Inhibitory control of locomotor statistics in walking Drosophila.

In order to forage for food, many animals regulate not only specific limb movements but the statistics of locomotor behavior over time, for example switching between long-range dispersal behaviors and more localized search depending on the availability of resources. How pre-motor circuits regulate such locomotor statistics is not clear. Here we took advantage of the robust changes in locomotor statistics evoked by attractive odors in walking Drosophila to investigate their neural control. We began by analyzing the statistics of ground speed and angular velocity during three well-defined motor regimes: baseline walking, upwind running during odor, and search behavior following odor offset. We find that during search behavior, flies adopt higher angular velocities and slower ground speeds, and tend to turn for longer periods of time in one direction. We further find that flies spontaneously adopt periods of different mean ground speed, and that these changes in state influence the length of odor-evoked runs. We next developed a simple physiologically-inspired computational model of locomotor control that can recapitulate these statistical features of fly locomotion. Our model suggests that contralateral inhibition plays a key role both in regulating the difference between baseline and search behavior, and in modulating the response to odor with ground speed. As the fly connectome predicts decussating inhibitory neurons in the lateral accessory lobe (LAL), a pre-motor structure, we generated genetic tools to target these neurons and test their role in behavior. Consistent with our model, we found that activation of neurons labeled in one line increased curvature. In a second line labeling distinct neurons, activation and inactivation strongly and reciprocally regulated ground speed and altered the length of the odor-evoked run. Additional targeted light activation experiments argue that these effects arise from the brain rather than from neurons in the ventral nerve cord, while sparse activation experiments argue that speed control in the second line arises from both LAL neurons and a population of neurons in the dorsal superior medial protocerebrum (SMP). Together, our work develops a biologically plausible computational architecture that captures the statistical features of fly locomotion across behavioral states and identifies potential neural substrates of these computations.

Ethanol Behavioral Responses in Drosophila.

Drosophila melanogaster is a powerful genetic model for investigating the mechanisms underlying ethanol-induced behaviors, metabolism, and preference. Ethanol-induced locomotor activity is especially useful for understanding the mechanisms by which ethanol acutely affects the brain and behavior. Ethanol-induced locomotor activity is characterized by hyperlocomotion and subsequent sedation with increased exposure duration or concentration. Locomotor activity is an efficient, easy, robust, and reproducible behavioral screening tool for identifying underlying genes and neuronal circuits as well as investigating genetic and molecular pathways. We introduce a detailed protocol for performing experiments investigating how volatilized ethanol affects locomotor activity using the fly Group Activity Monitor (flyGrAM). We introduce installation, implementation, data collection, and subsequent data-analysis methods for investigating how volatilized stimuli affect activity. We also introduce a procedure for how to optogenetically probe neuronal activity to identify the neural mechanisms underlying locomotor activity.

Methods for Exploring the Circuit Basis of Ethanol-Induced Changes in Drosophila Group Locomotor Activity.

Locomotion is a behavioral readout that can be used to understand responses to specific stimuli or perturbations. The fly Group Activity Monitor (flyGrAM) provides a high-throughput and high-content readout of the acute stimulatory and sedative effects of ethanol. The flyGrAM system is adaptable and seamlessly introduces thermogenetic or optogenetic stimulation to dissect neural circuits underlying behavior and tests responses to other volatilized stimuli (humidified air, odorants, anesthetics, vaporized drugs of abuse, etc.). The automated quantification and readout of activity provide users with a real-time representation of the group activity within each chamber throughout the experiment, helping users to quickly determine proper ethanol doses and duration, run behavioral screens, and plan follow-up experiments.

Circuits that encode and guide alcohol-associated preference.

A powerful feature of adaptive memory is its inherent flexibility. Alcohol and other addictive substances can remold neural circuits important for memory to reduce this flexibility. However, the mechanism through which pertinent circuits are selected and shaped remains unclear. We show that circuits required for alcohol-associated preference shift from population level dopaminergic activation to select dopamine neurons that predict behavioral choice in Drosophila melanogaster. During memory expression, subsets of dopamine neurons directly and indirectly modulate the activity of interconnected glutamatergic and cholinergic mushroom body output neurons (MBON). Transsynaptic tracing of neurons important for memory expression revealed a convergent center of memory consolidation within the mushroom body (MB) implicated in arousal, and a structure outside the MB implicated in integration of naïve and learned responses. These findings provide a circuit framework through which dopamine neuronal activation shifts from reward delivery to cue onset, and provide insight into the maladaptive nature of memory.

Cue-Induced Ethanol Seeking in Drosophila melanogaster Is Dose-Dependent.

Alcohol use disorder generates devastating social, medical and economic burdens, making it a major global health issue. The persistent nature of memories associated with intoxication experiences often induces cravings and triggers relapse in recovering individuals. Despite recent advances, the neural and molecular mechanisms underlying these memories are complex and not well understood. This makes finding effective pharmacological targets challenging. The investigation of persistent alcohol-associated memories in the fruit fly, Drosophila melanogaster, presents a unique opportunity to gain a comprehensive understanding of the memories for ethanol reward at the level of genes, molecules, neurons and circuits. Here we characterize the dose-dependent nature of ethanol on the expression of memory for an intoxication experience. We report that the concentration of ethanol, number of ethanol exposures, length of ethanol exposures, and timing between ethanol exposures are critical in determining whether ethanol is perceived as aversive or appetitive, and in how long the memory for the intoxicating properties of ethanol last. Our study highlights that fruit flies display both acute and persistent memories for ethanol-conditioned odor cues, and that a combination of parameters that determine the intoxication state of the fly influence the seemingly complex retention and expression of memories associated with intoxication. Our thorough behavioral characterization provides the opportunity to interrogate the biological underpinnings of these observed preference differences in future studies.

Cigarette Smoke-Induced Alterations in Frontal White Matter Lipid Profiles Demonstrated by MALDI-Imaging Mass Spectrometry: Relevance to Alzheimer's Disease.

BACKGROUND: Meta-analysis has shown that smokers have significantly increased risks for Alzheimer's disease (AD), and neuroimaging studies showed that smoking alters white matter (WM) structural integrity. OBJECTIVE: Herein, we characterize the effects of cigarette smoke (CS) exposures and withdrawal on WM myelin lipid composition using matrix assisted laser desorption and ionization-imaging mass spectrometry (MALDI-IMS). METHODS: Young adult male A/J mice were exposed to air (8 weeks; A8), CS (4 or 8 weeks; CS4, CS8), or CS8 followed by 2 weeks recovery (CS8 + R). Frontal lobe WM was examined for indices of lipid and protein oxidation and lipid profile alterations by MALDI-IMS. Lipid ions were identified by MS/MS with the LIPID MAPS prediction tools database. Inter-group comparisons were made using principal component analysis and R-generated heatmap. RESULTS: CS increased lipid and protein adducts such that higher levels were present in CS8 compared with CS4 samples. CS8 + R reversed CS8 effects and normalized the levels of oxidative stress. MALDI-IMS demonstrated striking CS-associated alterations in WM lipid profiles characterized by either reductions or increases in phospholipids (phosphatidylinositol, phosphatidylserine, phosphatidylcholine, or phosphatidylethanolamine) and sphingolipids (sulfatides), and partial reversal of CS's inhibitory effects with recovery. The heatmap hierarchical dendrogram and PCA distinguished CS exposure, duration, and withdrawal effects on WM lipid profiles. CONCLUSION: CS-mediated WM degeneration is associated with lipid peroxidation, protein oxidative injury, and alterations in myelin lipid composition, including shifts in phospholipids and sphingolipids needed for membrane integrity, plasticity, and intracellular signaling. Future goals are to delineate WM abnormalities in AD using MALDI-IMS, and couple the findings with MRI-mass spectroscopy to improve in vivo diagnostics and early detection of brain biochemical responses to treatment.

Potential contributions of the tobacco nicotine-derived nitrosamine ketone (NNK) in the pathogenesis of steatohepatitis in a chronic plus binge rat model of alcoholic liver disease.

AIMS: Alcoholic liver disease (ALD) is linked to binge drinking and cigarette smoking. Heavy chronic ± binge alcohol, or low-level exposures to dietary nitrosamines cause steatohepatitis with insulin resistance and oxidative stress in animal models. This study examines hepatotoxic effects of sub-mutagenic exposures to tobacco-specific nitrosamine (NNK) in relation to ALD. METHODS: Long Evans rats were fed liquid diets containing 0 or 26% (caloric) ethanol (EtOH) for 8 weeks. In Weeks 3 through 8, rats were treated with NNK (2 mg/kg) or saline by i.p. injection, 3×/week, and in Weeks 7 and 8, EtOH-fed rats were binge-administered 2 g/kg EtOH 3×/week; controls were given saline. RESULTS: EtOH ± NNK caused steatohepatitis with necrosis, disruption of the hepatic cord architecture, ballooning degeneration, early fibrosis, mitochondrial cytopathy and ER disruption. Severity of lesions was highest in the EtOH+NNK group. EtOH and NNK inhibited insulin/IGF signaling through Akt and activated pro-inflammatory cytokines, while EtOH promoted lipid peroxidation, and NNK increased apoptosis. O(6)-methyl-Guanine adducts were only detected in NNK-exposed livers. CONCLUSION: Both alcohol and NNK exposures contribute to ALD pathogenesis, including insulin/IGF resistance and inflammation. The differential effects of EtOH and NNK on adduct formation are critical to ALD progression among alcoholics who smoke.

Differential Sphingolipid and Phospholipid Profiles in Alcohol and Nicotine-Derived Nitrosamine Ketone-Associated White Matter Degeneration.

BACKGROUND: Alcohol-mediated neurodegeneration is associated with white matter (WM) atrophy due to targeting of myelin and oligodendrocytes. However, variability in disease severity suggests cofactors contribute to WM degeneration. We examined the potential cofactor role of the tobacco-specific nitrosamine, nicotine-derived nitrosamine ketone (NNK), because smoking causes WM atrophy and most heavy drinkers consume tobacco products. METHODS: This 8-week study of Long Evans rats had 4 treatment groups: control; NNK-2 mg/kg, 3×/wk in weeks 3 to 8; ethanol (EtOH) (chronic-26% caloric + binge-2 g/kg, 3×/wk in weeks 7 to 8); and EtOH + NNK. Exposure effects on WM lipid biochemical profiles and in situ distributions were examined using matrix-assisted laser desorption/ionization imaging mass spectrometry and tandem mass spectrometry. RESULTS: NNK mainly caused WM fiber degeneration and fiber loss, EtOH caused demyelination, and dual exposures had additive effects. EtOH and EtOH + NNK decreased WM (including corpus callosum) and/or gray matter (hypothalamus, cortex, medial temporal) levels of several phosphatidylserine, phosphatidylinositol, and sphingolipid (sulfatide [ST]) species, while NNK increased or had minimal effect on these lipids. EtOH + NNK had broader and larger inhibitory effects on phospholipids and ST than EtOH or NNK alone. Principal component analysis clustered control with NNK, and EtOH with EtOH + NNK groups, highlighting the independent EtOH- rather than NNK-driven responses. CONCLUSIONS: Chronic EtOH exposures decreased several phospholipid and sphingolipid species in brain, while concomitant NNK exposures exacerbated these effects. These findings support our hypothesis that tobacco smoking is a pathogenic cofactor in alcohol-mediated WM degeneration.