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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. In experimental animals, elevations in brain KYNA have been shown to impair their cognitive performance in an array of behavioral assays2,7,8,9,10. An emerging hypothesis suggests that KYNA plays an integral role in modulating cognitive functions by impacting sleep-wake behavior11, thus further supporting the role of astrocyte-derived molecules in modulating the neurobiology of sleep and cognition12.
Clinically, elevations in KYNA have been found in cerebrospinal fluid and post-mortem brain tissue from patients with schizophrenia13,14,15,16, a debilitating psychiatric disorder characterized by cognitive impairments. Patients with schizophrenia are also often plagued by sleep disturbances that may exacerbate the illness17. Understanding the role of KP metabolism and KYNA in modulating a relationship between sleep and cognition, particularly between learning and memory, may lead to the development of novel therapies for treating these poor outcomes in schizophrenia and other psychiatric illnesses.
A reliable and consistent method for the measurement of KP metabolites is important to assure that the research emerging from various institutions can be integrated into the scientific understanding of KP biology. Presently, we describe the methodology to measure kynurenine in rat plasma and KYNA in the rat brain by high-performance liquid chromatography (HPLC). The present protocol, which makes use of a fluorimetric detection in the presence of Zn2+, was first developed by Shibata18 and more recently adapted and optimized to derivatize with 500 mM zinc acetate as the post-column reagent, allowing for the detection of endogenous, nanomolar amounts of KYNA in the brain11.
To stimulate the de novo endogenous KYNA production as described in the present protocol, the direct bioprecursor kynurenine is injected intraperitoneally (i.p.) in rats. In combination with biochemical assessments to determine the degree of KYNA production, the impacts of a kynurenine challenge on the hippocampal-dependent memory (passive avoidance paradigm) and the sleep-wake architecture (EEG and EMG signals) is also investigated11. A combination of these techniques allows for the study of the biochemical and functional impact of a kynurenine challenge in vivo in rats.
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 Psychiatric Research Center. They were kept on a 12/12 h light-dark cycle, with lights on at zeitgeber time (ZT) 0 and lights off at ZT 12. The animals received ad libitum access to food and water during the experiments. The facility was fully accredited by the American Association for the Accreditation of Laboratory Animal Care.
1. Intraperitoneal Kynurenine Administration to Rats
Note: In this protocol, kynurenine was administered at ZT 0 (the beginning of the light phase) and tissue was collected at ZT 2 and ZT 4 to determine a time course for the kynurenine metabolism. Saline-injected animals were used as a control. For instance, if a rat weighs 500 g and the desired dose is 100 mg/kg, the rat should receive a 5 mL injection of a 10 mg/mL solution of kynurenine.
2. Kynurenine Measurements Using High-performance Liquid Chromatography
3. Passive Avoidance Paradigm
NOTE: These behavioral experiments were designed based on our biochemical findings with the acute kynurenine challenge. To maximize an increase in brain KYNA, kynurenine (100 mg/kg) was administered at ZT 0, 2 h prior to the training session in the passive avoidance paradigm to test hippocampal-mediated learning, that occurred at ZT 2. The apparatus consists of 2 equally sized compartments (21.3 cm high, 20.3 cm wide, and 15.9 cm deep) separated by a guillotine door and contained within a soundproof box. The two compartments of the testing apparatus are termed “light side” and “dark side”. The walls of the light side are clear and, during the trials, a light will turn on to further illuminate this compartment. The walls of the dark compartment are completely covered to maintain a black-out condition.
4. Sleep Analysis
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...
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...
The authors have nothing to disclose.
The present study was funded in part by the National Institutes of Health (R01 NS102209) and a donation from the Clare E. Forbes Trust.
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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