Hello, my name is Chiara Cirelli, I'm a professor of psychiatry at the University of Wisconsin in Madison. And I'm here to tell you about the molecular biology and genetics of sleep and my lecture is going to be divided into two main parts. First, I will review studies of molecular biology mainly transcriptomic studies showing that the behavioral states like sleep and wake, have a profound effect on the expression of hundreds of genes in the brain. And in the second part, I will review genetic studies in humans and animals showing the other side of the story, meaning that genes that can have a very profound effect on the sleep phenotype. The molecular biology studies so far have been based mainly on microarray analysis and have been done in several species, as you can see here in flies, which are diurnal animals, so they sleep mainly during the night. And in many rodents, hamsters, mice, and rats that are nocturnal animals and they sleep during the day. The electrophysiological signatures of sleep and wake in these species are quite different as you can see as schematicized here, but yet all these animals do sleep. The array analysis is quite powerful. It is possible to probe up to 15,000 different messenger RNAs, and the take home message from many of these studies has been that up to five percent of all the genes expressed in the brain regions for instance, the cerebral cortex or the forebrain, are affected in their expression by sleep and wake. Most importantly, the sleep and wake genes belong to different functional categories and very often as we will see, are the complementary kind of functions. Here is a schematic of the major categories of cellular processes that are affected by sleep and wake genes. And for sleep genes, I mean those genes whose messenger RNA is expressed at higher level after several hours of sleep relative to several hours or of either spontaneous wake or acute sleep deprivation. And vice versa of course, wake genes are those upregulated after spontaneous or forced wake relative to sleep. So our category of wake genes includes those implicated in synaptic plasticity and more specifically in synoptic potentiation. While in sleep there is upregulation of genes involved in synoptic depression, wake genes are also involved in energy metabolism and in the cellular stress response, while sleep genes are involved in proteins synthesis and in lipid metabolism. Let's focus first on the three major categories of wake genes and let's start with the synoptic potentiation. Follow up studies to those that I mentioned with transcriptomics have shown that in most cases, this plasticity related the genes are induced and upregulated in wake, not only the messenger RNA level but also at the protein level. Here is an example, this is phosphor creb and here you see a schematic to show you which region of the cerebral cortex of the rat we are looking at in these images, these are immunocyto chemicals staining for phosphor creb in the rat cerebral cortex in the parietal cortex and is quite obvious that the expression of phosphor creb, is very much increased after six hours of waking relative to six hours of sleep and this is true across all layers of the cortex and is also true in many other cortical areas. It is the phosphorelated form of creb that is changing between sleep and wake, it's not just the total amount of creb. Another example is BDNF, Brain Derived Neuraltrophic Factor. Again you see here in the same rat cortical area, the expression of BDNF in the cell bodies, here and in the fibers, in both cases there is much higher expression after wake than after sleep. And there are many other plasticity-related genes that are induced by wake including those listed here, NGFI-A, Homer, Narp, CamKII and Arc. There are also more direct markers of synaptic potentiation that we can study and look for their differential expression between sleep and wake. Perhaps the best characterized marker of synoptic strands is currently the expression of the GluR1-AMPA receptors. In the adult brain these receptors are tetramers, they are made up of two GluR1 and two GlueR2 subunits. And the expression of GluR1 containing excitatory antireceptors is increased in many conditions of increased synoptic strands and synaptic potentiation. Here you see just a few examples of the studies in vitro and in vivo that have provided the evidence for my statement. And this increase is in the number of these receptors as well as in the phosphorelation levels of the receptors of the GluR1 subunit, that specific serin residues such as 845 and 831. And vice versa conditions of synoptic depression are associated with reduced expression in the number and in the phosphorelation levels of these receptors. So in our study, animals were collected, rats after different behavioral stages. Here they were collected after several hours of sleep or sleep deprivation during the day or at night after several hours of spontaneous wake. The cerebral cortex was collected, synapto-neurosomes were prepared to enrich for synoptic proteins. And through Western Blot analysis, the expression of this GluR1 containing AMPA receptor was measured and you see here that there is a 30 to 40 percent increase in GluR1 protein levels after wake, relative to sleep. And the same change, although I'm not showing this here, is happening after shorter deprivation relative to sleep and similar changes that also according if we measure the phosphorelation levels. So let's now, after synaptic potentiation, let's now focus on the genes, the wake genes, that are related to energy metabolism.
