Neuropharmacology Core Services

Receptor Binding Assay – Screening % Displacement – Cell membranes are isolated from stable-cell lines overexpressing the receptors of interest. Extracts, fractions, or purified compounds are placed in a competition assay with a radiolabeled ligand known to bind to the given receptor. Compounds are screened at 1 or 10 μM, and extracts/fractions are screened at 10 μg/ml.

Receptor Binding Assay – Ki –  Full concentration % displacement curves can be performed to determine Ki, using the receptor cell membranes described above.

Neurite Outgrowth (primary neurons) – Cells are plated in the presence of potential growth-inhibiting or growth-stimulating compounds. Growth at the desired time point is assessed using Sholl analysis. The assay can be performed as a competition against a known inhibitor of growth. One to eight compounds can be run per assay.

Neuroprotection – Primary cultures of neurons are treated with one concentration of one to eight compounds or extracts in combination with an excitotoxic insult to assess the potential of neuroprotection. Viability assessed with MTS or Live/DEAD indicators.

Neuroprotection EC-50 – Primary cultures of neurons are treated with increasing concentrations of one compound in combination with an excitotoxic insult to assess the potential of neuroprotection. Viability assessed with MTS or Live/DEAD indicators.

Neurotoxicity – Primary cultures of neurons are treated with one to eight compounds/extracts to assess the potential of neurotoxicity. Viability assessed with MTS or Live/DEAD indicators.

Hotplate – The hotplate will be used to measure thermal nociception and can indicate if compounds have actions at supraspinal mechanisms.

Tail Flick – Tail-flick (TF) assay is used to quantify spinal responses to thermal nociception.

Acetic Acid Writing – The Acetic Acid (AA) Writhing test is used to quantify pain induced by peripheral origins.

von Frey – The electronic von Frey is used to evaluate drugs against mechanical allodynia.

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Nitroglycerin induced migraine – The nitroglycerin (NTG) model used repeated induction and measurement of clinically-relevant behavioral endpoints of a migraine simulation in rodents.

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Cisplatin Induced Neuropathy – Cisplatin Induced Neuropathy (CIN) is used to evaluate drugs against mechanical allodynia in a model of chronic nociception.

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Complete Freund’s Adjuvant – Complete Freund’s Adjuvant (CFA) induced inflammatory pain model is used to test acute and chronic effects of analgesic and anti-inflammatory drugs.

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Elevated Plus Maze – The Elevated Plus Maze (EPM) is used to screen anxiolytic activity of pharmacological agents. This test is performed using a plus shaped maze with two open arms and tow closed arms. This procedure is based on a rodent’s exploratory/avoidance behavior whereby animals will naturally avoid open and elevated environments while spending more time exploring closed environments. Administration of anxiolytics will increase the time an animal explores the open arms.

Forced Swim Test – The Forced Swim Test (FST) is used to assess behavioral despair or learned helplessness. Animals are placed in an inescapable container filled with water and their escape behavior is recorded. Over a test trial, animals will struggle to escape but eventually give up and display depressive-live behaviors (immobility/floating). Pharmacological agents with antidepressant activity will reverse this depressive-like behavior and increase the amount of time spent struggling/escaping.

Tail Suspension – The tail suspension test used to evaluate compounds with antidepressant activity. In this test, the extent of immobility is associated with a depressive-like behaviors and is significantly diminished with the use of antidepressant drugs.

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Conditioned Place Preference – The Condition Place Preference (CPP) paradigm is used to evaluate positively reinforcing and aversive properties of drugs. CPP is based on associative learning where animals prefer environments paired with rewarding properties and avoid environments paired with aversive properties.

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Morris Water Maze – The Morris Water Maze is used to study aspects of spatial memory. This assay relies on animals to explore their environment and escape from water with a minimum amount of effort used (swimming the shortest distance). The time it takes an animal to find a hidden platform in a water pool after previous exposure, using distinct external cues, is used as a measure of spatial memory. The water maze is sensitive to the effects of drugs on the acquisition and performance of this task as well as the effects of aging.

Barnes Maze – The Barnes Maze is commonly used to evaluate spatial learning and memory in mice. Through the use of spatial cues, mice are trained to find an escape hole on top of an open, brightly-illuminated surface. Mice have a natural tendency to avoid brightly-lit, open spaces, thus mice are motivated to find a dark hidden escape box. This task highlights the abilities of the mice to learning, remember, extinguish old memories, and re-learn new spatial memories.

Radial Arm Maze – The Radial Arm water maze is used to evaluate spatial learning and memory in mice. When placed in water, mice are motivated to find an escape platform for safety. This is a modified radial arm maze set to mimic the Morris Water Maze. While the Morris Water Maze is the gold standard for testing spatial learning and memory in rats, mice have a tendency to sink in the large pool of water. Thus, the more restricted zones of the radial arm water maze reduce the risk of drowning. Together, this task highlights the abilities of the mice to learning, remember, extinguish old memories, and re-learn new spatial memories.

Y-maze – The Y-maze is commonly used to evaluate working memory in rodents. Rodents, especially rats, have a natural curiosity to explore their environment. By changing the environment after an acclimation period, cognitively-intact animals will explore the novelty of the new environment. Thus, inferences regarding working memory, willingness to explore novelty, and spatial learning can be made with this maze.

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Open Field – The Open Field test evaluates compounds on locomotive, exploratory, and anxiety-like behaviors. This procedure is based on rodent’s innate exploratory and avoidance behavior. Animals will naturally spend more time in corners or periphery of an open field to protect itself rather than a center, which is the most anxiogenic area. Administration of anxiolytics increase time spent in the center of the arena while stressful stimuli decrease the number of visits to the center of the arena.

Rotarod – The Rotarod (RR) is used to assess motor coordination in rodents. Animals are trained to walk on a rotating rod at various speeds and motor coordination is tested by the latency for the animal to fall off the rotating rod. As some drugs cause motor impairment (ataxia), these animals will demonstrate an increase latency to fall.

Grip Strength – The rodent grip strength test is used to evaluate limb motor or muscular functions in rodents. This test uses the rodent’s natural tendency to grasp a bar or grid when suspended by its tail. The bar is attached to a force transducer that measures the force in grams produced during the pull of the bar. The purpose of this test is to evaluate forelimb strength and is used to monitor the progression of neuromuscular disorders as well as screen novel therapeutics.

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Zebrafish PTZ Seizure – The generalized epilepsy zebrafish model is used to identify novel compounds to treat seizures ideally with fewer side effects than current anticonvulsant drugs. In this model, pentylenetetrazole (PTZ) is used to chemically induce seizures in the zebrafish to mimic generalized epilepsy following treatment with novel compound. The ViewPoint Zebrabox measures activity of the larvae to determine if the seizures were reduced following treatment.

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Genetic Zebrafish Epilepsy (Dravet) – The Dravet syndrome epilepsy zebrafish model is used to identify novel compounds to treat seizures that do not respond to the typical anticonvulsants (pharmaco-resistant). In this model, scn1a homozygous mutant zebrafish are used to as a Dravet syndrome model. The ViewPoint Zebrabox measures activity of the larvae to determine if the seizures were reduced following treatment.

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Zebrafish Toxicity – The purpose of the zebrafish acute toxicity assay is to determine concentrations of novel compounds that are non-toxic to the developing zebrafish embryo/larva. With this assay, we can determine if a compound that was effective at reducing seizures is toxic during an acute exposure.

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Tissue Harvest – Harvesting of heart, lungs, liver, kidney, spleen, brain, blood, and other tissues will be carried out following an IACUC approved method of euthanasia.

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Rodent PTSD – The rodent PTSD model is used to evaluate interventions that can attenuate or alleviate symptoms of PTSD. This model is highly translational to symptoms humans diagnosed with PTSD experience. Animals will be exposed to an aversive stimulus and exposed to situational reminders of the aversive stimulus. This mirrors the clinical picture of PTSD where after a traumatic event cues in the environment related to the event precipitate features of avoidance, changes in arousal, anxiety, depression, and potential changes in cognition.

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Tetrad Assay – The tetrad assay is a series of behavioral tests that characterize the effects of cannabinoids. Cannabinoids such as Delta-9-teterahydrocannabinol (THC) have shown to produce decreases in spontaneous activity, catalepsy, hypothermia, and antinociception. These effects are evaluated in the tetrad assay.

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