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Improved methods for marking active neuron populations.

ABSTRACT: Marking functionally distinct neuronal ensembles with high spatiotemporal resolution is a key challenge in systems neuroscience. We recently introduced CaMPARI, an engineered fluorescent protein whose green-to-red photoconversion depends on simultaneous light exposure and elevated calcium, which enabled marking active neuronal populations with single-cell and subsecond resolution. However, CaMPARI (CaMPARI1) has several drawbacks, including background photoconversion in low calcium, slow kinetics and reduced fluorescence after chemical fixation. In this work, we develop CaMPARI2, an improved sensor with brighter green and red fluorescence, faster calcium unbinding kinetics and decreased photoconversion in low calcium conditions. We demonstrate the improved performance of CaMPARI2 in mammalian neurons and in vivo in larval zebrafish brain and mouse visual cortex. Additionally, we herein develop an immunohistochemical detection method for specific labeling of the photoconverted red form of CaMPARI. The anti-CaMPARI-red antibody provides strong labeling that is selective for photoconverted CaMPARI in activated neurons in rodent brain tissue.

SUBMITTER: Moeyaert B

PROVIDER: S-EPMC6202339 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Improved methods for marking active neuron populations.

Moeyaert Benjamien B Holt Graham G Madangopal Rajtarun R Perez-Alvarez Alberto A Fearey Brenna C BC Trojanowski Nicholas F NF Ledderose Julia J Zolnik Timothy A TA Das Aniruddha A Patel Davina D Brown Timothy A TA Sachdev Robert N S RNS Eickholt Britta J BJ Larkum Matthew E ME Turrigiano Gina G GG Dana Hod H Gee Christine E CE Oertner Thomas G TG Hope Bruce T BT Schreiter Eric R ER

Nature communications 20181025 1

Marking functionally distinct neuronal ensembles with high spatiotemporal resolution is a key challenge in systems neuroscience. We recently introduced CaMPARI, an engineered fluorescent protein whose green-to-red photoconversion depends on simultaneous light exposure and elevated calcium, which enabled marking active neuronal populations with single-cell and subsecond resolution. However, CaMPARI (CaMPARI1) has several drawbacks, including background photoconversion in low calcium, slow kinetic ...[more]

PMID: 30361563

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Project description:BACKGROUND:Understanding the dispersal dynamics of invasive mosquito species is fundamental to improve vector surveillance and to target control efforts. Aedes albopictus has been deemed a poor flyer as its range of active dispersal is generally assumed to be limited to a few hundred metres, while laboratory studies suggest this mosquito could actually fly several kilometres. The discrepancy may be due to differences in the local environment or to the methodological approach. In Switzerland, Ae. albopictus has been present since 2003 and has since then expanded its range. While passive dispersal is a key driver, it remains unclear how far this mosquito spreads through active flight and what the age structure and size of the local population are, all important parameters for vector surveillance and control. METHOD:We investigated the active dispersal, daily survival rate and population size of Ae. albopictus in mark-release-recapture studies in Coldrerio and Lugano, two areas of intensive control in Switzerland. To mark mosquitoes emerging from breeding sites, we used self-marking units with fluorescent pigment that have minimal impact on mosquito survival and behaviour. We recaptured the adult mosquitoes with BG-Sentinel traps within a radius of 1 km from the marking units over 22 consecutive days. RESULTS:We found that 77.5% of the recaptured Ae. albopictus individuals flew further than 250 m, the limit that is usually deemed sufficient for vector containment. The average age of females and males was 8.6 days and 7.8 days in Coldrerio and Lugano, respectively, while the estimated mosquito population densities were 134 mosquitoes/ha in Coldrerio and 767 mosquitoes/ha in Lugano. CONCLUSIONS:Self-marking units are an effective tool to mark wild mosquitoes. Using this approach, we found that mosquitoes survive long enough to potentially transmit arboviral disease in our study area and that host-seeking Ae. albopictus females may travel further than previously assumed for European mosquito populations. This finding has direct implications for vector control as emergency treatments around positive cases, as well as surveillance and control around detections of new infestations, might need to be extended beyond the usual recommended range of just a few hundred metres.

Project description:Sleep-active neurons depolarize during sleep to suppress wakefulness circuits. Wake-active wake-promoting neurons in turn shut down sleep-active neurons, thus forming a bipartite flip-flop switch. However, how sleep is switched on is unclear because it is not known how wakefulness is translated into sleep-active neuron depolarization when the system is set to sleep. Using optogenetics in Caenorhabditis elegans, we solved the presynaptic circuit for depolarization of the sleep-active RIS neuron during developmentally regulated sleep, also known as lethargus. Surprisingly, we found that RIS activation requires neurons that have known roles in wakefulness and locomotion behavior. The RIM interneurons-which are active during and can induce reverse locomotion-play a complex role and can act as inhibitors of RIS when they are strongly depolarized and as activators of RIS when they are modestly depolarized. The PVC command interneurons, which are known to promote forward locomotion during wakefulness, act as major activators of RIS. The properties of these locomotion neurons are modulated during lethargus. The RIMs become less excitable. The PVCs become resistant to inhibition and have an increased capacity to activate RIS. Separate activation of neither the PVCs nor the RIMs appears to be sufficient for sleep induction; instead, our data suggest that they act in concert to activate RIS. Forward and reverse circuit activity is normally mutually exclusive. Our data suggest that RIS may be activated at the transition between forward and reverse locomotion states, perhaps when both forward (PVC) and reverse (including RIM) circuit activity overlap. While RIS is not strongly activated outside of lethargus, altered activity of the locomotion interneurons during lethargus favors strong RIS activation and thus sleep. The control of sleep-active neurons by locomotion circuits suggests that sleep control may have evolved from locomotion control. The flip-flop sleep switch in C. elegans thus requires an additional component, wake-active sleep-promoting neurons that translate wakefulness into the depolarization of a sleep-active neuron when the worm is sleepy. Wake-active sleep-promoting circuits may also be required for sleep state switching in other animals, including in mammals.

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Improved methods for marking active neuron populations.

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Improved methods for marking active neuron populations.

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