Developmental patterns of sleep slow wave activity and synaptic density in adolescent mice.

STUDY OBJECTIVE: In humans sleep slow wave activity (SWA) declines during adolescence. It has been suggested that this decline reflects the elimination of cortical synapses, but this hypothesis has never been tested directly. DESIGN: We focused on mouse frontal cortex and collected data from early adolescence (∼postnatal day 20, P20) to adulthood (P60) of (1) SWA; (2) expression of synapsin I, a presynaptic marker; and (3) number of dendritic spines in layers I-II. SETTING: Basic sleep research laboratory. PATIENTS OR PARTICIPANTS: YFP-line H mice (n = 70; P15-87, all males) and GFP-line S mice (n = 14; P17-60, 8 females) were used for EEG recording. Forty-five YFP mice (P19-119, 12 females) and 42 GFP-S mice (P20-60, 14 females) were used for in vivo 2-photon imaging and ex vivo confocal microscopy, respectively. Other YGP mice (n = 57, P10-77) were used for western blot analysis of synapsin I. INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: As in humans, SWA in mice declined from early adolescence to adulthood. Synapsin I levels increased from P10 to P24, with little change afterwards. Mean spine density in apical dendrites of layer V pyramidal neurons (YFP-H) showed no change from P20 to P60. Spine number in layers I-II apical dendrites, belonging to layer III and V pyramidal neurons (GFP-S), increased slightly from P20 to P30 and decreased from P30 to P60; smaller spines decreased in number from P20 to P60, while bigger spines increased. CONCLUSIONS: In mice, it is unlikely that the developmental decrease in SWA can be accounted for by a net pruning of cortical synapses.

Effects of sleep and wake on oligodendrocytes and their precursors.

Previous studies of differential gene expression in sleep and wake pooled transcripts from all brain cells and showed that several genes expressed at higher levels during sleep are involved in the synthesis/maintenance of membranes in general and of myelin in particular, a surprising finding given the reported slow turnover of many myelin components. Other studies showed that oligodendrocyte precursor cells (OPCs) are responsible for the formation of new myelin in both the injured and the normal adult brain, and that glutamate released from neurons, via neuron-OPC synapses, can inhibit OPC proliferation and affect their differentiation into myelin-forming oligodendrocytes. Because glutamatergic transmission is higher in wake than in sleep, we asked whether sleep and wake can affect oligodendrocytes and OPCs. Using the translating ribosome affinity purification technology combined with microarray analysis in mice, we obtained a genome-wide profiling of oligodendrocytes after sleep, spontaneous wake, and forced wake (acute sleep deprivation). We found that hundreds of transcripts being translated in oligodendrocytes are differentially expressed in sleep and wake: genes involved in phospholipid synthesis and myelination or promoting OPC proliferation are transcribed preferentially during sleep, while genes implicated in apoptosis, cellular stress response, and OPC differentiation are enriched in wake. We then confirmed through BrdU and other experiments that OPC proliferation doubles during sleep and positively correlates with time spent in REM sleep, whereas OPC differentiation is higher during wake. Thus, OPC proliferation and differentiation are not perfectly matched at any given circadian time but preferentially occur during sleep and wake, respectively.

Changes in brain gene expression during migration in the white-crowned sparrow.

Long-term recordings of seasonal sleep patterns in captive white-crowned sparrows (Zonotrichia leucophrys gambelii) have shown that these birds markedly reduce sleep time during the migratory period relative to the non-migratory period. It was also found that, despite this sleep reduction, sparrows showed no evidence of neurobehavioral deficits in a standard operant task used to assess the effects of sleep loss. In this study, we performed an extensive microarray analysis of gene expression in the sparrow telencephalon during the migratory season (M), relative to a 78-h period of enforced sleep restriction during the non-migratory season (SR), and a 6-h period of normal wakefulness during the non-migratory season (W). Of the estimated 17,100 transcripts that were reliably detected, only 0.17% changed expression as a function of M (relative to both SR and W), and 0.11% as a function of SR (relative to both M and W). Brain transcripts whose expression increased during M include the facilitated glucose transporter GLUT1, the presenilin associated rhomboid-like protein PARL, and several members of the heat shock protein family, such as HSP70, HSP90, GRP78 and BiP. These data suggest that migration is associated with brain cellular stress and enhanced energetic demands.

Molecular and electrophysiological evidence for net synaptic potentiation in wake and depression in sleep.

Plastic changes occurring during wakefulness aid in the acquisition and consolidation of memories. For some memories, further consolidation requires sleep, but whether plastic processes during wakefulness and sleep differ is unclear. We show that, in rat cortex and hippocampus, GluR1-containing AMPA receptor (AMPAR) levels are high during wakefulness and low during sleep, and changes in the phosphorylation states of AMPARs, CamKII and GSK3beta are consistent with synaptic potentiation during wakefulness and depression during sleep. Furthermore, slope and amplitude of cortical evoked responses increase after wakefulness, decrease after sleep and correlate with changes in slow-wave activity, a marker of sleep pressure. Changes in molecular and electrophysiological indicators of synaptic strength are largely independent of the time of day. Finally, cortical long-term potentiation can be easily induced after sleep, but not after wakefulness. Thus, wakefulness appears to be associated with net synaptic potentiation, whereas sleep may favor global synaptic depression, thereby preserving an overall balance of synaptic strength.

Molecular correlates of sleep and wakefulness in the brain of the white-crowned sparrow.

In the mammalian brain, sleep and wakefulness are associated with widespread changes in gene expression. The extent to which the molecular correlates of vigilance state are conserved across phylogeny, however, is only beginning to be explored. The goal of this study was to determine whether sleep and wakefulness affect gene expression in the avian brain. To achieve this end we performed an extensive microarray analysis of gene expression during sleep, wakefulness, and short-term sleep deprivation in the telencephalon of the white-crowned sparrow (Zonotrichia leucophrys gambelii). We found that, as in the rodent cerebral cortex, behavioral state, independent of time of day, has widespread effects on avian brain gene expression, affecting the transcript levels of 255 genes (1.4% of all tested transcripts). Wakefulness-related transcripts (n = 114) code for proteins involved in energy metabolism and oxidative phosphorylation, immediate early genes and transcription factors associated with activity-dependent neural plasticity, as well as heat-shock proteins and molecular chaperones associated with the unfolded protein response. Sleep-related transcripts (n = 141) code for proteins involved in membrane trafficking, lipid/cholesterol synthesis, translational regulation, cellular adhesion, and cytoskeletal organization. Remarkably, despite the considerable differences in morphology and cytology between the mammalian neocortex and the avian telencephalon, the functional categories of transcripts identified in this study exhibit a significant degree of overlap with those identified in the rodent cortex.

Nucleoplasmin facilitates reprogramming and in vivo development of bovine nuclear transfer embryos.

Successful cloning by somatic cell nuclear transfer (NT) involves an oocyte-driven transition in gene expression from an inherited somatic pattern, to an embryonic form, during early development. This reprogramming of gene expression is thought to require the remodeling of somatic chromatin and as such, faulty and/or incomplete chromatin remodeling may contribute to the aberrant gene expression and abnormal development observed in NT embryos. We used a novel approach to supplement the oocyte with chromatin remodeling factors and determined the impact of these molecules on gene expression and development of bovine NT embryos. Nucleoplasmin (NPL) or polyglutamic acid (PGA) was injected into bovine oocytes at different concentrations, either before (pre-NT) or after (post-NT) NT. Pre-implantation embryos were then transferred to bovine recipients to assess in vivo development. Microinjection of remodeling factors resulted in apparent differences in the rate of blastocyst development and in pregnancy initiation rates in both NPL- and PGA-injected embryos, and these differences were dependent on factor concentration and/or the time of injection. Post-NT NPL-injected embryos that produced the highest rate of pregnancy also demonstrated differentially expressed genes relative to pre-NT NPL embryos and control NT embryos, both of which had lower pregnancy rates. Over 200 genes were upregulated following post-NT NPL injection. Several of these genes were previously shown to be downregulated in NT embryos when compared to bovine IVF embryos. These data suggest that addition of chromatin remodeling factors to the oocyte may improve development of NT embryos by facilitating reprogramming of the somatic nucleus.

Identification of differentially expressed genes in individual bovine preimplantation embryos produced by nuclear transfer: improper reprogramming of genes required for development.

Using an interwoven-loop experimental design in conjunction with highly conservative linear mixed model methodology using estimated variance components, 18 genes differentially expressed between nuclear transfer (NT)- and in vitro fertilization (IVF)-produced embryos were identified. The set is comprised of three intermediate-filament protein genes (cytokeratin 8, cytokeratin 19, and vimentin), three metabolic genes (phosphoribosyl pyrophosphate synthetase 1, mitochondrial acetoacetyl-coenzyme A thiolase, and alpha-glucosidase), two lysosomal-related genes (prosaposin and lysosomal-associated membrane protein 2), and a gene associated with stress responses (heat shock protein 27) along with major histocompatibility complex class I, nidogen 2, a putative transport protein, heterogeneous nuclear ribonuclear protein K, mitochondrial 16S rRNA, and ES1 (a zebrafish orthologue of unknown function). The three remaining genes are novel. To our knowledge, this is the first report comparing individual embryos produced by NT and IVF using cDNA microarray technology for any species, and it uses a rigorous experimental design that emphasizes statistical significance to identify differentially expressed genes between NT and IVF embryos in cattle.

Production of cloned cattle from in vitro systems.

The pregnancy initiation and maintenance rates of nuclear transfer embryos produced from several bovine cell types were measured to determine which cell types produced healthy calves and had growth characteristics that would allow for genetic manipulation. Considerable variability between cell types from one animal and the same cell type from different animals was observed. In general, cultured fetal cells performed better with respect to pregnancy initiation and calving than adult cells with the exception of cumulous cells, which produced the highest overall pregnancy and calving rates. The cell type that combined relatively high pregnancy initiation and calving rates with growth characteristics that allowed for extended proliferation in culture were fetal genital ridge (GR) cells. Cultured GR cells used in nuclear transfer and embryo transfer initiated pregnancies in 40% of recipient heifers (197), and of all recipients that received nuclear transfer embryos, 9% produced live calves. Cultured GR cells doubled as many as 85 times overall and up to 75 times after dilution to single-cell culture. A comparison between transfected and nontransfected cells showed that transfected cells had lower pregnancy initiation (22% versus 32%) and calving (3.4% versus 8.9%) rates.

Ontogeny of cloned cattle to lactation.

Central to the success of large animal cloning is the production of healthy animals that can provide products for human health, food, and other animal agriculture applications. We report development of cloned cattle derived from 34 genetically unique, nonembryonic cell lines using nuclear transfer performed between 1 January 1998 and 29 February 2000. Nearly 25% (535/2170) of the recipients receiving reconstructed embryos initiated pregnancy. Overall, 19.8% (106/535) of the initiated pregnancies resulted in live births, while 77% (82/106) of these cattle clones remain healthy and productive today. Although a wide variation in birth weight of clone calves was observed, their growth rates, reproductive performance, and lactation characteristics are similar to that found in noncloned dairy cattle. Our data represent the most comprehensive information on cattle derived from nuclear transfer procedures and indicate that this emerging reproductive technology offers unique opportunities to meet critical needs in both human health care and agriculture.