Ralph J. Greenspan

Stress response genes protect against lethal effects of sleep deprivation in Drosophila.

Sleep is controlled by two processes: a homeostatic drive that increases during waking and dissipates during sleep, and a circadian pacemaker that controls its timing. Although these two systems can operate independently, recent studies indicate a more intimate relationship. To study the interaction between homeostatic and circadian processes in Drosophila, we examined homeostasis in the canonical loss-of-function clock mutants period (per01), timeless (tim01), clock (Clkjrk) and cycle (cyc01). cyc01 mutants showed a disproportionately large sleep rebound and died after 10 hours of sleep deprivation, although they were more resistant than other clock mutants to various stressors. Unlike other clock mutants, cyc01 flies showed a reduced expression of heat-shock genes after sleep loss. However, activating heat-shock genes before sleep deprivation rescued cyc01 flies from its lethal effects. Consistent with the protective effect of heat-shock genes, was the observation that flies carrying a mutation for the heat-shock protein Hsp83 (Hsp8308445) showed exaggerated homeostatic response and died after sleep deprivation. These data represent the first step in identifying the molecular mechanisms that constitute the sleep homeostat.

The Brain Activity Map Project and the Challenge of Functional Connectomics

The function of neural circuits is an emergent property that arises from the coordinated activity of large numbers of neurons. To capture this, we propose launching a large-scale, international public effort, the Brain Activity Map Project, aimed at reconstructing the full record of neural activity across complete neural circuits. This technological challenge could prove to be an invaluable step toward understanding fundamental and pathological brain processes.

Dopaminergic Modulation of Arousal in Drosophila

BACKGROUND Arousal levels in the brain set thresholds for behavior, from simple to complex. The mechanistic underpinnings of the various phenomena comprising arousal, however, are still poorly understood. Drosophila behaviors have been studied that span different levels of arousal, from sleep to visual perception to psychostimulant responses. RESULTS We have investigated neurobiological mechanisms of arousal in the Drosophila brain by a combined behavioral, genetic, pharmacological, and electrophysiological approach. Administration of methamphetamine (METH) suppresses sleep and promotes active wakefulness, whereas an inhibitor of dopamine synthesis promotes sleep. METH affects courtship behavior by increasing sexual arousal while decreasing successful sexual performance. Electrophysiological recordings from the medial protocerebrum of wild-type flies showed that METH ingestion has rapid and detrimental effects on a brain response associated with perception of visual stimuli. Recordings in genetically manipulated animals show that dopaminergic transmission is required for these responses and that visual-processing deficits caused by attenuated dopaminergic transmission can be rescued by METH. CONCLUSIONS We show that changes in dopamine levels differentially affect arousal for behaviors of varying complexity. Complex behaviors, such as visual perception, degenerate when dopamine levels are either too high or too low, in accordance with the inverted-U hypothesis of dopamine action in the mammalian brain. Simpler behaviors, such as sleep and locomotion, show graded responses that follow changes in dopamine level.

Serotonin and neuropeptide F have opposite modulatory effects on fly aggression.

Both serotonin (5-HT) and neuropeptide Y have been shown to affect a variety of mammalian behaviors, including aggression. Here we show in Drosophila melanogaster that both 5-HT and neuropeptide F, the invertebrate homolog of neuropeptide Y, modulate aggression. We show that drug-induced increases of 5-HT in the fly brain increase aggression. Elevating 5-HT genetically in the serotonergic circuits recapitulates these pharmacological effects, whereas genetic silencing of these circuits makes the flies behaviorally unresponsive to the drug-induced increase of 5-HT but leaves them capable of aggression. Genetic silencing of the neuropeptide F (npf) circuit also increases fly aggression, demonstrating an opposite modulation to 5-HT. Moreover, this neuropeptide F effect seems to be independent of 5-HT. The implication of these two modulatory systems in fly and mouse aggression suggest a marked degree of conservation and a deep molecular root for this behavior.

Genetic feminization of brain structures and changed sexual orientation in male Drosophila

The neural basis of sexual orientation in Drosophila was studied by the production of males with regionally feminized brains. Such flies express the female form of the sex determination gene transformer in a limited number of neurons under the control of GAL4 enhancer trap inserts. This method facilitated the creation of lines with a stable pattern of feminization. In tests of sexual preferences, flies that were feminized in a portion of the antennal lobes or in a subset of the corpora pedunculata (mushroom bodies) courted both males and females. These two brain structures, both of which are involved in olfactory processing, may function in the recognition of sex-specific pheromones, in the control of sex-specific behaviors, or both.

Activation of EGFR and ERK by rhomboid signaling regulates the consolidation and maintenance of sleep in Drosophila

Epidermal growth factor receptor (EGFR) signaling in the mammalian hypothalamus is important in the circadian regulation of activity. We have examined the role of this pathway in the regulation of sleep in Drosophila melanogaster. Our results demonstrate that rhomboid (Rho)- and Star-mediated activation of EGFR and ERK signaling increases sleep in a dose-dependent manner, and that blockade of rhomboid (rho) expression in the nervous system decreases sleep. The requirement of rho for sleep localized to the pars intercerebralis, a part of the fly brain that is developmentally and functionally analogous to the hypothalamus in vertebrates. These results suggest that sleep and its regulation by EGFR signaling may be ancestral to insects and mammals.

A Proposal for a Coordinated Effort for the Determination of Brainwide Neuroanatomical Connectivity in Model Organisms at a Mesoscopic Scale

In this era of complete genomes, our knowledge of neuroanatomical circuitry remains surprisingly sparse. Such knowledge is critical, however, for both basic and clinical research into brain function. Here we advocate for a concerted effort to fill this gap, through systematic, experimental mapping of neural circuits at a mesoscopic scale of resolution suitable for comprehensive, brainwide coverage, using injections of tracers or viral vectors. We detail the scientific and medical rationale and briefly review existing knowledge and experimental techniques. We define a set of desiderata, including brainwide coverage; validated and extensible experimental techniques suitable for standardization and automation; centralized, open-access data repository; compatibility with existing resources; and tractability with current informatics technology. We discuss a hypothetical but tractable plan for mouse, additional efforts for the macaque, and technique development for human. We estimate that the mouse connectivity project could be completed within five years with a comparatively modest budget.

Electrophysiological Correlates of Rest and Activity in Drosophila melanogaster

Extended periods of rest in Drosophila melanogaster resemble mammalian sleep states in that they are characterized by heightened arousal thresholds and specific alterations in gene expression. Defined as inactivity periods spanning 5 or more min, amounts of this sleep-like state are, as in mammals, sensitive to prior amounts of waking activity, time of day, and pharmacological intervention. Clearly recognizable changes in the pattern and amount of brain electrical activity accompany changes in motor activity and arousal thresholds originally used to identify mammalian sleeping behavior. Electroencephalograms (EEGs) and/or local field potentials (LFPs) are now widely used to quantify sleep state amounts and define types of sleep. Thus, slow-wave sleep (SWS) is characterized by EEG spindles and large-amplitude delta-frequency (0-3.5 Hz) waves. Rapid-eye movement (REM) sleep is characterized by irregular gamma-frequency cortical EEG patterns and rhythmic theta-frequency (5-9 Hz) hippocampal EEG activity. It is unknown whether rest and activity in Drosophila are associated with distinct electrophysiological correlates. To address this issue, we monitored motor activity levels and recorded LFPs in the medial brain between the mushroom bodies, structures implicated in the modulation of locomotor activity, of Drosophila. The results indicate that LFPs can be reliably recorded from the brains of awake, moving fruit flies, that targeted genetic manipulations can be used to localize sources of LFP activity, and that brain electrical activity of Drosophila is reliably correlated with activity state.

Identification of genes involved in Drosophila melanogaster geotaxis, a complex behavioral trait

Identifying the genes involved in polygenic traits has been difficult. In the 1950s and 1960s, laboratory selection experiments for extreme geotaxic behavior in fruit flies established for the first time that a complex behavioral trait has a genetic basis. But the specific genes responsible for the behavior have never been identified using this classical model. To identify the individual genes involved in geotaxic response, we used cDNA microarrays to identify candidate genes and assessed fly lines mutant in these genes for behavioral confirmation. We have thus determined the identities of several genes that contribute to the complex, polygenic behavior of geotaxis.

The flexible genome

A principal assumption underlying contemporary genetic analysis is that the normal function of a gene can be inferred directly from its mutant phenotype. The interactivity among genes that is now being revealed calls this assumption into question and indicates that there might be considerable flexibility in the capacity of the genome to respond to diverse conditions. The reservoir for much of this flexibility resides in the nonspecificity and malleability of gene action.