REVIEW Temperature 2:3, 406--417; July/August/September 2015; Published with license by Taylor & Francis Group, LLC How genetically engineered systems are helping to define, and in some cases redefine, the neurobiological basis of sleep and wake Patrick M Fuller1,*, Akihiro Yamanaka2,*, and Michael Lazarus3,* 1Department of Neurology; Beth Israel Deaconess Medical Center; Division of Sleep Medicine; Harvard Medical School; Boston, MA USA; 2Department of Neuroscience II; Research Institute of Environmental Medicine; Nagoya University; Nagoya, Aichi, Japan; 3International Institute for Integrative Sleep Medicine; University of Tsukuba; Tsukuba, Ibaraki, Japan Keywords: adeno-associated virus, optogenetics, DREADD, RNA interference, sleep-wake regulation Abbreviations: AAV, adeno-associated viruses; GluClab, C. elegans glutamate- and ivermectin (IVM)-gated chloride channel subunits a and b; DOX, doxycycline; DREADD, designer receptors exclusively activated by designer drugs; EEG, electroencephalogram; MCH, melanin-concentrating hormone; NREM, non-rapid eye movement sleep; PG, Prostaglandin; REM, rapid eye movement sleep; RNAi, RNA interference; tTA, tetracycline-responsive transcription factor. The advent of genetically engineered systems, including transgenic animals and recombinant viral vectors, has facilitated a more detailed understanding of the molecular and cellular substrates regulating brain function. In this review we highlight some of the most recent molecular biology and genetic technologies in the experimental “systems neurosciences,” many of which are rapidly becoming a methodological standard, and focus in particular on those tools and techniques that permit the reversible and cell-type specific manipulation of neurons in behaving animals. These newer techniques encompass a wide range of approaches including conditional deletion of genes based on Cre/loxP technology, gene silencing using RNA interference, cell-type specific mapping or ablation and reversible manipulation (silencing and activation) of neurons in vivo. Combining these approaches with viral vector delivery systems, in particular adeno-associated viruses (AAV), has extended, in some instances greatly, the utility of these tools. For example, the spatially- and/or temporally-restricted transduction of specific neuronal cell populations is now routinely achieved using the combination of Cre-driver mice and stereotaxic-based delivery of AAV expressing Credependent cassettes. We predict that the experimental application of these tools, including creative combinatorial approaches and the development of even newer reagents, will prove necessary for a complete understanding of the neuronal circuits subserving most neurobiological functions, including the regulation of sleep and wake. Introduction Three decades ago Francis Crick envisioned technologies that would permit the inactivation of specified neuronal populations, in turn enabling scientists to determine how “function follows structure” (of the brain). To this end, one of the more enduring mysteries in the neurosciences is the brain mechanisms and substrates (i.e., key circuit nodes, their transmitters and their targets) that regulate sleep – a highly conserved and vital biological process. Over the last 2 decades, researchers have developed a wide range of molecular and electrophysiological techniques and tools for probing and perturbing neural circuitry, including the circuitry that regulates sleep and wake in mammals. And while these tools and techniques have provided significant insight into the molecular and neurotransmitter systems used by the brain to regulate sleep and wake (cf. section ‘Neuronal mechanisms of sleepwake regulation’), many of these approaches have non-trivial limitations, in particular with respect to data interpretation. For example, pharmacologic approaches such as receptor antagonists and protein inhibitors are often limited by low solubility, poor blood-brain-barrier permeability or other “off-target” side effects. Global knockout approaches have limited temporal and spatial resolution and can be confounded by ontogenetic issues. Even acute lesion approaches (including so-called “cell-specific” lesions) can produce collateral damage to adjacent brain structures that may, in turn, produce effects that are epiphenomenal to the lesion itself. Fortunately the emergence of newer conditional genomic models is helping to overcome many of these © Patrick M Fuller, Akihiro Yamanaka, and Michael Lazarus *Correspondence to: Patrick M Fuller; Email: pfuller@bidmc.harvard.edu; Akihiro Yamanaka; Email: yamank@riem.nagoya-u.ac.jp; Michael Lazarus; Email: lazarus. michael.ka@u.tsukuba.ac.jp Submitted: 05/22/2015; Revised: 07/13/2015; Accepted: 07/15/2015 http://dx.doi.org/10.1080/23328940.2015.1075095 This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted. 406 Temperature Volume 2 Issue 3 (e.g. fast Fourier transform) to determine the vigilance state of the subject under recording (Fig. 1).1,2,3 Over the years, and on the basis of EEG interpretation, several models of sleepwake regulation, both circuit- and humoralbased, have been proposed (Fig. 2). Humoral mechanisms of sleep- wake regulation The neural and cel- lular basis of sleep need Figure 1. Sleep bioassay system for rodents. (A) To monitor electroencephalogram (EEG) signals, stainless steel screws are or, alternatively, “sleep implanted epidurally over the frontal cortical and parietal areas of one hemisphere. In addition, electromyogram (EMG) drive” remains unre- activity is monitored by stainless steel, teflon-coated wires placed bilaterally within the trapezius muscles. (B) wakefulness (i) is characterized by low to moderate voltage EEG and the occurrence of EMG activity, whereas NREM sleep (ii) is identified by the appearance of large, slow brain waves with a rhythm below 0.5–4 Hz (orange frequencies in the fast Fourier transform, FFT, of the EEG) and REM sleep (iii), exhibits a shift back to a rapid low-voltage EEG and the appearance of brain waves in the theta range, i.e., 6–10 Hz (blue frequencies in FFT of the EEG). solved, but has been conceptualized as a homeostatic pressure that builds during the waking period and is dissipated by sleep. technical and interpretational concerns. Specific examples, which One theory is that endogenous somnogenic factors accumulate are discussed herein, include conditional deletion of genes based during wake and that their gradual accumulation is the underpin- on Cre/loxP technology, gene silencing using RNA interference, ning of sleep homeostatic pressure. And while the first formal cell-type specific mapping or ablation and genetically engineered hypothesis that sleep is regulated by humoral factors is credited receptor-channel systems (e.g. opsin-based optical switches, to Rosenbaum in 1892,4 it was Ishimori5,6 and Pieron7 who designer receptors exclusively activated by designer drugs and independently, and over 100 years ago, demonstrated the exis- mutated non-mammalian channel systems), the latter of which tence of sleep-promoting chemicals. Both researchers proposed, permit the “remote” and reversible manipulation of neuronal and indeed proved, that hypnogenic substances or “hypnotoxins” activity in behaving animals. In addition to providing an over- were present in the cerebral spinal fluid (CSF) of sleep-deprived view of these emerging molecular biological techniques, we will dogs.8 Over the past century several additional putative hypno- provide literature-based examples of their application in experiments seeking a detailed understanding of the anatomic and molecular mechanism governing behavioral state regulation. Models of sleep-wake regulation genic substances implicated in the sleep homeostatic process have been identified (for review, see9), including prostaglandin (PG) D210 (for review, see11), cytokines12 (for review, see13), adenosine14 (for review, see15), anandamide,16 and the urotensin II peptide.17 Additional clues into the “humoral mechanisms” Technical advances have often precipitated quantum leaps in underpinning sleep regulation have been gleaned from study of our understanding of neurobiological processes. For example, individuals exhibiting “sickness behavior”, i.e., the fever, malaise, Hans Berger’s discovery in 1929 that electrical potentials recorded increased pain sensitivity, anorexia, and changes in sleep–wake from the human scalp took the form of sinusoidal waves, the fre- typically observed during a bacterial or viral infection (for review, quency of which was directly related to the level of wakefulness of see18). Sickness behavior links to a cascade of pro-inflammatory the subject, led to rapid advances in our understanding of sleep- mediators, including a wide range of cytokines and PGs that trig- wake regulation, in both animals and humans alike. To this day ger an array of physiological responses termed the acute phase the electroencephlogram (EEG), in conjunction with the electro- reaction. It is now an accepted fact that sleep regulation and the myogram (i.e., electrical activity produced by skeletal muscles), production of pro-infammatory cytokines by the host defense represents the data “backbone” of nearly every experimental and (immune) system are strongly interrelated.19-21 The sleep pat- clinical assessment that seeks to correlate behavior and physiology terns of humans in response to elevated levels of cytokines are with the activity of cortical neurons in behaving animals, includ- complicated and dose-dependent, and include increased or ing humans. In most basic sleep research laboratories these EEG decreased non-rapid eye movement (NREM) sleep. Specifically, recordings are performed using a cable-based system wherein human studies have shown that increased levels of proinflamma- acquired data is subjected off-line to pattern and spectrum analysis tory cytokines (at plasma concentrations capable of inducing a www.tandfonline.com Temperature 407 fever) disrupt, rather than promote, sleep. On the other hand, smaller increases in plasma con- centrations of proinflam- matory cytokines, such as occurs during the circadian cycle, can increase NREM sleep and cortical slow wave intensity.22 By con- trast, rodents treated with proinflammatory cytokines exhibit an enhancement of NREM sleep and a decrease in rapid eye movement (REM) sleep. Although cytokine pro- duction is inevitably accompanied by the secre- tion of PGs, the increase of NREM sleep during an infection is independent of PGs,23 a surprising fact considering: 1) the fever response is completely dependent on PGE2 type EP3 receptor signaling24,25 and 2) PGs have been Figure 2. Circuit basis of sleep-wake regulation. Model 1 (shown in panel a): Adenosine inhibits the release of acetycholine from basal forebrain (BF) cholinergic neurons to produce slow-wave sleep. Model 2 (shown in panels b-d): a implicated in the regulation of sleep.11 How and why immune signaling molecules and other hyp- nogenic substances modu- late sleep remain flip–flop switching mechanism involving mutually inhibitory interactions between sleep-promoting neurons in the ventrolateral preoptic area (VLPO) and wake-promoting neurons in the hypothalamus [i.e., histaminergic tuberomam- milary nucleus (TMN)], and brainstem [i.e., noradrenergic locus coeruleus (LC), serotonergic dorsal raphe nucleus (DR), and cholinergic laterodorsal tegmental nucleus (LDT)]. The flip-flop switch between the VLPO and hypothalamus and brainstem is stabilized by orexin/hypocretin (OX/Hcrt) inputs. Adenosine is known to act as an endogenous somno- gen and promotes sleep via inhibitory A1 receptors (A1) in the basal forebrain, VLPO and TMN and excitatory A2A receptors (A2A) in the nucleus accumbens (NAc) and VLPO.98-101 Other Abbreviations: Ach, acetylcholine; 5-HT, seroto- incompletely understood nin, NE, norepinephrine. and are currently areas of active investigation. hypothalamus provide a stabilizing influence on the switch, to prevent unwanted state transitions such as occurs in narco- Circuit mechanisms of sleep-wake regulation lepsy.39-41 A similar mutually inhibitory interaction between the Experimental work by Economo,26-28 Ranson,29 Moruzzi and ventral periaqueductal gray, lateral pontine tegmentum, and sub- Magoun,30 and others in the early and mid 20th century pro- laterodorsal nucleus in the brainstem has been proposed for the duced findings that inspired circuit-based theories of sleep and switching in and out of REM sleep.42,43 And while these models wake and, to a certain degree, overshadowed the then prevailing have proven valuable heuristics and provided important interpre- humoral theory of sleep. To date, several “circuit models” have tative frameworks for studies in sleep research, a fuller under- been proposed, each informed by data of varying quality and standing of the circuits regulating sleep-wake will require a more quantity (for review, see31-33). One model, for example, proposes complete knowledge of its components. that slow wave sleep is generated by adenosine-driven inhibition It is also the case that a unified model accounting for the myr- of acetylcholine release from cholinergic neurons in the basal iad of state-dependent changes in physiology remains lacking. As forebrain (BF),34 although several studies has shown that cholin- an example, current models have (near uniformly) failed to ergic BF neurons are not essential for sleep induction.35,36 address the regulated reduction in body temperature that occurs Another contemporary circuit model posits a flip-flop switching with sleep. Indeed, a reduction in core body temperature facili- mechanism involving mutually inhibitory interactions between tates the entry into sleep and even modest changes to shell or sleep-promoting neurons in the ventrolateral preoptic area core body temperature during sleep can positively or adversely (VLPO) and wake-promoting neurons in the brain stem and affect sleep quality and consolidation,44,45 suggesting a direct hypothalamus.31,37,38 The flip-flop model further predicts that influence of thermosensory afferents on sleep circuits. Given orexin-/hypocretin-containing neurons of the lateral moreover that the regulation of sleep and body temperature is 408 Temperature Volume 2 Issue 3 non-dividing cells. Although isolation of an AAV serotype 2 clone facilitated development of stable vectors for trans- genic and therapeutic gene delivery, the level of gene expression in cells infected with AAV2 is generally low. As tissue specificity and affinity is determined by proteins that are present in the virus capsid (protein shell or viral envelope), successful attempts to pseudotype and engineer capsids have resulted in a great variety of AAV serotypes suitable for the infection of neurons in a wide range of animal species. For example, researchers can take advantage of AAV serotype variability to infect interdigitating populations of neurons in mice52 or deliver transgenes to brains of verte- brates (e.g. zebra finches) that cannot be genetically modified by reproductive technologies.53 Due to this great versa- tility, it is no surprise that AAV has become one of the most widely used Figure 3. Cre-lox system. (A) The 34-base pair loxP sequence consists of an 8-base pair core sequence biological tools of today’s neuroscience. where recombination takes place (white box) and 2 flanking 13-base pair inverted repeats. (B–D) The outcome of recombination by Cre, a P1 Bacteriophage enzyme that is encoded by the locus originally named as “causes recombination”, is determined by the location and orientation of lox sites. (B) Cre mediates the inversion of the floxed DNA segment, if lox sites are oriented in opposite directions. (C) Cre mediates a deletion of the floxed DNA segment, if lox sites are oriented in the same direction. (D) Cre mediates a translocation, if lox sites are located on different chromosomes. Conditional gene manipulations based on Cre/loxP technology and focal RNA interference Transgenic animals with constitutive gene disruptions have provided impor- tant insights into the in vivo roles of temporally coordinated, it is likely, at least to a certain extent, various genes (and their gene products) in sleep wake regulation. that these systems share common neuronal circuits. In general Interpretation of experimental data generated using constitutive support of this hypothesis, the preoptic forebrain contains neu- gene disruption does, however, warrant caution given several lim- rons that are important for both sleep and temperature con- iting features of these animals, including: (i) approximately 15% trol.46,47 And, similarly, the lateral parabrachial nucleus, which is of knockouts are developmentally lethal and so studies employing linked with arousal control, receives peripheral cutaneous ther- these mice are restricted to embryonic development, making it mosensory signals.48-50 Deconstructing these shared and distinct virtually impossible to relate the function of the deleted gene to circuits will undoubtedly be facilitated by the development of sleep-wake; (ii) ontogenetic complications that result in abnormal newer molecular-genetic tools, and ulitimately should inform a development of other systems or compensatory alterations (e.g. unified model of sleep-wake regulation. levels of neurotransmitters or their receptors), which may con- tribute to the development of a phenotype that is epiphenomenal Molecular biology and genetic technologies in sleep research to the knockout itself; and (iii) constitutive knockout animals are As indicated, the combinatorial application of transgenic mice limited in the ability to inform the localization of individual and viral vector delivery systems in systems-level neuroscience brain areas or neurons involved in sleep-wake regulation. research has become increasingly common. Indeed, the power of On the other hand, Cre or Flp recombinase mediated DNA this experimental approach is undeniable. And while a variety of recombination in genetically engineered mice, i.e., conditional viral vector systems have been harnessed for experimental pur- knockouts, has proven a powerful approach for evaluating the poses, we would highlight work using adeno-associated viruses role of genes, including “sleep genes”, in a spatially and tempo(AAV) as these have enabled scientist to address long-standing rally restricted manner.54 Cre recombination was originally dis- questions in neurobiology. AAV are single-stranded DNA-con- covered in the P1 bacteriophage as part of the virus’ life taining parvoviruses of 57 serotypes classified in 7 species51 that cycle.55,56 The Cre enzyme recombines a pair of short target are currently not known to cause disease. Recombinant AAV vec- sequences called the lox sites, a mechanism which the P1 phage tors are replication deficient and AAV-delivered transgenes (typi- uses to circularize and facilitate replication of its genomic DNA cally no larger than 5 kb) mostly persist as highly stable, actively during reproduction. Flp recombination is analogous to the Cre/ transcribed episomes enabling long-lasting gene expression in lox system but involves recombination of DNA sequences www.tandfonline.com Temperature 409 flanked by FRT (short for “flippase recognition target”) sites derived from baker’s yeast (Saccharomyces cerevisiae). For one reason or another, the Cre/lox recombination strategy has been the preferred recombi- nation approach for the vast majority of transgenic manipulations in mice and other organisms.57,58 And it is the orientation and location of the loxP sequen- ces that determine whether Cre catalyzes a deletion, inversion, or chromosomal translocation of DNA sequences (Fig. 3). By genetic targeting of Cre (or Flp) to discrete populations of neurons and crossing the mice harboring these transgenes with mice bearing loxP (or FRT) modified Figure 4. The arousal effects of caffeine are abolished in rats with site-specific deletion of A2A receptors (A2AR) in the shell of the nucleus accumbens (NAc). To identify the neurons on which caffeine acts to produce arousal, A2A receptors were focally depleted by bilateral injections of adeno-associated virus carrying short-hairpin RNA for the A2A receptor into the core (dashed green line in the left panel) or shell (dashed red line in the right panel) of the NAc of rats.61 Typical hypnograms that show changes in wakefulness and in rapid eye movement (REM) and non-REM alleles, it is possible to modulate these genes in a neuron-specific fashion.59 Alternatively, AAV express- (NREM) sleep after administration of caffeine at a dose of 15 mg/kg indicate that rats with a shell, but not a core, knockdown of the A2A receptors showed a strongly attenuated caffeine arousal. Green and red areas in the hypnograms represent wakefulness after caffeine administration that correspond to the depletion of A2A receptors in the respective core and shell of the NAc. ing either Cre or Flp can be stereotaxically-injected into specific nuclei in the brains of mice in an efficient and cost-effective manner. Moreover, and in con- bearing loxP- or FRT-modified alleles to restrict gene expression trast to Cre/loxP conditional knockout models, rats or even more to the site of AAV injection.25,60 The utility of this technical phylogenetically advanced organisms, such as non-human priapproach was elegantly illustrated in 2 recent studies.61,62 In the mates, which can provide a better approximation of human brain first study, Lazarus and colleagues employed, in combination, responses, can be used as experimental model systems. conditional A2A receptor knockout mice and stereotaxic-based Many labs have also developed lines of mice with loxP-flanked microinjections of Cre-expressing AAV into the nucleus accum- sequences that disrupt expression of genes (“transcriptional dis- bens to show that A2A receptors in the nucleus accumbens pro- ruptors”), effectively extending the utility of conditional knockmote sleep.61 In the second study, Anaclet and colleagues placed outs by addressing some of their limitations. In their basal state, microinjection of AAV-Cre into the parafacial zone of condi- transcriptional disruptor mice typically demonstrate a phenotype tional vesicular GABA transporter mice to show that GABAergic identical to that of constitutive knockout mice; after exposure to neurons of the medullary parafacial zone are required for normal Cre recombinase the loxP-flanked blocking sequence is removed amounts of slow-wave-sleep.62 and gene expression (and, typically, the phenotype) is normal- Also in the landmark Lazarus et al. paper on the arousal-pro- ized. Transcriptional disrupter mice are particularly useful in sitmoting effect of caffeine,61 the world’s most widely prescribed uations where the gene(s) of interest are more widely and psychoactive drug, the authors pioneered the use of RNA inter- diffusely expressed in the brain. In other words, gene re-expres- ference (RNAi) to silence focally the expression of A2A receptors sion can be targeted to discrete sites, which in turn greatly faciliin the brain of rats and show that deletion of A2A receptors in the tates functional analysis. Focal gene reactivation using shell of the nucleus accumbens is sufficient to abolish the arousal transcriptional disruptor mice also offers other potential advan- effect of caffeine (Fig. 4). RNAi is a system within living cells tages over traditional conditional knockout mice including: the that helps regulate which genes are active and also the magnitude ability to 1) perform a relatively quick anatomic survey for gene of their activity.63 This gene regulation process also includes the function in various brain regions across the neuraxis, and 2) nor- interaction of small interfering RNA with mRNA to prevent malize gene expression only in sites with a latent genetic capacity mRNA from producing a protein. The RNAi can be applied in to express the gene of interest, i.e., eutopic expression. As an any animal model and by using local infection with AAV carrying excellent experimental example of the gene “reactivation” short-hairpin RNA, focal RNAi can be produced in live animals approach, Scammell and colleagues placed focal injections of 410 Temperature Volume 2 Issue 3 AAV containing Cre into discrete brain regions of mice with fragment A of the diphtheria toxin (DTA-AAV). The original re-activatable orexin-2 receptors. In doing so, they found that diphtheria toxin, an exotoxin secreted by Corynebacterium diph- normal expression and function of orexin-2 receptors, and hence theriae, is a single polypeptide consisting of 2 fragments A and B. orexin signaling, in the posterior hypothalamus, including the Binding of fragment B to the cell surface allows fragment A to pen- tuberomammilary nucleus, plays an essential role in the wake- etrate the host cell and act as a potent RNA translational inhibitor. promoting effects of orexins.64 In combination with mice expressing Cre under the control of the vesicular glutamate transporter 2, this transcriptionally silenced Conditional tracing of long axonal pathways and lesioning DTA-AAV was recently used by Saper and colleagues to demon- of neuronal cell populations strate that glutamatergic neurons within the external lateral and lat- In addition to loxP-modified mice, investigators have also eral crescent subdivisions of the lateral pontine parabrachial taken advantage of the large number of transgenic Cre-expressing nucleus critically contribute to hypercapnia-induced arousal.49 mice available for use in a wide range of experiments, including As an alternative to using loxP-flanked neo cassettes as tran- the anterograde tracing of neural projections from cell-type spe- scriptional stop sequences, transgenes are now commonly cloned cific neuronal populations. For example, Gautron and colleagues into AAV plasmids in a double floxed inverted (FLEX) or double developed an AAV vector that encodes a humanized Renilla green inverted orientation (DIO). Within the FLEX or DIO orienta- fluorescent protein (hrGFP) whose expression is transcriptionally tion, transgenes are cloned in reverse orientation, so that a nonsilenced by a neo cassette flanked by loxP sites.65 This vector con- sense transcript is produced until the transgene is exposed to Cre struct, which results in hrGFP protein expression only in neurons recombinase, which flips the gene into the sense orientation. The with Cre recombinase activity, was used in combination with lep- advantage to FLEX/DIO cassette is that it confers great selectiv- tin receptor-Cre mice to define the efferent projections of leptin- ity, without transcriptional “leakage” in cells that lack Cre expres- responsive neurons in the hypothalamus. In another example of sion. We have, for example, recently generated a FLEX version of this approach, stereotaxic microinjections of conditional hrGFP- our DTA-AAV as well as adopted this system for all of our AAV- AAV into the nucleus accumbens of mice in which Cre expres- based genetically engineered receptor-channel systems (compare sion is driven by the promoter of the A2A receptor gene66 were ‘Reversible in vivo silencing and activation of neurons in freely used to trace axonal projections of A2A receptor-positive neurons behaving animals’). known to participate in sleep-wake regulation. With little modifications, the same approach can readily be Reversible in vivo silencing and activation of neurons adapted for the lesioning of cell-type specific neuronal population. in freely behaving animals Toward this end we (Fuller and Lazarus) developed an AAV-based Significant research efforts have recently been directed at system for the transgenic expression of the highly cell-toxic developing genetic-molecular tools to achieve reversible and cell- type specific in vivo silencing of neurons in awake, behaving animals. The obvi- ous goal in developing these tools is to help establish a causal relationship between the activity of specific neurons (or neuronal populations) and behav- ioral and physiological outcomes. While several genetic tools have been devel- oped for this purpose, including condi- tional blockade of neurotransmitter release and suppression of neuronal excitability, each method has distinct advantages as well as limitations. One tool that has been developed for acute and reversible in vivo silencing or activa- tion of neurons is optogenetics technologies.67 It would not be an exaggeration to state that optogenetics has ushered in a new era of neurobiology.68 Simply Figure 5. Photostimulation of neurons in behaving animals: combining Cre-driver mice and stereo- taxic-based delivery of adeno-associated virus (AAV) expressing Cre-dependent channelrhodopsin (ChR2). ChR2 are nonspecific cation channels, conducting HC, NaC, KC, and Ca2C ions. ChR2 absorbs blue light with an absorption spectrum maximum at 480 nm resulting in the opening of a pore in the trans-membrane protein and depolarization of neurons by allowing for the flow of ions according to their electrochemical gradient.102,103 Other Abbreviations: Cre, Cre recombinase; EF1a, archaeal elongation factor 1 a; loxP, locus of X-over P1; lox2272, variant of loxP; pA, poly A tail; WRPE, woodchuck hepatitis virus posttranscriptional regulatory element. put, an optically-driven “switch” can be placed into specific neurons of a living animal by the local microinjection of viral vectors expressing an opsin [e.g. excitatory channelrhodopsin-2 (ChR2), as shown in Fig. 5] in a Cre-dependent configuration. Alternatively, the opsin www.tandfonline.com Temperature 411 gene can be placed under a cell-type specific promoter, limiting neurons during normal waking time reduced the length of expression to, for example, only orexin-producing cells.69 Trans- waking bouts and increased both NREM and REM sleep. In genic mice have also been engineered to express opsins under var- the third, Tsunematsu and colleagues used a genetic approach ious gene promoters. For example, Tsunematsu and colleagues that faciliated the temporal and spatial control of the expresgenerated transgenic mice in which halorhodopsin, which is sion of opsins and other transgenes.76 They employed a tetra- orange light-driven chloride pump whose photoactivation results cycline-controlled transcriptional activation technique in the inhibition/silencing of neurons, was exclusively expressed (Fig. 6), which is a method of inducible expression wherein in orexin neurons. In these halorhodopsin-based experiments, transcription depends on the tetracycline-responsive transcrip- the authors showed that silencing of orexin neurons induced tion factor (tTA) and is turned off at the TetO promoter in NREM sleep, which confirmed a role for orexin neurons in the the presence of the antibiotic tetracycline [or its derivative, maintenance of wakefulness.70 It is also the case that optogenetic doxycycline (DOX); “Tet-Off”].77,78 Initially, Tsunematsu tools can be used to identify functional synaptic connectivity and colleagues expressed ChR2 (E128T/T159C) in MCH between specific neuronal populations, both in vivo and in brain neurons by generating mice with 2 transgenes in which the slices. Originally coined “ChR2-assisted circuit mapping,” or first transgene provides pro-MCH promoter-driven expression CRACM for short, this technique involves combining direct of tTA and the second enables tTA-dependent expression of photostimulation of presynaptic ChR2-expressing axons/termi- ChR2.76 Activation of MCH neurons in these mice increased nals with patch-clamp recordings of post-synpatic neurons. In time in REM sleep, whereas optogenetic inhibition, which this arrangement, specific inputs are activated to evoke neuro- was achieved through expression of tTA-dependent archaertransmitter release and establish functional synaptic connectivity. hodopsinT,79 did not affect the amount of REM sleep. Addi- This elegant technique was recently used by Arrigoni and col- tionally, mice with genetically ablated MCH neurons (Tet- leagues to show that release of histamine from neurons of the Off-controlled expression of DTA) showed an increase in tuberomammilllary nucleus (TMN) can disinhibit the TMN and wake and a decrease in NREM sleep without affecting REM suppress (indirectly) the activity of sleep-active VLPO neurons to sleep amount. Taken the together, the results from these 3 promote TMN neuronal firing, a finding that lends credence to optogenetic-based studies suggest that MCH neurons contribthe sleep-wake “flip-flop switch” hypothesis.71 Creative variants utes to both NREM and REM sleep, possibly in a state- and of this technique, including the combined application of time-of-day dependent manner. CRACM and retrogradely transported microspheres, have It is worth noting that, despite its undeniable contribution to enabled the mapping of circuits spanning 3 synaptically-coupled the experimental neurosciences, in vivo optogenetic tools are not sites within the brain.72 without limiting features. These limitations include invasive We next highlight 3 recently pub- lished (and related) studies in which optogenetic-based approaches were used to interrogate the neuronal circuitry sub- serving the regulation of REM and NREM sleep, with a particular emphasis on the role of lateral hypothalamic mela- nin-concentrating hormone (MCH) neurons.73 In the first, Adamatidis and colleagues used AAV to deliver Cre- dependent light-actviated opsins (ChETA and halorhopdsin) within the lateral hypothalamus of mice expressing Cre recombinase under the prepro- MCH promoter. In vivo activation and inhibition of these neurons revealed that these neurons are critical for maintenance of REM sleep,74 and that this links to GABA release onto TMN neu- Figure 6. Inducible gene expression by using tetracycline-controlled transcriptional activation (Tet rons by MCH neurons. The second expression systems). Gene transcription is reversibly turned on or off in the presence of the antibiotic study, by Shiromani and colleagues, also employed an AAV-based delivery approach, but instead used a MCH promoter system to drive expression of tetracycline (Tc) or doxycycline (DOX), a more stable tetracycline analog. In a Tet-Off system, tetracycline and its derivatives bind transactivator protein (tTA) and render it incapable of binding to the tetracycline response element (TRE) consisting of several TetO sequences and a minimal promoter, thereby preventing transcription of TRE-controlled genes. A Tet-On system works similarly, but the rtTA protein is capable of binding to the TRE operator, and hence initiating transcription of the trans- channelrhodposin in MCH neurons of wildtype mice.75 Optogenetic stimula- tion of the ChR2-expessing MCH gene, only when bound by DOX. For most purposes, there is no inherent advantage of using the TetOff system over the Tet-On system, although there is no apparent literature example in which the Tet-Off system has been used to study the regulation and function of sleep. 412 Temperature Volume 2 Issue 3 that suggests a key role for the mPFC in positive emotions that trigger cataplexy. Another recently developed system by Roth and colleagues permits the selective and “remote” manipulation (activation and silencing) of neuronal activity via all 3 major GPCR signaling pathways (Gi, Gs and Gq). These so- called “designer receptors exclusively activated by designer drugs” (DREADD) involve, broadly speaking, mutant GPCRs that do not respond to their endogenous ligands but are responsive to otherwise inert biological compounds (Fig. 7). The usefulness of these chemogenetic systems has been Figure 7. In vivo chemogenetic inhibition or activation of neurons in behaving animals: combining Cre-driver mice and stereotaxic-based delivery of adeno-associated virus (AAV) expressing Cre-dependent “designer receptor exclusively activated by designer drugs” (DREADD). DREADD permit temporal control of excitatory or inhibitory G-protein coupled receptor signaling in vivo by utilizing mutated human muscarinic acetylcholine (AcH) receptors.82,83 These AcH receptors, excitatory hM3 and inhibi- shown in various studies across the neurosciences and other fields.82-85 In an important proof-of-concept study for sleep biology, the laboratory of Takeshi Sakurai demonstrated that DREADD- tory hM4, are unresponsive to their natural ligand acetylcholine, but can be activated by nanomolar doses of the synthetic small-molecule clozapine-N-oxide (CNO). Other Abbreviations: Cre, Cre recombinase; hsyn, human synapsin promoter; loxP, locus of X-over P1; lox2272, variant of loxP; pA, poly A tail; WRPE, woodchuck hepatitis virus posttranscriptional regulatory element. driven changes in the activity of orexin neurons can alter behavioral state.86 More specifically, Gq-DREADD excitation of orexin neurons increased the amount of time spent in wakefulness, whereas Gi-DREADD inhibition of instrumentation, the challenge of light penetration to larger brain orexin neurons promoted NREM sleep. In related work, Inut- regions, scalability issues, lower throughput, artificial synchro- suka and colleagues activated orexin neurons, also using Gqnized patterns of activation/inhibition and limited direct evi- DREADD,87 and observed increases in food and water consump- dence that photo-evoked release of neuropeptides is possible. tion as well as locomotor activity and metabolic rate, suggesting And because of these limiting features, our labs and others have that orexin neurons also contribute to the regulation of energy begun exploring and developing alternative tools for achieving in homeostasis. More recently, Anaclet and colleagues generated vivo, reversible silencing that also offers ease of implementation, and experimentally deployed Cre-dependent versions of the Gq no cabling into the CNS and a ligand that can be delivered and Gi DREADD-AAV systems to establish necessity and suffi- peripherally or even in the drinking water. One such system was ciency of a node of GABAergic brainstem neurons in generating first introduced in 2007 by Andersen and colleagues,80 and slow-wave-sleep and cortical slow-wave-activity.72 involves the AAV-based delivery of 2 channel subunits (a and b) Newer generation DREADD systems are under development that comprise a modified C. elegans glutamate- and ivermectin with several examples appearing in the recent literature. For (IVM)-gated chloride channel (GluClab). This heteromeric example, a new Gi-coupled DREADD that uses the kappa-opi- channel prevents action potentials from firing by hyperpola- oid receptor as a template (KORD) and is activated by the phar- rizing the membrane in a ligand-dependent manner. In this macologically inert ligand salvinorin B was recently described.88 elegant proof-of-concept study, the authors showed that that Co-expression of the KORD and the Gq-coupled M3- the GluClab channel can be stably expressed in vivo without DREADD within the same neuronal population permits the neurotoxicity, that this channel can be activated by (dose- sequential and bidirectional control of the target neuronal popu- dependent) systemic administration of IVM in vivo (at dos- lation, and hence behavior. ages that do not cause organismal toxicity), channel activation Increasingly, the experimental framework in which both opto- is reversible in vivo and, finally, that the channel-IVM medi- and chemo-genetic techniques are being applied involves concur- ated neuronal silencing can be used to manipulate behavior rent electrophysiologic and imaging techniques, such as tetrode in awake, behaving animals. In a more recent study in sleep recording89 or deep-brain fiber-optic endomicroscopy,90 in biology, the technique was instrumental in defining the neu- behaving mice. Such innovative combinatorial approaches perral substrates of emotion-driven cataplexy.81 Here, Oishi and mit, for example, the simultaneous recording and perturbation of colleagues showed that the GluClab-IVM-mediated inhibi- genetically defined sets of neurons, even in regions of high cellu- tion of the medial prefrontal cortex (mPFC) prevented choc- lar heterogenecity. Hence the combined application of genetically olate-induced cataplexy in orexin knock-out mice, a finding driven system with in vivo electrophysiologic and/or imaging www.tandfonline.com Temperature 413 techniques will likely prove instrumental in elucidating the detailed circuit and synaptic basis of wake-sleep control.” Reversible neurotransmission blocking by the tetanus neurotoxin and tetracycline-controlled transcriptional activation Tetanus neurotoxin cleaves the synaptic vesicle-associated membrane protein and thus blocks vesicle-mediated neurotransmission.91 Local reversible silencing of neurotransmission can be achieved by the injection of tTA-expressing AAV into transgenic mice with tetanus neurotoxin expression under the control of tTA. Such an approach has recently been used to genetically dissect the circuit-function relationships within the basal ganglia.92 In this study, an AAV-tTA/tetanus neurotoxin approach was used to evaluate the functional roles of the direct (striatonigral) and indirect (striatopallidal) pathways in learning behaviors. Here the authors exploited the fact that substance P and enkephalin are selectively expressed in the direct or indirect pathway, respectively, and thus, AAV-mediated expression of tTA under the control of the substance P or enkephalin promoter induced specific tetanus neurotoxin-blocking of the striatonigral or striatopallidal neurons. In doing so, this study revealed that dopamine/D2R action on the indirect pathway is important for aversive, but not reward-based, learning. This same approach may also prove useful in determining the extent to which the 2 efferent pathways of the basal ganglia are required for the regulation of wakefulness. The Tet-off system has also been widely used in combination with genetically engineered receptor-channel systems including opsins or DREADDs. One particularly useful application of the Tet-Off system is the generation of synthetic activity-dependent transgene traces in the brains of mice in which, for example, ChR2 or Gq-DREADD are expressed in a behavior-dependent manner through c-fos (a marker of neuronal activity) promoterdriven tTA expression.93-95 To this end, a Tet-tagged version of the excitatory DREADD was recently used to selectively map and reactivate neuronal ensembles in the preoptic hypothalamus that were activated by the a2 adrenergic receptor agonist dexmedetomidine.96 In doing so, the authors were able to demonstrate that dexmedetomidine–induced sedation is achieved, in part, through engagement of sleep-promoting hypothalamic circuitry. Concluding remarks It is the authors’ surmise that the experimental application of the tools described in this review, including creative combinational applications, will prove critical to the process of elaborating the spatial and temporal properties of the circuitry mediating the transition between sleep and waking states, as well as developing a unified model for the humoral and neural mechanisms governing sleep-wake regulation. We also feel that “systems-level” sleep research will be greatly informed by large-scale gene network studies that employ functional genomics approaches such as transcriptome, proteome, and metabolome analysis for identifying “new targets” that might form the basis for the development of additional conditional transgenics. Indeed, the methods described herein or elsewhere97 will not only make it possible to determine the detailed anatomic and molecular bases of sleepwake regulation, but should also help to shed light on some of the greatest mysteries in systems somnology, including: “why do we sleep” and “what is the function of sleep?.” Disrupted sleep, including its voluntary loss and sleep disorders, are linked to traffic and work-related accidents as well as significant social losses due to an increased prevalence of mood and other neuropsychiatric disorders. Insufficient sleep is also an established independent risk factor for cardiovascular and metabolic diseases, such as diabetes and obesity, and is linked with increased cancer risk. Thus, while sleep has been a perpetual topic of scientific inquiry that continues to attract many scientists, it is also an important field that will greatly benefit society through the development of strategies to remedy sleep disorders and associated diseases. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Funding This work was supported by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (B) 24300129 (to M.L.) and 23300142 (to A.Y.); World Premier International Research Center Initiative (WPI) from the Ministry of Education, Culture, Sports, Science, and Technology (to M.L.); the National Institutes of Health (NS26837 to P.M.F.); and a grant from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (Grant-in-Aid for Scientific Research on Innovative Areas “Mesoscopic Neurocircuitry”, 23115103, to A.Y.). About the Authors Patrick M Fuller is a principal investigator at Harvard Medical School and Beth Israel Deaconess Medical Center. The investigative focus of his laboratory is the cellular and synaptic basis by which the brain regulates sleep and wakeful consciousness. 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