RESUMEN
The second-generation Drosophila traumatic brain injury (TBI) device dTBI2 improves Drosophila TBI administration by providing a moderate-throughput, tunable, head-specific injury. Our updated device design improves user-friendliness, eliminates inconsistencies in injury timing, and has an updated circuit design to extend the longevity of delicate electronic components. dTBI2 improves reproducibility across users and runs, and results in more consistent post-injury phenotypes. This protocol describes the construction, calibration, and use of the dTBI2 device, which uses an Arduino-controlled piezoelectric actuator to deliver a force that compresses a fly head against a metal collar. The duration and depth of head compression is tunable, allowing calibration of injury severity. All device components are commercially available, and the entire device can be constructed in under a week for less than $1000. The dTBI2 design will enable any lab to build a highly reliable, low-cost device for Drosophila TBI, facilitating increased adoption and ease of exploration of closed-head TBI in Drosophila for forward genetic screens. We describe below the three protocols necessary for constructing a dTBI2 device. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Construction of the dTBI2 control device Basic Protocol 2: Construction of the piezoelectric actuator housing Basic Protocol 3: Administration of dTBI2 injuries.
Asunto(s)
Lesiones Traumáticas del Encéfalo , Drosophila , Animales , Reproducibilidad de los Resultados , Modelos Animales de Enfermedad , Lesiones Traumáticas del Encéfalo/terapia , PresiónRESUMEN
Circadian cycles of sleep:wake and gene expression change with age in all organisms examined. Metabolism is also under robust circadian regulation, but little is known about how metabolic cycles change with age and whether these contribute to the regulation of behavioral cycles. To address this gap, we compared cycling of metabolites in young and old Drosophila and found major age-related variations. A significant model separated the young metabolic profiles by circadian timepoint, but could not be defined for the old metabolic profiles due to the greater variation in this dataset. Of the 159 metabolites measured in fly heads, we found 17 that cycle by JTK analysis in young flies and 17 in aged. Only four metabolites overlapped in the two groups, suggesting that cycling metabolites are distinct in young and old animals. Among our top cyclers exclusive to young flies were components of the pentose phosphate pathway (PPP). As the PPP is important for buffering reactive oxygen species, and overexpression of glucose-6-phosphate dehydrogenase (G6PD), a key component of the PPP, was previously shown to extend lifespan in Drosophila, we asked if this manipulation also affects sleep:wake cycles. We found that overexpression in circadian clock neurons decreases sleep in association with an increase in cellular calcium and mitochondrial oxidation, suggesting that altering PPP activity affects neuronal activity. Our findings elucidate the importance of metabolic regulation in maintaining patterns of neural activity, and thereby sleep:wake cycles.
Asunto(s)
Relojes Circadianos , Drosophila , Animales , Drosophila/metabolismo , Sueño , Especies Reactivas de Oxígeno/metabolismo , Vía de Pentosa Fosfato , Ritmo CircadianoRESUMEN
The gut microbiome is well known to impact host physiology and health. Given widespread control of physiology by circadian clocks, we asked how the microbiome interacts with circadian rhythms in the Drosophila gut. The microbiome did not cycle in flies fed ad libitum, and timed feeding (TF) drove limited cycling only in clockless per01 flies. However, TF and loss of the microbiome influenced the composition of the gut cycling transcriptome, independently and together. Moreover, both interventions increased the amplitude of rhythmic gene expression, with effects of TF at least partly due to changes in histone acetylation. Contrary to expectations, timed feeding rendered animals more sensitive to stress. Analysis of microbiome function in circadian physiology revealed that germ-free flies reset more rapidly with shifts in the light:dark cycle. We propose that the microbiome stabilizes cycling in the host gut to prevent rapid fluctuations with changing environmental conditions.
Asunto(s)
Relojes Circadianos , Microbioma Gastrointestinal , Animales , Ritmo Circadiano/genética , Drosophila/fisiología , FotoperiodoRESUMEN
Temperatures outside the preferred range require flies to acutely adjust their behavior. A new study finds that heat-sensing neurons provide input to fly circadian clock neurons to extend the daytime siesta, allowing flies to sleep through excessive daytime heat.
Asunto(s)
Relojes Circadianos , Proteínas de Drosophila , Animales , Ritmo Circadiano , Drosophila melanogaster/fisiología , Sueño/fisiología , TemperaturaRESUMEN
The relatively simple Drosophila circadian clock circuit consists of 150 clock neurons that coordinate rhythmic behavior and physiology, which are generally classified based on neuroanatomical location. Transcriptional and connectomic studies have identified novel subdivisions of these clock neuron populations, and identified neuropeptides not previously known to be expressed in the fly clock circuit. An additional feature of fly clock neurons is daily axonal remodeling, first noted in small ventrolateral neurons, but more recently also found in additional clock neuron groups. These findings raise new questions about the functional roles of clock neuron subpopulations and daily remodeling of network architecture in regulating circadian behavior and physiology.
Asunto(s)
Relojes Circadianos , Proteínas de Drosophila , Neuropéptidos , Animales , Ritmo Circadiano/fisiología , Drosophila/fisiología , Proteínas de Drosophila/genética , Neuropéptidos/fisiologíaRESUMEN
The ability to control transgene expression, both spatially and temporally, is essential for studying model organisms. In Drosophila, spatial control is primarily provided by the GAL4/UAS system, whilst temporal control relies on a temperature-sensitive GAL80 (which inhibits GAL4) and drug-inducible systems. However, these are not ideal. Shifting temperature can impact on many physiological and behavioural traits, and the current drug-inducible systems are either leaky, toxic, incompatible with existing GAL4-driver lines, or do not generate effective levels of expression. Here, we describe the auxin-inducible gene expression system (AGES). AGES relies on the auxin-dependent degradation of a ubiquitously expressed GAL80, and therefore, is compatible with existing GAL4-driver lines. Water-soluble auxin is added to fly food at a low, non-lethal, concentration, which induces expression comparable to uninhibited GAL4 expression. The system works in both larvae and adults, providing a stringent, non-lethal, cost-effective, and convenient method for temporally controlling GAL4 activity in Drosophila.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Animales Modificados Genéticamente , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Expresión Génica , Ácidos Indolacéticos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Sleep is controlled by homeostatic mechanisms, which drive sleep after wakefulness, and a circadian clock, which confers the 24-h rhythm of sleep. These processes interact with each other to control the timing of sleep in a daily cycle as well as following sleep deprivation. However, the mechanisms by which they interact are poorly understood. We show here that hugin+ neurons, previously identified as neurons that function downstream of the clock to regulate rhythms of locomotor activity, are also targets of the sleep homeostat. Sleep deprivation decreases activity of hugin+ neurons, likely to suppress circadian-driven activity during recovery sleep, and ablation of hugin+ neurons promotes sleep increases generated by activation of the homeostatic sleep locus, the dorsal fan-shaped body (dFB). Also, mutations in peptides produced by the hugin+ locus increase recovery sleep following deprivation. Transsynaptic mapping reveals that hugin+ neurons feed back onto central clock neurons, which also show decreased activity upon sleep loss, in a Hugin peptide-dependent fashion. We propose that hugin+ neurons integrate circadian and sleep signals to modulate circadian circuitry and regulate the timing of sleep.
Asunto(s)
Relojes Circadianos/fisiología , Proteínas de Drosophila/metabolismo , Neuronas/fisiología , Neuropéptidos/genética , Neuropéptidos/metabolismo , Sueño/fisiología , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Femenino , Homeostasis , Locomoción , Mutación , Privación de Sueño , Vigilia/fisiologíaRESUMEN
Regulation of circadian behavior and physiology by the Drosophila brain clock requires communication from central clock neurons to downstream output regions, but the mechanism by which clock cells regulate downstream targets is not known. We show here that the pars intercerebralis (PI), previously identified as a target of the morning cells in the clock network, also receives input from evening cells. We determined that morning and evening clock neurons have time-of-day-dependent connectivity to the PI, which is regulated by specific peptides as well as by fast neurotransmitters. Interestingly, PI cells that secrete the peptide DH44, and control rest:activity rhythms, are inhibited by clock inputs while insulin-producing cells (IPCs) are activated, indicating that the same clock cells can use different mechanisms to drive cycling in output neurons. Inputs of morning cells to IPCs are relevant for the circadian rhythm of feeding, reinforcing the role of the PI as a circadian relay that controls multiple behavioral outputs. Our findings provide mechanisms by which clock neurons signal to nonclock cells to drive rhythms of behavior.
Asunto(s)
Encéfalo/metabolismo , Ritmo Circadiano/fisiología , Neuronas/metabolismo , Animales , DrosophilaRESUMEN
Drosophila melanogaster is a powerful model organism used to study circadian rhythms, historically for elucidating the molecular basis of the clock and, more recently, for allowing for dissection of neural circuits underlying rhythmic behavior. The fly can be used to investigate the neuronal basis of complex behaviors at single-neuron resolution. Patch clamp electrophysiology permits single-neuron recording of resting membrane potential and action potential firing in response to genetic or environmental manipulations or application of drugs and neurotransmitters. Here we describe a protocol for dissecting Drosophila brains for electrophysiology, setting up and using a patch clamp system, and analyzing firing data around the circadian day and in stimulation-response experiments to test for functional neuronal connectivity in circadian circuits.
Asunto(s)
Ritmo Circadiano , Neuronas/fisiología , Técnicas de Placa-Clamp/métodos , Potenciales de Acción , Animales , Encéfalo/citología , Células Cultivadas , Drosophila melanogaster , Neuronas/citología , Cultivo Primario de Células/métodosRESUMEN
Circadian clocks monitor both light and temperature cycles to entrain behavior and physiology to the environment. Recently in Nature, Yadlapalli et al. (2018) identified a subgroup of Drosophila clock neurons that responds to temperature input with changes in intracellular calcium and mediates effects of temperature on circadian entrainment and sleep.
Asunto(s)
Relojes Circadianos , Animales , Ritmo Circadiano , Frío , Neuronas , TemperaturaRESUMEN
The mechanisms by which clock neurons in the Drosophila brain confer an â¼24-hr rhythm onto locomotor activity are unclear, but involve the neuropeptide diuretic hormone 44 (DH44), an ortholog of corticotropin-releasing factor. Here we identified DH44 receptor 1 as the relevant receptor for rest:activity rhythms and mapped its site of action to hugin-expressing neurons in the subesophageal zone (SEZ). We traced a circuit that extends from Dh44-expressing neurons in the pars intercerebralis (PI) through hugin+ SEZ neurons to the ventral nerve cord. Hugin neuropeptide, a neuromedin U ortholog, also regulates behavioral rhythms. The DH44 PI-Hugin SEZ circuit controls circadian locomotor activity in a daily cycle but has minimal effect on feeding rhythms, suggesting that the circadian drive to feed can be separated from circadian locomotion. These findings define a linear peptidergic circuit that links the clock to motor outputs to modulate circadian control of locomotor activity.
Asunto(s)
Relojes Circadianos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiología , Locomoción/genética , Neuropéptidos/genética , Receptores de Superficie Celular/genética , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Masculino , Neuropéptidos/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
Circadian clocks regulate much of behavior and physiology, but the mechanisms by which they do so remain poorly understood. While cyclic gene expression is thought to underlie metabolic rhythms, little is known about cycles in cellular physiology. We found that Drosophila insulin-producing cells (IPCs), which are located in the pars intercerebralis and lack an autonomous circadian clock, are functionally connected to the central circadian clock circuit via DN1 neurons. Insulin mediates circadian output by regulating the rhythmic expression of a metabolic gene (sxe2) in the fat body. Patch clamp electrophysiology reveals that IPCs display circadian clock-regulated daily rhythms in firing event frequency and bursting proportion under light:dark conditions. The activity of IPCs and the rhythmic expression of sxe2 are additionally regulated by feeding, as demonstrated by night feeding-induced changes in IPC firing characteristics and sxe2 levels in the fat body. These findings indicate circuit-level regulation of metabolism by clock cells in Drosophila and support a role for the pars intercerebralis in integrating circadian control of behavior and physiology.
Asunto(s)
Ritmo Circadiano/fisiología , Señales (Psicología) , Proteínas de Drosophila/genética , Drosophila/fisiología , Ingestión de Alimentos , Regulación de la Expresión Génica , Animales , Ritmo Circadiano/genética , Drosophila/citología , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Fenómenos Electromagnéticos , Cuerpo Adiposo/metabolismo , Insulina/metabolismo , Masculino , Neuronas/fisiologíaRESUMEN
General anesthesia is a relatively safe medical procedure, which for nearly 170 years has allowed life saving surgical interventions in animals and people. However, the molecular mechanism of general anesthesia continues to be a matter of importance and debate. A favored hypothesis proposes that general anesthesia results from direct multisite interactions with multiple and diverse ion channels in the brain. Neurotransmitter-gated ion channels and two-pore K+ channels are key players in the mechanism of anesthesia; however, new studies have also implicated voltage-gated ion channels. Recent biophysical and structural studies of Na+ and K+ channels strongly suggest that halogenated inhalational general anesthetics interact with gates and pore regions of these ion channels to modulate function. Here, we review these studies and provide a perspective to stimulate further advances.
Asunto(s)
Anestésicos por Inhalación/farmacología , Canales de Potasio con Entrada de Voltaje/química , Canales de Sodio Activados por Voltaje/química , Secuencia de Aminoácidos , Animales , Humanos , Activación del Canal Iónico/efectos de los fármacos , Datos de Secuencia Molecular , Canales de Potasio con Entrada de Voltaje/metabolismo , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
Halogenated inhaled general anesthetic agents modulate voltage-gated ion channels, but the underlying molecular mechanisms are not understood. Many general anesthetic agents regulate voltage-gated Na(+) (NaV) channels, including the commonly used drug sevoflurane. Here, we investigated the putative binding sites and molecular mechanisms of sevoflurane action on the bacterial NaV channel NaChBac by using a combination of molecular dynamics simulation, electrophysiology, and kinetic analysis. Structural modeling revealed multiple sevoflurane interaction sites possibly associated with NaChBac modulation. Electrophysiologically, sevoflurane favors activation and inactivation at low concentrations (0.2 mM), and additionally accelerates current decay at high concentrations (2 mM). Explaining these observations, kinetic modeling suggests concurrent destabilization of closed states and low-affinity open channel block. We propose that the multiple effects of sevoflurane on NaChBac result from simultaneous interactions at multiple sites with distinct affinities. This multiple-site, multiple-mode hypothesis offers a framework to study the structural basis of general anesthetic action.
Asunto(s)
Anestésicos por Inhalación/farmacología , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Éteres Metílicos/farmacología , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo , Anestésicos por Inhalación/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Fenómenos Electrofisiológicos , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Cinética , Éteres Metílicos/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Técnicas de Placa-Clamp , Conformación Proteica/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Sevoflurano , Canales de Sodio/químicaRESUMEN
Despite the clinical ubiquity of anesthesia, the molecular basis of anesthetic action is poorly understood. Amongst the many molecular targets proposed to contribute to anesthetic effects, the voltage gated sodium channels (VGSCs) should also be considered relevant, as they have been shown to be sensitive to all general anesthetics tested thus far. However, binding sites for VGSCs have not been identified. Moreover, the mechanism of inhibition is still largely unknown. The recently reported atomic structures of several members of the bacterial VGSC family offer the opportunity to shed light on the mechanism of action of anesthetics on these important ion channels. To this end, we have performed a molecular dynamics "flooding" simulation on a membrane-bound structural model of the archetypal bacterial VGSC, NaChBac in a closed pore conformation. This computation allowed us to identify binding sites and access pathways for the commonly used volatile general anesthetic, isoflurane. Three sites have been characterized with binding affinities in a physiologically relevant range. Interestingly, one of the most favorable sites is in the pore of the channel, suggesting that the binding sites of local and general anesthetics may overlap. Surprisingly, even though the activation gate of the channel is closed, and therefore the pore and the aqueous compartment at the intracellular side are disconnected, we observe binding of isoflurane in the central cavity. Several sampled association and dissociation events in the central cavity provide consistent support to the hypothesis that the "fenestrations" present in the membrane-embedded region of the channel act as the long-hypothesized hydrophobic drug access pathway.
Asunto(s)
Anestésicos Generales/farmacología , Anestésicos por Inhalación/farmacología , Bacterias/metabolismo , Activación del Canal Iónico , Canales de Sodio/efectos de los fármacos , Anestésicos Generales/metabolismo , Anestésicos por Inhalación/metabolismo , Simulación de Dinámica Molecular , Canales de Sodio/metabolismoRESUMEN
BACKGROUND: Halogenated inhaled anesthetics modulate voltage-gated ion channels by unknown mechanisms. RESULTS: Biophysical analyses revealed novel activation of K(v) channels by the inhaled anesthetic sevoflurane. CONCLUSION: K(v) channel activation by sevoflurane results from the positive allosteric modulation of activation gating. SIGNIFICANCE: The unique activation of K(v) channels by sevoflurane demonstrates novel anesthetic specificity and offers new insights into allosteric modulation of channel gating. Voltage-gated ion channels are modulated by halogenated inhaled general anesthetics, but the underlying molecular mechanisms are not understood. Alkanols and halogenated inhaled anesthetics such as halothane and isoflurane inhibit the archetypical voltage-gated Kv3 channel homolog K-Shaw2 by stabilizing the resting/closed states. By contrast, sevoflurane, a more heavily fluorinated ether commonly used in general anesthesia, specifically activates K-Shaw2 currents at relevant concentrations (0.05-1 mM) in a rapid and reversible manner. The concentration dependence of this modulation is consistent with the presence of high and low affinity interactions (K(D) = 0.06 and 4 mM, respectively). Sevoflurane (<1 mM) induces a negative shift in the conductance-voltage relation and increases the maximum conductance. Furthermore, suggesting possible roles in general anesthesia, mammalian Kv1.2 and Kv1.5 channels display similar changes. Quantitative description of the observations by an economical allosteric model indicates that sevoflurane binding favors activation gating and eliminates an unstable inactivated state outside the activation pathway. This study casts light on the mechanism of the novel sevoflurane-dependent activation of Kv channels, which helps explain how closely related inhaled anesthetics achieve specific actions and suggests strategies to develop novel Kv channel activators.
Asunto(s)
Anestésicos por Inhalación/farmacología , Éteres Metílicos/farmacología , Canales de Potasio con Entrada de Voltaje/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Humanos , Cinética , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/genética , Ratas , Sevoflurano , Xenopus laevisRESUMEN
Computational methods and experimental data are used to provide structural models for NaChBac, the homo-tetrameric voltage-gated sodium channel from the bacterium Bacillus halodurans, with a closed and partially open pore domain. Molecular dynamic (MD) simulations on membrane-bound homo-tetrameric NaChBac structures, each comprising six helical transmembrane segments (labeled S1 through S6), reveal that the shape of the lumen, which is defined by the bundle of four alpha-helical S6 segments, is modulated by hinge bending motions around the S6 glycine residues. Mutation of these glycine residues into proline and alanine affects, respectively, the structure and conformational flexibility of the S6 bundle. In the closed channel conformation, a cluster of stacked phenylalanine residues from the four S6 helices hinders diffusion of water molecules and Na(+) ions. Activation of the voltage sensor domains causes destabilization of the aforementioned cluster of phenylalanines, leading to a more open structure. The conformational change involving the phenylalanine cluster promotes a kink in S6, suggesting that channel gating likely results from the combined action of hinge-bending motions of the S6 bundle and concerted reorientation of the aromatic phenylalanine side-chains.
Asunto(s)
Canales de Sodio/metabolismo , Canales de Sodio Activados por Voltaje/química , Bacillus/metabolismo , Proteínas Bacterianas/metabolismo , Biofisica/métodos , Glicina/química , Activación del Canal Iónico/fisiología , Iones , Potenciales de la Membrana , Modelos Moleculares , Conformación Molecular , Simulación de Dinámica Molecular , Movimiento (Física) , Fenilalanina/química , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Sodio/química , Factores de TiempoRESUMEN
Several voltage-gated ion channels are modulated by clinically relevant doses of general anesthetics. However, the structural basis of this modulation is not well understood. Previous work suggested that n-alcohols and inhaled anesthetics stabilize the closed state of the Shaw2 voltage-gated (Kv) channel (K-Shaw2) by directly interacting with a discrete channel site. We hypothesize that the inhibition of K-Shaw2 channels by general anesthetics is governed by interactions between binding and effector sites involving components of the channel's activation gate. To investigate this hypothesis, we applied Ala/Val scanning mutagenesis to the S4-S5 linker and the post-PVP S6 segment, and conducted electrophysiological analysis to evaluate the energetic impact of the mutations on the inhibition of the K-Shaw2 channel by 1-butanol and halothane. These analyses identified residues that determine an apparent binding cooperativity and residue pairs that act in concert to modulate gating upon anesthetic binding. In some instances, due to their critical location, key residues also influence channel gating. Complementing these results, molecular dynamics simulations and in silico docking experiments helped us visualize possible anesthetic sites and interactions. We conclude that the inhibition of K-Shaw2 by general anesthetics results from allosteric interactions between distinct but contiguous binding and effector sites involving inter- and intrasubunit interfaces.