The invention provides a method of identifying a compound that alters vigilance. The method consists of contacting an invertebrate with a candidate compound, evaluating a vigilance property in the contacted invertebrate, and determining if the candidate compound alters the vigilance property in the contacted invertebrate. A candidate compound that alters the vigilance property in the contacted invertebrate is identified as a compound that alters vigilance. The invention also provides a method of identifying a vigilance enhancing compound that modulates homeostatic regulation or a vigilance diminishing compound that modulates homeostatic regulation. The method consists of contacting an invertebrate with a compound that increases or decreases vigilance, and determining the effect of the compound on a homeostatic regulatory property of vigilance. A compound that alters the homeostatic regulatory property is characterized as being a vigilance enhancing compound or a vigilance diminishing compound that modulates homeostatic regulation.

Description of the Invention

SUMMARY OF THE INVENTION

The invention provides a method of identifying a compound that alters vigilance. The method consists of contacting an invertebrate with a candidate compound, evaluating a vigilance property in the contacted invertebrate, and determining if the candidate compound alters the vigilance property in the contacted invertebrate. A candidate compound that alters the vigilance property in the contacted invertebrate is identified as a compound that alters vigilance.

In one embodiment, the vigilance property evaluated is a behavioral property, including activity, latency to sleep or arousal threshold. In another embodiment, the vigilance property evaluated is a molecular property, including expression of one or more vigilance-modulated genes.

The invention also provides a method of identifying a vigilance enhancing compound that modulates homeostatic regulation. The method consists of contacting an invertebrate with a compound that increases vigilance, and determining the effect of the compound on a homeostatic regulatory property of vigilance. A compound that alters the homeostatic regulatory property is characterized as being a vigilance enhancing compound that modulates homeostatic regulation.

Also provided is a method of identifying a vigilance diminishing compound that modulates homeostatic regulation. The method consists of contacting an invertebrate with a compound that decreases vigilance, and determining the effect of the compound on a homeostatic regulatory property of vigilance. A compound that alters the homeostatic regulatory property is characterized as being a vigilance diminishing compound that modulates homeostatic regulation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of rapidly and efficiently identifying compounds that alter vigilance, including compounds that promote sleep, prevent sleep, or increase vigilance. The compounds identified by the methods of the invention can thus be used to treat individuals suffering from psychological, physiological or genetic conditions that deprive them of restorative sleep or that cause excessive sleepiness. These compounds can also be used to prolong wakefulness, such as when it is desired to extend an individual's productivity, or to increase attentiveness, learning or memory.

Sleep in mammals has been defined by several criteria, including electrophysiological and behavioral criteria. Behavioral criteria for sleep include sustained quiescence, increased arousal threshold, and "sleep rebound," or increased sleep or increased sleep intensity following prolonged waking. The criterion of sleep rebound indicates that sleep is under homeostatic control and is thus distinguishable from mere inactivity.

Recently, physiological correlates of sleep in mammals have been extended to the level of gene expression. Molecular screening has revealed that brain levels of mitochondrial enzymes and of several genes implicated in neural plasticity are high during waking and low during sleep (see, for example, (see Cirelli et al., Mol. Brain Res. 56:293 (1998); Cirelli et al., Ann. Med. 31:117 (1999); and Cirelli et al., Sleep 22(S):113 (1999)). Therefore, sleep in mammals can also be characterized by a distinct pattern of gene expression.

Although it is well-known that most organisms exhibit circadian rest-activity cycles, prior to the present invention it was not known that invertebrates exhibit a sleep-like state that is comparable, by behavioral, physiological, developmental, molecular and genetic criteria, to mammalian sleep. This sleep-like state in invertebrates is henceforth referred to as "sleep."

As described herein, invertebrate sleep is very similar, by behavioral criteria, to mammalian sleep. More specifically, as shown in Example I, sleep in an exemplary invertebrate, Drosophila melanogaster, is associated with sustained behavioral quiescence and increased arousal threshold. Additionally, sleep deprivation during the normal sleep period led to a rebound effect comparable to sleep rebound in mammals, indicating that sleep is under similar homeostatic control in invertebrates.

Furthermore, as described herein, sleep in invertebrates is dependent on age, and follows a similar pattern of age dependency as mammalian sleep, indicating that sleep in invertebrates is developmentally regulated. Likewise, sleep remains homeostatically regulated in older invertebrates, as it is in older mammals (see Example II). Additionally, sleep and wake in invertebrates are subject to pharmacological manipulation using compounds that are known to act as stimulants or hypnotics in mammals (see Example III).

Furthermore, of importance to the determination that sleep and wake in invertebrates are truly similar to mammalian sleep and wake, it is also described herein that several classes of genes, and several individual genes, whose regulation is dependent on vigilance state in mammals are similarly regulated in invertebrates (see Example IV). Additionally, as disclosed herein, mutations in genes that regulate sleep in invertebrates affect vigilance properties, including homeostatic regulation of sleep (see Example IV). Likewise, mutations have been identified in mammalian genes that affect sleep, including orexin (see Chemelli et al., Cell 98:437-451 (1999)), indicating that in both invertebrates and mammals, vigilance is under genetic control.

The discovery that invertebrates exhibit sleep and wake states that are similar by behavioral, developmental, pharmacological, genetic and molecular criteria to mammalian sleep and wake, provides a basis for the methods disclosed herein of identifying novel compounds that can be used to modulate vigilance in mammals by screening compounds for their effect on vigilance properties in invertebrates.

The invention provides a method of identifying a compound that alters vigilance. The method consists of contacting an invertebrate with a candidate compound, evaluating a vigilance property in the contacted invertebrate, and determining if the candidate compound alters the vigilance property in the contacted invertebrate A candidate compound that alters the vigilance property in the contacted invertebrate is identified as a compound that alters vigilance.

As used herein, the term "vigilance" is intended to mean the degree or extent to which an organism exhibits sleep or wake behaviors. Thus, the term "altering vigilance" is intended to encompass a change in state from wake to sleep or vice-versa, as well as any increase or decrease in intensity or duration of behaviors associated with a sleep or wake state.

The methods of the invention can be used to identify compounds that either increase or decrease vigilance. A compound that increases vigilance can, for example, cause the animal to wake from sleep, prolong periods of wakefulness, prolong normal latency to sleep, restore normal sleep patterns following sleep deprivation, or enhance beneficial wake-like characteristics, such as alertness, responsiveness to stimuli, energy, and ability to learn and remember. In contrast, a compound that decreases vigilance can, for example, cause an animal to sleep, prolong periods of sleep, promote restful sleep, decrease latency to sleep, or decrease unwanted wake-like characteristics, such as anxiety and hyperactivity.

As used herein, the term "vigilance property" is intended to mean a behavioral, physiological or molecular property in invertebrates that is correlated with mammalian sleep and wake states. As described further below, invertebrates can exhibit a variety of behavioral properties that are closely correlated with mammalian sleep and wake states, including activity, arousal threshold and latency to sleep. Additionally, as described further below, invertebrates can exhibit a variety of molecular properties that are closely correlated with mammalian sleep and wake states, including expression of vigilance-modulated genes. Invertebrates can also exhibit physiological properties that are closely correlated with mammalian sleep, including the frequency, type and intensity of neuronal signals, heart rate, and the like.

Generally, invertebrates exhibit circadian patterns of rest and activity, with most rest occurring during the night in diurnal animals and most activity occurring during the day. In contrast, in nocturnal animals most rest occurs during the day, whereas most activity takes place during the night. Under laboratory conditions, it is possible to regulate the circadian rest-activity cycle by regulating the length of light and dark, and thus establish what are referred to herein as "normal wake periods" and "normal sleep periods." For example, in Drosophila melanogaster subjected to a 12 h:12 h light:dark cycle, the "normal wake period" is the 12 hour light period, whereas the "normal sleep period" is the 12 hour dark period. Those skilled in the art can readily determine or establish normal wake and sleep periods for other invertebrates.

An example of a behavioral vigilance property that can be evaluated in invertebrates is activity during all or part of a normal wake or sleep period. As used herein, the term "activity" is intended to encompass all behavioral activities normally exhibited by that invertebrate including, for example, locomoting, movements of body parts, grooming, eating, and the like, in contrast to "inactivity" or "rest." Activity can be evaluated throughout a normal wake period or throughout a normal sleep period, or both, or evaluated for only part of a normal wake or sleep period, such as for at least 10 minutes, 30 minutes, 1, 2, 4, 6, 8 or 12 hours. Once activity during a normal sleep period or normal wake period is established, those skilled in the art can readily evaluate whether a candidate compound increases or decreases intensity of activity or alters the pattern of activity during all or part of that period.

For certain applications of the method, it will be preferable to evaluate activity following sleep deprivation. As described previously, sleep rebound following sleep deprivation is a characteristic of homeostatically regulated sleep. Thus, by establishing the normal sleep rebound behavior of the invertebrate, those skilled in the art can readily evaluate whether a candidate compound affects the normal homeostatic regulation of sleep.

As used herein, the term "sleep deprivation" refers to depriving the animal of rest. This deprivation is generally for a sufficient period of time during a normal sleep period to result in a detectable decrease in activity, increase in sleep, or increase in intensity of sleep during the subsequeent period, also known as a "sleep rebound" effect. In general, sleep deprivation results from depriving the animal of rest during at least 10%, such as at least 25%, including from 50% to 100% of the normal sleep period.

Any method appropriate for the particular invertebrate can be used to deprive an animal of sleep. As described in Example I, Drosophila melanogaster can be sleep-deprived for the entire normal sleep period, using manual or automated physical stimulation, and the amount, pattern and intensity of activity indicative of sleep rebound evaluated (see FIG. 2A, see Original Patent). In other organisms, it may be preferable to sleep-deprive the animals using electrical stimulation, noise, or other stimuli, for longer or shorter periods. The time period and method for sleep-depriving an animal can be determined by those skilled in the art for a particular application.

Various manual and automated assays can be used to evaluate intensity and patterns of activity. For example, activity can be detected visually, either by direct observation or by time-lapse photography. Alternatively, an ultrasound monitoring system can be used, such as the system shown in FIG. 1A (see Original Patent) and described in Example I (see Original Patent). Such a system is advantageous in detecting very small movements of the animals' body parts and, as shown in FIG. 1B (see Original Patent), the output is closely correlated with visual observations. An example of the activity of Drosophila melanogaster during a normal wake period (12 hour light period) and a normal sleep period (12 hour dark period), as evaluated using an ultrasound monitoring system, is shown in FIG. 1C (see Original Patent).

As a further example, an infrared monitoring system, such as the infrared Drosophila Activity Monitoring System available from Trikinetics (described in M. Hamblen et al., J. Neurogen. 3:249 (1986)), can be used. As described in Example I, below, an infrared monitoring system is advantageous when simultaneously evaluating activity in large numbers of invertebrates. An example of the activity of a population of Drosophila melanogaster during a normal wake period (12 hour light period) and a normal sleep period (12 hour dark period), as evaluated using an ultrasound monitoring system, is shown in FIG. 1C (see Original Patent).

Those skilled in the art can determine an appropriate method to evaluate invertebrate activity in a particular application of the method, depending on considerations such as the size and number of invertebrates, their normal activity level, the intended number of data points, and whether a quantitative or qualitative assessment of activity is desired.

A further example of a behavioral vigilance property that can be evaluated in invertebrates is latency to sleep. As used herein, the term "latency to sleep" refers to the period of time to the first rest bout following the change from the normal wake period to the normal sleep period (ie. from light to dark in diurnal animals, or from dark to light in nocturnal animals). As shown in FIG. 4E (see Original Patent), latency to sleep in control Drosophila melanogaster was about 40 minutes. If desired, latency to sleep following sleep deprivation can also be established. Once normal latency to sleep, or latency to sleep following sleep deprivation are established for a particular invertebrate, one skilled in the art can evaluate whether a candidate compound increases or decreases this vigilance property.

Another example of a behavioral vigilance property that can be evaluated in invertebrates is arousal threshold. As used herein, the term "arousal threshold" refers to the amount of stimulation required to elicit a behavioral response, such as movement. Any reproducible stimulus can be used to evaluate arousal threshold including, for example, vibratory stimulus, noise, electrical stimulation, heat, or light.

Invertebrates that are in a wake state will exhibit a behavioral response at a lower level of stimulation than invertebrates that are in a sleep state. For example, as described in Example I, below, when subjected to vibratory stimuli of varying intensities, Drosophila melanogaster that were in a wake-like state, as determined by activity criteria, responded to low-level stimuli that did not elicit a response in flies that were in a sleep state. Furthermore, an animal that is deeply asleep will exhibit an increased arousal threshold compared to an animal that less deeply asleep. Accordingly, arousal threshold is a measure of sleep versus wake, as well as intensity of sleep. Once normal arousal threshold associated with sleep and wake are established for a particular invertebrate, those skilled in the art can readily evaluate whether a candidate compound increases or decreases this vigilance property.

Other vigilance properties that can be measured in invertebrates include molecular properties correlated with sleep and wake states. As used herein, the term "molecular property" refers to any property that can be evaluated in invertebrate tissues, cells or extracts, including, for example, production or turnover of a second messengers, GTP hydrolysis, influx or efflux of ions or amino acids, membrane voltage, protein phosphorylation or glycosylation, membrane voltage, enzyme activity, protein-protein interactions., protein secretion, and gene expression.

A specific example of a molecular vigilance property that can be evaluated in invertebrates is expression of one or more vigilance-modulated genes. As used herein, the term "expression" is intended to encompass expression at the mRNA or polypeptide level. Accordingly, expression of a vigilance-modulated gene can be evaluated by any qualitative or quantitative method that detects mRNA, protein or activity, including methods described further below. Once the abundance or pattern of expression of vigilance-modulated genes are established for a particular invertebrate, those skilled in the art can readily evaluate whether a candidate compound increases or decreases expression of one or more vigilance-modulated genes.

As used herein, the term "vigilance-modulated gene" refers to a gene whose expression level varies according to vigilance state. For example, the expression level of a vigilance-modulated gene can normally vary by at least about 10%, such as at least 25%, or at least about 50%, including at least about 100%, 250%, 500%, 1000% more between sleep and wake. As described herein, at least about 1% of the transcripts in invertebrates are modulated by vigilance state and, consequently, correspond to vigilance-modulated genes. Therefore, in the methods of the invention one can evaluate expression of at least one vigilance-modulated gene, such as at least 2, 5, 10, 20, 50, 100 or more vigilance-modulated genes. Although not necessary for the practice of the invention, as described below, these genes can be cloned and/or their sequences determined using standard molecular biology procedures.

If desired for a particular application of the method, genes whose expression is normally upregulated in the wake-like state, or genes whose expression is normally upregulated in sleep, or any combination, can be evaluated.

Exemplary vigilance-modulated genes in Drosophila melanogaster include a homolog of mammalian Fatty acid synthase (Fas); Cytochrome oxidase C, subunit I; Cytochrome p450 (Cyp4e2); BiP; and arylalkyamine N-acetyl transferase (Dat). Each of these genes was expressed at higher levels during waking than during sleep (see Example IV). In contrast, a gene designated "Rest" was 45% higher during sleep than during rest.

As disclosed herein, there is similarity between vigilance-modulated gene expression in rats and in Drosophila melanogaster, both in terms of number and type of genes that are modulated. For example, as described in Example IV, below, Cytochrome oxidase C, subunit I shows a rapid increase in expression during the first few hours of waking in both rats and Drosophila. Likewise, expression of a Drosophila and a rat Cytochrome P450 were similarly upregulated in waking and sleep deprivation. Therefore, vigilance-modulated genes in invertebrates include homologs of genes whose expression levels vary with the vigilance state of mammals.

A variety of vigilance-modulated genes in rats are described in Cirelli et al., Mol. Brain Res. 56, 293 (1998); Cirelli et al., Ann. Med. 31:117 (1999); Cirelli et al., Sleep 22(S):113 (1999) and include, for example, immediate-early genes, transcription factors and chaperones (e.g. NGFI-A, NGFI-B, Zn-15 related zinc finger, Arc, JunB and IER5); mitochondrial genes (e.g. Cytochrome oxidase C subunit 1, Cytochrome oxidase C subunit IV, NADH dehydrogenase subunit 2, 12S rRNA and F1-ATPase subunit alpha; and other genes, such as neurogranin, bone morphogenetic protein 2, glucose-regulated protein 78, brain-derived neurotrophic factor, interleukin-1.beta., dendrin, and Ca.sup.++/calmodulin-dependent protein kinase II (.alpha.-subunit) Other vigilance-modulated genes in rats include Cytochrome P450 (Cyp4F5), AA117313, aryl sulfotransferase IV, human breast autoantigen homolog, KIAA313 homolog, and membrane protein E25. Therefore, invertebrate homologs of each of these genes are considered to be vigilance-modulated genes.

Those skilled in the art can determine the extent of identity or similarity between two genes needed to establish that an invertebrate sequence is the homolog of a mammalian vigilance-modulated gene. Generally, homologous genes will encode polypeptides having at least about 25% identity, such as at least about 30%, 40%, 50%, 75% or greater identity across the entire sequence, or a functional domain thereof. Methods for cloning homologs from any invertebrate species, using PCR or library screening, are well known in the art, and are described, for example, in standard molecular biology manuals such as Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1992) and in Ansubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1998).

Another example of a molecular vigilance property that can be evaluated in invertebrates is function of one or more vigilance-altering genes. As used herein, the term "vigilance-altering gene" refers to a gene whose expression level can, but does not need to, vary with vigilance state, but whose function influences or is required for inducing or maintaining a vigilance level or a vigilance property. Exemplary functions of a vigilance-altering gene that can be evaluated include transcriptional or translational regulatory activity, and phosphorylation, dephosphorylation, glycosylation or other post-translational modification.

Vigilance-modulated genes and vigilance-altering genes can be identified, or their roles confirmed, by a variety of methods, including genetic methods. For example, animals can be generated or identified with mutations at selected or random loci, and their vigilance properties evaluated in order to determine whether vigilance-modulated or vigilance-altering genes map to these loci. For example, as described in Example IV, below, the gene for arylalkylamine N-acetyl transferase (also known as dopamine acetyltransferase, or Dat) is both a vigilance-modulated gene and a vigilance-altering gene in invertebrates. Drosophila homozygous for a naturally-occurring hypomorphic allele of this gene, Dat.sup.lo, exhibit a sleep rebound following sleep deprivation that is much greater than in wild-type flies, indicating that the Dat gene functions in the homeostatic regulation of sleep. Drosophila hemizygous for the Dat.sup.lo mutation, generated by crossing homozygotes with Drosophila deficient at the Dat locus (Df), exhibit an even more severe sleep rebound effect. Other vigilance modulated genes and vigilance-altering genes can be identified, or their roles confirmed, by similar methods.

As described in Example IV, below, Dopa decarboxylase (Ddc) is a further example of a vigilance-altering gene whose function affects homeostatic regulation of sleep. More specifically, the amount of Ddc enzymatic activity in the invertebrate is directly correlated with the amount of sleep rebound exhibited by the animal following sleep deprivation, with animals severely mutant at the Ddc locus exhibiting less rebound than more mildly affected flies, and mildly affected flies exhibiting less rebound than wild-type flies.

Genetic methods of identifying new vigilance-modulated or vigilance-altering genes that are applicable to a variety of invertebrates are known in the art. For example, the invertebrate can be mutagenized using chemicals, radiation or insertions (e.g. transposons, such as P element mutagenesis), appropriate crosses performed, and the progeny screened for phenotypic differences in vigilance properties compared with normal controls. The gene can then be identified by a variety of methods including, for example, linkage analysis or rescue of the gene targeted by the inserted element. Genetic methods of identifying genes are described for Drosophila, for example, in Greenspan, Fly Pushing: The Theory and Practice of Drosophila Genetics, Cold Spring Harbor Laboratory Press (1997).

There is a distinction between genes that are modulated by vigilance state and genes that are modulated by circadian rhythms. Thus, a gene that is modulated by vigilance state will have a particular expression level during a normal wake period that is similar to the expression level following sleep deprivation, and a different expression level during a normal sleep period. In contrast, a gene that is modulated by circadian rhythms will have a particular expression level during the light period, and a different expression level during the dark period, independent of the vigilance state of the animal. As shown in Example IV, below, D-fos is an example of a gene whose expression is modulated by circadian rhythm rather than by vigilance state.

Assays to evaluate expression of vigilance-modulated genes can involve sacrificing the animal at the appropriate time, such as during a normal wake period, during a normal sleep period or following sleep deprivation, homogenizing the entire animal, or a portion containing the brain or sensory organs, and extracting either mRNA or proteins therefrom. Alternatively, such assays can be performed in biopsied tissue from the invertebrate.

A variety of assays well known in the art can be used to evaluate expression of particular vigilance-modulated genes, including the genes described above. Assays that detect mRNA expression generally involve hybridization of a detectable agent, such as a complementary primer or probe, to the nucleic acid molecule. Such assays include, for example, Northern or dot blot analysis, primer extension, RNase protection assays, reverse-transcription PCR, competitive PCR, real-time quantitative PCR (TaqMan PCR), and nucleic acid array analysis.

Additionally, constructs containing the promoter of a vigilance-modulated gene and a reporter gene (e.g. .beta.-galactosidase, green fluorescent protein, luciferase) can be made by known methods, and used to generate transgenic invertebrates. In such transgenic invertebrates, expression of the reporter gene is a marker for expression of the vigilance-modulated gene.

Assays that detect protein expression can also be used to evaluate expression of particular vigilance-modulated genes. Such assays generally involve binding of a detectable agent, such as an antibody or selective binding agent, to the polypeptide in a sample of cells or tissue from the animal. Protein assays include, for example, immunohistochemistry, immunofluorescence, ELISA assays, immunoprecipitation, and immunoblot analysis.

Those skilled in the art will appreciate that the methods of the invention can be practiced in the absence of knowledge of the sequence or function of the vigilance-modulated genes whose expression is evaluated. Expression of vigilance-modulated genes can thus be evaluated using assays that examine overall patterns of gene expression characteristic of vigilance state. It will be understood that as these vigilance-modulated genes are identified or sequenced, specific probes, primers, antibodies and other binding agents can be used to evaluate their expression more specifically using any of the above detection methods.

One assay to examine-patterns of expression of vigilance-modulated genes, that does not require prior knowledge of their sequence, is mRNA differential display, which is described, for example, in Cirelli et al., Mol. Brain Res. 56:293 (1998) and exemplified in invertebrates in Example IV, below. In such a method, RNA from the animal is reverse-transcribed and amplified by PCR using a particular combination of arbitrary primers. A detectable label, such as an enzyme, biotin, fluorescent dye or a radiolabel, is incorporated into the amplification products. The labeled products are then separated by size, such as on acrylamide gels, and detected by any method appropriate for detecting the label, including autoradiography, phosphoimaging or the like.

Such a method allows concurrent examination of expression of thousands of RNA species, the vast majority of which are expected not to be modulated by vigilance state. However, as described in Example IV, below, there will be a characteristic, reproducible banding pattern associated with vigilance state. It can be readily determined whether a particular candidate compound alters this pattern of gene expression, such as by increasing or decreasing the intensity of vigilance-modulated bands.

A further assay to examine patterns of expression of vigilance-modulated genes is array analysis, in which nucleic acids representative of all or a portion of the genome of the invertebrate, or representative of all or a portion of expressed genes of the invertebrate, are attached to a solid support, such as a filter, glass slide or a chip. Detectably labeled probes, such as cDNA probes, are then prepared from mRNA of an animal, and hybridized to the array to generate a characteristic, reproducible pattern of spots associated with vigilance state. It can be readily determined whether a particular candidate compound alters this pattern of gene expression, such as by increasing or decreasing the intensity of vigilance-modulated spots.

Following identification of patterns of vigilance-modulated gene expression, those skilled in the art can clone the genes, if desired, using standard molecular biology approaches. For example, a vigilance-modulated band identified by differential display can be eluted from a gel and sequenced, or used to probe a library to identify the corresponding cDNA or genomic DNA. Likewise, a vigilance-modulated gene from an array can be identified based on its known position on the array, or cloned by PCR or by probing a library.

Given the teachings described herein that behavioral vigilance properties are closely correlated with molecular vigilance properties, and that behavioral and molecular properties are highly conserved across disparate species, for example, mammals and flies, it is understood that the invention can be practiced using any invertebrate that exhibits at least one behavioral or one molecular vigilance property that is susceptible to evaluation or measurement.

As disclosed herein, Drosophila melanogaster is an example of an invertebrate that exhibits a variety of vigilance properties that can be evaluated, including homeostatically regulated activity, arousal threshold, latency to sleep, and expression of vigilance-modulated genes. Those skilled in the art understand that other Drosophila species are also likely to exhibit similar vigilance properties, including D. simulans, D. virilis, D. pseudoobscura D. funebris, D. immigrans, D. repleta, D. affinis, D. saltans, D. sulphurigaster albostrigata and D. nasuta albomicans. Likewise, other flies, including, sand flies, mayflies, blowflies, flesh flies, face flies, houseflies, screw worm-flies, stable flies, mosquitos, northern cattle grub, and the like will also exhibit vigilance properties.

Furthermore, insects other than flies can also exhibit behavioral and molecular vigilance properties. For example, species of cockroach exhibit rest rebound following rest deprivation, as well as a higher arousal threshold correlated with rest (Tobler et al., Sleep Res. 1:231-239 (1992)). Thus, the invention can also be practiced with insects such as cockroaches, honeybees, wasps, termites, grasshoppers, moths, butterflies, fleas, lice, boll weevils and beetles.

Arthropods other than insects also can exhibit behavioral and molecular vigilance properties. For example, scorpions exhibit rest rebound following rest deprivation, as well as a characteristic arousal threshold and heart rate associated with rest (Tobler et al., J. Comp. Physiol. 163:227-235 (1988)). Thus, the invention can also be practiced using arthropods such as scorpions, spiders, mites, crustaceans, centipedes and millipedes.

Due to the high degree of genetic similarity across invertebrate species, invertebrates other than arthropods, such as flatworms, nematodes (e.g. C. elegans), mollusks (e.g. Aplysia or Hermissenda), echinoderms and annelids will exhibit behavioral and molecular properties correlated with vigilance state, and can be used in the methods of the invention.

Those skilled in the art can determine, using the assays described herein, whether a particular invertebrate exhibits behavioral or molecular properties correlated with vigilance state and, therefore, would be applicable for use in the methods of the invention. The choice of invertebrate will also depend on additional factors, for example, such as the availability of the animals, the normal activity levels of the animals, the availability of molecular probes for vigilance-modulated genes, the number of animals and compounds one intends to screen, the ease and cost of maintaining the animals in a laboratory setting, the method of contacting and type of compounds being tested, and the particular property being evaluated. Those skilled in the art can evaluate these factors in determining an appropriate invertebrate to use in the screening methods.

For example, if it is desired to evaluate molecular properties in the methods of the invention, an invertebrate that is genetically well-characterized, such that homologs of vigilance-modulate genes are known or can be readily determined, may be preferred. Thus, appropriate invertebrates in which to evaluate molecular properties of vigilance can include, for example, Drosophila, and C. elegans. If it desired to evaluate behavioral properties in the methods of the invention, an invertebrate that exhibits one or more behavioral properties now known to be consistent with sleep, such as fruit flies, cockroaches, honeybees, wasps, moths, mosquitos, scorpions, may be preferred.

As disclosed herein, invertebrate sleep exhibits an age-dependence similar to mammalian sleep. Therefore, it may be desirable to practice the methods of the invention using invertebrates of different ages so as to identify compounds that alter vigilance in the very young or very old. Such compounds can be tailored for use in pediatric or geriatric patients.

As also disclosed herein, invertebrate sleep patterns differ between females and males. Therefore, it may be desirable to practice the methods of the invention using invertebrates of both genders separately to identify compounds appropriate for use in females, males, or both females and males.

If desired, invertebrates that contain mutations of varying degrees of severity in vigilance-altering genes can be used in the screening methods described herein, and compounds identified that correct these defects. In such screens, a vigilance property is evaluated in mutant invertebrates and in normal invertebrates. A compound that alters the vigilance property in the mutant invertebrate to a level or amount more similar to the property in the normal animal can thus be identified. For example, a screen can be conducted in a Drosophila that is mutant at the Dat locus or the Ddc locus, both of which, as shown in Example IV, alter, in different directions, the amount of sleep rebound exhibited by the animal following sleep deprivation. Accordingly, a compound that alters homeostatic regulation of sleep can be identified as a compound that restores more normal sleep rebound in a Dat or a Ddc mutant animal. Animals mutant in other vigilance-modulated or vigilance-altering genes can similarly be identified or generated, and used to identify compounds that affect a particular function implicated in vigilance (e.g. neurotransmitter synthesis or degradation), or a particular property of vigilance, including a homeostatically regulated property of vigilance.

The methods of the invention are practiced by contacting an invertebrate with a candidate compound, and evaluating a vigilance property. Appropriate invertebrates, candidate compounds and vigilance properties to evaluate for various applications of the method have been described above. As used herein, the term "contacting" refers to any method of administering a candidate compound to an invertebrate such that the compound, or a metabolite thereof, is introduced into the invertebrate in an effective amount so as to act on its nervous system.

Exemplary methods of contacting an invertebrate with a candidate compound include feeding the compound to the animal, topical administration of the compound, administration by aerosol spray, immersion of the animal in a solution containing the compound, and injection of the compound. An appropriate method of contacting an invertebrate with a compound can be determined by those skilled in the art and will depend, for example, on the type and developmental stage of the invertebrate, whether the invertebrate is sleeping or awake at the time of contacting, the number of animals being assayed, and the chemical and biological properties of the compound (e.g. solubility, digestibility, bioavailability, stability and toxicity). For example, as shown in Example IV below, Drosophila melanogaster can be contacted with stimulants or hypnotics by dissolving the drugs in fly food and providing the food to the flies.

A "candidate compound" used to contact the invertebrate can be any molecule that potentially alters vigilance. A candidate compound can be a naturally occurring macromolecule, such as a peptide, nucleic acid, carbohydrate, lipid, or any combination thereof, or a partially or completely synthetic derivative, analog or mimetic of such a macromolecule. A candidate compound can also be a small organic or inorganic molecule, either naturally occurring, or prepared partly or completely by synthetic methods. If desired, a candidate compound can be combined with, or dissolved in, an agent that facilitates uptake of the compound by the invertebrate, such as an organic solvent (e.g. DMSO, ethanol), aqueous solvent (e.g. water or a buffer), or food.

A candidate compound can be tested at a single dose, or at a range of doses. It is expected that the effects on properties correlated with vigilance will be dose dependent, as demonstrated with caffeine and hydroxyzine in Example III, below. Appropriate concentrations of candidate compound to test in the methods of the invention can be determined by those skilled in the art, and will depend on the chemical and biological properties of the compound and the method of contacting. Exemplary concentration ranges to test include from about 10 .mu.g/ml to about 500 mg/ml, such as from about 100 .mu.g/ml to 250 mg/ml, including from about 1 mg/ml to 200 mg/ml.

The number of different compounds to screen in the methods of the invention can be determined by those skilled in the art depending on the application of the method. For example, a smaller number of candidate compounds would generally be used if the type of compound that is likely to alter vigilance is known or can be predicted, such as when derivatives of a lead compound are being tested. However, when the type of compound that is likely to alter vigilance is unknown, it is generally understood that the larger the number of candidate compounds screened, the greater the likelihood of identifying a compound that alters vigilance. Therefore, the methods of the invention can employ screening individual compounds separately or populations of compounds including small populations and large or diverse populations, to identify a compound that alters vigilance.

The appropriate time and duration to administer the compound can be determined by those skilled in the art depending on the application of the method. For example, it may be desirable to administer a compound at the beginning or end of the normal wake or sleep period, continuously throughout a normal wake or sleep period, or prior to, during, or after sleep deprivation, depending on the vigilance property being evaluated and the desired effect of the compound. As exemplified in Example III, below, compounds that either increase or decrease vigilance can be administered in the last hour of the normal wake period, and their effect on activity during the next sleep period or on latency to sleep can be readily observed.

Methods for producing libraries of candidate compounds to use in the methods of the invention, including chemical or biological molecules such as simple or complex organic molecules, metal-containing compounds, carbohydrates, peptides, proteins, peptidomimetics, glycoproteins, lipoproteins, nucleic acids, antibodies, and the like, are well known in the art. Libraries containing large numbers of natural and synthetic compounds also can be obtained from commercial sources.

Following contacting the invertebrate with the candidate compound, any of the vigilance properties described above can be evaluated, and a determination made as to whether the compound alters, such as increases or decreases, the vigilance property compared to a baseline or established value for the property in an untreated control. Such a compound will similarly alter vigilance in mammals. However, it will be understood that the efficacy and safety of the compound in laboratory mammals can be further evaluated before administering the compound to humans or veterinary animals. For example, the compound can be tested for its maximal efficacy and any potential side-effects using several different invertebrates or laboratory mammals, across a range of doses, in a range of formulations, and at various times during the normal sleep and wake periods.

Additionally, a compound that alters vigilance can be tested for its effects on one or more additional vigilance properties in order to determine its most effective application in therapy. For example, it may be desirable to determine whether a compound that increases vigilance does so without significantly altering latency to sleep when the effect of the compound wears off. Such a compound would be an improvement over many of the currently known vigilance-enhancing drugs that cause a characteristic "crash" afterwards. It may also be desirable to determine whether the compound that alters vigilance does so without a compensatory sleep rebound effect.

Therefore, once a compound is identified that alters a desirable vigilance property, the methods of the invention can be used to determine other vigilance characteristics of the compound. Such other characteristics can be assessed either simultaneously with the initial screen, or alternatively they can be assessed in or more separate screens to identify or characterize other vigilance properties of the compound. For example, a vigilance altering compound identified that promotes sleep can be further assessed to determine whether that compound additionally reduces arousal threshold to normal sleep levels, while preserving the ability of the animal to be wakened normally, and with subsequent normal wake-like behaviors. Such a compound would be an improvement over many of the currently available sleep-inducing drugs, which may not promote truly restorative sleep or normal function on awakening. Similarly, a vigilance altering compound identified that promotes wakefulness can be further assessed, as described above, to determine whether that compound additionally reduces the rate or extent of the wake-sleep transition, or "crash," following the vigilance enhancing effects of the compound.

The methods of the invention are therefore applicable for screening and identifying compounds that exhibit preferred vigilance altering effects as well as for identifying compounds that exhibit a combination of preferred vigilance altering effects to yield optimal vigilance altering compounds. Such optimal vigilance altering compounds can be identified which combine preferred effects on vigilance levels together with maintaining some or all hemostatic regulatory properties of vigilance.

As used herein, "homestatic regulatory properties of vigilance" or "homeostatic regulatory properties" is intended to mean those vigilance properties that are compensatory changes in vigilance resulting from, or correlating with, the quantity or quality of vigilance from a previous time period. Homeostatic regulatory properties are therefore vigilance properties when viewed in light of the vigilance state of a previous period. Such properties include, for example, vigilance properties such as sleep rebound, wake period, latency to sleep, the rate of the sleep-wake transition, alertness or drowsiness when there has been a corresponding and opposite change in vigilance in the immediate, prior period, or when there has been a correlative effect in the immediate, prior period.

For the specific homeostatic regulatory property referred to as sleep rebound, prolonged or more intense sleep periods occur as a compensatory change to prior increases in vigilance periods. For the remaining homeostatic regulatory properties specifically exemplified above, such properties are, for example, compensatory changes due to correlative effects in the prior period. For example, the transition rate between wake and sleep states will be correspondingly increased or decreased depending on the amount and quality of the previous wake or sleep vigilance state. Similarly, an animal will be more alert following a more restful period and will be more drowsy following a less restful period. Such compensatory vigilance states arise from the quality and nature of vigilance state of the previous time period. Homeostatic regulatory properties of vigilance other than those described above also exist and are well known to those skilled in the art.

Preferred or optimal vigilance altering compounds can be identified using the methods of the invention which exhibit, for example, predetermined effects on the magnitude of vigilance levels or on the period and duration of the effect. For example, vigilance altering compounds can be identified that either increase or decrease vigilance levels in small or large increments or to a specified degree. Vigilance altering compounds similarly can be identified that increase or decrease vigilance levels to a maximum amount allowable without affecting other vital or relevant physiological processes. Preferred or optimal compounds also can be selected that modulate the duration of the vigilance altering effect for a predetermined period, including maximal durations, without adversely affecting other vital or relevant physiological processes.

Compounds exhibiting one or more combinations of the above effects can similarly be identified using the methods of the invention. A specific example of one such preferred or optimal combination is a compound that alters vigilance, either by increasing or decreasing vigilance, to its maximal extent, but for a short and specified time. Another example is a compound that results in small alterations in vigilance levels but exhibits a relatively prolonged, and predetermined duration of the effect. Vigilance altering compounds exhibiting other combinations of preferred or optimal vigilance effects can similarly be selected using the methods of the invention, given the teachings and descriptions herein.

Additionally, preferred or optimal vigilance altering compounds can be identified using the methods of the invention which modulate, for example, one or more homeostatic regulatory properties of vigilance following a prior perturbation in vigilance levels or periods. For example, vigilance altering compounds can be identified that modulate the sleep rebound, wake period, latency to sleep, the rate of the sleep-wake transition, alertness or drowsiness. Vigilance altering compounds can be identified, for example, that increase or decrease the period or amount of sleep rebound following prolonged periods of increased vigilance. Similarly, vigilance altering compounds can be identified, for example, that increase or decrease the period or amount of wake period as well as the level of vigilance following prolonged periods of sleep. Such compounds can be preferred because they increase the animal's alertness and therefore decrease lethargic periods during the wake state. Finally, vigilance altering compounds can be identified that, for example, decrease the rate of the wake-to-sleep transition so as to prevent a crash following prolonged waking periods as well as increase the rate of the sleep-to-wake transitions so as to achieve normal levels of vigilance following prolonged or induced periods of sleep.

Vigilance altering compounds exhibiting one or more combinations of the above modulatory effects on homeostatic regulatory properties can similarly be identified using the methods of the invention. One specific example is a compound that prevents or reduces sleep rebound to a specified extent and maintains normal vigilance levels following prolonged wake periods. Another specific example is a compound that increases the rate of the sleep-to-wake transition while also preventing lethargic periods during the wake state following prolonged or induced sleep.

Likewise, the methods of the invention are also applicable to identifying compounds that maintain or mimic, for example, one or more homeostatic regulatory properties following a prior perturbation. For example, it can be desirable to maintain or induce normal homeostatic regulatory properties following prior preturbation of vigilance levels or periods. In such instances, the methods of the invention can be used to identify compounds that cause such effects following a prior modulation of vigilance.

Finally, preferred or optimal vigilance altering compounds can be identified using the methods of the invention which exhibit combinations, including optimal combinations, of one or more preferred vigilance altering effects and modulation or maintenance of one or more homeostatic regulatory properties of vigilance. For example, vigilance altering compounds can be identified that induce specific magnitudes or durations of vigilance levels and which alter homeostatic regulatory properties following the induced changes in vigilance levels. One specific example, is a compound that maximally increases vigilance levels over prolonged periods without a subsequent sleep rebound effect. Alternatively, such a vigilance increasing compound can also result in little or no crash following the prolonged wake period. Another example is a compound that decreases vigilance, such as induces restful sleep states, for a predetermined period without a lethargic vigilance states following the sleep period. Similarly, vigilance altering compounds can be identified that induce specific magnitudes or durations of vigilance levels and which alter homeostatic regulatory properties simultaneously with the induced changes in vigilance levels. Compounds exhibiting various other combinations of vigilant altering effects and modulation, or maintenance, of homeostatic regulatory properties can similarly be identified using the method and teachings described herein.

Therefore, the invention allows the identification of compounds that alter vigilance levels and modulate or maintain homeostatic regulatory properties of vigilance. Such compounds can be identified in the initial screen, or alternatively, such compounds can be identified step-wise by first identifying compounds that alter vigilance and subsequently determining whether such identified compounds affect homeostatic regulatory properties of vigilance, such as sleep rebound and latency to sleep. Similarly, compounds can be identified either in the initial screen or in step-wise procedures that alter vigilance properties and are devoid of deleterious side-effects, such as a precipitous crash after the drug wears off or lack of restfulness following drug induced sleep. Therefore, the methods of the invention are applicable for identifying compounds that alter vigilance in mammals, as well as to identifying compounds that alter vigilance levels with concomitant homeostatic regulatory properties. Similarly, the methods of the invention are also applicable to identifying compounds that alter vigilance in mammals that are devoid of deleterious and unwanted side-effects.

Compounds identified by the methods of the invention as compounds that alter vigilance can also have an effect on neuronal plasticity, or the ability to learn and form memories. Learning is not possible during sleep in mammals, whereas learning and memory are positively associated with the level of vigilance during waking. Thus, by increasing vigilance, it is also possible to increase learning and memory. Accordingly, in one embodiment, the invertebrate is contacted with a candidate compound, a vigilance property is evaluated, and learning or memory is also evaluated.

A variety of assays are known in the art that can be used to evaluate learning and either short-term or long-term memory in invertebrates, including habituation and sensitization assays, and conditioning assays. Habituation refers to a decrease, and sensitization refers to an increase, in a behavioral response on repeated presentation of the same stimulus, and can be considered rudimentary forms of learning. Exemplary habituation assays that can be readily adapted for use in a variety of invertebrates are described, for example, for C. elegans in Rankin et al., Behav. Brain Res. 37:89-92 (1990); for Drosophila in Boynton et al., Genetics 131:655-672 (1992); and for Aplysia in Kandel et al., Cold Spring Harb. Symp. Quant. Biol. 40:465-482 (1976).

Classical (Pavlovian) conditioning is an accepted behavioral paradigm for learning and memory. In an exemplary conditioning assay, invertebrates can be exposed to two different stimuli, such as two odorants or two colors of light, one of which is associated with negative reinforcement, such as an electric shock. The animals are then removed and tested in a new apparatus, similar to the training arrangement but without reinforcement. Avoidance behavior is scored as learning, and retention time of the learned behavior is scored as memory. Exemplary conditioning assays that can be readily adapted for use in a variety of invertebrates are described, for example, for Drosophila in Quinn et al., Proc. Natl. Acad. Sci. USA 71:708-712 (1974); for cockroach in Mizunami et al., J. Comp. Neruol. 402:520-537 (1998); and for crab in Hoyle, Behav. Biol. 18:147-163 (1976).
 

Claim 1 of 2 Claims

1. A method of identifying a vigilance enhancing compound that modulates homeostatic regulation, comprising: (a) contacting an invertebrate with a candidate vigilance enhancing compound, (b) determining the effect of said candidate compound on a homeostatic regulatory property of vigilance selected from the group consisting of sleep rebound, wake period, latency to sleep, rate of sleep wake transition, alertness and drowsiness by comparing said effect to a baseline or established value for the property in an untreated control, wherein a compound that alters said homeostatic regulatory property is characterized as being a vigilance enhancing compound that modulates homeostatic regulation.

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