Spaceflight and hind limb unloading induce similar changes in electrical impedance characteristics of mouse gastrocnemius muscle.

OBJECTIVE: To assess the potential of electrical impedance myography (EIM) to serve as a marker of muscle fiber atrophy and secondarily as an indicator of bone deterioration by assessing the effects of spaceflight or hind limb unloading. METHODS: In the first experiment, 6 mice were flown aboard the space shuttle (STS-135) for 13 days and 8 earthbound mice served as controls. In the second experiment, 14 mice underwent hind limb unloading (HLU) for 13 days; 13 additional mice served as controls. EIM measurements were made on ex vivo gastrocnemius muscle. Quantitative microscopy and areal bone mineral density (aBMD) measurements of the hindlimb were also performed. RESULTS: Reductions in the multifrequency phase-slope parameter were observed for both the space flight and HLU cohorts compared to their respective controls. For ground control and spaceflight groups, the values were 24.7±1.3°/MHz and 14.1±1.6°/MHz, respectively (p=0.0013); for control and HLU groups, the values were 23.9±1.6°/MHz and 19.0±1.0°/MHz, respectively (p=0.014). This parameter also correlated with muscle fiber size (ρ=0.65, p=0.011) for spaceflight and hind limb aBMD (ρ=0.65, p=0.0063) for both groups. CONCLUSIONS: These data support the concept that EIM may serve as a useful tool for assessment of muscle disuse secondary to immobilization or microgravity.

Embryonic disruption of the candidate dyslexia susceptibility gene homolog Kiaa0319-like results in neuronal migration disorders.

Developmental dyslexia, the most common childhood learning disorder, is highly heritable, and recent studies have identified KIAA0319-Like (KIAA0319L) as a candidate dyslexia susceptibility gene at the 1p36-34 (DYX8) locus. In this experiment, we investigated the anatomical effects of knocking down this gene during rat corticogenesis. Cortical progenitor cells were transfected using in utero electroporation on embryonic day (E) 15.5 with plasmids encoding either: (1) Kiaa0319l small hairpin RNA (shRNA), (2) an expression construct for human KIAA0319L, (3) Kiaa0319l shRNA+KIAA0319L expression construct (rescue), or (4) controls (scrambled Kiaa0319l shRNA or empty expression vector). Mothers were injected with 5-bromo-2-deoxyuridine (BrdU) at either E13.5, E15.5, or E17.5. Disruption of Kiaa0319l function (by knockdown, overexpression, or rescue) resulted in the formation of large nodular periventricular heterotopia in approximately 25% of the rats, which can be seen as early as postnatal day 1. Only a small subset of heterotopic neurons had been transfected, indicating non-cell autonomous effects of the transfection. Most heterotopic neurons were generated in mid- to late-gestation, and laminar markers suggest that they were destined for upper cortical laminae. Finally, we found that transfected neurons in the cerebral cortex were located in their expected laminae. These results indicate that KIAA0319L is the fourth of four candidate dyslexia susceptibility genes that is involved in neuronal migration, which supports the association of abnormal neuronal migration with developmental dyslexia.

Electrical impedance alterations in the rat hind limb with unloading.

OBJECTIVES: Methods are needed for quantifying muscle deconditioning due to immobilization, aging, or spaceflight. Electrical impedance myography (EIM) is one technique that may offer easy-to-follow metrics. Here, we evaluate the time course and character of the change in single- and multi-frequency EIM parameters in the hind-limb suspension model of muscle deconditioning in rats. METHODS: Sixty-two rats were studied with EIM during a two-week period of hind limb unloading followed by a two-week recovery period. Random subsets of animals were sacrificed at one-week time intervals to measure muscle fiber size. RESULTS: Significant alterations were observed in nearly all impedance parameters. The 50 kHz phase and multi-frequency phase-slope, created by taking the slope of a line fitted to the impedance values between 100-500 kHz, appeared most sensitive to disuse atrophy, the latter decreasing by over 33.0±6.6% (p<0.001), a change similar to the maximum reduction in muscle fiber size. Impedance alterations, however, lagged changes in muscle fiber size. CONCLUSIONS: EIM is sensitive to disuse change in the rat, albeit with a delay relative to alterations in muscle fiber size. Given the rapidity and simplicity of EIM measurements, the technique could prove useful in providing a non-invasive approach to measuring disuse change in animal models and human subjects.

Bilateral subcortical heterotopia with partial callosal agenesis in a mouse mutant.

Cognition and behavior depend on the precise placement and interconnection of complex ensembles of neurons in cerebral cortex. Mutations that disrupt migration of immature neurons from the ventricular zone to the cortical plate have provided major insight into mechanisms of brain development and disease. We have discovered a new and highly penetrant spontaneous mutation that leads to large nodular bilateral subcortical heterotopias with partial callosal agenesis. The mutant phenotype was first detected in a colony of fully inbred BXD29 mice already known to harbor a mutation in Tlr4. Neurons confined to the heterotopias are mainly born in midgestation to late gestation and would normally have migrated into layers 2-4 of overlying neocortex. Callosal cross-sectional area and fiber number are reduced up to 50% compared with coisogenic wildtype BXD29 substrain controls. Mutants have a pronounced and highly selective defect in rapid auditory processing. The segregation pattern of the mutant phenotype is most consistent with a two-locus autosomal recessive model, and selective genotyping definitively rules out the Tlr4 mutation as a cause. The discovery of a novel mutation with strong pleiotropic anatomical and behavioral effects provides an important new resource for dissecting molecular mechanisms and functional consequences of errors of neuronal migration.

Persistent spatial working memory deficits in rats following in utero RNAi of Dyx1c1.

Disruptions in the development of the neocortex are associated with cognitive deficits in humans and other mammals. Several genes contribute to neocortical development, and research into the behavioral phenotype associated with specific gene manipulations is advancing rapidly. Findings include evidence that variants in the human gene DYX1C1 may be associated with an increased risk of developmental dyslexia. Concurrent research has shown that the rat homolog for this gene modulates critical parameters of early cortical development, including neuronal migration. Moreover, recent studies have shown auditory processing and spatial learning deficits in rats following in utero transfection of an RNA interference (RNAi) vector of the rat homolog Dyx1c1 gene. The current study examined the effects of in utero RNAi of Dyx1c1 on working memory performance in Sprague-Dawley rats. This task was chosen based on the evidence of short-term memory deficits in dyslexic populations, as well as more recent evidence of an association between memory deficits and DYX1C1 anomalies in humans. Working memory performance was assessed using a novel match-to-place radial water maze task that allows the evaluation of memory for a single brief (∼4-10 seconds) swim to a new goal location each day. A 10-min retention interval was used, followed by a test trial. Histology revealed migrational abnormalities and laminar disruption in Dyx1c1 RNAi-treated rats. Dyx1c1 RNAi-treated rats exhibited a subtle, but significant and persistent impairment in working memory as compared to Shams. These results provide further support for the role of Dyx1c1 in neuronal migration and working memory.

The effects of embryonic knockdown of the candidate dyslexia susceptibility gene homologue Dyx1c1 on the distribution of GABAergic neurons in the cerebral cortex.

Developmental dyslexia is a language-based learning disability, and a number of candidate dyslexia susceptibility genes have been identified, including DYX1C1, KIAA0319, and DCDC2. Knockdown of function by embryonic transfection of small hairpin RNA (shRNA) of rat homologues of these genes dramatically disrupts neuronal migration to the cerebral cortex by both cell autonomous and non-cell autonomous effects. Here we sought to investigate the extent of non-cell autonomous effects following in utero disruption of the candidate dyslexia susceptibility gene homolog Dyx1c1 by assessing the effects of this disruption on GABAergic neurons. We transfected the ventricular zone of embryonic day (E) 15.5 rat pups with either Dyx1c1 shRNA, DYX1C1 expression construct, both Dyx1c1 shRNA and DYX1C1 expression construct, or a scrambled version of Dyx1c1 shRNA, and sacrificed them at postnatal day 21. The mothers of these rats were injected with BrdU at either E13.5, E15.5, or E17.5. Neurons transfected with Dyx1c1 shRNA were bi-modally distributed in the cerebral cortex with one population in heterotopic locations at the white matter border and another migrating beyond their expected location in the cerebral cortex. In contrast, there was no disruption of migration following transfection with the DYX1C1 expression construct. We found untransfected GABAergic neurons (parvalbumin, calretinin, and neuropeptide Y) in the heterotopic collections of neurons in Dyx1c1 shRNA treated animals, supporting the hypothesis of non-cell autonomous effects. In contrast, we found no evidence that the position of the GABAergic neurons that made it to the cerebral cortex was disrupted by the embryonic transfection with any of the constructs. Taken together, these results support the notion that neurons within heterotopias caused by transfection with Dyx1c1 shRNA result from both cell autonomous and non-cell autonomous effects, but there is no evidence to support non-cell autonomous disruption of neuronal position in the cerebral cortex itself.

Genetic modulation of striatal volume by loci on Chrs 6 and 17 in BXD recombinant inbred mice.

Natural variation in the absolute and relative size of different parts of the human brain is substantial, with a range that often exceeds a factor of 2. Much of this variation is generated by the cumulative effects of sets of unknown gene variants that modulate the proliferation, growth and death of neurons and glial cells. Discovering and testing the functions of these genes should contribute significantly to our understanding of differences in brain development, behavior and disease susceptibility. We have exploited a large population of genetically well-characterized strains of mice (BXD recombinant inbred strains) to map gene variants that influence the volume of the dorsal striatum (caudate-putamen without nucleus accumbens). We used unbiased methods to estimate volumes bilaterally in a sex-balanced sample taken from the Mouse Brain Library (www.mbl.org). We generated a matched microarray data set to efficiently evaluate candidate genes (www.genenetwork.org). As in humans, volume of the striatum is highly heritable, with greater than twofold differences among strains. We mapped a locus that modulates striatal volume on chromosome (Chr) 6 at 88 +/- 5 Mb. We also uncovered an epistatic interaction between loci on Chr 6 and Chr 17 that modulates striatal volume. Using bioinformatic tools and the corresponding expression database, we have identified positional candidates in these quantitative trait locus intervals.

Air pollution induces enhanced mitochondrial oxidative stress in cystic fibrosis airway epithelium.

We studied the effects of airborne particulate matters (PM) on cystic fibrosis (CF) epithelium. We noted that PM enhanced human CF bronchial epithelial apoptosis, activated caspase-9 and PARP-1; and reduced mitochondrial membrane potential. Mitochondrial inhibitors (4,4-diisothiocyanatostilbene-2,2'disulfonic acid, rotenone and thenoyltrifluoroacetone) blocked PM-induced generation of reactive oxygen species and apoptosis. PM upregulated pro-apoptotic Bad, Bax, p53 and p21; and enhanced mitochondrial localization of Bax. The anti-apoptotic Bcl-2, Bcl-xl, Mcl-1 and Xiap remained unchanged; however, overexpression of Bcl-xl blocked PM-induced apoptosis. Accordingly, we provide the evidence that PM enhances oxidative stress and mitochondrial signaling mediated apoptosis via the modulation of Bcl family proteins in CF.

Spectral processing deficits in belt auditory cortex following early postnatal lesions of somatosensory cortex.

Induced or genetically based cortical laminar malformations in somatosensory cortex have been associated with perceptual and acoustic processing deficits in mammals. Perinatal freeze-lesions of developing rat primary somatosensory (S1) cortex induce malformations resembling human microgyria. Induced microgyria located in parietal somatosensory cortex have been linked to reduced behavioral detection of rapid sound transitions and altered spectral processing in primary auditory cortex (A1). Here we asked whether belt auditory cortex function would be similarly altered in rats with S1 microgyria (MG+). Pure-tone acoustic response properties were assessed in A1 and ventral auditory (VAF) cortical fields with Fourier optical imaging and multi-unit recordings. Three changes in spectral response properties were observed in both A1 and VAF in MG+ rats: 1) multi-unit response magnitudes were reduced 2) optical and multi-unit frequency responses were more variable; 3) at high sound levels units responded to a broader range of pure-tone frequencies. Optical and multi-unit pure-tone response magnitudes were both reduced for low sound levels in VAF but not A1. Sound level "tuning" was reduced in VAF but not in A1. Finally, in VAF frequency tuning and spike rates near best frequency were both altered for mid- but not high-frequency recording sites. These data suggest that VAF belt auditory cortex is more vulnerable than A1 to early postnatal induction of microgyria in neighboring somatosensory cortex.

Postnatal analysis of the effect of embryonic knockdown and overexpression of candidate dyslexia susceptibility gene homolog Dcdc2 in the rat.

Embryonic knockdown of candidate dyslexia susceptibility gene (CDSG) homologs in cerebral cortical progenitor cells in the rat results in acute disturbances of neocortical migration. In the current report we investigated the effects of embryonic knockdown and overexpression of the homolog of DCDC2, one of the CDSGs, on the postnatal organization of the cerebral cortex. Using a within-litter design, we transfected cells in rat embryo neocortical ventricular zone around embryonic day (E) 15 with either 1) small hairpin RNA (shRNA) vectors targeting Dcdc2, 2) a DCDC2 overexpression construct, 3) Dcdc2 shRNA along with DCDC2 overexpression construct, 4) an overexpression construct composed of the C terminal domain of DCDC2, or 5) an overexpression construct composed of the DCX terminal domain of DCDC2. RNAi of Dcdc2 resulted in pockets of heterotopic neurons in the periventricular region. Approximately 25% of the transfected brains had hippocampal pyramidal cell migration anomalies. Dcdc2 shRNA-transfected neurons migrated in a bimodal pattern, with approximately 7% of the neurons migrating a short distance from the ventricular zone, and another 30% migrating past their expected lamina. Rats transfected with Dcdc2 shRNA along with the DCDC2 overexpression construct rescued the periventricular heterotopia phenotype, but did not affect the percentage of transfected neurons that migrate past their expected laminar location. There were no malformations associated with any of the overexpression constructs, nor was there a significant laminar disruption of migration. These results support the claim that knockdown of Dcdc2 expression results in neuronal migration disorders similar to those seen in the brains of dyslexics.

Early cortical damage in rat somatosensory cortex alters acoustic feature representation in primary auditory cortex.

Early postnatal freeze-lesions to the cortical plate result in malformations resembling human microgyria. Microgyria in primary somatosensory cortex (S1) of rats are associated with a reduced behavioral detection of rapid auditory transitions and the loss of large cells in the thalamic nucleus projecting to primary auditory cortex (A1). Detection of slow transitions in sound is intact in animals with S1 microgyria, suggesting dissociation between responding to slow versus rapid transitions and a possible dissociation between levels of auditory processing affected. We hypothesized that neuronal responses in primary auditory cortex (A1) would be differentially reduced for rapid sound repetitions but not for slow sound sequences in animals with S1 microgyria. We assessed layer IV cortical responses in primary auditory cortex (A1) to single pure-tones and periodic noise bursts (PNB) in rats with and without S1 microgyria. We found that responses to both types of acoustic stimuli were reduced in magnitude in animals with microgyria. Furthermore, spectral resolution was degraded in animals with microgyria. The cortical selectivity and temporal precision were then measured with conventional methods for PNB and tone-stimuli, but no significant changes were observed between microgyric and control animals. Surprisingly, the observed spike rate reduction was similar for rapid and slow temporal modulations of PNB stimuli. These results suggest that acoustic processing in A1 is indeed altered with early perturbations of neighboring cortex. However, the type of deficit does not affect the temporal dynamics of the cortical output. Instead, acoustic processing is altered via a systematic reduction in the driven spike rate output and spectral integration resolution in A1. This study suggests a novel form of plasticity, whereas early postnatal lesions of one sensory cortex can have a functional impact on processing in neighboring sensory cortex.

Bim mediates mitochondria-regulated particulate matter-induced apoptosis in alveolar epithelial cells.

We studied the role of Bim, a pro-apoptotic BCL-2 family member in Airborne particulate matter (PM 2.5 microm)-induced apoptosis in alveolar epithelial cells (AEC). PM induced AEC apoptosis by causing significant reduction of mitochondrial membrane potential and increase in caspase-9, caspase-3 and PARP-1 activation. PM upregulated pro-apoptotic protein Bim and enhanced translocation of Bim to the mitochondria. ShRNABim blocked PM-induced apoptosis by preventing activation of the mitochondrial death pathway suggesting a role of Bim in the regulation of mitochondrial pathway in AEC. Accordingly, we provide the evidence that Bim mediates PM-induced apoptosis via mitochondrial pathway.

Quantitative trait loci linked to thalamus and cortex gray matter volumes in BXD recombinant inbred mice.

To investigate whether there are separate or shared genetic influences on the development of the thalamus and cerebral cortex, we identified quantitative trait loci (QTLs) for relevant structural volumes in BXD recombinant inbred (RI) strains of mice. In 34 BXD RI strains and two parental strains (C57BL/6J and DBA/2J), we measured the volumes of the entire thalamus and cortex gray matter using point counting and Cavalieri's rule. Heritability was calculated using analysis of variance (ANOVA), and QTL analysis was carried out using WebQTL (http://www.genenetwork.org). The heritability of thalamus volume was 36%, and three suggestive QTLs for thalamus volume were identified on chromosomes 10, 11 and 16. The heritability of cortical gray matter was 43%, and four suggestive QTLs for cortex gray matter volume were identified on chromosomes 2, 8, 16 and 19. The genetic correlation between thalamus and cortex gray matter volumes was 0.64. Also, a single QTL on chromosome 16 (D16Mit100) was identified for thalamus volume, cortex gray matter volume and Morris water maze search-time preference (r=0.71). These results suggest that there are separate and shared genetic influences on the development of the thalamus and cerebral cortex.

Holo-analysis of the efficacy of Bio-Mos in broiler nutrition.

1. This first holo-analysis of the efficacy in broiler nutrition of the saccharide product, Bio-Mos(R) (BM), is part of a comprehensive empirical modelling research programme quantifying and comparing the efficacies and future research needs of the diverse candidates offered as replacements for antibiotics. 2. The data used are from 32 publications with broiler performance data from a world-wide literature collection of 124 (1997-2003) on the use of BM as a broiler feed additive. It contains the results of 82 negatively controlled feeding tests from 21 countries using a total of 85 142 broilers, averaging 401 per treatment. 3. The 82 feed intake, liveweight gain, feed conversion ratio and 44 mortality responses to BM average -12.2 g, 27.6 g, -0.0391 and 0.0311%, respectively, with coefficients of variation of 828, 227, 192 and 8392%. Respective beneficial response frequencies for gain, conversion and mortality are 65, 70 (52% jointly) and 52%. 4. Holo-analytical multiple regression models were elaborated for the effects of BM on feed intake, liveweight gain, feed conversion ratio and mortality, using conventional Por=0.10 out and less stringent Por=0.34 out partial regression coefficient standards, with and without>or=3xroot mean square error outliers, in order to quantify the influences of negative control performance, test duration, BM dosage, cage housing, processed feed, antibiotic feed, year of test and USA test. To date they account for only 5-36% of response variations, due primarily to 52 of the 82 tests having no reported feed formulations. 5. These exploratory models confirm beneficial responses to BM for current broiler performance levels. They also indicate a need to clarify BM dose-response relationships in order to maximise efficient use in praxis and to facilitate cogent comparisons with other pronutrients, including other saccharide products.

Holo-analysis of the efficacy of Bio-Mos in turkey nutrition.

1. This holo-analysis of the effects in turkey nutrition of Bio-Mos(R) (BM) (Alltech Inc., Nicholasville, KY) aims to elaborate comprehensive empirical models for the assessment of responses under practical conditions and for its comparison with other potential pronutrient antibiotic replacements. 2. The data bank utilised was obtained from 54 publications on BM in turkeys from a total collection of 57 papers (1995-2003) on the use of saccharide products as turkey feed additives. These yielded 33 start-to-finish negatively-controlled feed intake, liveweight gain and feed conversion and 24 mortality effects in the USA, France and Poland, using 12,723 birds with a mean of 212 birds per treatment. 3. The feed, gain, conversion and mortality responses to BM compared with the relevant negative controls average -243 g, 57.0 g, -0.0156 and 1.29% with coefficients of variation of 451, 451, 705 and 426%, respectively. Beneficial gain, conversion and mortality effects were found in 52, 52 (33% jointly) and 52% of the tests, respectively. 4. Holo-analytical multiple regression models comparing conventional Por=0.10 out and less stringent Por=0.34 out partial regression coefficient standards, with and without>or=1.5xroot mean square error outliers, contain significant independent variables for negative control performance, duration, year of test, not-USA test, discontinuous dosage, cage housing, mash feed, feed antibiotic, feed anticoccidial, disease condition and high control (>10%) mortality. 5. These developmental models indicate fruitful pathways for future developments with an accent on the definition of specific dose-response relationships, whilst also confirming the value and future potential for BM in turkey production in praxis.

Fibroblast growth factor-10 prevents H2O2-induced cell cycle arrest by regulation of G1 cyclins and cyclin dependent kinases.

We studied the effects of fibroblast growth factor (FGF-10) on H2O2-induced alveolar epithelial cell (AEC) G1 arrest and the role of G1 cyclins. FGF-10 prevented H2O2-induced AEC G1 arrest. FGF-10 induced 2-4-fold increase in cyclin E, cyclin A and CDKs (2,4) alone and in AEC treated with H2O2. H2O2 downregulated cyclin D1; FGF-10 blocked these effects. FGF-10 prevented H2O2-induced upregulation of CDK inhibitor, p21. SiRNAp21 blocked H2O2-induced downregulation of cyclins, CDKs and AEC G1 arrest. Accordingly, we provide first evidence that FGF-10 regulates G1 cyclins and CDKs, and prevents H2O2-induced AEC G1 arrest.

DYX1C1 functions in neuronal migration in developing neocortex.

Rodent homologues of two candidate dyslexia susceptibility genes, Kiaa0319 and Dcdc2, have been shown to play roles in neuronal migration in developing cerebral neocortex. This functional role is consistent with the hypothesis that dyslexia susceptibility is increased by interference with normal neural development. In this study we report that in utero RNA interference against the rat homolog of another candidate dyslexia susceptibility gene, DYX1C1, disrupts neuronal migration in developing neocortex. The disruption of migration can be rescued by concurrent overexpression of DYX1C1, indicating that the impairment is not due to off-target effects. Transfection of C- and N-terminal truncations of DYX1C1 shows that the C-terminal TPR domains determine DYX1C1 intracellular localization to cytoplasm and nucleus. RNAi rescue experiments using truncated versions of DYX1C1 further indicate that the C-terminus of DYX1C1 is necessary and sufficient to DYX1C1's function in migration. In conclusion, DYX1C1, similar to two other candidate dyslexia susceptibility genes, functions in neuronal migration in rat neocortex.

Holo-analysis.

Meta-analysis is a vague descriptor used to encompass very diverse methods of data collection analysis, ranging from simple averages to more complex statistical methods. Holo-analysis is a fully comprehensive statistical analysis of all available data and all available variables in a specified topic, with results expressed in a holistic factual empirical model. The objectives and applications of holo-analysis include software production for prediction of responses with confidence limits, translation of research conditions to praxis (field) circumstances, exposure of key missing variables, discovery of theoretically unpredictable variables and interactions, and planning future research. Holo-analyses are cited as examples of the effects on broiler feed intake and live weight gain of exogenous phytases, which account for 70% of variation in responses in terms of 20 highly significant chronological, dietary, environmental, genetic, managemental, and nutrient variables. Even better future accountancy of variation will be facilitated if and when authors of papers routinely provide key data for currently neglected variables, such as temperatures, complete feed formulations, and mortalities.

Histometric changes and cell death in the thalamus after neonatal neocortical injury in the rat.

Freezing injury to the developing cortical plate results in a neocortical malformation resembling four-layered microgyria. Previous work has demonstrated that following freezing injury to the somatosensory cortex, males (but not females) have more small and fewer large cells in the medial geniculate nucleus. In the first experiment, we examined the effects of induced microgyria to the somatosensory cortex on neuronal numbers, neuronal size, and nuclear volume of three sensory nuclei: ventrobasal complex, dorsal lateral geniculate nucleus, and medial geniculate nucleus. We found that there was a decrease in neuronal number and nuclear volume in ventrobasal complex of microgyric rats when compared with shams, whereas there were no differences in these variables in the dorsal lateral geniculate nucleus or medial geniculate nucleus. We also found that there were more small and fewer large neurons in both ventrobasal complex and medial geniculate nucleus. In experiment 2, we attempted to determine the role of cell death in the thalamus on these histometric measures. We found that cell death peaked within 24 h of the freezing injury and was concentrated mostly in ventrobasal complex. In addition, there was evidence of greater cell death in males at this age. Taken together, these results support the notion that males are more severely affected by early injury to the cerebral cortex than females.