The identification and validation of candidate causal genes regulating sleep and wake may provide new therapeutic targets for insomnia as well as other diseases in which sleep is dysregulated. The approach used in our studies is direct; causal inference generated a comprehensive list of candidate genes, which we used to identify pharmacological reagents specific to these targets. Such an approach allowed for the alignment of genetic causality with pharmacodynamics using small molecule tools in preclinical sleep studies. The objective of confirming associated sleep/wake traits identified from a genetically segregating population is met with certain challenges. In our studies, causal inference identified candidate genes that govern sleep in an N2 cross involving mice, while validation studies were conducted on Sprague-Dawley rats. For each genetic target, select molecules had significant effects on rat sleep architecture that were in alignment with associated sleep traits discovered in mice. Validating genetic targets in rats that were originally identified in mice is a testament to the strength and potential of these targets as being impactful on sleep and arousal across species. Further work may determine if these results translate to humans.
Behavioral state as determined by EEG provides a complex readout on the overall electrical activity in the brain that includes information on many complex and inter-related traits including sleep state, total time spent in each state, and fragmentation of sleep. Identified candidate genes may primarily impact a single sleep-wake trait, which at the same time may affect sleep architecture due to compensation; a loss or gain in a single sleep state affects total time spent in other sleep states. Therefore, alignment of associated sleep-wake traits with effects of target engagement on rat electroencephalography is not straightforward. Our criterion for alignment is therefore either positive (e.g. gain of time spent) or negative (e.g. loss of time spent) effects on the associated sleep-wake trait, with the expectation of consequential compensatory effects on other parameters.
Reagent polypharmacology and pharmacokinetics are other considerations and potential challenges to this approach. Even molecules with high specificity for target gene products have the potential for low affinity off-target interactions that produce effects on behavioral state. Selection of reagents that have complementary or contrasting pharmacology offers the best approach to differentiating target and off-target effects. Additionally, certain small molecules may not sufficiently penetrate the central nervous system to produce significant effects on behavioral state. Whenever possible, in the present study we utilized doses that had demonstrated central effects on other behaviors. A negative finding, however, is inconclusive in the absence of in depth characterization, such as receptor occupancy, and may indicate the compound did not have effects at selected doses due to poor CNS bioavailability. Our choice of reagents targeting CHRM3, CHRNA4, HTR1D, DRD5, GLP1R and CACNA1I satisfied the above selection requirements, and demonstrated effects on rat sleep architecture that aligned with associated sleep-wake traits as elaborated on below.
The cholinergic receptors, nicotinic acetycholine receptor alpha4 subunit and the muscarinic acetycholine receptor M3 subunit, were both associated with total sleep, wake and non-REM sleep traits. The selective pharmacological tools had the same overall impact on rat sleep architecture. Scopolamine, darifenacin, cytisine, and varenicline significantly promote wake at the expense of SWS and REM sleep. The selective muscarinic M3 antagonist, darifenacin, and more potent nicotinic alpha4beta2 agonist, varenicline, has more substantial, quantifiable, and prolonged impact on wake, total sleep and non-REM sleep. These findings are consistent with the observation that cholinergic neurotransmission provides input for the Ascending Arousal System, neuronal circuitry that induces cortical activity and modulate behavioral state. Major cholinergic contributors to this pathway originate in the mesopontine area and activate thalamic relay neurons, which in turn promote cortical activity and wake. Secondary contribution bypasses thalamic control of wakefulness, and originates in the basal forebrain (Saper et al., 2005
). Muscarinic M3 receptor containing neurons are localized to the mesopontine region, in the pedunculopontine and laterodorsal tegmental nuclei and may contribute to both branches of the Ascending Arousal System (Vilaro et al., 1994
). These regions of the mesopontine area fire rapidly during wake and REM sleep (Saper et al., 2005
In addition to promoting wake through the Ascending Arousal System, cholinergic signaling also inhibits key neurons in the ventrolateral preoptic nucleus that promote sleep (Saper et al., 2005
). The nicotinic acetylcholine receptor, alpha 4 subunit may contribute to this sleep effect (Dani & Bertrand, 2007
). In this way, modulating cholinergic neurotransmission may primarily inhibit sleep and induce a compensatory shift in sleep architecture favoring light sleep and wake.
We studied effects of modulating monoaminergic neurotransmission on rat sleep. Genes that encode serotonin 1D receptor and dopamine D5 receptor were both identified in our causality study (associated with duration between REM bouts, and non-REM sleep respectively). Monoaminergic signaling pathways feed into thalamic relay neurons that ultimately stimulate cortical activity and arousal, including those that originate from the ventral tegmental area (dopaminergic), and the raphe nuclei (serotonergic) (Saper et al., 2005
). Certain psychiatric and neurological pathology are attributed to dysfunction in the serotinergic and dopaminergic pathways that also display co-morbidity with disordered sleep. Depression, Parkinson’s disease, and schizophrenia are all conditions that exhibit altered monoaminergic signaling, and are also associated with disruptions in sleep architecture (Peterson & Benca, 2006
; Cohrs, 2008
; Compta et al., 2009
Dopamine receptor D5 was identified as a candidate gene associated with non-REM sleep. Subtypes D1 and D5, are classified D1-like receptors, while dopamine receptors D2, D3, and D4 are considered D2-like. The two subfamilies of dopamine receptors have differing pharmacology, while, within subfamilies, receptor subtypes are nearly indistinct (Missale et al., 1998
). SCH23390 is a potent dopamine D1 and D5 (D1-like) receptor antagonist (Bourne, 2001
). In contrast, risperidone, an atypical antipsychotic, was examined for its high affinity for the dopamine D2 (D2-like) receptor. In our studies, both risperidone and SCH23390 impact non-REM sleep. However, SCH23390, the D1-like specific antagonist impacted latency to non-REM sleep, and had profound day after effects on non-REM and other sleep parameters as compared to the D2-like compound, risperidone. This day after effect is paradoxical, since the compound has a short half life of 25 minutes in rats, and may result from rapid neuronal expression changes or another compensatory or anticipatory mechanism(Bourne, 2001
The Htr1d gene was associated with duration between bouts of REM sleep (REM consolidation/fragmentation). Studies involving 5-HT1D receptor antagonism detected acute effects on cumulative time spent and average bout duration of REM sleep. Within 30 minutes of dosing, mean number of entries into REM sleep decreased. REM effects were specific to BRL-15572, a 5-HT1D receptor antagonist, and indicate reduction of the number of cycles and the average duration of REM sleep. And while the magnitude was relatively small but significant, and brief in duration, causality does not necessarily discriminate effect size. Dosing BRL-15572 early in the active period may reveal more prolonged effects on REM sleep.
We have previously reported effects of T-type calcium channel (Cacna1i
) antagonism on rat electroencephalography (Uebele et al., 2009
). The Cacna1i
gene was identified as a candidate causal gene for average bout duration of total sleep and SWS. TTA-A2 promotes SWS at the expense of active wake, light and REM sleep. TTA-A2 not only increases total sleep, but it specifically increases bout duration of SWS sleep, demonstrating remarkable alignment with the associated trait.
Glucagon-like peptide-1 (GLP1) functions as an incretin, to stimulate insulin and inhibit glucagon (Kanoski et al., 2011
). Both liraglutide and exendin-4 are incretin mimetics that also demonstrate central effects on appetite control when administered peripherally (Kanoski et al., 2011
). The Glp1r
gene was associated with wake, total sleep, non-REM sleep and latency to non-REM sleep. Both GLP1r agonists are sleep promoting compounds that specifically induce light sleep over SWS and REM sleep. Glucagon-like peptide-1 receptors are found throughout the hypothalamus, in regions that impact arousal, and when taken together with the observed pharmacology of liraglutide and exendin-4, GLP-1 receptors may play a role in regulating sleep and wake homeostasis.
In summary, pharmacological validation of candidate genetic targets is a direct approach that offers advantages over characterization of target genes in knockout or transgenic models. Interrogating gene function using small molecule tools provides an accelerated route to evaluate potential new targets for sleep disorders as well as for psychiatric diseases. In addition, small molecules test the targets in an organism that developed normally, without any genetic lesion, that could have led to abnormal physiology via compensatory gene regulation during development. This approach many not only validate or invalidate candidate genes as targets for drug discovery, but may also provide insight into the potential causal or reactive nature of specific genes and networks for regulating behavior. Taken together, the results from the present study demonstrate robust agreement between genetic causality in mice and pharmacologically induced sleep effects in rats, providing novel insights into the underlying physiology of sleep and wake regulation conserved between these species as well as the identification of new avenues for therapeutic investigation.