Drosophila Brain Research Articles

Mating modifies oxidative stress in the brain and confers protection against Parkinson's Disease in a Drosophila model

Mating exerts profound and multifaceted effects on the physiology of female insects, particularly influencing metabolic alterations and bolstering stress resilience. Drosophila melanogaster has emerged as an excellent model to investigate the mechanism of neurodegenerative diseases. However, interplay between mating and its impact on the Drosophila brain remains a tantalizing enigma, awaiting elucidation. Herein, we reported that mating significantly improved the climbing and jumping activity in mated females compared to the virgins in Drosophila. Mating also reduced oxidative stress in the brain. Based on the results, we found that, mated females exhibited better behavioral performance and fewer loss of dopaminergic (DA) neurons than unmated females in PINK1 RNAi flies, a well-established Parkinson's disease (PD) model. Further study demonstrated that mating led to decreased iron content in the brain, a process associated with decreased Transferrin 1 (Tsf1) and Malvolio (Mvl) and increased ferritin. Additionally, mating inhibited expression of Duox and Nox, two NADPH oxidases in Drosophila. Furthermore, Kr-h1, a transcription factor of JH, acted downstream of mating to regulate genes involved in iron metabolism and NADPH oxidases. Collectively, the findings suggested a pivotal role of mating in regulating iron metabolism and NADPH oxidases in the brain of Drosophila. Consequently, considering mating status is imperative in scientific research, particularly in the context of neurological disorders.

Accumulation of F-actin drives brain aging and limits healthspan in Drosophila

The actin cytoskeleton is a key determinant of cell structure and homeostasis. However, possible tissue-specific changes to actin dynamics during aging, notably brain aging, are not understood. Here, we show that there is an age-related increase in filamentous actin (F-actin) in Drosophila brains, which is counteracted by prolongevity interventions. Critically, decreasing F-actin levels in aging neurons prevents age-onset cognitive decline and extends organismal healthspan. Mechanistically, we show that autophagy, a recycling process required for neuronal homeostasis, is disabled upon actin dysregulation in the aged brain. Remarkably, disrupting actin polymerization in aged animals with cytoskeletal drugs restores brain autophagy to youthful levels and reverses cellular hallmarks of brain aging. Finally, reducing F-actin levels in aging neurons slows brain aging and promotes healthspan in an autophagy-dependent manner. Our data identify excess actin polymerization as a hallmark of brain aging, which can be targeted to reverse brain aging phenotypes and prolong healthspan.

Loss of function in Drosophila transcription factor Dif delays brain development in larvae resulting in aging adult brain

Drosophila NF-κB transcription factor Dif has been well known for its function in innate immunity, and recent study also reveals its role in neuronal cells. However, the underlying mechanisms of Dif in the brain remain elusive. In this study, we aim to investigate the function of Dif in Drosophila brain development and how Dif regulates structure and plasticity of the brain to affect aging and behaviors. Based on the analysis of differentially expressed genes, we identified key genes associated with cell division, development and aging in the brain of Dif1 loss of function mutant. In Dif1 larvae, we found that the metamorphosis and brain development were delayed, and cell division was decreased. In Dif1 adults, the number of neuron cells was reduced in the brain, the lifespan and locomotor activity were decreased, protein markers associated with aging-related neurodegenerative diseases in the brain were altered in abundance or activity. Our results indicated that Dif plays a crucial role in brain plasticity and neurogenesis, dysfunction of Dif delays larval brain development and impacts proliferation of neuronal cells, resulting in aging adult brain by regulating expression of key genes in multiple signaling pathways involved in cell division, neurogenesis and aging.

Transposable Element (TE) insertion predictions from RNAseq inputs and TE impact on RNA splicing and gene expression in Drosophila brain transcriptomes

Recent studies have suggested that Transposable Elements (TEs) residing in introns frequently splice into and alter primary gene-coding transcripts. To re-examine the exonization frequency of TEs into protein-coding gene transcripts, we re-analyzed a Drosophila neuron circadian rhythm RNAseq dataset and a deep long RNA fly midbrain RNAseq dataset using our Transposon Insertion and Depletion Analyzer (TIDAL) program. Our TIDAL results were able to predict several TE insertions from RNAseq data that were consistent with previous published studies. However, we also uncovered many discrepancies in TE-exonization calls, such as reads that mainly support intron retention of the TE and little support for chimeric mRNA spliced to the TE. We then deployed rigorous genomic DNA-PCR (gDNA-PCR) and RT-PCR procedures on TE-mRNA fusion candidates to see how many of bioinformatics predictions could be validated. By testing a w1118 strain from which the deeper long RNAseq data was derived and comparing to an OreR strain, only 9 of 23 TIDAL candidates (< 40%) could be validated as a novel TE insertion by gDNA-PCR, indicating that deeper study is needed when using RNAseq data as inputs into current TE-insertion prediction programs. Of these validated calls, our RT-PCR results only supported TE-intron retention. Lastly, in the Dscam2 and Bx genes of the w1118 strain that contained intronic TEs, gene expression was 23 times higher than the OreR genes lacking the TEs. This study's validation approach indicates that chimeric TE-mRNAs are infrequent and cautions that more optimization is required in bioinformatics programs to call TE insertions using RNAseq datasets.

A Drosophila computational brain model reveals sensorimotor processing

The recent assembly of the adult Drosophila melanogaster central brain connectome, containing more than 125,000 neurons and 50 million synaptic connections, provides a template for examining sensory processing throughout the brain1,2. Here we create a leaky integrate-and-fire computational model of the entire Drosophila brain, on the basis of neural connectivity and neurotransmitter identity3, to study circuit properties of feeding and grooming behaviours. We show that activation of sugar-sensing or water-sensing gustatory neurons in the computational model accurately predicts neurons that respond to tastes and are required for feeding initiation4. In addition, using the model to activate neurons in the feeding region of the Drosophila brain predicts those that elicit motor neuron firing5—a testable hypothesis that we validate by optogenetic activation and behavioural studies. Activating different classes of gustatory neurons in the model makes accurate predictions of how several taste modalities interact, providing circuit-level insight into aversive and appetitive taste processing. Additionally, we applied this model to mechanosensory circuits and found that computational activation of mechanosensory neurons predicts activation of a small set of neurons comprising the antennal grooming circuit, and accurately describes the circuit response upon activation of different mechanosensory subtypes6–10. Our results demonstrate that modelling brain circuits using only synapse-level connectivity and predicted neurotransmitter identity generates experimentally testable hypotheses and can describe complete sensorimotor transformations.

Regulation of Drosophila brain development and organ growth by the Minibrain/Rala signaling network.

The human dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) is implicated in the pathology of Down syndrome, microcephaly, and cancer, however the exact mechanism through which it functions is unknown. Here, we have studied the role of the Drosophila ortholog of DYRK1A, Minibrain (Mnb), in brain development and organ growth. The neuroblasts (neural stem cells) that eventually give rise to differentiated neurons in the adult brain are formed from a specialized tissue in the larval optic lobe called the neuroepithelium, in a tightly regulated process. Molecular marker analysis of mnb mutants revealed alterations in the neuroepithelium and neuroblast regions of developing larval brains. Using affinity purification-mass spectrometry (AP-MS), we identified the novel Mnb binding partners Ral interacting protein (Rlip) and RALBP1 associated Eps domain containing (Reps). Rlip and Reps physically and genetically interact with Mnb, and the three proteins may form a ternary complex. Mnb phosphorylates Reps, and human DYRK1A binds to the Reps orthologs REPS1 and REPS2. Mnb also promotes re-localization of Rlip from the nucleus to the cytoplasm in cultured cells. Furthermore, Mnb engages the small GTPase Ras-like protein A (Rala) to regulate brain and wing development. This work uncovers a previously unrecognized role of Mnb in the neuroepithelium and defines the functions of the Mnb/Reps/Rlip/Rala signaling network in organ growth and neurodevelopment.

Chemical Characterization and Beneficial Effects of Walnut Oil on a Drosophila melanogaster Model of Parkinson's Disease.

A nutritional approach could be a promising strategy to prevent or decrease the progression of neurodegenerative disorders such as Parkinson's disease (PD). The neuroprotective role of walnut oil (WO) was investigated in Drosophila melanogaster treated with rotenone (Rot), as a PD model, WO, or their combination, and compared to controls. WO reduced mortality and improved locomotor activity impairment after 3 and 7 days, induced by Rot. LC-MS analyses of fatty acid levels in Drosophila heads showed a significant increase in linolenic (ALA) and linoleic acid (LA) both in flies fed with the WO-enriched diet and in those treated with the association of WO with Rot. Flies supplemented with the WO diet showed an increase in brain dopamine (DA) level, while Rot treatment significantly depleted dopamine content; conversely, the association of Rot with WO did not modify DA content compared to controls. The greater intake of ALA and LA in the enriched diet enhanced their levels in Drosophila brain, suggesting a neuroprotective role of polyunsaturated fatty acids against Rot-induced neurotoxicity. The involvement of the dopaminergic system in the improvement of behavioral and biochemical parameters in Drosophila fed with WO is also suggested.

Synaptic connectome of a neurosecretory network in the Drosophila brain.

Hormones mediate inter-organ signaling which is crucial in orchestrating diverse behaviors and physiological processes including sleep and activity, feeding, growth, metabolism and reproduction. The pars intercerebralis and pars lateralis in insects represent major hubs which contain neurosecretory cells (NSC) that produce various hormones. To obtain insight into how hormonal signaling is regulated, we have characterized the synaptic connectome of NSC in the adult Drosophila brain. Identification of neurons providing inputs to multiple NSC subtypes implicates diuretic hormone 44-expressing NSC as a major coordinator of physiology and behavior. Surprisingly, despite most NSC having dendrites in the subesophageal zone (primary taste processing center), gustatory inputs to NSC are largely indirect. We also deciphered pathways via which diverse olfactory inputs are relayed to NSC. Further, our analyses revealed substantial inputs from descending neurons to NSC, suggesting that descending neurons regulate both endocrine and motor output to synchronize physiological changes with appropriate behaviors. In contrast to NSC inputs, synaptic output from NSC is sparse and mostly mediated by corazonin NSC. Therefore, we additionally determine putative paracrine interconnectivity between NSC subtypes and hormonal pathways from NSC to peripheral tissues by analyzing single-cell transcriptomic datasets. Our comprehensive characterization of the Drosophila neurosecretory network connectome provides a platform to understand complex hormonal networks and how they orchestrate animal behaviors and physiology.

Transcriptional repression and enhancer decommissioning silence cell cycle genes in postmitotic tissues.

The mechanisms that maintain a non-cycling status in postmitotic tissues are not well understood. Many cell cycle genes have promoters and enhancers that remain accessible even when cells are terminally differentiated and in a non-cycling state, suggesting their repression must be maintained long term. In contrast, enhancer decommissioning has been observed for rate-limiting cell cycle genes in the Drosophila wing, a tissue where the cells die soon after eclosion, but it has been unclear if this also occurs in other contexts of terminal differentiation. In this study, we show that enhancer decommissioning also occurs at specific, rate-limiting cell cycle genes in the long-lived tissues of the Drosophila eye and brain, and we propose this loss of chromatin accessibility may help maintain a robust postmitotic state. We examined the decommissioned enhancers at specific rate-limiting cell cycle genes and showed that they encode for dynamic temporal and spatial expression patterns that include shared, as well as tissue-specific elements, resulting in broad gene expression with developmentally controlled temporal regulation. We extend our analysis to cell cycle gene expression and chromatin accessibility in the mammalian retina using a published dataset and find that the principles of cell cycle gene regulation identified in terminally differentiating Drosophila tissues are conserved in the differentiating mammalian retina. We propose a robust, non-cycling status is maintained in long-lived postmitotic tissues through a combination of stable repression at most cell cycle genes, alongside enhancer decommissioning at specific rate-limiting cell cycle genes.

A LAT1-Like Amino Acid Transporter Regulates Neuronal Activity in the Drosophila Mushroom Bodies.

The proper functioning of neural circuits that integrate sensory signals is essential for individual adaptation to an ever-changing environment. Many molecules can modulate neuronal activity, including neurotransmitters, receptors, and even amino acids. Here, we ask whether amino acid transporters expressed by neurons can influence neuronal activity. We found that minidiscs (mnd), which encodes a light chain of a heterodimeric amino acid transporter, is expressed in different cell types of the adult Drosophila brain: in mushroom body neurons (MBs) and in glial cells. Using live calcium imaging, we found that MND expressed in α/β MB neurons is essential for sensitivity to the L-amino acids: Leu, Ile, Asp, Glu, Lys, Thr, and Arg. We found that the Target Of Rapamycin (TOR) pathway but not the Glutamate Dehydrogenase (GDH) pathway is involved in the Leucine-dependent response of α/β MB neurons. This study strongly supports the key role of MND in regulating MB activity in response to amino acids.

Exploring the Asymmetric Body's Influence on Interval Timing Behaviors of Drosophila melanogaster.

The roles of brain asymmetry in Drosophila are diverse, encompassing the regulation of behavior, the creation of memory, neurodevelopment, and evolution. A comprehensive examination of the Drosophila brain has the potential to enhance our understanding of the functional significance of brain asymmetry in cognitive and behavioral processes, as well as its role in evolutionary perspectives. This study explores the influence of brain asymmetry on interval timing behaviors in Drosophila, with a specific focus on the asymmetric body (AB) structure. Despite being bilaterally symmetric, the AB exhibits functional asymmetry and is located within the central complex of the fly brain. Interval timing behaviors, such as rival-induced prolonged mating duration: longer mating duration behavior (LMD) and sexual experience-mediated shorter mating duration behavior (SMD), are essential for Drosophila. We utilize genetic manipulations to selectively activate or inhibit AB neurons and evaluates their impact on LMD and SMD behaviors. The results indicate that specific populations of AB neurons play unique roles in orchestrating these interval timing behaviors. Notably, inhibiting GAL4R38D01-labeled AB neurons disrupts both LMD and SMD, while GAL4R42C09 neuron inhibition affects only LMD. Moreover, hyperexcitation of GAL4R72A10-labeled AB neurons perturbs SMD. Our study identifies NetrinB (NetB) and Abdominal-B (Abd-B) are important genes for AB neurons in LMD and highlights the role of 5-HT1B neurons in generating LMD through peptidergic Pigment-dispersing factor (PDF) signaling. In summary, this study underscores the importance of AB neuron asymmetry in mediating interval timing behaviors and provides insights into the underlying mechanisms of memory formation and function in Drosophila.

Translational regulation enhances distinction of cell types in the nervous system.

Multicellular organisms are composed of specialized cell types with distinct proteomes. While recent advances in single-cell transcriptome analyses have revealed differential expression of mRNAs, cellular diversity in translational profiles remains underinvestigated. By performing RNA-seq and Ribo-seq in genetically defined cells in the Drosophila brain, we here revealed substantial post-transcriptional regulations that augment the cell-type distinctions at the level of protein expression. Specifically, we found that translational efficiency of proteins fundamental to neuronal functions, such as ion channels and neurotransmitter receptors, was maintained low in glia, leading to their preferential translation in neurons. Notably, distribution of ribosome footprints on these mRNAs exhibited a remarkable bias toward the 5' leaders in glia. Using transgenic reporter strains, we provide evidence that the small upstream open-reading frames in the 5' leader confer selective translational suppression in glia. Overall, these findings underscore the profound impact of translational regulation in shaping the proteomics for cell-type distinction and provide new insights into the molecular mechanisms driving cell-type diversity.

Translational regulation enhances distinction of cell types in the nervous system

Multicellular organisms are composed of specialized cell types with distinct proteomes. While recent advances in single-cell transcriptome analyses have revealed differential expression of mRNAs, cellular diversity in translational profiles remains underinvestigated. By performing RNA-seq and Ribo-seq in genetically defined cells in the Drosophila brain, we here revealed substantial post-transcriptional regulations that augment the cell-type distinctions at the level of protein expression. Specifically, we found that translational efficiency of proteins fundamental to neuronal functions, such as ion channels and neurotransmitter receptors, was maintained low in glia, leading to their preferential translation in neurons. Notably, distribution of ribosome footprints on these mRNAs exhibited a remarkable bias toward the 5′ leaders in glia. Using transgenic reporter strains, we provide evidence that the small upstream open-reading frames in the 5’ leader confer selective translational suppression in glia. Overall, these findings underscore the profound impact of translational regulation in shaping the proteomics for cell-type distinction and provide new insights into the molecular mechanisms driving cell-type diversity.

Molecular and cellular mechanisms of teneurin signaling in synaptic partner matching

In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionarily conserved teneurins are transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurins' functions is unclear. Here, we use in situ proximity labeling to obtain the intracellular interactome of a teneurin (Ten-m) in the Drosophila brain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced our understanding of both cellular and molecular mechanisms of synaptic partner matching.

Changes in mitochondrial distribution occur at the axon initial segment in association with neurodegeneration in Drosophila.

Changes in mitochondrial distribution are a feature of numerous age-related neurodegenerative diseases. In Drosophila, reducing the activity of Cdk5 causes a neurodegenerative phenotype and is known to affect several mitochondrial properties. Therefore, we investigated whether alterations of mitochondrial distribution are involved in Cdk5-associated neurodegeneration. We find that reducing Cdk5 activity does not alter the balance of mitochondrial localization to the somatodendritic versus axonal neuronal compartments of the mushroom body, the learning and memory center of the Drosophila brain. We do, however, observe changes in mitochondrial distribution at the axon initial segment (AIS), a neuronal compartment located in the proximal axon involved in neuronal polarization and action potential initiation. Specifically, we observe that mitochondria are partially excluded from the AIS in wild-type neurons, but that this exclusion is lost upon reduction of Cdk5 activity, concomitant with the shrinkage of the AIS domain that is known to occur in this condition. This mitochondrial redistribution into the AIS is not likely due to the shortening of the AIS domain itself but rather due to altered Cdk5 activity. Furthermore, mitochondrial redistribution into the AIS is unlikely to be an early driver of neurodegeneration in the context of reduced Cdk5 activity.

3D‐Printed Carbon Nanoneedle Electrodes for Dopamine Detection in Drosophila

AbstractIn vivo electrochemistry in small brain regions or synapses requires nanoelectrodes with long straight tips for submicron scale measurements. Nanoelectrodes can be fabricated using a Nanoscribe two‐photon printer, but annealed tips curl if they are long and thin. We propose a new pulling‐force strategy to fabricate a straight carbon nanoneedle structure. A micron‐width bridge is printed between two blocks. The annealed structure shrinks during pyrolysis, and the blocks create a pulling force to form a long, thin, and straight carbon bridge. Parameterization study and COMSOL modeling indicate changes in the block size, bridge size and length affect the pulling force and bridge shrinkage. Electrodes were printed on niobium wires, insulated with aluminum oxide, and the bridge cut with focused ion beam (FIB) to expose the nanoneedle tip. Annealed needle diameters ranged from 400 nm to 5.25 μm and length varied from 50.5 μm to 146 μm. The electrochemical properties are similar to glassy carbon, with good performance for dopamine detection with fast‐scan cyclic voltammetry. Nanoelectrodes enable biological applications, such as dopamine detection in a specific Drosophila brain region. Long and thin nanoneedles are generally useful for other applications such as cellular sensing, drug delivery, or gas sensing.

The astrocyte-enriched gene deathstar plays a crucial role in the development, locomotion, and lifespan of D. melanogaster

ABSTRACT The Drosophila melanogaster brain is a complex organ with various cell types, orchestrating the development, physiology, and behaviors of the fly. While each cell type in Drosophila brain is known to express a unique gene set, their complete genetic profile is still unknown. Advances in the RNA sequencing techniques at single-cell resolution facilitate identifying novel cell type markers and/or re-examining the specificity of the available ones. In this study, exploiting a single-cell RNA sequencing data of Drosophila optic lobe, we categorized the cells based on their expression pattern for known markers, then the genes with enriched expression in astrocytes were identified. CG11000 was identified as a gene with a comparable expression profile to the Eaat1 gene, an astrocyte marker, in every individual cell inside the Drosophila optic lobe and midbrain, as well as in the entire Drosophila brain throughout its development. Consistent with our bioinformatics data, immunostaining of the brains dissected from transgenic adult flies showed co-expression of CG11000 with Eaat1 in a set of single cells corresponding to the astrocytes in the Drosophila brain. Physiologically, inhibiting CG11000 through RNA interference disrupted the normal development of male D. melanogaster, while having no impact on females. Expression suppression of CG11000 in adult flies led to decreased locomotion activity and also shortened lifespan specifically in astrocytes, indicating the gene’s significance in astrocytes. We designated this gene as ‘deathstar’ due to its crucial role in maintaining the star-like shape of glial cells, astrocytes, throughout their development into adult stage.

Fruit ripening retardant Daminozide induces cognitive impairment, cell specific neurotoxicity, and genotoxicity in Drosophila melanogaster

BackgroundWe explored neurotoxic and genotoxic effects of Daminozide, a fruit ripening retardant, on the brain of Drosophila melanogaster, based on our previous finding of DNA fragmentation in larval brain cell in the flies experimentally exposed to this chemicals. MethodsAdult flies were subjected to two distinct concentrations of daminozide (200 mg/L and 400 mg/L) mixed in culture medium, followed by an examination of specific behaviors such as courtship conditioning and aversive phototaxis, which serve as indicators of cognitive functions. We investigated brain histology and histochemistry to assess the overall toxicity of daminozide, focusing on neuron type-specific effects. Additionally, we conducted studies on gene expression specific to neuronal function. Statistical comparisons were then made between the exposed and control flies across all tested attributes. ResultsThe outcome of behavioral assays suggested deleterious effects of Daminozide on learning, short term and long term memory function. Histological examination of brain sections revealed cellular degeneration, within Kenyon cell neuropiles in Daminozide-exposed flies. Neurone specific Immuno-histochemistry study revealed significant reduction of dopaminergic and glutaminergic neurones with discernible reduction in cellular counts, alteration in cell and nuclear morphology among daminozide exposed flies. Gene expression analyses demonstrated upregulation of rutabaga (rut), hb9 and down regulation of PKa- C1, CrebB, Ace and nAchRbeta-1 in exposed flies which suggest dysregulation of gene functions involved in motor neuron activity, learning, and memory. ConclusionTaken together, our findings suggests that Daminozide induces multifaceted harmful impacts on the neural terrain of Drosophila melanogaster, posing a threat to its cognitive abilities.

Hue selectivity from recurrent circuitry in Drosophila

In the perception of color, wavelengths of light reflected off objects are transformed into the derived quantities of brightness, saturation and hue. Neurons responding selectively to hue have been reported in primate cortex, but it is unknown how their narrow tuning in color space is produced by upstream circuit mechanisms. We report the discovery of neurons in the Drosophila optic lobe with hue-selective properties, which enables circuit-level analysis of color processing. From our analysis of an electron microscopy volume of a whole Drosophila brain, we construct a connectomics-constrained circuit model that accounts for this hue selectivity. Our model predicts that recurrent connections in the circuit are critical for generating hue selectivity. Experiments using genetic manipulations to perturb recurrence in adult flies confirm this prediction. Our findings reveal a circuit basis for hue selectivity in color vision.

Synapse-Specific Trapping of SNARE Machinery Proteins in the Anesthetized Drosophila Brain.

General anesthetics disrupt brain network dynamics through multiple pathways, in part through postsynaptic potentiation of inhibitory ion channels as well as presynaptic inhibition of neuroexocytosis. Common clinical general anesthetic drugs, such as propofol and isoflurane, have been shown to interact and interfere with core components of the exocytic release machinery to cause impaired neurotransmitter release. Recent studies however suggest that these drugs do not affect all synapse subtypes equally. We investigated the role of the presynaptic release machinery in multiple neurotransmitter systems under isoflurane general anesthesia in the adult female Drosophila brain using live-cell super-resolution microscopy and optogenetic readouts of exocytosis and neural excitability. We activated neurotransmitter-specific mushroom body output neurons and imaged presynaptic function under isoflurane anesthesia. We found that isoflurane impaired synaptic release and presynaptic protein dynamics in excitatory cholinergic synapses. In contrast, isoflurane had little to no effect on inhibitory GABAergic or glutamatergic synapses. These results present a distinct inhibitory mechanism for general anesthesia, whereby neuroexocytosis is selectively impaired at excitatory synapses, while inhibitory synapses remain functional. This suggests a presynaptic inhibitory mechanism that complements the other inhibitory effects of these drugs.