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

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

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

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

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

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.

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