Thomas Scammell Laboratory
The video on the right hand side of your screen shows cataplexy in narcoleptic mice. All mice in this video lack the gene coding for the orexin peptides. Mice were filmed under infrared light during the night when they are active. Before having cataplexy, each mouse is engaged in active behaviors such as grooming and running. The mouse then becomes abruptly immobile and falls prone or to its side. After 30-60 seconds of immobility, the mouse abruptly resumes active behaviors. Onsets of cataplexy are shown with filled arrowheads; offsets with open arrowheads.
Research in the Scammell Laboratory
Our current work focuses on the neurobiology of sleep and the neural basis of narcolepsy. Narcolepsy is caused by an extensive and selective loss of the hypothalamic neurons that produce the orexin neuropeptides (also known as hypocretins). This cell loss generally occurs in the teens or young adulthood and results in lifelong sleepiness and cataplexy, brief episodes of muscle weakness that are similar to the paralysis that occurs during REM sleep. Much of our current research focuses on mouse models of narcolepsy because mice lacking orexins also have sleepiness and frequent episodes of cataplexy. We hypothesize that orexins normally stabilize the activity of wake-promoting brain regions, but absence of orexins produces behavioral state instability, with rapid transitions from wakefulness into sleep, and intrusions into wakefulness of REM sleep elements such as cataplexy or hallucinations.
Our major goals are to identify the neural mechanisms through which the orexin system controls sleep and wakefulness and to determine how loss of the orexin peptides results in sleepiness and cataplexy. We are pursuing these questions in several ongoing studies:
1) To identify the critical pathways through which orexins promote wakefulness and suppress cataplexy, we have produced mice in which local expression of Cre recombinase induces local expression of the orexin receptors. Using adeno-associated viral vectors and genetic techniques, we induce expression of orexin receptors in specific brain regions such as the basal forebrain or in neurochemically specific nuclei such as the histamine-producing neurons. We have found that orexin signaling through the basal forebrain or posterior hypothalamus substantially improves the sleepiness of narcolepsy, and we are now identifying the specific cells that mediate this improvement.
2) We are examining the roles of several wake-promoting systems using optogenetic techniques. In these experiments, we microinject an adeno-associated viral vector that drives expression of channelrhodopsin into mice that express Cre recombinase in select neurons such as the histamine neurons of the tuberomammillary nucleus. Channelrhodopsin is then expressed only in these cells, and we use pulses of blue laser light to study how these cells promote arousal.
3) In humans, cataplexy is triggered by strong, positive emotions, and we seek to identify the mechanisms through which emotions trigger cataplexy. We have found that cataplexy in narcoleptic mice is increased by rewarding stimuli such as running wheels, group housing, and highly palatable food such as chocolate. We have found that the medial prefrontal cortex and amygdala are necessary for cataplexy, and we are now mapping the specific neural pathways through which this occurs.
4) We have found that the orexin neurons also produce dynorphin and glutamate, and we are using electrophysiologic techniques, mathematical modeling, and new lines of mice to determine the functions of these co-neurotransmitters.
5) In collaboration with Dr Elda Arrigoni's lab, we are examining the electrophysiologic effects of orexin and dynorphin peptides on neurons of the basal forebrain and other regions. These studies use patch clamp recordings and channelrhodopsins to identify the precise mechanisms through which these peptides influence their targets.
6) We have found that the orexin-producing neurons are active during wakefulness, and we are using novel methods to trace these the inputs and electrophysiologic mechanisms through which this occurs.
7) In studies of human brains, we have found that loss of the orexin neurons in narcolepsy is also accompanied by a large increase in the number of neurons producing histamine. This may be a compensatory response that helps produce wakefulness after loss of the orexin neurons. In related work, we are also examining whether loss of the orexin neurons and other wake-promoting systems contributes to the sleepiness often seen after traumatic brain injury.
8) Other new projects are determining how cholinergic neurons of the pons regulate sleep and how pain disrupts sleep.
Our lab uses a variety of anatomic, physiologic, and molecular techniques. We frequently study sleep/wake behavior in mice using detailed analysis of the electroencephalogram in conjunction with recordings of muscle activity, locomotion, behavior, and body temperature. We have also developed new mathematical techniques for analysis of the transitions between behavioral states and examination of intermediate states. We trace neural pathways using novel and conventional anterograde and retrograde tracers, and we perform immunostaining and in situ hybridization histochemistry to map the distribution of neurotransmitters, receptors, and other molecules. We also use a variety of molecular techniques to design and produce novel recombinant mice.
Through these approaches, we hope to gain a detailed understanding of the neurobiology of sleep and wakefulness that will result in highly effective therapies for patients with narcolepsy and other sleep disorders.
1. Some of the key pathways that promote wakefulness. Monoaminergic systems (green) diffusely innervate the forebrain, and cholinergic signals (blue) project to the forebrain and thalamus from the pons and basal forebrain.
2. The orexin neurons promote wakefulness and regulate REM sleep, but these cells are lost in narcolepsy. This loss of orexin signaling probably causes sleepiness through reduced activity of the basal forebrain and monoaminergic systems. In the absence of orexins, REM sleep is poorly controlled, resulting in episodes of paralysis and dream-like hallucinations during wakefulness.
3. Noradrenergic neurons of the locus coeruleus (brown) in humans are heavily innervated by orexin-containing nerve terminals (black)
4. Double-label in situ hybridization shows that the orexin neurons (prepro-orexin mRNA labeled red) also contain dynorphin (pre-prodynorphin labeled with silver grains, white dots).
5. Microinjection of an adeno-associated viral vector (AAV) to induce local expression of Cre recombinase enables one to focally manipulate the expression of loxP-flanked genes. This photo shows in situ hybridization for the adenosine A1 receptor in a mouse with loxP sites flanking the major A1 receptor exons. Local injection of AAV-Cre reduced A1 mRNA expression by about 90% (deleted region indicated by arrows).