Neonatal and Juvenile Ocular Development in Sprague-Dawley Rats: A Histomorphological and Immunohistochemical Study.

As in many altricial species, rats are born with fused eyelids and markedly underdeveloped eyes. While the normal histology of the eyes of mature rats is known, the histomorphological changes occurring during postnatal eye development in this species remain incompletely characterized. This study was conducted to describe the postnatal development of ocular structures in Sprague-Dawley (SD) rats during the first month of age using histology and immunohistochemistry (IHC). Both eyes were collected from 51 SD rats at 13 time points between postnatal day (PND)1 and PND30. Histologic examination of hematoxylin and eosin-stained sections was performed, as well as IHC for cleaved-caspase-3 and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) to evaluate apoptosis, and IHC for Ki-67 and phospho-histone-H3 to evaluate cell proliferation. Extensive ocular tissue remodeling occurred prior to the eyelid opening around PND14 and reflected the interplay between apoptosis and cell proliferation. Apoptosis was particularly remarkable in the maturing subcapsular anterior epithelium of the lens, the inner nuclear and ganglion cell layers of the developing retina, and the Harderian gland, and was involved in the regression of the hyaloid vasculature. Nuclear degradation in the newly formed secondary lens fibers was noteworthy after birth and was associated with TUNEL-positive nuclear remnants lining the lens organelle-free zone. Cell proliferation was marked in the developing retina, cornea, iris, ciliary body and Harderian gland. The rat eye reached histomorphological maturity at PND21 after a rapid phase of morphological changes characterized by the coexistence of cell death and proliferation.

Methylation pattern variation between goats and rats during the onset of puberty.

Puberty is initiated by increased pulsatile gonadotropin-releasing hormone (GnRH) release from the hypothalamus. Epigenetic repression is thought to play a crucial role in the initiation of puberty, although the existence of analogous changes in methylation patterns across species is unclear. We analysed mRNA expression of DNA methyltransferases (DNMTs) and methyl-binding proteins (MBPs) in goats and rats by quantitative real-time PCR (qRT-PCR). DNA methylation profiles of hypothalamic were determined at the pre-pubertal and pubertal stages by bisulphite sequencing. In this study, expression of DNMTs and MBPs mRNA showed different patterns in goats and rats. Global methylation variation was low in goats and rats, and the profile remained stable during puberty. Gene ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis revealed the involvement of 62 pathways in puberty in goats and rats including reproduction, type I diabetes mellitus and GnRH signalling pathways and found that Edn3, PTPRN2 and GRID1 showed different methylation patterns during puberty in goats and rats and similar variation patterns for Edn3 and PTPRN2 were showed. These indicated that Edn3 and PTPRN2 would play a role in the timing of puberty. This study provides evidence of the epigenetic control of puberty.

[Effects of icariin total flavonoids capsule on bone mineral density and bone histomorphometry in growing rats].

Objective: To investigate the effect of icariin total flavonoids capsules (ITFC) on bone mineral density (BMD) and bone histomorphometry in growing rats and its anti-osteoporosis mechanism. Methods: Thirty female SD rats were randomly divided into 3 groups:normal control group, ITFC-1 group and ITFC-2 group. Rats in ITFC-1 group and ITFC-2 group were fed with 50 mg·kg-1·d-1 or 100 mg·kg-1·d-1 ITFC, respectively, and those in normal control group were fed with equal volume of distilled water. The whole body BMD was measured after 4, 8 and 12 weeks, and BMDs of the right femur and lumbar vertebrae were measured after 12 weeks. The serum levels of tartaric acid phosphatase 5b (TRACP 5b) and bone alkaline phosphatase (BALP) were measured by ELISA. Bone morphometry was performed on the right tibia. Results: There were no significant differences in the body weight increase between normal control group and two ITFC groups (all P>0.05). There were also no significant differences in whole body BMDs after 4 and 8 weeks between normal control group and ITFC groups (all P>0.05). After 12 weeks, the whole body BMD, BMD of bone in vitro, serum BALP level and trabecular area in ITFC-1 group and ITFC-2 group were significantly higher, trabecular separation was significantly lower than that in normal control group (all P<0.05); and the trabecular width and the number in ITFC-2 group were also significantly higher, and serum TRACP 5b level was significantly lower than that in normal control group (all P<0.05). The BMD of bone in vitro, serum BALP level, trabecular number and area in ITFC-2 group were significantly higher, and serum TRACP 5b level was significantly lower than that in ITFC-1 group (all P<0.05). Conclusion: ITFC can prevent osteoporosis by increasing bone density and bone formation, decreasing bone resorption and improving microstructure of bone.

Evaluation of the impact of transgenic maize BT799 on growth, development and reproductive function of Sprague-Dawley rats in three generations.

BT799 was Bacillus thuringiensis-genetic modified (GM) maize, and Sprague-Dawley (SD) rats were treated with different diet formulations containing BT799 maize grain (33% and 66%) or its non-transgenic Zhengdan 958 (ZD958, 33% and 66%). The feeding lasted for 10 (P)/14 (F1 and F2) weeks. The reproductive capacity and pathological responses were detected in each generation of rats fed with BT799 and ZD958. During the growth and development of parental rats, each group showed the same trend in body weight gain and food intake, with a few fluctuations at individual time points. No statistically significant difference was observed in reproductive data (copulation index, fertility index, and live birth rate) of rats fed with transgenic maize compared with non-transgenic maize. We observed some apparent changes in reproductive data (sperm numbers and motility) and pathological responses (organ relative weights, hematological parameters, serum chemistry parameters, and sex hormone levels) among rats fed with BT799 maize grain. However, these differences were within the laboratory's historical normal range of control SD rats and not maize grain dose-dependent. These changes were not considered to be adverse or toxic. No significant difference in macroscopic or histological adverse effects was observed between rats consuming transgenic BT799 diet and non-transgenic diet. In conclusion, the long-term intake of BT799 maize was as safe as the corresponding non-transgenic maize for three-generation SD rats.

Urinary Metabolic Profiling via LC-MS/MS Reveals Impact of Bovine Lactoferrin on Bone Formation in Growing SD Rats.

Lactoferrin (LF) exerts a promoting bone health function. The effects of LF on bone formation at the metabolic level have been less explored. Urinary metabolic profiling of growing Sprague-Dawley (SD) rats LF-supplemented (1000 mg/kg bw) for four weeks were explored by Liquid chromatography-tandem mass spectrometry (LC-MS/MS). The serum markers of bone formation and bone resorption, the bone mass, and the osteogenesis markers of femur were measured by an enzyme-linked immunosorbent assay, micro-computerized tomography, and immunohistochemistry, respectively. Compared with the control, LF supplementation improved bone formation (p < 0.05), reduced bone resorption (p < 0.05), enhanced femoral bone mineral density and microarchitecture (p < 0.05), and upregulated osteocalcin, osterix, and Runx-2 expression (p < 0.05) of femur. LF upregulated 69 urinary metabolites. KEGG and pathway enrichment analyses of those urinary metabolites, and the Person's correlation analyses among those urinary metabolites and bone status revealed that LF impacted on bone formation via regulatory comprehensive pathways including taurine and hypotaurine metabolism, arginine and proline metabolism, cyanoamino acid metabolism, nitrogen metabolism, nicotinate and nicotinamide metabolism, and fatty acid biosynthesis. The present study indicated the metabolomics is a useful and practical tool to elucidate the mechanisms by which LF augments bone mass formation in growing animals.

Altered motor development following late gestational alcohol and cannabinoid exposure in rats.

Cannabis is the most commonly used illicit drug among pregnant women, and rates are likely to increase given recent legalization. In addition, half of pregnant women who report consuming cannabis also report drinking alcohol. However, little is known about the consequences of prenatal cannabis alone or in combination with alcohol, particularly with cannabis products that are continually increasing in potency of the primary psychoactive constituent in cannabis, Δ9-tetrahydrocannabinol (THC). The current study investigated the effects of early exposure to cannabinoids during the brain growth spurt on early physical and motor development alone (Experiment 1) or in combination with alcohol (Experiment 2). In Experiment 1, Sprague-Dawley rat pups were exposed to a cannabinoid receptor agonist (CP-55,940 [CP]; 0.1, 0.25, 0.4 mg/kg/day), the drug vehicle, or a saline control from postnatal days (PD) 4-9. In Experiment 2, rat pups were exposed to CP (0.4 mg/kg/day) or the vehicle, and were additionally intubated with alcohol (11.9% v/v; 5.25 g/kg/day) or received a sham intubation. Subjects in both experiments were tested on a motor development task (PD 12-20) and a motor coordination task during adolescence (PD 30-32). Both developmental cannabinoid and alcohol exposure separately decreased body growth throughout development, and combined exposure exacerbated these effects, although only alcohol exposure induced long-term body weight reductions. Developmental cannabinoid exposure advanced early motor development, whereas alcohol exposure delayed development, and subjects given combined exposure did not differ from controls on some measures. Alcohol exposure impaired motor coordination later in life. In contrast, cannabinoid exposure, by itself, did not significantly affect long-term motor coordination, but did exacerbate alcohol-related impairments in motor coordination among females. These results suggest that cannabinoid exposure may not only alter development by itself, but may exacerbate alcohol's teratogenic effects in specific behavioral domains. These findings have important implications not only for individuals affected by prenatal exposure, but also for establishing public policy for women regarding cannabis use during pregnancy.

Effect of radioactive iodine-induced hypothyroidism on longitudinal bone growth during puberty in immature female rats.

Thyroid cancer in children, the most common endocrine malignancy, shows aggressive behavior and has a high recurrence rate after surgical ablation. Radioactive iodine (RAI) treatment is the most effective primary modality for medical ablation of juvenile thyroid cancer, and leads to intentional hypothyroidism. Although several negative impacts of hypothyroidism have been reported in children in response to other antithyroid agents, the combined effects of RAI exposure and hypothyroidism, on growing bones specifically, are unknown. In this study, we investigated the effect of RAI-induced hypothyroidism on the long bones during the pubertal growth spurt using immature female rats. Female Sprague-Dawley rats were randomly divided into a control group, and an RAI-treated group fed with RAI (0.37 MBq/g body weight) twice via gavage. After 4 weeks, we observed a significantly-reduced serum free thyroxine level in the RAI group. The latter group also displayed decreased body weight gain compared to the control. In addition, the lengths of long bones, such as the leg bones and vertebral column, as well as bone mineral content, were reduced in the RAI-treated animals. Our results confirm the negative impacts of RAI-induced thyroid deficiency during puberty on longitudinal bone growth and bone mineralization.

Organ growth functions in maturing male Sprague-Dawley rats based on a collective database.

Ten different organ weights (liver, spleen, kidneys, heart, lungs, brain, adrenals, testes, epididymes, and seminal vesicles) of male Sprague-Dawley (S-D) rats of different ages (1-280 d) were extracted based on a thorough literature survey database. A generalized Michaelis-Menten (GMM) model, used to fit organ weights versus age in a previous study (Schoeffner et al., 1999) based on a limited data, was used to find the best fit model for the present expanded data compilation. The GMM model has the functional form: Wt = (Wt(o).K(gamma) + Wt(max).Age(gamma))/(K(gamma) + Age(gamma)) where Wt is organ/tissue weight at a specified age, Wt(o) and Wt(max) are weight at birth and maximal growth, respectively, and K and gamma are constants. Organ weights were significantly correlated with their respective ages for all organs and tissues. GMM-derived organ growth and percent body weight (%BW) fractions of different tissues were plotted against animal age and compared with experimental values as well as previously published models. The GMM-based organ growth and %BW fraction profiles were in general agreement with our empirical data as well as with previous studies. The present model was compared with the GMM model developed previously for six organs--liver, spleen, kidneys, heart, lungs, and brain--based on a limited data, and no significant difference was noticed between the two sets of predictions. It was concluded that the GMM models presented herein for different male S-D rats organs (liver, spleen, kidneys, heart, lungs, brain, adrenals, testes, epididymes, and seminal vesicles) are capable of predicting organ weights and %BW ratios accurately at different ages.

The role of microbiota in compensatory growth of protein-restricted rats.

Compensatory growth is a physiological phenomenon found in both humans and animals. However, the underlying mechanisms are unclear. In this study, for the first time, we investigated the role of microbiota in compensatory growth induced by protein restriction using a rat model. Weaned Sprague-Dawley rats were fed a low protein diet (L group), a normal protein diet (N group) and a low protein diet for 2 weeks followed by a normal protein diet (LN group). The results showed that in contrast with the inhibited growth of rats in the L group, compensatory growth was observed in the LN group. Meanwhile, rats in the LN group had increased concentrations of total short chain fatty acids, particularly butyrate, and an altered bacterial composition with modified abundances of Peptostreptococcaceae, Bifidobacteriaceae, Porphyromonadaceae and Prevotellaceae in the colonic content. Furthermore, gene expression analysis indicated that the rats that experienced compensatory growth had improved barrier function and innate immune function in the colon. Our data revealed the importance of colonic microbiota in achieving compensatory growth.

Can a Small Intestine Segment Be an Alternative Biological Conduit for Peripheral Nerve Regeneration?

BACKGROUND: Autologous nerve grafts are used to bridge peripheral nerve defects. Limited sources and donor site morbidity are the major problems with peripheral nerve grafts. Although various types of autologous grafts such as arteries, veins and muscles have been recommended, an ideal conduit has not yet been described. AIMS: To investigate the effectiveness of a small intestinal conduit for peripheral nerve defects. STUDY DESIGN: Animal experimentation. METHODS: Twenty-one rats were divided into three groups (n=7). Following anaesthesia, sciatic nerve exploration was performed in the Sham group. The 10 mm nerve gap was bridged with a 15 mm ileal segment in the small intestinal conduit group and the defect was replaced with orthotopic nerve in autologous nerve graft group. The functional recovery was tested monthly by walking-track analysis and the sciatic functional index. Histological evaluation was performed on the 12th week. RESULTS: Sciatic functional index tests are better in autologous nerve graft group (-55.09±6.35); however, during follow-up, progress in sciatic functional index was demonstrated, along with axonal regeneration and innervation of target muscles in the small intestinal conduit group (-76.36±12.08) (p<0.05). In histologic sections, distinctive sciatic nerve regeneration was examined in the small intestinal conduit group. The expression of S-100 and neurofilament was observed in small intestinal conduit group but was less organised than in the autologous nerve graft group. Although the counted number (7459.79±1833.50 vs. 4226.51±1063.06 mm2), measured diameter [2.19 (2.15-2.88) vs. 1.74 (1.50-2.09) µm] and myelin sheath thickness [1.18 (1.09-1.44) vs. 0.66 (0.40-1.07) µm] of axons is significantly high in the middle sections of autologous nerve graft compared to the small intestinal conduit group, respectively (p<0.05), the peripheral nerve regeneration was also observed in the small intestinal conduit group. CONCLUSION: Small intestinal conduit should not be considered as an alternative to autologous nerve grafts in its current form; however, the results are promising. Even though the results are no better than autologous nerve grafts, with additional procedures, it might be a good alternative due to harvesting abundant sources without donor site morbidity.

Beneficial effects of a novel shark-skin collagen dressing for the promotion of seawater immersion wound healing.

BACKGROUND: Wounded personnel who work at sea often encounter a plethora of difficulties. The most important of these difficulties is seawater immersion. Common medical dressings have little effect when the affected area is immersed in seawater, and only rarely dressings have been reported for the treatment of seawater-immersed wounds. The objective of this study is to develop a new dressing which should be suitable to prevent the wound from seawater immersion and to promote the wound healing. METHODS: Shark skin collagen (SSC) was purified via ethanol de-sugaring and de-pigmentation and adjusted for pH. A shark skin collagen sponge (SSCS) was prepared by freeze-drying. SSCS was attached to an anti-seawater immersion polyurethane (PU) film (SSCS + PU) to compose a new dressing. The biochemical properties of SSC and physicochemical properties of SSCS were assessed by standard methods. The effects of SSCS and SSCS + PU on the healing of seawater-immersed wounds were studied using a seawater immersion rat model. For the detection of SSCS effects on seawater-immersed wounds, 12 SD rats, with four wounds created in each rat, were divided into four groups: the 3rd day group, 5th day group, 7th day group and 12th day group. In each group, six wounds were treated with SSCS, three wounds treated with chitosan served as the positive control, and three wounds treated with gauze served as the negative control. For the detection of the SSCS + PU effects on seawater-immersed wounds, 36 SD rats were divided into three groups: the gauze (GZ) + PU group, chitosan (CS) + PU group and SSCS + PU group, with 12 rats in each group, and two wounds in each rat. The wound sizes were measured to calculate the healing rate, and histomorphology and the immunohistochemistry of the CD31 and TGF-ß expression levels in the wounded tissues were measured by standard methods. RESULTS: The results of Ultraviolet-visible (UV-vis) spectrum, Fourier-transform infrared (FTIR) spectrum, circular dichroism (CD) spectra, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and amino acid composition analyses of SSC demonstrated that SSC is type I collagen. SSCS had a homogeneous porous structure of approximately 200 µm, porosity rate of 83.57% ± 2.64%, water vapor transmission ratio (WVTR) of 4500 g/m2, tensile strength of 1.79 ± 0.41 N/mm, and elongation at break of 4.52% ± 0.01%. SSCS had significant beneficial effects on seawater-immersed wound healing. On the 3rd day, the healing rates in the GZ negative control, CS positive control and SSCS rats were 13.94% ± 5.50%, 29.40% ± 1.10% and 47.24% ± 8.40%, respectively. SSCS also enhanced TGF-ß and CD31 expression in the initial stage of the healing period. The SSCS + PU dressing effectively protected wounds from seawater immersion for at least 4 h, and accelerated re-epithelialization, vascularization and granulation formation of seawater-immersed wounds in the earlier stages of wound healing, and as well as significantly promoted wound healing. The SSCS + PU dressing also enhanced expression of TGF-ß and CD31. The effects of SSCS and SSCS + PU were superior to those of both the chitosan and gauze dressings. CONCLUSIONS: SSCS has significant positive effects on the promotion of seawater-immersed wound healing, and a SSCS + PU dressing effectively prevents seawater immersion, and significantly promotes seawater-immersed wound healing.

High-Iron Consumption Impairs Growth and Causes Copper-Deficiency Anemia in Weanling Sprague-Dawley Rats.

Iron-copper interactions were described decades ago; however, molecular mechanisms linking the two essential minerals remain largely undefined. Investigations in humans and other mammals noted that copper levels increase in the intestinal mucosa, liver and blood during iron deficiency, tissues all important for iron homeostasis. The current study was undertaken to test the hypothesis that dietary copper influences iron homeostasis during iron deficiency and iron overload. We thus fed weanling, male Sprague-Dawley rats (n = 6-11/group) AIN-93G-based diets containing high (~8800 ppm), adequate (~80) or low (~11) iron in combination with high (~183), adequate (~8) or low (~0.9) copper for 5 weeks. Subsequently, the iron- and copper-related phenotype of the rats was assessed. Rats fed the low-iron diets grew slower than controls, with changes in dietary copper not further influencing growth. Unexpectedly, however, high-iron (HFe) feeding also impaired growth. Furthermore, consumption of the HFe diet caused cardiac hypertrophy, anemia, low serum and tissue copper levels and decreased circulating ceruloplasmin activity. Intriguingly, these physiologic perturbations were prevented by adding extra copper to the HFe diet. Furthermore, higher copper levels in the HFe diet increased serum nonheme iron concentration and transferrin saturation, exacerbated hepatic nonheme iron loading and attenuated splenic nonheme iron accumulation. Moreover, serum erythropoietin levels, and splenic erythroferrone and hepatic hepcidin mRNA levels were altered by the dietary treatments in unanticipated ways, providing insight into how iron and copper influence expression of these hormones. We conclude that high-iron feeding of weanling rats causes systemic copper deficiency, and further, that copper influences the iron-overload phenotype.

The alpha- and beta-isoforms of the inhibitor protein of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase: characteristics and tissue- and developmental-specific expression.

The inhibitor protein (PKI) of the cAMP-dependent protein kinase was first characterized from rabbit skeletal muscle. More recently a form of PKI was isolated and cloned from rat testis which shares relatively limited amino acid sequence with the rabbit skeletal muscle form. We have now isolated a cDNA from rat brain which encodes a protein corresponding to the rabbit skeletal muscle PKI. This establishes the presence of the "skeletal muscle" and "testis" proteins in the same species and therefore that they clearly represent distinct isoforms. We have also demonstrated that the isoform from testis, like the skeletal muscle isoform, is specific for the cAMP-dependent protein kinase and that it is able to inhibit this enzyme when expressed in cultured JEG-3 cells. Both forms contain the five specific amino acid recognition determinants which have been shown to be required for high affinity binding to the protein kinase catalytic site, although there is some noted lack of conservation of codons used for these residues. Overall, the two rat isoforms are only 41% identical at the amino acid level and 46% at the level of coding nucleotides. We propose that the rabbit skeletal muscle and rat testis forms be designated PKI alpha and PKI beta, respectively. Using Northern blot analysis, we have examined the tissue distribution of the two forms in the rat and their relative expression during development. In the adult rat, mRNA of the PKI alpha species is highest in muscle (both skeletal and cardiac) and brain (cortex and cerebellum).(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of red light exposure at night on circadian metabolism and physiology in Sprague-Dawley rats.

Early studies on rodents showed that short-term exposure to high-intensity light (> 70 lx) above 600 nm (red-appearing) influences circadian neuroendocrine and metabolic physiology. Here we addressed the hypothesis that long-term, low-intensity red light exposure at night (rLEN) from a 'safelight' emitting no light below approximately 620 nm disrupts the nocturnal circadian melatonin signal as well as circadian rhythms in circulating metabolites, related regulatory hormones, and physi- ologic parameters. Male Sprague-Dawley rats (n = 12 per group) were maintained on control 12:12-h light:dark (300 lx; lights on, 0600) or experimental 12:12 rLEN (8.1 lx) lighting regimens. After 1 wk, rats underwent 6 low-volume blood draws via cardiocentesis (0400, 0800, 1200, 1600, 2000, and 2400) over a 4-wk period to assess arterial plasma melatonin, total fatty acid, glucose, lactic acid, pO2, pCO2, insulin, leptin and corticosterone concentrations. Results revealed plasma melatonin levels (mean ± 1 SD) were high in the dark phase (197.5 ± 4.6 pg/mL) and low in the light phase (2.6 ± 1.2 pg/mL) of control condi- tions and significantly lower than controls under experimental conditions throughout the 24-h period (P < 0.001). Prominent circadian rhythms of plasma levels of total fatty acid, glucose, lactic acid, pO2, pCO2, insulin, leptin, and corticosterone were significantly (P < 0.05) disrupted under experimental conditions as compared with the corresponding entrained rhythms under control conditions. Therefore, chronic use of low-intensity rLEN from a common safelight disrupts the circadian organization of neuroendocrine, metabolic, and physiologic parameters indicative of animal health and wellbeing.

Borna disease virus infection impacts microRNAs associated with nervous system development, cell differentiation, proliferation and apoptosis in the hippocampi of neonatal rats.

MicroRNAs (miRNAs) regulate gene expression by inhibiting transcription or translation and are involved in diverse biological processes, including development, cellular differentiation and tumor generation. miRNA microarray technology is a high­throughput global analysis tool for miRNA expression profiling. Here, the hippocampi of four borna disease virus (BDV)­infected and four non­infected control neonatal rats were selected for miRNA microarray and bioinformatic analysis. Reverse transcription quantitative polymerase chain reaction (RT­qPCR) analysis was subsequently performed to validate the dysregulated miRNAs. Seven miRNAs (miR­145*, miR­146a*, miR­192*, miR­200b, miR­223*, miR­449a and miR­505), showed increased expression, whereas two miRNAs (miR­126 and miR­374) showed decreased expression in the BDV­infected group. By RT­qPCR validation, five miRNAs (miR­126, miR­200b, miR­374, miR­449a and miR­505) showed significantly decreased expression (P<0.05) in response to BDV infection. Biocarta pathway analysis predicted target genes associated with 'RNA', 'IGF1mTOR', 'EIF2', 'VEGF', 'EIF', 'NTHI', 'extrinsic', 'RB', 'IL1R' and 'IGF1' pathways. Gene Ontology analysis predicted target genes associated with 'peripheral nervous system development', 'regulation of small GTPase-mediated signal transduction', 'regulation of Ras protein signal transduction', 'aerobic respiration', 'membrane fusion', 'positive regulation of cell cycle', 'cellular respiration', 'heterocycle metabolic process', 'protein tetramerization' and 'regulation of Rho protein signal transduction' processes. Among the five dysregulated miRNAs identified by RT­qPCR, miR­126, miR­200b and miR­449a showed a strong association with nervous system development, cell differentiation, proliferation and apoptosis.

Patterns of Spontaneous Local Network Activity in Developing Cerebral Cortex: Relationship to Adult Cognitive Function.

Detecting neurodevelopµental disorders of cognition at the earliest possible stages could assist in understanding them mechanistically and ultimately in treating them. Finding early physiological predictors that could be visualized with functional neuroimaging would represent an important advance in this regard. We hypothesized that one potential source of physiological predictors is the spontaneous local network activity prominent during specific periods in development. To test this we used calcium imaging in brain slices and analyzed variations in the frequency and intensity of this early activity in one area, the entorhinal cortex (EC), in order to correlate early activity with level of cognitive function later in life. We focused on EC because of its known role in different types of cognitive processes and because it is an area where spontaneous activity is prominent during early postnatal development in rodent models of cortical development. Using rat strains (Long-Evans, Wistar, Sprague-Dawley and Brattleboro) known to differ in cognitive performance in adulthood we asked whether neonatal animals exhibit corresponding strain-related differences in EC spontaneous activity. Our results show significant differences in this activity between strains: compared to a high cognitive-performing strain, we consistently found an increase in frequency and decrease in intensity in neonates from three lower performing strains. Activity was most different in one strain considered a model of schizophrenia-like psychopathology. While we cannot necessarily infer a causal relationship between early activity and adult cognition our findings suggest that the pattern of spontaneous activity in development could be an early predictor of a developmental trajectory advancing toward sub-optimal cognitive performance in adulthood. Our results further suggest that the strength of dopaminergic signaling, by setting the balance between excitation and inhibition, is a potential underlying mechanism that could explain the observed differences in early spontaneous activity patterns.

Distribution of corticospinal motor neurons in the postnatal rat: quantitative evidence for massive collateral elimination and modest cell death.

The postnatal development of rat corticospinal motor neurons (CSMN) was studied by retrograde tracing with cholera toxin B subunit (CTB) injected into the upper cervical dorsal spinal cord on the first postnatal day (P0), P3, P10, P20, and at adulthood. CTB-labeled neurons were visualized by immunocytochemistry and extensively quantified throughout the cortex. At P0, CSMN were found to an extent similar to that reported in P3 animals with other neuronal tracers, now permitting in vitro studies of neonatal CSMN. Between P0 and P3, the number of labeled neurons increased by 30% to a total maximum of approximately 185,000 in both cortices. The increase occurred throughout the cortex. At P10, the number of labeled CSMN had decreased to 60% of the number at P3. Fewer CSMN were evident particularly in the perirhinal cortex. Between P10 and P20, the number of CSMN decreased further to 52% of the maximal number at P3. This decrease occurred predominantly in the cingulate and parietal cortex. The number of labeled CSMN in rats injected at P0 and analyzed at P20 was 10% lower than the number in P0-injected littermates that were analyzed at P3, which suggests that only a small portion of the "disappearing" CSMN undergoes developmental neuronal death. Thus, the spinal projection of the remaining 38% is apparently eliminated between P3 and P20. Detailed quantitative analysis of the CSMN distribution demonstrated that neuronal death occurs predominantly in the perirhinal cortex. In contrast, axonal elimination of corticospinal projections occurred throughout the CSMN field, i.e., primarily in the frontal, occipital, and perirhinal cortex between P3-P10 and in the cingulate and parietal cortex between P10-P20.

Genesis and fate of the perireticular thalamic nucleus during early development.

A striking feature of the internal capsule during early development is that it is full of small neurones. Later, this group of neurones, called the perireticular thalamic nucleus, appears to have reduced in size, and only a few scattered cells are seen. In an effort to understand better the developmental history of the perireticular nucleus this study examines: i) the period of cell generation in the nucleus, ii) the magnitude of cell loss in the nucleus, and iii) the subsequent fate of cells in the nucleus during development. The perireticular cells are generated very early in development, being among the first generated in the thalamus (rats: E13-14; cats: E21-30). In rats, the first perireticular cells are generated at about the same developmental stage as the first subplate cells, which are among the first generated cells of the cortex: in cats, the first perireticular cells are generated well before those in the subplate (E24-30). In rats, the number of perireticular cells during developmental peaks at P5 (approximately 30,000) and then declines sharply (approximately 98%) by P15 (approximately 750), when adult-like patterns are seen. This dramatic loss of perireticular cells is due to both cell death and a migration of cells into the adjacent globus pallidus. The majority of the perireticular cells which migrate into the globus pallidus, however, are likely to die also. The presence of pyknotic profiles (indicators of dying cells) in the rat perireticular nucleus points to cell death as a contributor to the reduction in cell number during development. In this study, a period of relatively high pyknotic profile incidence (number of pyknotic cells per 1,000 "living" cells) is recorded in the perireticular nucleus over a 5 day period, from P2 to P7 (13.5-15.5). Similar values and patterns are recorded in the reticular nucleus and globus pallidus, except that in these structures, a period of relatively high pyknotic profile incidence (15-20) occurs over a shorter period (3 days; P2-5). Previous studies have suggested that some perireticular cells migrate into and settle within the adjacent globus pallidus. This study, with the use of long-term survivals after tracer injections in rats, shows that none (or very few) of these perireticular cells which migrate into the globus pallidus survive into more mature postnatal stages. Tracer (biotinylated dextran) was injected into the sensory nuclei of the dorsal thalamus at early stages (P7) and the rats were allowed to survive for either a day thereafter (to P8) or until well after the period of cell death was complete (to P16 or P21). In the short-term survivals (to P8), there are many dextran-labelled cells seen in the globus pallidus and in the perireticular nucleus. In the long-term survivals (to P16 or P21), by contrast, there are no dextran-labelled cells apparent in the globus pallidus or in the perireticular nucleus. It is likely that these cells in the globus pallidus, as with those in the perireticular nucleus, undergo cell death during development.

Expression of two forms of glutamic acid decarboxylase (GAD67 and GAD65) during postnatal development of rat somatosensory barrel cortex.

The postnatal development of glutamic acid decarboxylase (GAD; GAD67 and GAD65) expression was studied in the rat somatosensory cortex. Delineation of barrels in layer IV by GAD67 immunoreactivity occurred between postnatal days P3 and P6 and remained evident into adulthood. At birth, a band of GAD67-positive elements was already present in superficial layer V. This band was prominent until P6 and gradually disappeared after P9. In parallel, there was a gradual appearance of GAD67-immunoreactive cells neuropil and puncta, which began in layer VI/subplate at P1 and achieved the adult laminar pattern by about P13. This later GAD67 immunoreactivity was responsible for the demarcation of barrels in layer IV. Development of GAD65 immunoreactivity was delayed relative to GAD67. GAD65 immunoreactivity, which was in little evidence before P6, increased markedly in density and in delineation of cell bodies over the next several weeks. During this prolonged developmental process, GAD65 first showed a negative image of the barrels compared with the septae and the surrounding cortex. Subsequently, there was a filling in of the barrels resulting in rather uniform GAD65 immunoreactivity across the barrel field and surrounding cortex. These results suggest that the development of the gamma-aminobutyric acid (GABA) synthetic system in the barrel cortex involves several processes: the disappearance of a precocious GAD67 system in layer V, the temporally overlapping maturation of the mature GAD67 system in an inside-outside manner, and the delayed and prolonged development of the GAD65 system.

Estrogen receptor beta immunoreactivity in differentiating cells of the developing rat cerebellum.

Estrogen receptors (ER) play a significant role in the development of some regions of the mammalian brain. Recently, ER-beta (ERbeta) mRNA and protein were shown to be expressed in the rat cerebellum. In the present study, the ontogeny of ERbeta protein expression was examined in the rat cerebellum during postnatal development. Western blot analysis indicated that a single ERbeta-like immunoreactive species of approximately 55 kDa was present in protein lysates prepared from the cerebella of female and male Sprague-Dawley rat pups. Immunocytochemical analysis of cerebellar sections from the midline vermis revealed that during development, the expression of ERbeta varied with age and cell-type, but not sex. In the developing cerebellum, highest levels of ERbeta-immunoreactivity (IR) were detected in neurons during neurite growth, and in some glia during migration. Throughout the first postnatal week, ERbeta-IR was localized to differentiating granule cells in the external germinal layer and to migrating glia. Differentiating granule cells expressed detectable levels of ERbeta throughout development. In Purkinje cells, ERbeta-IR was first detected on postnatal day 6 (P6), with peak intensities of immunostaining coinciding with the initiation of axonal and dendritic growth that occurs between P7 and P8. Expression of ERbeta-IR remained high during maturation of Purkinje cell dendrites, and then decreased to a lower level maintained in the adult. From the third postnatal week, ERbeta-IR was also detected in the later developing Golgi, stellate, and basket neurons. These results suggest that ERbeta may play a role in growth-related mechanisms during differentiation of cerebellar neurons and glia.