Reviewer
Andrew L. Eagle
  • Faculty, Michigan State University
Research fields
  • Neuroscience
Fluorescence Screens for Identifying Central Nervous System–Acting Drug–Biosensor Pairs for Subcellular and Supracellular Pharmacokinetics

Subcellular pharmacokinetic measurements have informed the study of central nervous system (CNS)–acting drug mechanisms. Recent investigations have been enhanced by the use of genetically encoded fluorescent biosensors for drugs of interest at the plasma membrane and in organelles. We describe screening and validation protocols for identifying hit pairs comprising a drug and biosensor, with each screen including 13–18 candidate biosensors and 44–84 candidate drugs. After a favorable hit pair is identified and validated via these protocols, the biosensor is then optimized, as described in other papers, for sensitivity and selectivity to the drug. We also show sample hit pair data that may lead to future intensity-based drug-sensing fluorescent reporters (iDrugSnFRs). These protocols will assist scientists to use fluorescence responses as criteria in identifying favorable fluorescent biosensor variants for CNS-acting drugs that presently have no corresponding biosensor partner.


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Cortical Laminar Recording of Multi-unit Response to Distal Forelimb Electrical Stimulation in Rats
Authors:  Charles-Francois V. Latchoumane, Rameen Forghani and Lohitash Karumbaiah, date: 11/20/2021, view: 2156, Q&A: 0

Severe traumatic brain injury (sTBI) survivors experience permanent functional disabilities due to significant volume loss and the brain’s poor capacity to regenerate. Chondroitin sulfate glycosaminoglycans (CS-GAGs) are key regulators of growth factor signaling and neural stem cell homeostasis in the brain. In this protocol, we describe how to perform recordings to quantify the neuroprotective and regenerative effect of implanted engineered CS-GAG hydrogel (eCS) on brain tissue. This experiment was performed in rats under three conditions: healthy without injury (Sham), controlled cortical impact (CCI) injury on the rostral forelimb area (RFA), and CCI-RFA with eCS implants. This protocol describes the procedure used to perform the craniotomy, the positioning of the cortical recording electrode, the positioning of the stimulation electrode (contralateral paw), and the recording procedure. In addition, a description of the exact electrical setup is provided. This protocol details the recordings in the brain of injured animals while preserving most of the uninjured tissue intact, with additional considerations for intralesional and laminar recordings of multi-unit response.


Graphic abstract:



Sensorimotor response to paw stimulation using cortical laminar recordings.

Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Protein Vesicle Tracking in HeLa Cells
Authors:  Natalia Pacheco-Fernandez, Mehrshad Pakdel and Julia von Blume, date: 03/05/2021, view: 6690, Q&A: 0

Monitoring vesicle trafficking is an excellent tool for the evaluation of protein dynamics in living cells. Such study is key for the understanding of protein sorting and secretion. Recent developments in microscopy, as well as new methodologies developed to study synchronized trafficking of proteins, allowed a better understanding of signaling, regulation and trafficking dynamics at the secretory pathway. One of the most helpful tools so far developed is the Retention Using Selective Hooks (RUSH) system, a methodology that facilitates the evaluation of synchronized cargo trafficking by monitoring fluorescent vesicles in cells upon biotin addition. Here we present a protocol that allows the quantitative evaluation of protein cargo trafficking at different fixed time points and an analytic approach that enables a better examination of specific cargo trafficking dynamics at the secretory pathway.


Graphic abstract:



Schematic representation of RUSH sorting assay in mammalian cells


Measuring Breathing Patterns in Mice Using Whole-body Plethysmography
Respiratory dysfunction is among the main cause of severe and fatal pathologies worldwide. The use of effective experimental models and methodologies for the study of the pulmonary pathophysiology is necessary to prevent, control and cure these diseases. Plethysmography, a technique for the assessment of lung function, has been widely applied in mice for the characterization of respiratory physiology. However, classical plethysmography methods present technical limitations such as the use of anesthesia and animal immobilization. Whole-body plethysmography (WBP) avoids these issues providing a non-invasive approach for the assessment of the respiratory function in conscious animals. WBP relies on the recording of pressure changes that are produced by the spontaneous breathing activity of an animal placed inside an airtight chamber. During normal respiration, pressure variation is directly proportional to the respiratory pattern of the animal allowing the measurement of the respiratory rate and tidal volume. These parameters are commonly used to evaluate pulmonary function in different physiological and disease models. In contrast to classical plethysmography methods, WBP technique allows reproducible serial measurements as it avoids animal restraint or the use of anesthesia. These key features rend WBP a suitable approach for longitudinal studies allowing the assessment of progressive respiratory alterations in physiological and pathological conditions. This protocol describes the procedures for the measurement of the breathing patterns in mice using the WBP method, the data analysis and results interpretation.
Quantitative Determination of Ca2+-binding to Ca2+-sensor Proteins by Isothermal Titration Calorimetry
Authors:  Seher Abbas and Karl-Wilhelm Koch, date: 04/05/2020, view: 4865, Q&A: 0
Diverse and complex molecular recognitions are central elements of signal transduction cascades. The strength and nature of these interaction modes can be determined by different experimental approaches. Among those, Isothermal titration calorimetry (ITC) offers certain advantages by providing binding constants and thermodynamic parameters from titration series without a need to label or immobilize one or more interaction partners. Furthermore, second messenger homeostasis involving Ca2+-ions requires in particular knowledge about stoichiometries and affinities of Ca2+-binding to Ca2+-sensor proteins or Ca2+-dependent regulators, which can be obtained by employing ITC. We used ITC to measure these parameters for a set of neuronal Ca2+-sensor proteins operating in photoreceptor cells. Here, we present a step wise protocol to (a) measure Ca2+ interaction with the Ca2+-sensor guanylate cyclase-activating protein 1, (b) to design an ITC experiment and prepare samples, (c) to remove Ca2+ nearly completely from Ca2+ binding proteins without using a chelating agent like EGTA.
SarkoSpin: A Technique for Biochemical Isolation and Characterization of Pathological TDP-43 Aggregates
Authors:  Manuela Pérez-Berlanga, Florent Laferrière and Magdalini Polymenidou, date: 11/20/2019, view: 5413, Q&A: 0
TDP-43 is the main aggregating protein in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Aggregated TDP-43 is resistant to diverse detergent solubilization, yet physiological TDP-43 and other abundant proteins commonly co-purify with pathological TDP-43. This mixed isolation has precluded the elucidation of the biochemical and structural features of the pathological TDP-43 and its role in disease. Here we describe SarkoSpin, a method for the isolation of pure pathological TDP-43 from patient autopsy brain by sample solubilization with Sarkosyl after nuclease treatment. This purification, which is also applicable to cell culture material, permits the study of biochemical properties of exclusively pathological TDP-43, allowing for the first time the determination of their link to the clinical presentation of FTLD. This method opens up a path for the study of pathological TDP-43 at the molecular and structural level in the heterogeneous spectrum of ALS and FTLD cases.
Assessing Olfaction Using Ultrasonic Vocalization Recordings in Mouse Pups with a Sono-olfactometer
Authors:  Sébastien Wagner, Pierre-Marie Lledo and Françoise Lazarini, date: 02/20/2019, view: 5505, Q&A: 0
Olfaction is the first sensory modality to develop during fetal life in mammals, and plays a key role in the various behaviors of neonates such as feeding and social interaction. Odorant cues (i.e., mother or predator scents) can trigger potentiation or inhibition of ultrasonic vocalizations (USV) emitted by pups following their isolation. Here, we report how USV are inhibited by olfactory cues using a sono-olfactometer that has been designed to quantify precisely olfaction in pups congenitally infected by cytomegalovirus. This olfactory-driven behavioral test assesses the USV emitted in presence of unfamiliar odorants such as citral scent or adult male mouse scent. We measure the number of USV emitted as an index of odorant detection during the three periods of the 5-min isolation time of the pup into the sono-olfactometer: first period without any odorant, second period with odorant exposure and last period with exhaust odorant. This protocol can be easily used to reveal olfactory deficits in pups with altered olfactory system due to toxic lesions or infectious diseases.
Assessing Classical Olfactory Fear Conditioning by Behavioral Freezing in Mice
Authors:  Jordan M. Ross and Max L. Fletcher, date: 09/20/2018, view: 5112, Q&A: 0
Classical fear conditioning typically involves pairing a discrete cue with a foot shock. Quantifying behavioral freezing to the learned cue is a crucial assay for neuroscience studies focused on learning and memory. Many paradigms utilize discrete stimuli such as tones; however, given mice are odor-driven animals and the wide variety of odorants commercially available, using odors as conditioned stimuli presents advantages for studies involving learning. Here, we describe detailed procedures for assembling systems for presenting discrete odor cues during single-day fear conditioning and subsequent analysis of freezing behavior to assess learning.
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