Collapse of Global Neuronal States in Caenorhabditis elegans under Isoflurane Anesthesia.

BACKGROUND: A comprehensive understanding of how anesthetics facilitate a reversible collapse of system-wide neuronal function requires measurement of neuronal activity with single-cell resolution. Multineuron recording was performed in Caenorhabditis elegans to measure neuronal activity at varying depths of anesthesia. The authors hypothesized that anesthesia is characterized by dyssynchrony between neurons resulting in a collapse of organized system states. METHODS: Using light-sheet microscopy and transgenic expression of the calcium-sensitive fluorophore GCaMP6s, a majority of neurons (n = 120) in the C. elegans head were simultaneously imaged in vivo and neuronal activity was measured. Neural activity and system-wide dynamics were compared in 10 animals, progressively dosed at 0%, 4%, and 8% isoflurane. System-wide neuronal activity was analyzed using principal component analysis. RESULTS: Unanesthetized animals display distinct global neuronal states that are reflected in a high degree of correlation (R = 0.196 ± 0.070) between neurons and low-frequency, large-amplitude neuronal dynamics. At 4% isoflurane, the average correlation between neurons is significantly diminished (R = 0.026 ± 0.010; P < 0.0001 vs. unanesthetized) and neuron dynamics shift toward higher frequencies but with smaller dynamic range. At 8% isoflurane, interneuronal correlations indicate that neuronal activity remains uncoordinated (R = 0.053 ± 0.029; P < 0.0001 vs. unanesthetized) with high-frequency dynamics that are even further restricted. Principal component analysis of unanesthetized neuronal activity reveals distinct structure corresponding to known behavioral states. At 4% and 8% isoflurane this structure is lost and replaced with randomized dynamics, as quantified by the percentage of total ensemble variance captured by the first three principal components. In unanesthetized worms, this captured variance is high (88.9 ± 5.4%), reflecting a highly organized system, falling significantly at 4% and 8% isoflurane (57.9 ± 11.2%, P < 0.0001 vs. unanesthetized, and 76.0 ± 7.9%, P < 0.001 vs. unanesthetized, respectively) and corresponding to increased randomization and collapse of system-wide organization. CONCLUSIONS: Anesthesia with isoflurane in C. elegans corresponds to high-frequency randomization of individual neuron activity, loss of coordination between neurons, and a collapse of system-wide functional organization.

Video-rate large-scale imaging with Multi-Z confocal microscopy.

Fast, volumetric imaging over large scales has been a long-standing challenge in biological microscopy. To address this challenge, we report an augmented variant of confocal microscopy that uses a series of reflecting pinholes axially distributed in the detection space, such that each pinhole probes a different depth within the sample. We thus obtain simultaneous multiplane imaging without the need for axial scanning. Our microscope technique is versatile and configured here to provide two-color fluorescence imaging with a field of view larger than a millimeter at video rate. Its general applicability is demonstrated with neuronal imaging of both Caenorhabditis elegans and mouse brains in vivo.

O-GlcNAc Signaling Orchestrates the Regenerative Response to Neuronal Injury in Caenorhabditis elegans.

Regrowth of an axon after injury is an inherently metabolic undertaking. Yet the mechanisms of metabolic regulation that influence repair following injury are not well understood. O-linked ß-N-acetylglucosamine (O-GlcNAc) is a post-translational modification of serines and threonines that functions as a sensor of cellular nutrients. Performing in vivo laser axotomies in Caenorhabditis elegans, we find that neuronal regeneration is substantially increased by disruptions of either the O-GlcNAc transferase or the O-GlcNAcase that decrease and increase O-GlcNAc levels, respectively. A lack of O-GlcNAc induces the AKT-1 branch in the insulin-signaling pathway to use glycolysis. In contrast, increased O-GlcNAc levels activate an opposing branch of the insulin-signaling pathway whereby SGK-1 modulates the FOXO transcription factor DAF-16 to influence mitochondrial function. The existence of this toggle-like mechanism between metabolic pathways suggests that O-GlcNAc signaling conveys cellular nutrient status to orchestrate metabolism in a damaged neuron and maximize the regenerative response.

Breakdown of Neural Function under Isoflurane Anesthesia: In Vivo, Multineuronal Imaging in Caenorhabditis elegans.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Previous work on the action of volatile anesthetics has focused at either the molecular level or bulk neuronal measurement such as electroencephalography or functional magnetic resonance imaging. There is a distinct gulf in resolution at the level of cellular signaling within neuronal systems. The authors hypothesize that anesthesia is caused by induced dyssynchrony in cellular signaling rather than suppression of individual neuron activity. METHODS: Employing confocal microscopy and Caenorhabditis elegans expressing the calcium-sensitive fluorophore GCaMP6s in specific command neurons, the authors measure neuronal activity noninvasively and in parallel within the behavioral circuit controlling forward and reverse crawling. The authors compare neuronal dynamics and coordination in a total of 31 animals under atmospheres of 0, 4, and 8% isoflurane. RESULTS: When not anesthetized, the interneurons controlling forward or reverse crawling occupy two possible states, with the activity of the "reversal" neurons AVA, AVD, AVE, and RIM strongly intercorrelated, and the "forward" neuron AVB anticorrelated. With exposure to 4% isoflurane and onset of physical quiescence, neuron activity wanders rapidly and erratically through indeterminate states. Neuron dynamics shift toward higher frequencies, and neuron pair correlations within the system are reduced. At 8% isoflurane, physical quiescence continues as neuronal signals show diminished amplitude with little correlation between neurons. Neuronal activity was further studied using statistical tools from information theory to quantify the type of disruption caused by isoflurane. Neuronal signals become noisier and more disordered, as measured by an increase in the randomness of their activity (Shannon entropy). The coordination of the system, measured by whether information exhibited in one neuron is also exhibited in other neurons (multiinformation), decreases significantly at 4% isoflurane (P = 0.00015) and 8% isoflurane (P = 0.0028). CONCLUSIONS: The onset of anesthesia corresponds with high-frequency randomization of individual neuron activity coupled with induced dyssynchrony and loss of coordination between neurons that disrupts functional signaling.

Novel DLK-independent neuronal regeneration in Caenorhabditis elegans shares links with activity-dependent ectopic outgrowth.

During development, a neuron transitions from a state of rapid growth to a stable morphology, and neurons within the adult mammalian CNS lose their ability to effectively regenerate in response to injury. Here, we identify a novel form of neuronal regeneration, which is remarkably independent of DLK-1/DLK, KGB-1/JNK, and other MAPK signaling factors known to mediate regeneration in Caenorhabditis elegans, Drosophila, and mammals. This DLK-independent regeneration in C. elegans has direct genetic and molecular links to a well-studied form of endogenous activity-dependent ectopic axon outgrowth in the same neuron type. Both neuron outgrowth types are triggered by physical lesion of the sensory dendrite or mutations disrupting sensory activity, calcium signaling, or genes that restrict outgrowth during neuronal maturation, such as SAX-1/NDR kinase or UNC-43/CaMKII. These connections suggest that ectopic outgrowth represents a powerful platform for gene discovery in neuronal regeneration. Moreover, we note numerous similarities between C. elegans DLK-independent regeneration and lesion conditioning, a phenomenon producing robust regeneration in the mammalian CNS. Both regeneration types are triggered by lesion of a sensory neurite via reduction of neuronal activity and enhanced by disrupting L-type calcium channels or elevating cAMP. Taken as a whole, our study unites disparate forms of neuronal outgrowth to uncover fresh molecular insights into activity-dependent control of the adult nervous system's intrinsic regenerative capacity.

Pharmacological chaperones stabilize retromer to limit APP processing.

Retromer is a multiprotein complex that trafficks cargo out of endosomes. The neuronal retromer traffics the amyloid-precursor protein (APP) away from endosomes, a site where APP is cleaved into pathogenic fragments in Alzheimer's disease. Here we determined whether pharmacological chaperones can enhance retromer stability and function. First, we relied on the crystal structures of retromer proteins to help identify the 'weak link' of the complex and to complete an in silico screen of small molecules predicted to enhance retromer stability. Among the hits, an in vitro assay identified one molecule that stabilized retromer against thermal denaturation. Second, we turned to cultured hippocampal neurons, showing that this small molecule increases the levels of retromer proteins, shifts APP away from the endosome, and decreases the pathogenic processing of APP. These findings show that pharmacological chaperones can enhance the function of a multiprotein complex and may have potential therapeutic implications for neurodegenerative diseases.