Post-Stroke Depression and Cognitive Aging: A Multicenter, Prospective Cohort Study.

Background: This study investigated the impact of post-stroke depression (PSD) on cognitive aging in elderly stroke patients. Methods: This study was an interim analysis of the Korean Stroke Cohort for Functioning and Rehabilitation. Among 10,636 patients with first-ever stroke, a total of 3215 patients with normal cognitive function three months post-stroke were included in the analysis. PSD was defined using the Korean Geriatric Depression Scale Short Form (K-GDS-SF) at three months. Cognitive aging was defined as a decline in the Korean version of the Mini-Mental Status Examination (K-MMSE) score to less than the second percentile. Results: The hazard ratio (HR) of PSD for cognitive decline was 2.16 (95% CI, 1.34−3.50, p < 0.01) in the older group (age ≥65 years), and 1.02 (95% CI, 0.50−2.07, n.s.) in the younger group (age <65 years). When the older group was divided by sex, the HR was 2.50 (95% CI, 1.26−4.96, p < 0.01) in male patients and 1.80 (95% CI, 0.93−3.51, n.s.) in female patients. However, women showed a higher incidence of cognitive decline in both the PSD and no PSD groups. Among K-GDS-SF factors, "Negative judgment about the past, present, and future" increased the HR of PSD in older male patients. Conclusions: Early PSD increased the HR for cognitive decline in older stroke patients, mainly in males. Specifically, older male patients with negative thinking were at increased risk of cognitive decline. The findings also suggest that older women may be at risk for cognitive decline. Therefore, preventive interventions for cognitive decline should be tailored differently for men and women.

Association of rheumatoid arthritis and high sodium intake with major adverse cardiovascular events: a cross-sectional study from the seventh Korean National Health and Nutrition Examination Survey.

OBJECTIVES: High salt intake has a harmful effect on hypertension; however, the association between major adverse cardiovascular events (MACE) and salt intake is still controversial. Rheumatoid arthritis (RA) is also characterised by excess cardiovascular risk. However, few studies have investigated the combined role of salt intake and RA in MACE in the general Korean population. Here, we evaluated this relationship among the Korean adult population. DESIGN: Retrospective, cross-sectional. SETTING: Population-based survey in Korea. METHODS: This study was based on the data of the seventh Korean National Health and Nutrition Examination Survey (2016-2018). The estimated 24-hour urinary sodium excretion (24HUNa), a surrogate marker for daily sodium intake, was calculated using the Tanaka equation and was stratified into five groups (<3, 3-3.999, 4-4.999, 5-5.999 and ≥6 g/day). Finally, data from 13 464 adult participants (weighted n=90 425 888) were analysed; all analyses considered a complex sampling design. Multivariable logistic regression for MACE as primary dependent variable was performed and adjusted for potential covariates. RESULTS: Participants with MACE had higher 24HUNa levels and RA proportion than those without MACE (p<0.001). The association of MACE with 24HUNa was J-shaped with a gradual increase from about 3 g/day. The highest 24HUNa (≥6 g/day) group was significantly associated with increased prevalence of MACE compared with the reference group (3-3.999 g/day) after adjusting for all associated covariates (OR 6.75, 95% CI 1.421 to 32.039). In the multivariate logistic regression analysis, RA (OR 2.05, 95% CI 1.283 to 3.264) and the highest 24HUNa group (OR 6.35, 95% CI 1.337 to 30.147) were significantly associated with MACE even after adjusting for baseline covariates. CONCLUSIONS: These nationally representative data suggest that RA and extremely high sodium intake are associated with MACE in the general adult Korean population. Avoiding extremely high salt intake and considering RA as an important risk factor for MACE might help promote public cardiovascular health.

Depressive symptoms with cognitive dysfunction increase the risk of cognitive impairment: analysis of the Korean Longitudinal Study of Aging (KLoSA), 2006-2018.

OBJECTIVES: Geriatric depression complicates cognitive health in older adults. This study aims to investigate the impact of depressive symptoms on cognitive impairment in community-dwelling older adults, depending on whether cognitive dysfunction accompanied. DESIGN: A community-based longitudinal cohort study. SETTING: This study analyzed data from the Korean Longitudinal Study of Aging (KLoSA) from 2006 to 2018. PARTICIPANTS: Among 10,254 individuals who were registered in the KLoSA study, a total of 9119 subjects met the criteria, and 4547 subjects were included in the final analysis. The subjects were grouped into 4 categories based on depressive symptoms and cognitive dysfunction at baseline assessment: "normal control" (NC, n = 3341), "depression only" (Dep-only, n = 652), "cognitive dysfunction only" (CD-only, n = 393), and "depression with cognitive dysfunction" (Dep-CD, n = 161). MEASUREMENTS: Cognitive impairment 10 years later was defined as K-MMSE scores below two percentile on demographically adjusted norms. RESULTS: Ten-year survival, that is, not experiencing cognitive impairment, was 80 $$ \pm \,$$1% in NC group, 72 $$ \pm $$ 2% in Dep-only group, 52 $$ \pm $$ 3% in CD-only group, and 44 $$ \pm $$ 5% in Dep-CD group. The hazard ratio (HR) of the Dep-only group (HR = 1.18, 95% CI, 0.97-1.43, n.s.) did not differ from that of the NC group, but the HR of the Dep-CD group was significantly higher (HR = 2.85, 95% CI, 2.23-3.66, p < 0.001) than the NC group. When the Dep-CD group was compared to the CD-only group, the HR was 1.13 (95% CI, 0.85-1.49, n.s.), which indicates that it did not significantly differ from the CD-only group. CONCLUSIONS: Our findings suggest that depressive symptoms with cognitive dysfunction are associated with a higher risk of cognitive impairment. Furthermore, cognitive dysfunction occurring with depressive symptoms is as much a risk for cognitive impairment as is pure cognitive dysfunction. Thus, healthcare providers should pay close attention to the community-dwelling elderly when depressive symptoms occur with cognitive dysfunction.

Effects of Process-Based Cognitive Training on Memory in the Healthy Elderly and Patients with Mild Cognitive Impairment: A Randomized Controlled Trial.

OBJECTIVE: This study investigated the effects of process-based cognitive training that targets working memory and cognitive control on memory improvement in healthy elderly individuals and patients with mild cognitive impairment (MCI). METHODS: Forty healthy subjects and 40 patients with MCI were randomly assigned to either the intervention or control group. The intervention group received 12 sessions of designated cognitive training. The control group did not receive cognitive training. A memory test was administered pre-intervention, post-intervention, and 4 weeks after the intervention. Additional comprehensive neuropsychological tests were also administered including a depression scale questionnaire. RESULTS: Performance in attention and working memory, which are directly related to the training domains, and global cognitive function were improved in the intervention group after training. In memory tests, interference by irrelevant stimuli was reduced and recognition memory was improved after the intervention. Furthermore, cognitive training ameliorated depressive symptoms. These training effects were not dependent on MCI status. CONCLUSION: Process-based cognitive training that targets working memory and cognitive control effectively improves memory processes including retrograde interference and recognition, as well as depressive symptoms associated with aging in healthy elderly individuals and patients with MCI.

Moderating effect of cognitive reserve on the association between grey matter atrophy and memory varies with age in older adults.

BACKGROUND: Educational attainment and verbal intelligence, which indirectly reflect an individual's cognitive reserve (CR), is suggested to buffer the effect of late-life brain degradation on cognitive performance outcome. We aimed to explore how the relationship between whole grey matter volume (GMV) and episodic memory function is altered by CR proxy as well as age in healthy older adults. METHODS: Elderly Verbal Learning Test (EVLT) and structural magnetic resonance imaging were administered to 110 community-residing older adults. Moderated moderation model tested whether the association between whole GMV and episodic memory was moderated by both CR and chronological age. RESULTS: The results showed that the moderating effect of CR on Immediate Recall, Short-delay Recall, and Recognition scores of EVLT differed across age groups. The elderly with higher CR showed steeper GMV effect on EVLT at the Age-Younger condition, while such moderating effect was reversed in the Age-Older condition, suggesting an alleviated brain atrophy effect in higher CR elderly. CONCLUSION: These findings suggest that although higher CR elderly may exhibit earlier GMV-related memory decline, the buffering effect of CR on the cognitive decline due to brain atrophy would become more evident in old-old elderly people who are likely to have accumulated more neuropathological changes. This study underscores chronological age as an important moderating factor in examining the moderating role of CR in late-life memory function.

Effect of Cognitive Reserve on Risk of Cognitive Impairment and Recovery After Stroke: The KOSCO Study.

Background and Purpose- The theory of cognitive reserve (CR) was introduced to account for individual differences in the clinical manifestation of neuropathology. This study investigated whether CR has a modulating effect on cognitive impairment and recovery after stroke. Methods- This study is an interim analysis of the Korean Stroke Cohort for Functioning and Rehabilitation. A total of 7459 patients with first-ever stroke were included for analysis. Education, occupation, and composite CR scores derived from those 2 variables were used as CR proxies. Scores from the Korean version of the Mini-Mental State Examination analyzed for 30 months after stroke onset were analyzed. Results- Lower CR increased the risk of cognitive impairment after stroke. The odds ratio was 1.89 (95% CI, 1.64-2.19) in patients with secondary education and 2.42 (95% CI, 2.03-2.90) in patients with primary education compared with patients with higher education. The odds ratio was 1.48 (95% CI, 1.23-1.98) in patients with a skilled manual occupation and 2.01 (95% CI, 1.42-2.83) in patients with a nonskilled manual occupation compared with patients with a managerial or professional occupation. In the multilevel model analysis, the Korean version of the Mini-Mental State Examination total score increased during the first 3 months (1.93 points per month) and then plateaued (0.02 point per month). The slopes were moderated by the level of education, occupation, and composite CR score: the higher the level of education, occupation, or CR score, the faster the recovery. In the older adult group, the Korean version of the Mini-Mental State Examination scores showed a long-term decline that was moderated by education level. Conclusions- Education and occupation can buffer an individual against cognitive impairment caused by stroke and promote rapid cognitive recovery early after stroke. In addition, higher education minimizes long-term cognitive decline after stroke, especially in older patients. Clinical Trial Registration- URL: https://www.clinicaltrials.gov. Unique identifier: NCT03402451.

Graded Coexpression of Ion Channel, Neurofilament, and Synaptic Genes in Fast-Spiking Vestibular Nucleus Neurons.

Computations that require speed and temporal precision are implemented throughout the nervous system by neurons capable of firing at very high rates, rapidly encoding and transmitting a rich amount of information, but with substantial metabolic and physical costs. For economical fast spiking and high throughput information processing, neurons need to optimize multiple biophysical properties in parallel, but the mechanisms of this coordination remain unknown. We hypothesized that coordinated gene expression may underlie the coordinated tuning of the biophysical properties required for rapid firing and signal transmission. Taking advantage of the diversity of fast-spiking cell types in the medial vestibular nucleus of mice of both sexes, we examined the relationship between gene expression, ionic currents, and neuronal firing capacity. Across excitatory and inhibitory cell types, genes encoding voltage-gated ion channels responsible for depolarizing and repolarizing the action potential were tightly coexpressed, and their absolute expression levels increased with maximal firing rate. Remarkably, this coordinated gene expression extended to neurofilaments and specific presynaptic molecules, providing a mechanism for coregulating axon caliber and transmitter release to match firing capacity. These findings suggest the presence of a module of genes, which is coexpressed in a graded manner and jointly tunes multiple biophysical properties for economical differentiation of firing capacity. The graded tuning of fast-spiking capacity by the absolute expression levels of specific ion channels provides a counterexample to the widely held assumption that cell-type-specific firing patterns can be achieved via a vast combination of different ion channels.SIGNIFICANCE STATEMENT Although essential roles of fast-spiking neurons in various neural circuits have been widely recognized, it remains unclear how neurons efficiently coordinate the multiple biophysical properties required to maintain high rates of action potential firing and transmitter release. Taking advantage of diverse fast-firing capacities among medial vestibular nucleus neurons of mice, we identify a group of ion channel, synaptic, and structural genes that exhibit mutually correlated expression levels, which covary with firing capacity. Coexpression of this fast-spiking gene module may be a basic strategy for neurons to efficiently and coordinately tune the speed of action potential generation and propagation and transmitter release at presynaptic terminals.

Fibroblast Growth Factor 10 Enhances the Developmental Efficiency of Somatic Cell Nuclear Transfer Embryos by Accelerating the Kinetics of Cleavage During In Vitro Maturation.

Somatic cell nuclear transfer (SCNT) is required for the generation of transgenic animals as disease models. During the in vitro development of SCNT embryos, the quality of matured oocytes is one of the major factors regulating the developmental potential of embryos. Time-lapse monitoring systems are new tools that assess the developmental capacity of embryos for use in embryo transfer. In this study, we investigated the effect of fibroblast growth factor 10 (FGF 10) on the developmental potential of SCNT embryos. After the in vitro maturation (IVM) of oocytes in IVM medium containing 10 ng/mL FGF 10 (10 F), the polar body extrusion rate was significantly higher than in the control. However, there was no difference in the percentage of fused embryos between the groups. The cleavage and blastocyst formation rates of embryos were significantly increased in the 10 F compared with the control. In addition, the total cell number was higher and the apoptotic index was lower in the 10 F than control at day 7. The messenger RNA (mRNA) expression of genes involved in apoptosis (baculoviral inhibitor of apoptosis repeat containing 5 [BIRC5] and caspase 3 [CASP3]) and development (octamer-binding transcription factor 4 [POU5F1] and sex determining region Y box 2 [SOX2]) increased after 10 F treatment. Furthermore, the kinetics of the first cleavage was faster and the percentage of embryos at cell block was significantly lower in the 10 F group than in the control. These results demonstrate that exposure of oocytes to FGF 10 during IVM promotes developmental competence.

Antioxidant ß-cryptoxanthin enhances porcine oocyte maturation and subsequent embryo development in vitro.

Oxidative stress is partly responsible for the poor quality of IVM oocytes. The present study investigated the effects of the antioxidant ß-cryptoxanthin on the IVM of porcine oocytes and the in vitro development of the ensuing embryos. Oocytes were matured in IVM medium containing different concentrations of ß-cryptoxanthin (0, 0.1, 1, 10 or 100µM). Treatment with 1µM ß-cryptoxanthin (Group 1B) improved polar body extrusion and the expression of maturation-related genes in cumulus cells and oocytes compared with control. In addition, levels of reactive oxygen species decreased significantly in Group 1B, whereas there were significant increases in glutathione levels and expression of the antioxidant genes superoxide dismutase 1 and peroxiredoxin 5 in this group. After parthenogenetic activation, although the cleavage rate did not differ between the control and 1B groups, the blastocyst formation rate was higher in the latter. Moreover, the total number of cells per blastocyst and relative mRNA levels of pluripotency marker and antioxidant genes were significantly higher in the 1B compared with control group. These results demonstrate that ß-cryptoxanthin decreases oxidative stress in porcine oocytes and improves their quality and developmental potential.

Short Telomeres Induce p53 and Autophagy and Modulate Age-Associated Changes in Cardiac Progenitor Cell Fate.

Aging severely limits myocardial repair and regeneration. Delineating the impact of age-associated factors such as short telomeres is critical to enhance the regenerative potential of cardiac progenitor cells (CPCs). We hypothesized that short telomeres activate p53 and induce autophagy to elicit the age-associated change in CPC fate. We isolated CPCs and compared mouse strains with different telomere lengths for phenotypic characteristics of aging. Wild mouse strain Mus musculus castaneus (CAST) possessing short telomeres exhibits early cardiac aging with cardiac dysfunction, hypertrophy, fibrosis, and senescence, as compared with common lab strains FVB and C57 bearing longer telomeres. CAST CPCs with short telomeres demonstrate altered cell fate as characterized by cell cycle arrest, senescence, basal commitment, and loss of quiescence. Elongation of telomeres using a modified mRNA for telomerase restores youthful properties to CAST CPCs. Short telomeres induce autophagy in CPCs, a catabolic protein degradation process, as evidenced by reduced p62 and increased accumulation of autophagic puncta. Pharmacological inhibition of autophagosome formation reverses the cell fate to a more youthful phenotype. Mechanistically, cell fate changes induced by short telomeres are partially p53 dependent, as p53 inhibition rescues senescence and commitment observed in CAST CPCs, coincident with attenuation of autophagy. In conclusion, short telomeres activate p53 and autophagy to tip the equilibrium away from quiescence and proliferation toward differentiation and senescence, leading to exhaustion of CPCs. This study provides the mechanistic basis underlying age-associated cell fate changes that will enable identification of molecular strategies to prevent senescence of CPCs. Stem Cells 2018;36:868-880.

Lysophosphatidic acid accelerates development of porcine embryos by activating formation of the blastocoel.

Culture media modifications, including the addition of various factors, are important for the in vitro production of oocytes and embryos. In this study, we investigated the effects of lysophosphatidic acid (LPA) on porcine embryo development. Porcine parthenogenetic embryos were cultured with 0, 0.1, 1, and 10 µM LPA for 7 days, or cultured in basic medium until Day 4 and then treated with LPA from Days 4 to 7. No difference in the in vitro development of embryos cultured with LPA for 7 days was observed. Conversely, rates of blastocyst and over-expanded blastocyst formation were higher in the 0.1 and 1 µM LPA-treated versus the other groups of embryos treated from Days 4 to 7. Moreover, formation of early blastocysts occurred earlier and embryo size was larger in LPA-treated compared to control embryos. Expression of Connexin 43 and gap junction and cell adhesion-related genes (GJC1 and CDH1, respectively) was also higher in LPA-treated compared to control embryos. Despite no difference in the blastocyst total cell number between groups, the apoptotic index was lower in the LPA-treated group than in the control group; indeed, BCL2L1 (B-cell lymphoma 2-like protein 1) expression increased while BAK (Bcl-2 homologous antagonist killer) decreased in the LPA-treated group. Thus, addition of LPA to the medium from Days 4 to 7 of culture improves blastocyst formation and aids the development of preimplantation embryos.

Treatment of allicin improves maturation of immature oocytes and subsequent developmental ability of preimplantation embryos.

Allicin (AL) regulates the cellular redox, proliferation, viability, and cell cycle of different cells against extracellular-derived stress. This study investigated the effects of allicin treatment on porcine oocyte maturation and developmental competence. Porcine oocytes were cultured in medium supplemented with 0 (control), 0.01, 0.1, 1, 10 or 100 µM AL, respectively, during in vitro maturation (IVM). The rate of polar body emission was higher in the 0.1 AL-treated group (74.5% ± 2.3%) than in the control (68.0% ± 2.6%) (P < 0.1). After parthenogenetic activation, the rates of cleavage and blastocyst formation were significantly higher in the 0.1 AL-treated group than in the control (P < 0.05). The reactive oxygen species level at metaphase II did not significantly differ among all groups. In matured oocytes, the expression of both BAK and CASP3, and BIRC5 was significantly lower and higher, respectively, in the 0.1 AL-treated group than in the control. Similarly, the expression of BMP15 and CCNB1, and the activity of phospho-p44/42 mitogen-activated protein kinase (MAPK), significantly increased. These results indicate that supplementation of oocyte maturation medium with allicin during IVM improves the maturation of oocytes and the subsequent developmental competence of porcine oocytes.

Production of transgenic pig as an Alzheimer's disease model using a multi-cistronic vector system.

Alzheimer's disease (AD) is a progressive neurodegenerative disease associated with memory loss and cognitive impairments. An AD transgenic (Tg) pig model would be useful for preclinical testing of therapeutic agents. We generated an AD Tg pig by somatic cell nuclear transfer (SCNT) using a multi-cistronic vector that harbored three AD-related genes with a total of six well-characterized mutations: hAPP (K670N/M671L, I716V, and V717I), hTau (P301L), and hPS1 (M146V and L286P). Four AD Tg cell lines were established from Jeju black pig ear fibroblasts (JB-PEFs); the resultant JB-PEFAD cells harbored transgene integration, expressed transgene mRNAs, and had normal karyotypes. Tg line #2-1, which expressed high levels of the transgenes, was used for SCNT; cleavage and blastocyst rates of embryos derived from this line were lower than those of Non-Tg. These embryos yielded three piglets (Jeju National University AD-Tg pigs, JNUPIGs) revealed by microsatellite testing to be genetically identical to JB-PEFAD. Transgenes were expressed in multiple tissues, and at especially high levels in brain, and Aß-40/42, total Tau, and GFAP levels were high in brains of the Tg animals. Five or more copies of transgenes were inserted into chromosome X. This is the first report of an AD Tg pig derived from a multi-cistronic vector.

Fibroblast growth factor 10 markedly improves in vitro maturation of porcine cumulus-oocyte complexes.

Growth factors synthesized by ovarian somatic cells affect cumulus cell expansion and oocyte maturation in vitro. Fibroblast growth factor 10 (FGF10), for example, is a known regulator of mammalian cumulus-oocyte complex maturation. In this study, we investigated the effects of 0, 5, 10, 50, and 100 ng/mL FGF10 (5F, 10F, 50F, and 100F, respectively) on in vitro cumulus cell expansion, oocyte maturation, and embryo development. The percentage of fully expanded cumulus cells at the oocyte's metaphase-II (MII) stage was significantly higher in the 10F-treated group than in the control. Transcript abundance of the cumulus cell expansion-related gene encoding hyaluronian synthase 2 (HAS2) in cumulus cells at oocyte germinal vesicle breakdown (GVBD) was significantly higher in the 10F- and 50F-treated groups compared to untreated controls, whereas the mRNA abundance of the protease cathepsin B (CTSB) at the oocyte MII stage was remarkably decreased in the 10F-treated group. The percentage of oocytes with normal spindles was greater in the 10F- and 50F-treated group at GVBD than in the other groups; the 5F-, 10F-, and 100F-treated groups were higher than the control; and the 50F-treated group was highest at MII. The abundance of GDF9 and BMP15 transcript at GVBD and BMP15 and CCNB1 transcripts at MII increased in the 10F-treated group. Cleavage rate, blastocyst formation rate, and total cell number were significantly higher in the 5F- to 50F-treated groups. These results demonstrate that FGF10 markedly improves cumulus cell expansion, oocyte maturation, and subsequent embryo development. Mol. Reprod. Dev. 84: 67-75, 2017. © 2016 Wiley Periodicals, Inc.

Cell Synchronization by Rapamycin Improves the Developmental Competence of Porcine SCNT Embryos.

The cell cycle stage of donor cells influences the success of somatic cell nuclear transfer (SCNT). This study investigated the effects of rapamycin treatment on synchronization of porcine fibroblasts in comparison with control and serum-starved cells, SCNT donor cell viability, and SCNT-derived embryo development. Porcine fibroblasts were treated with 0.1, 1, 10, and 100 µM rapamycin for 1 or 3 days. The proportion of cells in G0/G1 phase was significantly higher among cells treated with 1 µM rapamycin for 3 days (D3-1R) than among control and serum-starved cells (p < 0.05). In comparison with control cells, rapamycin-treated cells exhibited reduced proliferation, similar to serum-starved cells. The viability (as assessed by the MTT assay) of D3-1R-treated cells was good, similar to control cells, showing their quality was maintained. To confirm nutrient regulation by rapamycin treatment, we checked the transcript levels of nutrient transporter genes (SLC2A2, SLC2A4, SLC6A14, and SLC7A1). These levels were significantly lower in D3-1R-treated cells than in control cells (p < 0.01). We performed SCNT with D3-1R-treated cells (SCNT(D3-1R)) to confirm the effect of cell cycle synchronization by rapamycin treatment. Although SCNT(D3-1R) embryos did not have an increased fusion rate, their cleavage and blastocyst formation rates were significantly higher than those of control embryos (p < 0.05). Regarding embryo quality, the numbers of total and apoptotic cells per blastocyst were increased and decreased, respectively, in SCNT(D3-1R) blastocysts. The mRNA levels of developmental (CDX2 and CDH1) and proapoptotic (FAS and CASP3) genes were significantly higher and lower, respectively, in SCNT(D3-1R) blastocysts than in control blastocysts (p < 0.05). These results demonstrate that rapamycin treatment affects the cell cycle synchronization of donor cells and enhances the developmental potential of porcine SCNT embryos.

Effects of Feeder Cell Types on Culture of Mouse Embryonic Stem Cell In Vitro.

The suitable feeder cell layer is important for culture of embryonic stem (ES) cells. In this study, we investigated the effect of two kinds of the feeder cell, MEF cells and STO cells, layer to mouse ES (mES) cell culture for maintenance of stemness. We compare the colony formations, alkaline phosphatase (AP) activities, expression of pluripotency marker genes and proteins of D3 cell colonies cultured on MEF feeder cell layer (D3/MEF) or STO cell layers (D3/STO) compared to feeder free condition (D3/-) as a control group. Although there were no differences to colony formations and AP activities, interestingly, the transcripts level of pluripotency marker genes, Pou5f1 and Nanog were highly expressed in D3/MEF (79 and 93) than D3/STO (61and 77) or D3/- (65 and 81). Also, pluripotency marker proteins, NANOG and SOX-2, were more synthesized in D3/MEF (72.8±7.69 and 81.2±3.56) than D3/STO (32.0±4.30 and 56.0±4.90) or D3/- (55.0±4.64 and 62.0±6.20). These results suggest that MEF feeder cell layer is more suitable to mES cell culture.

Neuronal classification and marker gene identification via single-cell expression profiling of brainstem vestibular neurons subserving cerebellar learning.

Identification of marker genes expressed in specific cell types is essential for the genetic dissection of neural circuits. Here we report a new strategy for classifying heterogeneous populations of neurons into functionally distinct types and for identifying associated marker genes. Quantitative single-cell expression profiling of genes related to neurotransmitters and ion channels enables functional classification of neurons; transcript profiles for marker gene candidates identify molecular handles for manipulating each cell type. We apply this strategy to the mouse medial vestibular nucleus (MVN), which comprises several types of neurons subserving cerebellar-dependent learning in the vestibulo-ocular reflex. Ion channel gene expression differed both qualitatively and quantitatively across cell types and could distinguish subtle differences in intrinsic electrophysiology. Single-cell transcript profiling of MVN neurons established six functionally distinct cell types and associated marker genes. This strategy is applicable throughout the nervous system and could facilitate the use of molecular genetic tools to examine the behavioral roles of distinct neuronal populations.

Multiple types of cerebellar target neurons and their circuitry in the vestibulo-ocular reflex.

The cerebellum influences behavior and cognition exclusively via Purkinje cell synapses onto neurons in the deep cerebellar and vestibular nuclei. In contrast with the rich information available about the organization of the cerebellar cortex and its synaptic inputs, relatively little is known about microcircuitry postsynaptic to Purkinje cells. Here we examined the cell types and microcircuits through which Purkinje cells influence an oculomotor behavior controlled by the cerebellum, the horizontal vestibulo-ocular reflex, which involves only two eye muscles. Using a combination of anatomical tracing and electrophysiological recordings in transgenic mouse lines, we identified several classes of neurons in the medial vestibular nucleus that receive Purkinje cell synapses from the cerebellar flocculus. Glycinergic and glutamatergic flocculus target neurons (FTNs) with somata densely surrounded by Purkinje cell terminals projected axons to the ipsilateral abducens and oculomotor nuclei, respectively. Of three additional types of FTNs that were sparsely innervated by Purkinje cells, glutamatergic and glycinergic neurons projected to the contralateral and ipsilateral abducens, respectively, and GABAergic neurons projected to contralateral vestibular nuclei. Densely innervated FTNs had high spontaneous firing rates and pronounced postinhibitory rebound firing, and were physiologically homogeneous, whereas the intrinsic excitability of sparsely innervated FTNs varied widely. Heterogeneity in the molecular expression, physiological properties, and postsynaptic targets of FTNs implies that Purkinje cell activity influences the neural control of eye movements in several distinct ways. These results indicate that the cerebellum regulates a simple reflex behavior via at least five different cell types that are postsynaptic to Purkinje cells.

Alternatively spliced isoforms of TRIP8b differentially control h channel trafficking and function.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (h channels) are the molecular basis for the current, I(h), which contributes crucially to intrinsic neuronal excitability. The subcellular localization and biophysical properties of h channels govern their function, but the mechanisms controlling these characteristics, and especially the potential role of auxiliary subunits or other binding proteins, remain unclear. We focused on TRIP8b, an h channel-interacting protein that colocalizes with HCN1 in cortical and hippocampal pyramidal neuron dendrites, and found that it exists in multiple alternative splice variants with distinct effects on h channel trafficking and function. The developmentally regulated splice variants of TRIP8b all shared dual, C terminus-located interaction sites with HCN1. When coexpressed with HCN1 in heterologous cells individual TRIP8b isoforms similarly modulated gating of I(h), causing a hyperpolarizing shift in voltage dependence of channel activation, but differentially upregulated or downregulated I(h) current density and HCN1 surface expression. In hippocampal neurons, coexpression of TRIP8b isoforms with HCN1 produced isoform-specific changes of HCN1 localization. Interestingly, the TRIP8b isoforms most abundant in the brain are those predicted to enhance h channel surface expression. Indeed, shRNA knockdown of TRIP8b in hippocampal neurons significantly reduced native I(h). Thus, although TRIP8b exists in multiple splice isoforms, our data suggest that the predominant role of this protein in brain is to promote h channel surface expression and enhance I(h). Because I(h) expression is altered in models of several diseases, including temporal lobe epilepsy, TRIP8b may play a role in both normal neuronal function and in aberrant neuronal excitability associated with neurological disease.

Absence epilepsy in apathetic, a spontaneous mutant mouse lacking the h channel subunit, HCN2.

Analysis of naturally occurring mutations that cause seizures in rodents has advanced understanding of the molecular mechanisms underlying epilepsy. Abnormalities of I(h) and h channel expression have been found in many animal models of absence epilepsy. We characterized a novel spontaneous mutant mouse, apathetic (ap/ap), and identified the ap mutation as a 4 base pair insertion within the coding region of Hcn2, the gene encoding the h channel subunit 2 (HCN2). We demonstrated that Hcn2(ap) mRNA is reduced by 90% compared to wild type, and the predicted truncated HCN2(ap) protein is absent from the brain tissue of mice carrying the ap allele. ap/ap mice exhibited ataxia, generalized spike-wave absence seizures, and rare generalized tonic-clonic seizures. ap/+ mice had a normal gait, occasional absence seizures and an increased severity of chemoconvulsant-induced seizures. These findings help elucidate basic mechanisms of absence epilepsy and suggest HCN2 may be a target for therapeutic intervention.