scholarly journals The BioLuminescent-OptoGenetic in vivo Response to Coelenterazine is Proportional, Sensitive and Specific in Neocortex

2019 ◽  
Author(s):  
Manuel Gomez-Ramirez ◽  
Alexander I. More ◽  
Nina G. Friedman ◽  
Ute Hochgeschwender ◽  
Christopher I. Moore
Keyword(s):  
Neural Activity ◽  
Bioluminescence Imaging ◽  
Photon Emission ◽  
Gaussia Luciferase ◽  
Light Production ◽  
Electrophysiological Recordings ◽  
Dynamic Concentration ◽  
And Behavior ◽  
The Relationship

ABSTRACTBioLuminescent (BL) light production can modulate neural activity and behavior through coexpressed OptoGenetic (OG) elements, an approach termed ‘BL-OG’. Yet, the relationship between BL-OG effects and bioluminescent photon emission has not been characterized in vivo. Further, the degree to which BL-OG effects strictly depend on optogenetic mechanisms driven by bioluminescent photons is unknown. Crucial to every neuromodulation method is whether the activator shows a dynamic concentration range driving robust, selective, and non-toxic effects. We systematically tested the effects of four key components of the BL-OG mechanism (luciferin, oxidized luciferin, luciferin vehicle, and bioluminescence), and compared these against effects induced by the Luminopsin-3 (LMO3) BL-OG molecule, a fusion of slow burn Gaussia luciferase (sbGLuc) and Volvox ChannelRhodopsin-1 (VChR1). We performed combined bioluminescence imaging and electrophysiological recordings while injecting specific doses of Coelenterazine (substrate for sbGluc), Coelenteramide (CTM, the oxidized product of CTZ), or CTZ vehicle. CTZ robustly drove activity in mice expressing LMO3, with photon production proportional to firing rate. In contrast, low and moderate doses of CTZ, CTM, or vehicle did not modulate activity in mice that did not express LMO3. We also failed to find bioluminescence effects on neural activity in mice expressing an optogenetically non-sensitive LMO3 variant. We observed weak responses to the highest dose of CTZ in control mice, but these effects were significantly smaller than those observed in the LMO3 group. These results show that in neocortex in vivo, there is a large CTZ range wherein BL-OG effects are specific to its active chemogenetic mechanism.

scholarly journals Enhancing GABAergic Tone in the Rostral Nucleus of the Solitary Tract Reconfigures Sensorimotor Neural Activity

2020 ◽  
Author(s):  
Joshua D. Sammons ◽  
Caroline E. Bass ◽  
Jonathan D. Victor ◽  
Patricia M. Di Lorenzo
Keyword(s):  
Neural Activity ◽  
Inhibitory Activity ◽  
Nucleus Tractus Solitarius ◽  
Artificial Saliva ◽  
Experimental Chamber ◽  
Response Patterns ◽  
Related Activity ◽  
Electrophysiological Recordings ◽  
Rostral Nucleus ◽  
The Relationship

ABSTRACTRecent work has shown that most cells in the rostral, gustatory portion of the nucleus tractus solitarius (rNTS) in awake, freely licking rats show lick-related firing. However, the relationship between taste-related and lick-related activity in rNTS remains unclear. Here, we tested if GABA-derived inhibitory activity regulates the balance of lick- and taste-driven neuronal activity. Combinatorial viral tools were used to restrict expression of ChR2-EYFP to GAD1+ GABAergic neurons. Viral infusions were bilateral in rNTS. 2-4wks later, an optical fiber attached to 8-16 drivable microwires was implanted into the rNTS. After recovery, water-deprived rats were presented with taste stimuli in an experimental chamber. Trials were 5 consecutive taste licks [NaCl, KCl, NH4Cl, sucrose, MSG/IMP, citric acid, quinine, or artificial saliva (AS)] separated by 5 AS licks on a VR5 schedule. Each taste lick triggered a 1s train of laser light (25Hz; 473nm; 8-10mW) in a random half of the trials. In all, 113 cells were recorded in the rNTS, 50 responded to one or more taste stimuli without GABA enhancement. Selective changes in response magnitude (spike count) within cells shifted across unit patterns but preserved inter-stimulus relationships. Cells where enhanced GABAergic tone increased lick coherence conveyed more information distinguishing basic taste qualities and different salts than other cells. In addition, GABA activation significantly amplified the amount of information that discriminated palatable vs. unpalatable tastants. By dynamically regulating lick coherence and remodeling the across-unit response patterns to taste, enhancing GABAergic tone in rNTS reconfigures the neural activity reflecting sensation and movement.Significance StatementThe rostral nucleus tractus solitarius (rNTS) is the first structure in the central gustatory pathway. Electrophysiological recordings from the rNTS in awake, freely-licking animals show that cells in this area have lick- as well as taste-related activity, but the relationship between these characteristics is not well understood. Here, we showed evidence that GABA activation can dynamically regulate both of these two properties in rNTS cells to enhance the information conveyed, especially about palatable vs. unpalatable tastants. These data provide insights into the role of inhibitory activity in the rNTS.

Ex Vivo Brain Preparation to Analyze Vocal Pathways of Xenopus Frogs

2021 ◽  
Vol 2021 (9) ◽  
pp. pdb.prot106872
Author(s):  
Ayako Yamaguchi
Keyword(s):  
Neural Activity ◽  
Ex Vivo ◽  
Neural Circuitry ◽  
Neural Basis ◽  
Vocal Production ◽  
Basic Principles ◽  
Electrophysiological Recordings ◽  
The Brain

Understanding the neural basis of behavior is a challenging task for technical reasons. Most methods of recording neural activity require animals to be immobilized, but neural activity associated with most behavior cannot be recorded from an anesthetized, immobilized animal. Using amphibians, however, there has been some success in developing in vitro brain preparations that can be used for electrophysiological and anatomical studies. Here, we describe an ex vivo frog brain preparation from which fictive vocalizations (the neural activity that would have produced vocalizations had the brain been attached to the muscle) can be elicited repeatedly. When serotonin is applied to the isolated brains of male and female African clawed frogs, Xenopus laevis, laryngeal nerve activity that is a facsimile of those that underlie sex-specific vocalizations in vivo can be readily recorded. Recently, this preparation was successfully used in other species within the genus including Xenopus tropicalis and Xenopus victorianus. This preparation allows a variety of techniques to be applied including extracellular and intracellular electrophysiological recordings and calcium imaging during vocal production, surgical and pharmacological manipulation of neurons to evaluate their impact on motor output, and tract tracing of the neural circuitry. Thus, the preparation is a powerful tool with which to understand the basic principles that govern the production of coherent and robust motor programs in vertebrates.

scholarly journals Rapid Association Learning in the Primate Prefrontal Cortex in the Absence of Behavioral Reversals

2011 ◽  
Vol 23 (7) ◽  
pp. 1823-1828 ◽  
Author(s):  
Jason A. Cromer ◽  
Michelle Machon ◽  
Earl K. Miller
Keyword(s):  
Prefrontal Cortex ◽  
Neural Activity ◽  
Association Learning ◽  
Conditional Association ◽  
And Behavior ◽  
The Relationship ◽  
Behavioral Improvement ◽  
Previous Learning

The PFC plays a central role in our ability to learn arbitrary rules, such as “green means go.” Previous experiments from our laboratory have used conditional association learning to show that slow, gradual changes in PFC neural activity mirror monkeys' slow acquisition of associations. These previous experiments required monkeys to repeatedly reverse the cue–saccade associations, an ability known to be PFC-dependent. We aimed to test whether the relationship between PFC neural activity and behavior was due to the reversal requirement, so monkeys were trained to learn several new conditional cue–saccade associations without reversing them. Learning-related changes in PFC activity now appeared earlier and more suddenly in correspondence with similar changes in behavioral improvement. This suggests that learning of conditional associations is linked to PFC activity regardless of whether reversals are required. However, when previous learning does not need to be suppressed, PFC acquires associations more rapidly.

scholarly journals A calibrated optogenetic toolbox of stable zebrafish opsin lines

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Paride Antinucci ◽  
Adna Dumitrescu ◽  
Charlotte Deleuze ◽  
Holly J Morley ◽  
Kristie Leung ◽  
...  
Keyword(s):  
High Throughput ◽  
Neural Activity ◽  
Ion Selectivity ◽  
Transgenic Zebrafish ◽  
Substantial Variation ◽  
Opsin Expression ◽  
Electrophysiological Recordings ◽  
Subcellular Resolution ◽  
Kinetics Of

Optogenetic actuators with diverse spectral tuning, ion selectivity and kinetics are constantly being engineered providing powerful tools for controlling neural activity with subcellular resolution and millisecond precision. Achieving reliable and interpretable in vivo optogenetic manipulations requires reproducible actuator expression and calibration of photocurrents in target neurons. Here, we developed nine transgenic zebrafish lines for stable opsin expression and calibrated their efficacy in vivo. We first used high-throughput behavioural assays to compare opsin ability to elicit or silence neural activity. Next, we performed in vivo whole-cell electrophysiological recordings to quantify the amplitude and kinetics of photocurrents and test opsin ability to precisely control spiking. We observed substantial variation in efficacy, associated with differences in both opsin expression level and photocurrent characteristics, and identified conditions for optimal use of the most efficient opsins. Overall, our calibrated optogenetic toolkit will facilitate the design of controlled optogenetic circuit manipulations.

scholarly journals Evaluating Brightness and Spectral Properties of Click Beetle and Firefly Luciferases Using Luciferin Analogues: Identification of Preferred Pairings of Luciferase and Substrate for In Vivo Bioluminescence Imaging

2020 ◽  
Vol 22 (6) ◽  
pp. 1523-1531
Author(s):  
Giorgia Zambito ◽  
Natasa Gaspar ◽  
Yanto Ridwan ◽  
Mary P. Hall ◽  
Ce Shi ◽  
...  
Keyword(s):  
Near Infrared ◽  
Bioluminescence Imaging ◽  
Spectral Characteristics ◽  
Photon Emission ◽  
Ccd Camera ◽  
Spectral Unmixing ◽  
Tissue Imaging ◽  
Click Beetle

Abstract Purpose Currently, a variety of red and green beetle luciferase variants are available for bioluminescence imaging (BLI). In addition, new luciferin analogues providing longer wavelength luminescence have been developed that show promise for improved deep tissue imaging. However, a detailed assessment of these analogues (e.g., Akalumine-HCl, CycLuc1, and amino naphthyl luciferin (NH2-NpLH2)) combined with state of the art luciferases has not been performed. The aim of this study was to evaluate for the first time the in vivo brightness and spectral characteristics of firefly (Luc2), click beetle green (CBG99), click beetle red 2 (CBR2), and Akaluc luciferases when paired with different d-luciferin (d-LH2) analogues in vivo. Procedures Transduced human embryonic kidney (HEK 293T) cells expressing individual luciferases were analyzed both in vitro and in mice (via subcutaneous injection). Following introduction of the luciferins to cells or animals, the resulting bioluminescence signal and photon emission spectrum were acquired using a sensitive charge-coupled device (CCD) camera equipped with a series of band pass filters and spectral unmixing software. Results Our in vivo analysis resulted in four primary findings: (1) the best substrate for Luc2, CBG99, and CBR2 in terms of signal strength was d-luciferin; (2) the spectra for Luc2 and CBR2 were shifted to a longer wavelength when Akalumine-HCl was the substrate; (3) CBR2 gave the brightest signal with the near-infrared substrate, NH2-NpLH2; and (4) Akaluc was brighter when paired with either CycLuc1 or Akalumine-HCl when paired with d-LH2. Conclusion We believe that the experimental results described here should provide valuable guidance to end users for choosing the correct luciferin/luciferase pairs for a variety of BLI applications.

scholarly journals Bioluminescent Detection of Endotoxin Effects on HIV-1 LTR-driven Transcription in Vivo

2003 ◽  
Vol 51 (6) ◽  
pp. 741-749 ◽  
Author(s):  
Fiona E. Yull ◽  
Wei Han ◽  
E. Duco Jansen ◽  
M. Brett Everhart ◽  
Ruxana T. Sadikot ◽  
...  
Keyword(s):  
Bioluminescence Imaging ◽  
Luciferase Activity ◽  
Ex Vivo ◽  
Photon Emission ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Reporter Mice ◽  
Hiv 1

We investigated the effects of Gram-negative bacterial lipopolysaccharide (LPS) on luciferase expression in transgenic reporter mice in which luciferase expression is driven by the nuclear factor κB (NF-κB)-dependent portion of the human immunodeficiency virus-1 (HIV-1) long terminal repeat (HIV-1 LTR). Using these mice, we dissected the sources of luciferase activity at the organ level by (a) assessing luciferase activity in organ homogenates, (b) bioluminescence imaging in vivo, and (c) bioluminescence imaging of individual organs ex vivo. Luciferin dosage was a critical determinant of the magnitude of photon emission from these reporter mice. Photon emission increased at doses from 0.5–6 mg of luciferin given by intraperitoneal (IP) injection. The differential between basal and LPS-induced bioluminescence was maximal at 3–6 mg of luciferin. Luciferase expression was highly inducible in lungs, liver, spleen, and kidneys after a single IP injection of LPS, as assessed by luciferase activity measurements in organ homogenates. Luciferase activity was also induced in the forebrain by treatment with IP LPS. In contrast, aerosolized LPS produced a response localized to the lungs as assessed by both bioluminescence and ex vivo luciferase assay measurements. These studies demonstrate the utility of luciferase reporter mice for determining organ-specific gene expression in response to local and systemic stimuli.

There Are No Killer Apps but Connecting Neural Activity to Behavior through Computation Is Still a Good Idea

2016 ◽  
Author(s):  
P. Read Montague
Keyword(s):  
Decision Making ◽  
Cognitive Function ◽  
Simple Model ◽  
Behavioral Economics ◽  
Cognitive Science ◽  
Neural Activity ◽  
Mental States ◽  
And Behavior ◽  
The Relationship ◽  
The Brain

The quest to understand the relationship between neural activity and behavior has been ongoing for well over a hundred years. Although research based on the stimulus-and-response approach to behavior, advocated by behaviorists, flourished during the last century, this view does not, by design, account for unobservable variables (e.g., mental states). Putting aside this approach, modern cognitive science, cognitive neuroscience, neuroeconomics, and behavioral economics have sought to explain this connection computationally. One major hurdle lies in the fact that we lack even a simple model of cognitive function. This chapter sketches an application that connects neuromodulator function to decision making and the valuation that underlies it. The nature of this hypothesized connection offers a fruitful platform to understand some of the informational aspects of dopamine function in the brain and how it exposes many different ways of understanding motivated choice.

scholarly journals Proximal and distal modulation of neural activity by spatially confined optogenetic activation with an integrated high-density optoelectrode

2018 ◽  
Vol 120 (1) ◽  
pp. 149-161 ◽  
Author(s):  
Sarah Libbrecht ◽  
Luis Hoffman ◽  
Marleen Welkenhuysen ◽  
Chris Van den Haute ◽  
Veerle Baekelandt ◽  
...  
Keyword(s):  
Neural Activity ◽  
Large Scale ◽  
Heat Dissipation ◽  
Extracellular Recording ◽  
Cell Types ◽  
Cortical Layer ◽  
Neural Firing ◽  
Optical Stimulation ◽  
Electrophysiological Recordings

Optogenetic manipulations are widely used for investigating the contribution of genetically identified cell types to behavior. Simultaneous electrophysiological recordings are less common, although they are critical for characterizing the specific impact of optogenetic manipulations on neural circuits in vivo. This is at least in part because combining photostimulation with large-scale electrophysiological recordings remains technically challenging, which also poses a limitation for performing extracellular identification experiments. Currently available interfaces that guide light of the appropriate wavelength into the brain combined with an electrophysiological modality suffer from various drawbacks such as a bulky size, low spatial resolution, heat dissipation, or photovoltaic artifacts. To address these challenges, we have designed and fabricated an integrated ultrathin neural interface with 12 optical outputs and 24 electrodes. We used the device to measure the effect of localized stimulation in the anterior olfactory cortex, a paleocortical structure involved in olfactory processing. Our experiments in adult mice demonstrate that because of its small dimensions, our novel tool causes far less tissue damage than commercially available devices. Moreover, optical stimulation and recording can be performed simultaneously, with no measurable electrical artifact during optical stimulation. Importantly, optical stimulation can be confined to small volumes with approximately single-cortical layer thickness. Finally, we find that even highly localized optical stimulation causes inhibition at more distant sites. NEW & NOTEWORTHY In this study, we establish a novel tool for simultaneous extracellular recording and optogenetic photostimulation. Because the device is built using established microchip technology, it can be fabricated with high reproducibility and reliability. We further show that even very localized stimulation affects neural firing far beyond the stimulation site. This demonstrates the difficulty in predicting circuit-level effects of optogenetic manipulations and highlights the importance of closely monitoring neural activity in optogenetic experiments.

Bioluminescence imaging of Arc gene expression detects neural activity-dependent changes in vivo

2010 ◽  
Vol 68 ◽  
pp. e445
Author(s):  
Hironori Izumi ◽  
Tetsuya Ishimoto ◽  
Hiroshi Yamamoto ◽  
Hisao Nishijo ◽  
Hisashi Mori
Keyword(s):  
Gene Expression ◽  
Neural Activity ◽  
Bioluminescence Imaging ◽  
Activity Dependent ◽  
Arc Gene

scholarly journals A calibrated optogenetic toolbox of stable zebrafish opsin lines

2020 ◽  
Author(s):  
P Antinucci ◽  
AS Dumitrescu ◽  
C Deleuze ◽  
HJ Morley ◽  
K Leung ◽  
...  
Keyword(s):  
High Throughput ◽  
Neural Activity ◽  
Ion Selectivity ◽  
Transgenic Zebrafish ◽  
Substantial Variation ◽  
Opsin Expression ◽  
Electrophysiological Recordings ◽  
Subcellular Resolution ◽  
Kinetics Of

AbstractOptogenetic actuators with diverse spectral tuning, ion selectivity and kinetics are constantly being engineered providing powerful tools for controlling neural activity with subcellular resolution and millisecond precision. Achieving reliable and interpretable in vivo optogenetic manipulations requires reproducible actuator expression and calibration of photocurrents in target neurons. Here, we developed nine transgenic zebrafish lines for stable opsin expression and calibrated their efficacy in vivo. We first used high-throughput behavioural assays to compare opsin ability to elicit or silence neural activity. Next, we performed in vivo whole-cell electrophysiological recordings to quantify the amplitude and kinetics of photocurrents and test opsin ability to precisely control spiking. We observed substantial variation in efficacy, associated with differences in both opsin expression level and photocurrent characteristics, and identified conditions for optimal use of the most efficient opsins. Overall, our calibrated optogenetic toolkit will facilitate the design of controlled optogenetic circuit manipulations.

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