages without a concomitant increase in BrdU positive SC. In contrast, SC numbers in similarly treated WT mice are depleted over the same time period. Stereological analyses further show that although the overall number of SC increases in both genotypes during pre-pubertal stages, increase in SC numbers is significantly higher in Fstl3 KO males. We also found by immunofluorescence that Sycp3 organization appears early (postnatal day 7) in Fstl3 KO mouse testes indicating early meiotic entry. To identify signalling pathways associated with SC proliferation and maturation that are affected by Fstl3 deletion we performed total mRNA sequencing and compared the findings between WT and Fstl3 KO testes of 3 day and 8 weeks mice. Our results show that more than 1000 genes are differentially expressed between WT and KO testes. Pathway analyses reveal that the “Sertoli-to-Sertoli cell Communication” pathway is one of the most relevant enriched canonical pathways in 3 day old Fstl3 KO mice compared to WT. We are currently investigating the mechanisms for early establishment of BTB in Fstl3 KO testes. The results of the study presented suggests that loss of Fstl3 promotes the establishment of SC-SC interactions that support spermatogenesis during the proliferative phase of these cells.

Down-Regulation of miRNA-708 Promotes Aberrant Calcium Signaling by Targeting Neuronatin in a Mouse Model of Angelman Syndrome.

The expression of ubiquitin ligase UBE3A is paternally imprinted in neurons and loss of function of maternally inherited UBE3A causes Angelman syndrome (AS), a neurodevelopmental disorder characterized by severe intellectual disability and motor disturbances. Over activation of UBE3A is also linked with autism. Mice deficient for maternal Ube3a (AS mice) exhibit various behavioral features of AS including cognitive and motor deficits although the underlying molecular mechanism is poorly understood. Here, we investigated possible involvement of miRNA in AS pathogenesis and identified miR-708 as one of the down-regulated miRNA in the brain of AS mice. This miR-708 targets endoplasmic reticulum resident protein neuronatin (a developmentally regulated protein in the brain) leading to decrease in intracellular Ca2+. Suppression of miR-708 or ectopic expression of neuronatin increased the level of intracellular Ca2+ and phosphorylation of CaMKIIα at Thr286. Neuronatin level was significantly increased in various brain regions of AS mice during embryonic and early postnatal days as well as in parvalbumin-positive GABAergic neurons during adulthood with respect to age-matched wild type controls. Differentiated cultured primary cortical neurons obtained from AS mice brain also exhibited higher expression of neuronatin, increased intracellular basal Ca2+ along with augmented phosphorylation of CaMKIIα at Thr286. These results indicate that miR-708/neuronatin mediated aberrant calcium signaling might be implicated in AS pathogenesis.

Pulmonary Hypertension in Newborn Infants with Vein of Galen Malformation

Objective: Vein of Galen malformation (VGM) is a rare cerebrovascular malformation. Children with VGM presenting in newborn period demonstrate various clinical courses from normal development to severe neurologic impairment or death. The aim of our study was to describe the characteristics of pulmonary hypertension and its treatment response in newborn infants with VGM. Methods: We retrospectively reviewed medical records of newborn infants with VGM who were antenatal diagnosed and treated with endovascular embolization in a single tertiary center. Clinical characteristics including echocardiographic data were described. Results: A total of six patients were diagnosed with VGM. Endovascular embolization was performed early postnatal days (median, 6.5 days) in four patients who required cardiorespiratory support. Echocardiography revealed pulmonary hypertension in the four cases and two of them received targeted therapy against pulmonary hypertension including inhaled nitric oxide and other pulmonary vasodilators. Incomplete occlusion of VGM failed to reverse the pulmonary hypertension. Mortality occurred in two patients with severe pulmonary hypertension in whom incomplete occlusion for VGM lesion. The cause of death was multi-organ failure secondary to right heart failure. Conclusion: Pulmonary hypertension was a common manifestation in newborn case with symptomatic VGM. The efficacy of early embolization and management for pulmonary hypertension in the newborn cases with persistent pulmonary hypertension of the newborn requires further large studies.

The spatial distribution pattern of Connexin26 expression in supporting cells and its role in outer hair cell survival

Mutations in the GJB2 gene (which encodes Connexin26 (Cx26)) account for about a quarter of all cases of non-syndromic deafness. Previous studies have indicated that knockout (KO) of Gjb2 gene during early postnatal days can cause outer hair cell (OHC) loss in mouse models. However, the postnatal spatial distribution pattern of Cx26 in different types of supporting cells (SCs) and the role of such distributions for the survival of OHCs is still obscure. In this study, the spatial distribution patterns of Cx26 in SCs were observed, and based on these observations different spatial Cx26-null mouse models were established in order to determine the effect of changes in the spatial distribution of Cx26 in SCs on the survival of OHCs. At postnatal day (P)3, unlike the synchronous expression of Cx26 along both longitudinal and radial boundaries of most types of SCs, Cx26 expression was primarily observed along the longitudinal boundaries of rows of Deiter’s cells (DCs). From P5 to P7, radial expression of Cx26 was gradually observed between adjacent rows of DCs. When Gjb2 gene was knocked out at random in different types of SCs, about 40% of the total DCs lost Cx26 expression and these Cx26-null DCs were distributed randomly in all three rows of DCs. The mice in this randomly Cx26-null group showed normal hearing and no significant OHC loss. When using a longitudinal KO pattern to induce knockout of Gjb2 gene specifically in the third row of DCs, about 33% of the total DCs lost Cx26 expression in this specific longitudinally Cx26-null group. The mice in this group showed late-onset hearing loss and significant OHC loss, however, the morphology of corresponding DCs was slightly altered. In both experimental groups, no substantial DC loss was observed. These results indicate that longitudinal Cx26-based channels are predominant in DCs during P3–P5. The Cx26 expression along rows of DCs might play a key role in the survival of OHCs, but this longitudinal KO pattern in DCs has a limited effect on DC survival or on its postnatal development.

Is caseloading Sustainable? 25-year History of Caseloading at King’s College Hospital

Caseload midwifery is defined as follows: When a midwife carries a caseload she is the primary provider of midwifery care (the named midwife) during pregnancy, birth and the early postnatal days for an agreed number of women. She may be providing care to women wherever they are: at home, in the community or in a maternity unit. She has responsibility for the planning and monitoring of care throughout for the women on her list. She liaises with medical colleagues and social agencies as appropriate (National Childbirth Trust 1995). Caseloading is a relational model of care provided by a small team of midwives who do on-calls in order to provide a 24/7 service. Midwives have autonomy over their workload, allowing them to respond to the individual needs of women and their families. Mixed-risk caseloading has been an important element of maternity care at King’s College Hospital for 25 years, where currently almost 20 per cent of the 5-6,000 women who give birth there each year receive caseloading care.

Fast Ca2+ Transients of Inner Hair Cells Arise Coupled and Uncoupled to Ca2+ Waves of Inner Supporting Cells in the Developing Mouse Cochlea.

Before the onset of hearing, which occurs around postnatal day 12 (P12) in mice, inner hair cells (IHCs) of the immature cochlea generate sound-independent Ca2+ action potentials (APs), which stimulate the auditory pathway and guide maturation of neuronal circuits. During these early postnatal days, intercellular propagating Ca2+ waves elicited by ATP-induced ATP release are found in inner supporting cells (ISCs). It is debated whether IHCs are able to fire Ca2+ APs independently or require a trigger by an ISC Ca2+ wave. To identify the Ca2+ transients of IHCs underlying Ca2+ APs and to analyze their dependence on ISC Ca2+ waves, we performed fast Ca2+ imaging of Fluo-8 AM-loaded organs of Corti at P4/P5. Fast Ca2+ transients (fCaTs) generated by IHCs were simultaneously imaged with Ca2+ waves in ISCs. ISC Ca2+ waves frequently evoked bursts consisting of >5 fCaTs in multiple adjacent IHCs. Although Ca2+ elevations of small amplitude appeared to be triggered by ISC Ca2+ waves in IHCs of Cav1.3 knockout mice we never observed fCaTs, indicating their requirement for Ca2+ influx through Cav1.3 channels. The Ca2+ wave-triggered Ca2+ upstroke in wildtype IHCs occurred 0.52 ± 0.27 s later than the rise of the Ca2+ signal in the adjacent ISCs. In comparison, superfusion of 1 μM ATP elicited bursts of fCaTs in IHCs starting 0.99 ± 0.34 s prior to Ca2+ elevations in adjacent ISCs. PPADS irreversibly abolished Ca2+ waves in ISCs and reversibly reduced fCaTs in IHCs indicating differential involvement of P2 receptors. IHC and ISC Ca2+ signals were however unaltered in P2X2R/P2X3R double knockout or in P2X7R knockout mice. Together, our data revealed a fairly similar occurrence of fCaTs within a burst (56.5%) compared with 43.5% as isolated single fCaTs or in groups of 2–5 fCaTs (minibursts). We provide evidence that IHCs autonomously generate single fCaTs and minibursts whereas bursts synchronized between neighboring IHCs were mostly triggered by ISC Ca2+ waves. Neonatal IHCs thus spontaneously generate electrical and Ca2+ activity, which is enhanced and largely synchronized by activity of ISCs of Kölliker’s organ indicating two sources of spontaneous activity in the developing auditory system.

Retinal Pericytes: Characterization of Vascular Development-Dependent Induction Time Points in an Inducible NG2 Reporter Mouse Model

ABSTRACTPurpose/aim of the study: In the retina, defects in pericytes (PCs) function/loss are associated with various complications; however, the exact pathological mechanisms are still not fully elucidated. Following the behavior of retina-resident PCs during health and disease will reveal new insights for both the understanding of pathological mechanisms and the development of new regenerative therapies for the treatment of retinopathies. The main goal of this study is to determine whether the NG2-reporter mouse (NG2CreERTM-eGFP) is a suitable model to study the fate of retina-resident PCs.Material and methods: Vascular development-dependent reporter induction in retinal PCs was evaluated at different time points [(a) > P21, (b) < P21, and (c) P1 to > P21)] and additionally four different modes of application were tested. Reporter expression was evaluated by enhanced green fluorescent protein (eGFP) immunofluorescence by confocal microscopy and induction efficiency was calculated by analyzing NG2-expressing PCs in comparison to eGFP-labeled PCs in the three capillary layers.Results: eGFP-positive PCs were detected in the three retinal capillary layers at all time points and administration routes tested. Multiple tamoxifen (TAM) applications in adult (> P21) NG2CreERTM-eGFP mice resulted in 3.59% eGFP-positive PCs. 2.37% eGFP-labeled PCs were detected after single intraperitoneal TAM injections at early postnatal days (P2/P5); however, just 1.61% PCs revealed reporter expression upon activation via the lactating mother (P4–P7). The highest number of eGFP-labeled PCs (7.09%) was detected following triple TAM administrations (P10–P12). The number of reporter-positive PCs doubled using homozygous animals.Conclusion: Despite low recombination efficiency in the used PC-specific fate mapping mouse model, changes in NG2 promoter activity of PCs during vascular development are indicated by single and multiple TAM inductions at different developmental time points. Nevertheless, these findings need further confirmation in up-coming studies by using homozygous NG2CreERTM-eGFP mice and additionally by mating the NG2CreERTM with a different reporter mouse to increase the low recombination efficiency.

Effect of Maternal Hypothyroidism on BDNF Expression in Developing Rat Brain

Thyroid hormone is essential for proper development of brain and modest degrees of developmental thyroid hormone insufficiency can result severe neurological deficits. We used an animal model to study the role of thyroid hormones (THs) during the gestation days and early postnatal days on developing rat brain. Dose-dependent thyroid hormone insufficiency in dams was induced by methimazole (MMI). In the first study-group thyroid hormone deficiency was induced by delivery of methimazole (MMI) to dams via drinking water at the doses of 50, 75 and 100 ppm from early gestation (GD 3) until weaning of the pups (PN20). In the second study-group hypothyroid rats were treated with 200 μg of thyroxine hormone. Pups were sacrificed on postnatal days (PN) 20. Maternal blood collected for thyroid hormone analysis. Cerebellum, medulla, hippocampus, cortex, olfactory bulbs were separated from dissected brain and relative expression of BDNF gene transcripts known to play critical roles in developing rat brain, were determined by RT-PCR. Daily body weight in dams and pups, the number of pups at birth, the eye –closure opening day and BDNF expression in brain extract was determined in the preweaning rats as a function of MMI exposure. The results indicate that genes driving important developmental processes during early brain development are sensitive to perturbations of the thyroid axis function.

Brainstem catecholaminergic neurones and breathing control during postnatal development in male and female rats

The brainstem catecholaminergic (CA) modulation on ventilation changes with development. We determined the role of the brainstem CA system in ventilatory control under normocapnic and hypercapnic conditions during different phases of development [postnatal day (P)7-8, P14-15 and P20-21] in male and female Wistar rats. Brainstem CA neurones produce a tonic inhibitory drive that affects breathing frequency in P7-8 rats and provide an inhibitory drive during hypercapnic conditions in both males and females at P7-8 and P14-15. In pre-pubertal rats, brainstem CA neurones become excitatory for the CO2 ventilatory response in males but remain inhibitory in females. Diseases such as sudden infant death syndrome, congenital central hypoventilation syndrome and Rett syndrome have been associated with abnormalities in the functioning of CA neurones; therefore, the results of the present study contribute to a better understanding of this system. The respiratory network undergoes significant development during the postnatal phase, including the maturation of the catecholaminergic (CA) system. However, postnatal development of this network and its effect on the control of pulmonary ventilation ( ) is not fully understood. We investigated the involvement of brainstem CA neurones in respiratory control during postnatal development [postnatal day (P)7-8, P14-15 and P20-21], in male and female rats, through chemical injury with conjugated saporin anti-dopamine β-hydroxylase (DβH-SAP). Thus, DβH-SAP (420ngμL-1 ), saporin (SAP) or phosphate buffered solution (PBS) was injected into the fourth ventricle of neonatal Wistar rats of both sexes. and oxygen consumption were recorded 1week after the injections in unanaesthetized neonatal and juvenile rats during room air and hypercapnia. The resting ventilation was higher in both male and female P7-8 lesioned rats by 33%, with a decrease in respiratory variability being observed in males. The hypercapnic ventilatory response (HCVR) was altered in male and female lesioned rats at all postnatal ages. At P7-8, the HCVR for males and females was increased by 37% and 30%, respectively. For both sexes at P14-15 rats, the increase in during hypercapnia was 37% higher for lesioned rats. A sex-specific difference in HCRV was observed at P20-21, with lesioned males showing a 33% decrease, and lesioned females showing an increase of 33%. We conclude that brainstem CA neurones exert a tonic inhibitory effect on in the early postnatal days of the life of a rat, increase variability in P7-8 males and modulate HCRV during the postnatal phase.

P2Y12 but not P2Y13 Purinergic Receptor Controls Postnatal Rat Retinogenesis In Vivo.

Adenine nucleotides through P2Y1 receptor stimulation are known to control retinal progenitor cell (RPC) proliferation by modulating expression of the p57KIP2, a cell cycle regulator. However, the role of Gi protein-coupled P2Y12 and P2Y13 receptors also activated by adenine nucleotides in RPC proliferation is still unknown. Gene expression of the purinergic P2Y12 subtype was detected in rat retina during early postnatal days (P0 to P5), while expression levels of P2Y13 were low. Immunohistochemistry assays performed with rat retina on P3 revealed P2Y12 receptor expression in both Ki-67-positive cells in the neuroblastic layer and Ki-67-negative cells in the ganglion cell layer and inner nuclear layer. Nonetheless, P2Y13 receptor expression could not be detected in any stratum of rat retina. Intravitreal injection of PSB 0739 or clopidogrel, both selective P2Y12 receptor antagonists, increased by 20 and 15%, respectively, the number of Ki-67-positive cells following 24h of exposure. Moreover, the P2Y12 receptor inhibition increased cyclin D1 and decreased p57KIP2 expression. However, there were no changes in the S phase of the cell cycle (BrdU-positive cells) or in mitosis (phospho-histone-H3-positive cells). Interestingly, an increase in the number of cyclin D1/TUNEL-positive cells after treatment with PSB 0739 was observed. These data suggest that activation of P2Y12 receptors is required for the successful exit of RPCs from cell cycle in the postnatal rat retina.

Repetitive Brain Injury of Juvenile Mice Impairs Environmental Enrichment-Induced Modulation of REM Sleep in Adulthood

Traumatic brain injuries (TBIs) are a common and costly ongoing public health concern. Injuries that occur during childhood development can have particularly profound and long-lasting effects. One common consequence and potential mediator of negative outcomes of TBI is sleep disruption which occurs in a substantial proportion of TBI patients. These individuals report greater incidences of insomnia and sleep fragmentation combined with a greater overall sleep requirement meaning that many patients are chronically sleep-deprived. We sought to develop an animal model of developmental TBI-induced sleep dysfunction. Specifically, we tested the hypothesis that early (postnatal day 21), repeated closed head injuries in Swiss-Webster mice, would impair basal and homeostatic sleep responses in adulthood. Further, we asked whether environmental enrichment (EE), a manipulation that improves functional recovery following TBI and has been shown to alter sleep physiology, would prevent TBI-induced sleep dysfunction and alter sleep-modulatory peptide expression. In contrast to our hypothesis, the mild, repeated head injury that we used did not significantly alter basal or homeostatic sleep responses in mice housed in standard laboratory conditions. Sham-injured mice housed in enriched environments exhibited enhanced rapid eye movement (REM) sleep and expression of the REM-promoting peptide pro-melanin-concentrating hormone, an effect that was not apparent in TBI mice housed in enriched environments. Thus, TBI blocked the REM-enhancing effects of EE. This work has important implications for the management and rehabilitation of the TBI patient population.

Degeneration of short-wavelength cone cells in rd12 mice

Background Retinitis pigmentosa (RP) is one of the causes of congenital blindness.It is well known that the degeneration process of rod cells is difficult to detect in RP.Retinal degeneration 12 (rd12) mice is a new, spontaneously arising mouse model for human Leber congenital amaurosis (LCA), and it is helpful for us to explore the pathogenesis and determine the treating target of RP. Objective This study was to investigate the natural disease process of short-length sensitive cone cells in rd12 mice, a LCA Rpe65rd12 (B6[A]-Rpe65rd12/J) mouse. Methods The rd12 mice at postnatal (P) 14, P21, P35 and P90 were selected (5 mice for each), and the wild-type C57BL/6J (B6) mice with matched ages were included as controls.Photopic full-field electroretinogram (ERG) was recorded with Roland Q450SC UV visual physiology instrument.Cone response was recorded using single white light-emitting diode (LED) stimulation with the flash intensity of 1.00 cds/m2 and 1.96 cds/m2, and short wave-length sensitive cone response was recorded using ultraviolet light ([363±6]nm) stimulation with the flash intensity of 2.0 mWs/m2 and 3.0 mW/m2.The mice were sacrificed and retinal whole-mounts were prepared.The distribution and number of cone cells and UV-sensitive cone cells were detected by FITC-peanut agglutinin (FITC-PNA) and Cy3 immunofluorescence stainning, respectively. Results In P14 rd12 mice, the ERG responses of overall cone cells presented the negative waveform and the latency was delayed, and UV-sensitive cone response was unrecordable.The b-wave amplitude of overall cone cells reduced by 75% in P21 rd12 mice compared with wild-type B6 mice, and the mean latency of b-wave in the P21 rd12 mice was significantly longer than that in the wild-type B6 mice ([102.80±11.39]ms vs.[43.40±5.60]ms) (t=-8.106, P=0.001). The mean b-wave amplitudes of UV-sensitive cone cells were (59.60±36.00), (82.40±12.22) and (68.43±17.63)μV in the wild-type B6 mice, and those in the rd12 mice were unrecordable.Immunofluorescence showed that a lager number of cone cells with green fluorescence were seen, and the expression of opsin with red fluorescence was displayed in the UV-sensitive cone cells of nasal lateral on retinal ventral side in P14 wild-type B6 mice; while only a few opsin positive-response cells were seen in P14, P21 and P35 rd12 mice. Conclusions In rd12 mice, the functional abnormality and quantitative reduction of cone cells appear in the early postnatal days, and the loss of UV-sensitive cone cells is earlier and more obvious. Key words: Retinal degeneration/congenital; Eye proteins/metabolism; Photoreceptor/pathology; Electroretinography; Fluorescent antibody technique; Leber congenital amaurosis; Mice, inbred C57BL; Disease models, animal

Ethanol-induced damage to the developing spinal cord: The involvement of CCR2 signaling

Ethanol exposure during development causes fetal alcohol spectrum disorders (FASD). A large body of evidence shows that ethanol produces multiple abnormalities in the developing central nervous system (CNS), such as smaller brain size, reduced volume of cerebral white matter, permanent loss of neurons, and alterations in synaptogenesis and myelinogenesis. The effects of ethanol on the developing spinal cord, however, receive little attention and remain unclear. We used a third trimester equivalent mouse model to investigate the effect of ethanol on the developing spinal cord. Ethanol caused apoptosis and neurodegeneration in the dorsal horn neurons of mice of early postnatal days, which was accompanied by glial activation, macrophage infiltration, and increased expression of CCR2, a receptor for monocyte chemoattractant protein 1 (MCP-1). Ethanol-induced neuronal death during development resulted in permanent loss of spinal cord neurons in adult mice. Ethanol stimulated endoplasmic reticulum (ER) stress and oxidative stress, and activated glycogen synthase kinase 3β (GSK3β) and c-Jun N-terminal kinase (JNK) pathways. Knocking out MCP-1 or CCR2 made mice resistant to ethanol-induced apoptosis, ER stress, glial activation, and activation of GSK3β and JNK. CCR2 knock out offered much better protection against ethanol-induced damage to the spinal cord. Thus, developmental ethanol exposure caused permanent loss of spinal cord neurons and CCR2 signaling played an important role in ethanol neurotoxicity.

Restoring auditory cortex plasticity in adult mice by restricting thalamic adenosine signaling.

Circuits in the auditory cortex are highly susceptible to acoustic influences during an early postnatal critical period. The auditory cortex selectively expands neural representations of enriched acoustic stimuli, a process important for human language acquisition. Adults lack this plasticity. Here we show in the murine auditory cortex that juvenile plasticity can be reestablished in adulthood if acoustic stimuli are paired with disruption of ecto-5'-nucleotidase-dependent adenosine production or A1-adenosine receptor signaling in the auditory thalamus. This plasticity occurs at the level of cortical maps and individual neurons in the auditory cortex of awake adult mice and is associated with long-term improvement of tone-discrimination abilities. We conclude that, in adult mice, disrupting adenosine signaling in the thalamus rejuvenates plasticity in the auditory cortex and improves auditory perception.

Descending Systems Direct Development of Key Spinal Motor Circuits.

The formation of mature spinal motor circuits is dependent on both activity-dependent and independent mechanisms during postnatal development. During this time, reorganization and refinement of spinal sensorimotor circuits occurs as supraspinal projections are integrated. However, specific features of postnatal spinal circuit development remain poorly understood. This study provides the first detailed characterization of rat spinal sensorimotor circuit development in the presence and absence of descending systems. We show that the development of proprioceptive afferent input to motoneurons (MNs) and Renshaw cells (RCs) is disrupted by thoracic spinal cord transection at postnatal day 5 (P5TX). P5TX also led to malformation of GABApre neuron axo-axonic contacts on Ia afferents and of the recurrent inhibitory circuit between MNs and RCs. Using a novel in situ perfused preparation for studying motor control, we show that malformation of these spinal circuits leads to hyperexcitability of the monosynaptic reflex. Our results demonstrate that removing descending input severely disrupts the development of spinal circuits and identifies key mechanisms contributing to motor dysfunction in conditions such as cerebral palsy and spinal cord injury.SIGNIFICANCE STATEMENT Acquisition of mature behavior during postnatal development correlates with the arrival and maturation of supraspinal projections to the spinal cord. However, we know little about the role that descending systems play in the maturation of spinal circuits. Here, we characterize postnatal development of key spinal microcircuits in the presence and absence of descending systems. We show that formation of these circuits is abnormal after early (postnatal day 5) removal of descending systems, inducing hyperexcitability of the monosynaptic reflex. The study is a detailed characterization of spinal circuit development elucidating how these mechanisms contribute to motor dysfunction in conditions such as cerebral palsy and spinal cord injury. Understanding these circuits is crucial to developing new therapeutics and improving existing ones in such conditions.

Temporal relation between neural activity and neurite pruning on a numerical model and a microchannel device with micro electrode array

Synapse elimination and neurite pruning are essential processes for the formation of neuronal circuits. These regressive events depend on neural activity and occur in the early postnatal days known as the critical period, but what makes this temporal specificity is not well understood. One possibility is that the neural activities during the developmentally regulated shift of action of GABA inhibitory transmission lead to the critical period. Moreover, it has been reported that the shifting action of the inhibitory transmission on immature neurons overlaps with synapse elimination and neurite pruning and that increased inhibitory transmission by drug treatment could induce temporal shift of the critical period. However, the relationship among these phenomena remains unclear because it is difficult to experimentally show how the developmental shift of inhibitory transmission influences neural activities and whether the activities promote synapse elimination and neurite pruning. In this study, we modeled synapse elimination in neuronal circuits using the modified Izhikevich's model with functional shifting of GABAergic transmission. The simulation results show that synaptic pruning within a specified period like the critical period is spontaneously generated as a function of the developmentally shifting inhibitory transmission and that the specific firing rate and increasing synchronization of neural circuits are seen at the initial stage of the critical period. This temporal relationship was experimentally supported by an in vitro primary culture of rat cortical neurons in a microchannel on a multi-electrode array (MEA). The firing rate decreased remarkably between the 18–25 days in vitro (DIV), and following these changes in the firing rate, the neurite density was slightly reduced. Our simulation and experimental results suggest that decreasing neural activity due to developing inhibitory synaptic transmission could induce synapse elimination and neurite pruning at particular time such as the critical period. Additionally, these findings indicate that we can estimate the maturity level of inhibitory transmission and the critical period by measuring the firing rate and the degree of synchronization in engineered neural networks.

Expression of odontogenic ameloblast-associated protein in the dental follicle and its role in osteogenic differentiation of dental follicle stem cells

ObjectiveOdontogenic Ameloblast-Associated Protein (ODAM) is encoded by a secretory calcium-binding phosphoprotein cluster gene, which generally plays an important role for mineralization. Dental follicle (DF) is essential in regulating bone formation for tooth eruption. This study aims to reveal ODAM expression in the DFs of developing and erupting molars, and to determine the possible role of ODAM. DesignDFs were collected from human third molars and rat mandibular molars for gene expression assessment and for establishment of cell cultures. RT-PCR and western blot were conducted to determine ODAM expression. Over- or silencing expression of ODAM in the dental follicle stem cells (DFSCs) was done by transfecting the cells with ODAM plasmid or siRNA to evaluate ODAM effects on osteogenesis. ResultsRat DFs weakly expressed ODAM at early-postnatal days, but a chronological increment of ODAM expression from days 1 to 11 was observed. Differences in expression of ODAM were seen in the human DFs of different individuals. In vitro, ODAM was expressed in DFSCs, but almost no expression in DF-derived fibroblast-like cells. Forcing the DFSCs to overexpress ODAM accelerated osteogenesis, whereas continuously silencing the ODAM in the DFSCs reduced osteogenesis only at 2 weeks of osteogenic induction. ConclusionsODAM is differentially expressed in the DFs of different age molars. Its expression is coincident with the increased bone formation of tooth crypt during tooth eruption in rat DFs. Increase of ODAM expression may accelerate osteogenic differentiation of DFSCs. Thus, ODAM expression in the DF may regulate bone formation for timely tooth eruption.

Characterization of vocalizations emitted in isolation by California mouse (Peromyscus californicus) pups throughout the postnatal period.

Rodent species, such as monogamous and biparental California mice, produce vocalizations as a means of communication. A temporal examination of vocalizations produced by California mice pups in isolation was performed. Pup recordings were performed for 3 min at ∼10.00 and 14.00 hrs on early postnatal days (PND) 2-4, 7, 21, and 28. Once initial recordings were finished, pups were returned to the home cage with parents and any siblings for 5 minutes to determine if active biparental responses resulted in an enhanced vocalization response when pups were isolated and retested. We also sought to determine whether potential reduction in vocalizations by older pups might be due to procedure-habituation procedure associated with less anxiety and/or possibly decreased need for parental care. Vocalizations were measured in weanling (30 days of age) "naïve" pups not previously isolated. Results show older pups took significantly longer to vocalize, indicated by increased latency before producing their initial syllable compared to earlier ages. With increasing age, pups demonstrated decreased syllable duration, reduced number and duration of phrases, and decreased number of syllables per phrase. No differences in pup vocalizations were observed before and after being placed back with parents, suggestive biparental potentiation may not exist in California mice pups. Comparison of the naïve to habituated weanling pups indicated the former group had more total calls but no other differences in vocalization parameters were detected between these 2 groups. Collectively, the findings suggest that as California mice pups mature and approach weaning they generally vocalize less in isolation. (PsycINFO Database Record

Early neonatal loss of inhibitory synaptic input to the spinal motor neurons confers spina bifida-like leg dysfunction in a chicken model.

ABSTRACTSpina bifida aperta (SBA), one of the most common congenital malformations, causes lifelong neurological complications, particularly in terms of motor dysfunction. Fetuses with SBA exhibit voluntary leg movements in utero and during early neonatal life, but these disappear within the first few weeks after birth. However, the pathophysiological sequence underlying such motor dysfunction remains unclear. Additionally, because important insights have yet to be obtained from human cases, an appropriate animal model is essential. Here, we investigated the neuropathological mechanisms of progression of SBA-like motor dysfunctions in a neural tube surgery-induced chicken model of SBA at different pathogenesis points ranging from embryonic to posthatch ages. We found that chicks with SBA-like features lose voluntary leg movements and subsequently exhibit lower-limb paralysis within the first 2 weeks after hatching, coinciding with the synaptic change-induced disruption of spinal motor networks at the site of the SBA lesion in the lumbosacral region. Such synaptic changes reduced the ratio of inhibitory-to-excitatory inputs to motor neurons and were associated with a drastic loss of γ-aminobutyric acid (GABA)ergic inputs and upregulation of the cholinergic activities of motor neurons. Furthermore, most of the neurons in ventral horns, which appeared to be suffering from excitotoxicity during the early postnatal days, underwent apoptosis. However, the triggers of cellular abnormalization and neurodegenerative signaling were evident in the middle- to late-gestational stages, probably attributable to the amniotic fluid-induced in ovo milieu. In conclusion, we found that early neonatal loss of neurons in the ventral horn of exposed spinal cord affords novel insights into the pathophysiology of SBA-like leg dysfunction.

Early Postnatal Lipopolysaccharide Exposure Leads to Enhanced Neurogenesis and Impaired Communicative Functions in Rats.

Perinatal infection is a well-identified risk factor for a number of neurodevelopmental disorders, including brain white matter injury (WMI) and Autism Spectrum Disorders (ASD). The underlying mechanisms by which early life inflammatory events cause aberrant neural, cytoarchitectural, and network organization, remain elusive. This study is aimed to investigate how systemic lipopolysaccharide (LPS)-induced neuroinflammation affects microglia phenotypes and early neural developmental events in rats. We show here that LPS exposure at early postnatal day 3 leads to a robust microglia activation which is characterized with mixed microglial proinflammatory (M1) and anti-inflammatory (M2) phenotypes. More specifically, we found that microglial M1 markers iNOS and MHC-II were induced at relatively low levels in a regionally restricted manner, whereas M2 markers CD206 and TGFβ were strongly upregulated in a sub-set of activated microglia in multiple white and gray matter structures. This unique microglial response was associated with a marked decrease in naturally occurring apoptosis, but an increase in cell proliferation in the subventricular zone (SVZ) and the dentate gyrus (DG) of hippocampus. LPS exposure also leads to a significant increase in oligodendrocyte lineage population without causing discernible hypermyelination. Moreover, LPS-exposed rats exhibited significant impairments in communicative and cognitive functions. These findings suggest a possible role of M2-like microglial activation in abnormal neural development that may underlie ASD-like behavioral impairments.