Dormivigilia

We’ve been training for this for a little over a year (the prospect of re-qualifying for Regionals), and it’s finally here. At the end of this week, I will be travelling to West Palm Beach, Florida to attempt to qualify for the 2013 Reebok Crossfit Games…if we finish in the top three. We are ranked first going in, but recognize that it is going to be a fight for the podium. So, I will be taking a near week hiatus from science-based blogging and even some research. However, I do have an early career grant due on Wednesday and a travel application on Friday so I guess there will be work here and there : ) Here is a list of the seven workouts that I will do over three days with my teammates at Crossfit RX.

This week, our lab published a paper that we’ve been working on for some time (and one that I’m continuing). It’s also kind of hilarious that Montegraphia made it onto a paper from my postdoctoral lab (see acknowledgments section) before me! He only beat me by a few weeks though….

Our lab is interested in sex differences in sleep/wake before and after short-term sleep deprivation. In fact, my mentor has previously shown that males with circulating androgens sleep more often during the dark (active) period than females with circulating sex steroids. Removing the circulation of sex steroids, simply through castration or an ovarectomy, removes these sex differences. However, sleep rebounds following a period of short-term sleep deprivation were less sensitive to hormonal removal and replacement, suggesting that there are other physiological substrates that determine one’s sex that contribute to sex differences in sleep. One candidate is sex chromosomes.

This was essentially the specific aim of our recent paper–to differentiate between hormonal versus chromosomal sex differences in sleep. To accomplish this, we used the four core genotype mice which I described in a very recent post. These mice have been used to differentiate hormonal versus chromosomal sex differences in circadian timekeeping (though subtle) and alcohol drinking behaviors (extremely compelling). To recap, the sex chromosome opposes the gonadal makeup due to a spontaneous deletion of the testes-determining gene Sry. Through a complicated scheme of Mendelian genetics, hermaphroditic mice are produced–XY females and XX males. The mice in this study were also castrated and ovarectomized in order to remove the additional confound of circulating sex steroids. In reproductive physiology research, this will also help determine if some trait is possibly organized by hormones (ie. hard-wired) or activated by hormones.

Prior to sleep deprivation, there was only one time point in which the amount of sleep varied across a 24 hour period;  midnight. Here, normal and hermaphroditic (XXM) males had significantly more sleep than typical females (hermaphroditic females were somewhere in the middle). Further, after the animals were sleep-deprived, the hermaphroditic males had a greater propensity to sleep (deeply), particularly during the dark phase. This suggests that the chromosomal ‘male’  sleep homeostat is set higher (sucks for them).

While we did not report the neural substrates and mechanisms of these sleep phenotypes in this paper, we do have some evidence, but you’ll have to wait until the Society for Neuroscience meeting in San Diego to find out.

Ehlen, J., Hesse, S., Pinckney, L., & Paul, K. (2013). Sex Chromosomes Regulate Nighttime Sleep Propensity during Recovery from Sleep Loss in Mice PLoS ONE, 8 (5) DOI: 10.1371/journal.pone.0062205

We’ve known in the sleep community for quite some time that REM sleep is important for the consolidation and recall of memories, albeit declarative (facts), procedural (motor skills), and emotional. We also know, or at least can postulate that the hippocampus plays an important role wherein there are actually changes in neurons at global and local levels. In a recent article in the Journal of Neuroscience, scientists have studied the molecular signaling that underlies memory consolidation and recall during REM sleep, and it’s no surprise that changes in cAMP, MAPK, and CREB which are the main components of most major (metabotropic) signaling cascades preclude memory consolidation. These are the same molecular factors involved with long-term potentiation and depression that Dr. Eric Kandel and colleagues discovered in the Aplasia (sea slug) decades ago. You can clearly see changes in cAMP, MAPK, and CREB during REM sleep (dark bars) in these Western blots from the paper

These researchers also used mutant mice that lacked adenylyl cyclases–a complex upstream of cAMP–which are able to learn but their memories can’t be consolidated via the hippocampus. As shown in this paper, these mice also don’t have increases in cAMP, MAPK, and CREB, providing further evidence that this trifecta of molecular factors are important for memory consolidation driven by REM sleep. Maybe the brain is simpler than we think……not…..

Luo, J., Phan, T., Yang, Y., Garelick, M., & Storm, D. (2013). Increases in cAMP, MAPK Activity, and CREB Phosphorylation during REM Sleep: Implications for REM Sleep and Memory Consolidation Journal of Neuroscience, 33 (15), 6460-6468 DOI: 10.1523/JNEUROSCI.5018-12.2013

This paper represents my official graduation from the (Dave) Glass lab (note: thanks to my labmates, DG and his money, and the pursuit of some cool projects, grad school was an awesome ride that will never be forgotten). In this last hoorah, I examined how mice that cannot express the clock protein Per2 responded to cocaine. In a previous report, we found that systemic and intracranial administrations of cocaine at midday can advance rhythms of locomotor activity by 1-3 hours in wild-type mice. 

Using similar experimental techniques, we recapitulated our previous findings;  the wild-type controls for the Per2-mutants also shifted their rhythms in response to systemic and intracranial administrations of cocaine. Further, systemic administrations of cocaine at night impaired the ability of rhythms to shift in response to a pulse of light. The circadian system of Per2-mutants, however, was hypersensitized in response to systemic administrations of cocaine–their behavioral rhythms shifted 3x as much as wild-types and were comparable to shifts found in Per2-mutants and wild-types after cocaine was perfused into the brain through a probe targeting the SCN. Per2-mutants were also hypersensitized to light at night in that mutants not treated with cocaine had larger shifts compared with wild-types, and mutants treated with cocaine had basically no shift in response to light.

In lieu of these results, we now know that differences in cocaine-induced shifts driven by PER2 probably manifest from cocaine binding in some structure or action in some pathway that lies outside the SCN. This hypothesis is further supported by data showing changes in the intensity of locomotor activity (or lack thereof) in response to cocaine such that only the wild-types were sensitive to the (behavioral) stimulating effects of cocaine despite having smaller shifts of behavioral rhythms compared with Per2 mutants.

I’m out!

Brager AJ, Stowie AC, Prosser RA, & Glass JD (2013). The mPer2 clock gene modulates cocaine actions in the mouse circadian system. Behavioural brain research, 243, 255-60 PMID: 23333842

I apologize for discontinuing Neury Thursday (plus or minus a few days) for some time. Quite frankly, there was a drop in sleep, circadian, and drug-related publications in the Journal of Neuroscience. But I have a new line-up of J of N publications to keep me busy for the next month. The first article that I am going to discuss was done in collaboration with a ‘bigwig’ in the field of drug addiction and current director of the National Institute of Drug Abuse (NIDA) who is known for identifying structures responsible for drug-seeking, craving, and relapse through the utilities of neuroimaging (Dr. Nora Volkow). Well, her and colleagues are back at it, but have moved on from the study of humans to the study of rodents using nearly identical neuroimaging techniques! Yes, it is possible to do a PET (positron emission tomography) scan in rats. In brief, PET detects changes in metabolic activity in the brain (i.e.: glucose uptake) through the utility of a radioactive tracer. This study seems to be more or less a pilot study to test the validity of PET imaging in rodents and whether documented changes in activity correspond with previously established techniques for detecting heightened brain activity– quantification of cfos by means of immunohistochemistry– or stimulating brain areas– optogenetics.

The hypothesis was simple; identify brain areas with increased or decreased activity in awake rats. I do have one problem with the study, however.  I could not find any documentation of when during the animal’s light-dark photocycle that these procedures were executed (i.e. it’s evident that none of the reviewers are in the field of sleep and circadian rhythms). This is important because the accumulation of sleepiness across an animal’s dark period could have confounded the extent of change in brain activity. Despite this, the results are consistent with previous reports of which brain areas show heightened activity during wakefulness; areas of the striatum (nucleus accumbens, ventral pallidum, caudate putamen), limbic system (amygdala and hippocampus), and the somatosensory cortex, to name a few. It’s weird that the cingulate gyrus showed decreased activity during wakefulness because this is a major attention processing area, which I guess could be one indication of sleep and circadian systems confounding the results.

Overall, changes in metabolic activity detected with PET corresponded with changes detected by immunohistochemistry following optogenetic stimulation of these same brain areas. While I consider this to be more of a methods paper, I imagine that there will be many other studies relevant to the neurobiology of drug addiction using these techniques from this group in the near future. This is probably part of the reason why they probably did not choose to do a whole-brain examination because there are many more areas regulating wakefulness deep in the brainstem that they missed.

Thanos PK, Robison L, Nestler EJ, Kim R, Michaelides M, Lobo MK, & Volkow ND (2013). Mapping Brain Metabolic Connectivity in Awake Rats with μPET and Optogenetic Stimulation. The Journal of neuroscience : the official journal of the Society for Neuroscience, 33 (15), 6343-6349 PMID: 23575833

For the past two weeks, I’ve come home from a day of grueling benchwork  to decompress with Reno 911! In college, I used to think the show was idiotic, but now I find it to be hilarious. In a recent episode, the officers do a PSA about the dangers of drowsy driving. While drowsy driving is a serious concern and is responsible for thousands of accidents a year, it’s about time that the public recognizes that turkey does not directly cause sleepiness. In fact, the ‘active’ ingredient in turkey–tryptophan–is present in comparable doses in other meats like beef and chicken. Further, serotonin–a neurotransmitter promoting wakefulness (in most cases)–is derived from tryptophan. The likely physiological explanation for sleepiness induced by turkey is that blood flow and oxygen uptake by the brain is reduced in order to focus on digestion.

http://www.comedycentral.com/video-clips/q0ms4j/reno-911–dwt

Before the widespread use of transgenic mice, reproductive physiologists had limited means of studying the effects of sex (or gender) on behavioral and physiological processes; most studies would remove an animal’s gonads, causing the (sex) hormonal environment to be somewhat ‘equal,’ and then would restore the circulation or do a swap replacement (androgens to females, estrogens to males). Nowadays, we have access to the four core genotype line (well, some of us do) in which the sex chromosome complement can oppose the gonadal phenotype, essentially creating mice with gender identity disorder–XX mice with testes and XY mice with ovaries. This is due to a spontaneous deletion of the Sry gene, which dictates the development of testes. I’ve discussed alcohol-related research in these mice prior.

Very recently, some West Coast circadian biologists examined circadian rhythms in this line at behavioral, physiological, and molecular levels. Although the differences were subtle, there were certain time points and circumstances wherein the influences of sex chromosomes or sex hormones were apparent. First, the mice were put on running wheels, and it was found that the mice with female sex chromosomes (XX males and XX females) were active longer during the night compared to mice with male sex chromosomes (XY females and XY males) when the animals were housed under constant darkness. This is interesting because wild-type females with circulating estrogens also have longer periods of nighttime activity under constant darkness, suggesting that this sex difference may be mediated by the sex chromosomes. The researchers also investigated how the animals would shift (delay) their rhythms of wheel running in response to light in the early part of the night. There were apparent differences in XX and XY females versus XX and XY males with intact gonads, with the females having larger phase-delays, but these differences completely disappeared when the gonads were removed. Under this circumstance, this would suggest that sex differences in response to light may be mediated by sex hormones.

The researchers also looked at in vitro rhythms of the master circadian clock (the SCN) and peripheral tissues (adrenals and liver). But this wasn’t in the transgenic mice, just regular wild-types. In summary, the males had higher rates of neuronal activity in a particular section of the SCN at midday and their genetic rhythms in the peripheral tissues peaked at different times. All in all, sex differences in the circadian system are evident and can be influenced by difference components of sexual makeup (chromosomal vs hormonal) even if the differences aren’t striking.

Kuljis, D., Loh, D., Truong, D., Vosko, A., Ong, M., McClusky, R., Arnold, A., & Colwell, C. (2013). Gonadal- and Sex-Chromosome-Dependent Sex Differences in the Circadian System Endocrinology, 154 (4), 1501-1512 DOI: 10.1210/en.2012-1921

This weekend, my teammates and I finished our 5 weeks of worldwide (crossfit) competition; every Wednesday evening, a workout was released and we had until Sunday evening to complete it as many times as we chose. Most of us did it once although I chose to re-do the third one because I had complete muscle failure on the first try. Each workout was designed to test different metabolic pathways and skill sets–some had heavy Olympic lifts performed over a short duration whereas others had a mixture of light to heavy gymnastics and neurological-based exercises across a longer duration—but all of them hurt; some were lung burners, others caused intense muscle fatigue, while others completely drained your ability to move efficiently. All of us pushed ourselves to new physiological and mental limits and our hours of training paid off because we advanced to the next competition which is a three-day event in West Palm Beach. But we not only advanced, we dominated, having placed first in the Southeast (comprised of Florida, Georgia, South Carolina, and Alabama) and ninth in the world! If we place in the top three at the regional event in WPB, then we will compete in the ‘Fittest on Earth’ competition (ie. the Crossfit Games) outside of LA and can guarantee to see some air time on ESPN. Until then, I’ll be balancing training with research and teaching loads, although I am one of those weird people who manages to stay focused and become even more productive throughout the process…..or so I tell myself…..

I spent the latter part of last week in basketball country; Bloomington, Indiana. Aside from cursing over the loss of Indiana to Syracuse (only to preserve my first place ranking in the fantasy college basketball), I attended an animal behavior conference that was very multidisciplinary. Most of the presenters were graduate students and postdocs but their disciplines ranged from ecology, to computational biology, to neuroscience. The keynote speaker of the conference was Dr. Frances Champagne from Columbia University who does some really exciting (and media-friendly) research in epigenetics. She examines how behaviors linked to anxiety and depressive-states in rodents (high licking) can be passed on to several generations by means of pathological changes in the genetic architecture. One of the most common pathologies studied by her lab is DNA methylation which involves the addition of a methyl group to a cysteine or adenine, causing a change in gene expression and subsequent change in cell division or development. Dr. Champagne’s research has also been featured on one of my favorite science-based podcasts: Radiolab.

In addition to Dr. Champagne’s talk, I learned a substantial amount about social aggression and stress in hamsters; male hamsters have a very complex hierarchy of winners and losers with losers showing high levels of anxiety for a month after they have been defeated. The mechanism behind this social stress likely involves the monoaminergic pathway (serotonin, dopamine, norephinephrine), particularly in structures of the midbrain and hindbrain. I had the unfortunate opportunity to see this type of male-on-male (and even female) aggression in graduate school (though unprovoked). I say unfortunate because my fingers lost the fight (don’t worry, only a bite or two!).

I also had the opportunity to visit the Kinsey Institute when I was on campus. Alfred Kinsey was the pioneer of research on human sexuality and as expected, had been a target for many right-winged, religious groups given that he viewed homosexuality to be under the influences of both one’s biology and social environment. He also adopted a viewpoint that every human is bisexual in some regard with women displaying more bisexual tendencies than men. I often give students in my neuroendocrine course this study published by the Kinsey Institute to read as it examines the powerful influence of pheromones on reproductive (in some sense) behavior.

Overall, I enjoyed my trip. I’d like to thank my mentor and the Center for Behavioral Neuroscience at Georgia State for providing travel funds.

A few years ago, I met Dr. David Raizen at a small sleep and circadian rhythm retreat hosted by my undergraduate advisor (even though I’ve summarized talks given at these retreats in the past, David’s year as a young investigator was before the inception of my blog). David is a neuroscientist at UPENN who studies the mechanisms of sleep in a very basic animal model: C.elegans. Before you discredit his lab’s findings because of difficulty seeing the translational potential of this work, you may recall that many of the esteemed Nobel Laureates in the field of neuroscience worked on simple model organisms to identify the mechanisms of neuron communication (the lobster, studied by Ed Kravitz, Steven Kuffler, and colleagues at Harvard) and learning and memory (the sea slug, studied by Eric Kandel and colleagues at Columbia). Although classic identifications of sleep/wake derived from EEG waveforms are not undertaken in c.elegans, the worms do show sleep-like states which fit the four basic behavioral criteria of sleep; 1. species-specific posture; 2. increased arousal threshold; 3. rapidly reversible state; and 4. the obvious, behavioral quiescence.  In c. elegans, this sleep-like state is called lethargus quiescence and is observed through video recording.

In this particular study, Raizen and colleagues manipulated a transcriptional complex known as DAF-16/FOXO which is activated under conditions of stress like sleep (lethargus quiescence) deprivation. By depriving the worms of sleep, it was found that the DAF-16 complex migrated (translocated) from the cytoplasm to the nucleus in many cell types, including the intestine and muscle. Following sleep deprivation, the complex would then gradually return to the cytoplasm. Below is a visual representation of this translocation.

What happens if DAF-16 expression is arrested? In the absence of DAF-16, the worms failed to show a classic homeostatic response to sleep deprivation (more sleep during the recovery period, relative to baseline amounts). However, if this DAF-16 expression was restored to the muscle or the neurons, something interesting occurred; the normal homeostatic response was restored, but only when DAF-16 was rescued in the muscle (and not the central nervous system). Yes, folks, this is the first piece of evidence for muscle regulation of sleep, at least in a simple model organism. Whether this has replication potential in mammalian species remains to be determined. At any rate, I look forward to it. Before you start labeling this finding as a fluke, you should consider that there are vast physiological and molecular changes in the muscle in response to exercise and rest with the former requiring massive recruitment of energy sources (ATP, glucose) and coordination in neuronal communication. Much like the brain needs to ‘take a break’ during sleep as evidenced by neurons going offline, wouldn’t it make sense that the muscles need to ‘reboot’  during sleep too?

Driver, R., Lamb, A., Wyner, A., & Raizen, D. (2013). DAF-16/FOXO Regulates Homeostasis of Essential Sleep-like Behavior during Larval Transitions in C. elegans Current Biology, 23 (6), 501-506 DOI: 10.1016/j.cub.2013.02.009