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Abstract

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Protocol

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Behavior

A High-performance Liquid Chromatography Measurement of Kynurenine and Kynurenic Acid: Relating Biochemistry to Cognition and Sleep in Rats

Published: August 19th, 2018

DOI:

10.3791/58129

1Maryland Psychiatric Research Center, Department of Psychiatry, University of Maryland School of Medicine, 2Department of Pharmacology, University of Maryland School of Medicine

Alterations in the kynurenine pathway (KP) neuroactive metabolites are implicated in psychiatric illnesses. Investigating the functional outcomes of an altered kynurenine pathway metabolism in vivo in rodents may help elucidate novel therapeutic approaches. The current protocol combines biochemical and behavioral approaches to investigate the impact of an acute kynurenine challenge in rats.

The kynurenine pathway (KP) of tryptophan degradation has been implicated in psychiatric disorders. Specifically, the astrocyte-derived metabolite kynurenic acid (KYNA), an antagonist at both N-methyl-d-aspartate (NMDA) and α7 nicotinic acetylcholine (α7nACh) receptors, has been implicated in cognitive processes in health and disease. As KYNA levels are elevated in the brains of patients with schizophrenia, a malfunction at the glutamatergic and cholinergic receptors is believed to be causally related to cognitive dysfunction, a core domain of the psychopathology of the illness. KYNA may play a pathophysiologically significant role in individuals with schizophrenia. It is possible to elevate endogenous KYNA in the rodent brain by treating animals with the direct bioprecursor kynurenine, and preclinical studies in rats have demonstrated that acute elevations in KYNA may impact their learning and memory processes. The current protocol describes this experimental approach in detail and combines a) a biochemical analysis of blood kynurenine levels and brain KYNA formation (using high-performance liquid chromatography), b) behavioral testing to probe the hippocampal-dependent contextual memory (passive avoidance paradigm), and c) an assessment of sleep-wake behavior [telemetric recordings combining electroencephalogram (EEG) and electromyogram (EMG) signals] in rats. Taken together, a relationship between elevated KYNA, sleep, and cognition is studied, and this protocol describes in detail an experimental approach to understanding function outcomes of kynurenine elevation and KYNA formation in vivo in rats. Results obtained through variations of this protocol will test the hypothesis that the KP and KYNA serve pivotal roles in modulating sleep and cognition in health and disease states.

The KP is responsible for degrading nearly 95% of the essential amino acid tryptophan1. In the mammalian brain, kynurenine taken into astrocytes is metabolized into the neuroactive small molecule KYNA primarily by the enzyme kynurenine aminotransferase (KAT) II2. KYNA acts as an antagonist at NMDA and α7nACh receptors in the brain2,3,4, and also targets signaling receptors including the aryl hydrocarbon receptor (AHR) and the G-protein coupled receptor 35 (GPR35)5,6....

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Our experimental protocols were approved by the University of Maryland Institutional Animal Care and Use Committee and followed the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

NOTE: Adult male Wistar rats (250–350 g) were used in all experiments. Separate cohorts of animals were used for biochemical analysis, behavioral experiments, and sleep-wake recordings. The animals were housed in a temperature-controlled facility at the Maryland Psychiatr.......

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To validate the use of an intraperitoneal kynurenine injection as a method to elevate the brain KYNA, an HPLC analysis of tissue was performed. Standard curves (Figure 1) were constructed using the associated software and allowed for the quantification of the tissue samples. Representative chromatograms for kynurenine and KYNA are presented in Figure 2. Kynurenine was observed at a retention time of 6 min, and KYNA had a retentio.......

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For a reliable assessment of KYNA in the brain after a peripheral kynurenine administration, it is critical to combine and interpret biochemical and functional experiments. Here, we present a detailed protocol that permits new users to establish effective methods for measuring the plasma kynurenine and brain KYNA of rats. The measurement of kynurenine in the plasma confirmed the accurate injection and the measurement of the metabolite KYNA confirms the de novo synthesis in the brain. There are several advantages.......

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The present study was funded in part by the National Institutes of Health (R01 NS102209) and a donation from the Clare E. Forbes Trust.

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Name Company Catalog Number Comments
Wistar rats Charles River Laboratories adult male, 250-350 g
L-kynurenine sulfate Sai Advantium
ReproSil-Pur C18 column (4 x 150 mm) Dr. Maisch GmbH
EZ Clips Stoelting Co. 59022
Mounting materials screws PlasticsOne 00-96 X 1/16
Nonabsorbable Sutures MedRep Express 699B CP Medical Monomid Black Nylon Sutures, 4-0, P-3, 18", BOX of 12
Absorbable Sutures Ethicon J310H 4-0 Coated Vicryl Violet 1X27'' SH-1
Dental Cement Stoelting Co. 51458
Drill Bit Stoelting Co. 514551 0.45 mm
Name Company Catalog Number Comments
Alliance HPLC system
E2695 separation module Waters 176269503
2475 fluorescence detector Waters 186247500
post-column reagent manager Waters 725000556
Lenovo computer Waters 668000249
Empower software Waters 176706100
Name Company Catalog Number Comments
Passive avoidance box for rat
Extra tall MDF sound attenuating cubicle MedAssociates ENV-018MD Interior: 22"W x 22"H x 16"D
Center channel modulator shuttle box chamber MedAssociates ENV-010MC
Stainless steel grid floor for rat MedAssociates ENV-010MB-GF
Auto guillotine door MedAssociates ENV-010B-S
Quick disconnect shuttle grid floor harness for rat MedAssociates ENV-010MB-QD
Stimulus light, 1" white lens, mounted on modular panel MedAssociates ENV-221M
Sonalert module with volume control for rat chamber MedAssociates ENV-223AM
SmartCtrl 8 input/16 output package MedAssociates DIG-716P2
8 Channel IR control for shuttle boxes MedAssociates ENV-253C
Infrared source and dectector array strips MedAssociates ENV-256
Tabletop interface cabinet, 120 V 60 Hz MedAssociates SG-6080C
Dual range constant current aversive stimulation module MedAssociates ENV-410B
Solid state grid floor scrambler module MedAssociates ENV-412
Dual A/B shock control module MedAssociates ENV-415
2' 3-Pin mini-molex extension MedAssociates SG-216A-2
10' Shock output cable, DB-9 M/F MedAssociates SG-219G-10
Shuttle shock control cable 15', 6 MedAssociates SG-219SA
Small tabletop cabinet and power supply, 120 V 60 Hz MedAssociates SG-6080D
PCI interface package MedAssociates DIG-700P2-R2
Shuttle box avoidance utility package MedAssociates SOF-700RA-7
Name Company Catalog Number Comments
Sleep-Wake Monitoring Equipment
Ponehmah software Data Sciences International (DSI) PNP-P3P-610
MX2 8 Source Acquisition interface Data Sciences International (DSI) PNM-P3P-MX204
Dell computer, Optiplex 7020, Windows 7, 64 bit Data Sciences International (DSI) 271-0112-013
Dell 19" computer monitor Data Sciences International (DSI) 271-0113-001
Receivers for plastic cages, 8x Data Sciences International (DSI) 272-6001-001
Cisco RV130 VPN router Data Sciences International (DSI) RV130
Matrix 2.0 Data Sciences International (DSI) 271-0119-001
Network switch Data Sciences International (DSI) SG200-08P
Neuroscore software Data Sciences International (DSI) 271-0171-CFG
Two biopotential channels transmitter, model TL11M2-F40-EET Data Sciences International (DSI) 270-0134-001

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