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 retention time of 11 min. To observe the KP dynamics, 100 mg/kg of kynurenine was administered in this protocol, and tissue was collected immediately following the injection, or 2 or 4 h post-injection (Figure 3a). Plasma kynurenine (Figure 3b) and hippocampal KYNA (Figure 3c) were quantified. The specific parameters used in the HPLC determination of kynurenine metabolites are outlined in Table 1. The dose-response curve produced by this protocol supports its description of an acute kynurenine injection as a method to increase brain KYNA levels, with a peak achieved at 2 h post-injection and the levels of KYNA returned to their baseline 4 h post-injection.
Based on the aforementioned biochemical findings, the training in the passive avoidance paradigm was performed 2 h after the kynurenine injection (Figure 4a). No group differences were observed during the training trial (Figure 4b). During the testing trial, the control animals displayed a significant increase in latency to enter the dark side, indicating contextual learning. Animals injected with kynurenine on the previous day did not display the same increase in latency, demonstrating a deficit in learning. Based on these findings, we can conclude that acute kynurenine elevation, during the time of memory acquisition and consolidation, results in an impaired performance in a hippocampus-mediated task.
To investigate the impact of an acute kynurenine elevation during the light phase on sleep-wake architecture, animals were injected with saline on day 1 and with kynurenine on day 2 at ZT 0 (Figure 5a). EEG/EMG signals were recorded telemetrically for the entire 48 h period. Comparisons were made between the saline injection (vehicle) on day 1 and the kynurenine injection on day 2. The results indicated a reduction in total REM sleep duration (presented as a percentage change from the baseline) during the first 2 h (Figure 5b) after an injection. This was mirrored by an increase in wake duration during these time periods and a slight reduction in NREM sleep. These results demonstrate that an acute kynurenine elevation causes disturbances in sleep-wake dynamics.
Figure 1: Standard curves for a kynurenine and KYNA injection. 20 µL of each standard was injected to create the standard curve for (A) kynurenine and (B) KYNA. Please click here to view a larger version of this figure.
Figure 2: Representative chromatograms for kynurenine and KYNA. The standards were run prior to a sample to confirm their retention times. These panels show representative samples for (A) serum kynurenine and (B) brain KYNA. Please click here to view a larger version of this figure.
Figure 3: Analysis of serum and hippocampal tissue by HPLC following a kynurenine challenge. (A) Kynurenine (100 mg/kg) or saline were administered by an intraperitoneal injection at zeitgeber time (ZT) 0. Tissues were collected either 2 or 4 h post-injection. The following panels show exposure to (B) kynurenine-elevated serum kynurenine and (C) hippocampal KYNA levels 2 h after injection. **** P <0.001 indicates the significance by a two-way analysis of variance (ANOVA) followed by a Bonferroni t-test correction. N = 4–5 per group. All data are mean ± SEM. This figure has been modified from Pocivavsek et al.11. Please click here to view a larger version of this figure.
Figure 4: Effect of a kynurenine elevation on hippocampal-dependent memory in the passive avoidance paradigm. (A) Kynurenine (100 mg/kg) or saline were administered by intraperitoneal injection at zeitgeber time (ZT) 0. The animals were trained on the task at ZT 2. 24 h after the training, the animals were tested for memory formation. (B) An exposure to kynurenine prior to the training reduced the latency to avoid the aversive dark compartment on the testing day. ** P <0.01, *** P <0.001 indicate the significance by a two-way analysis of variance (ANOVA) followed by a Bonferroni t-test correction. N = 8–14 per group. All data are mean ± SEM. This figure has been modified from Pocivavsek et al.11. Please click here to view a larger version of this figure.
Figure 5: Effect of a kynurenine elevation on sleep-wake architecture. (A) Saline (day 1) and kynurenine (100 mg/kg; day 2) were administered by an intraperitoneal injection at zeitgeber time (ZT) 0. The sleep-wake behavior was recorded for the subsequent 24 h. (B) The rapid eye movement (REM) sleep duration was reduced and the wake duration was increased in the first 2 h following the kynurenine injection. * P <0.05 indicates the significance by a two-way analysis of variance (ANOVA) followed by a Bonferroni t-test correction. N = 6–8 per group. All data are mean ± SEM. This figure has been modified from Pocivavsek et al.11. Please click here to view a larger version of this figure.
Mobile phase composition | 50 mM sodium acetate, pH 6.2 |
5% acetonitrile | |
Mobile phase flow rate | 0.5 mL/min |
Post-column derivatization reagent | 500 mM zinc acetate |
Post-column derivatization flow rate | 0.1 ml/min |
Column type | ReproSil-Pur C18 |
Column dimensions | 4 x 150 mm2 |
Detector temperature | 4.0 ºC |
Kynurenine wavelength | Ex: 365, Em: 480 |
Kynurenine retention time | ~6 min |
KYNA wavelength | Ex: 344, Em: 398 |
KYNA retention time | ~11 min |
Table 1: Parameters for an HPLC determination of kynurenine pathway metabolites.
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 of the described fluorometric HPLC method, adapted from Shibata18, to derivatize KYNA in the presence of Zn2+, including the ability to precisely detect endogenous amounts of KYNA in the brain in the nanomolar range. In addition, the method has been adapted to detect the bioprecursor kynurenine in the plasma in the micromolar range by adjusting the fluorometric wavelengths of excitation and emission, as described in the protocol.
Critical steps within the protocol, such as specific times between the treatment and the euthanasia, are described to accurately determine the time course of the KYNA formation in the brain and guide the design and analysis of the behavioral and sleep-wake monitoring experiments. Hippocampal-mediated learning was assessed using the passive avoidance paradigm and a sleep-wake analysis was conducted with telemetric recordings of EEG/EMG data. As we determined that the brain KYNA levels were highest 2 h post-injection, we timed the behavioral paradigm training session accordingly, so that the acquisition in the passive avoidance task occurred when the KYNA levels were highest, at ZT 2. In addition, we also focused the analysis of the sleep-wake behavior on the critical ZT 0 to ZT 2 time frame during which the KYNA levels were highest in the brain. Taken together, the carefully considered experimental design has allowed us to bridge together findings from biochemical and functional experiments, introducing KYNA as a modulator of both cognition and sleep-wake behavior11.
A number of limitations should be considered in the described protocol. To stimulate the endogenous production of KYNA, the direct bioprecursor kynurenine was injected peripherally into rats. Kynurenine readily crosses the blood-brain barrier and the stimulated KP metabolism occurs in a variety of brain regions2. We presently focus on the elevation in astrocyte-derived KYNA in the hippocampus; however, it is important to consider that systemic injections of kynurenine also elevate metabolites of the neurotoxic branch of the KP, including 3-hydroxykynurenine (3-HK) and quinolinic acid. To tease apart the effects caused by the elevations in 3-HK compared to those caused by KYNA elevations, the methodology should be adjusted. Additionally, the perfuse elevation in KP metabolism throughout the brain11 provides a limited insight into the specific brain regions that are mediating the alterations in behavior.
When designing the behavioral experiment described here, we optimized the passive avoidance paradigm with respect to existing methods, as described in detail, to engage hippocampal-mediated contextual memory. Memory is comprised of three major subprocesses: encoding, consolidation, and retrieval. Optimal conditions for memory consolidation processes occur during sleep when newly encoded memory is integrated into long-term storage19,20. As studies in rodents have focused on narrow time windows of memory consolidation and identified that memory appears most sensitive when sleep is delayed after the acquisition, we chose to elevate the kynurenine and KYNA formation in the brain during this sensitive time window, impacting the acquisition and the consolidation, to test the hypothesis of this article. We did not, however, presently test if the retrieval of the memory was impacted by kynurenine elevations, as previously shown21. It is critically important to also consider the brain regions engaged in rodents to perform a task. While for this study, we were mostly interested in the role of the hippocampus in modulating cognition, modifications to the protocol may be useful to test animals in alternative behavioral paradigms that have been shown to be impacted by an acute kynurenine challenge, such as fear conditioning and working memory tasks2,7,8,9,10.
Additionally, in lieu of systemic injections of kynurenine to elevate the brain KYNA, alternative strategies to consider include the direct infusion of KYNA into an area of interest or the targeted inhibition of the synthesizing enzyme KAT II by pharmacological tools or molecular knock-down approaches in specific brain regions, to test mechanistic hypotheses. While these approaches may also introduce potentially invasive procedures that independently impact the functional outcomes measured, it is critical to design experiments with optimal control conditions.
Lastly, the sleep recording EEG/EMG protocol described here has the advantage of being telemetric rather than tethered, eliminating electrical noise, movement artifacts, and the risk of injury in an animal that has pulled the tethered cables. The physical size and light weight of the telemetric device, and its placement subcutaneously rather than intraperitoneally in the rat to optimize the post-surgical recovery, enables wireless recordings that capture naturalistic sleep-wake behavior with a free range of movement. However, an important consideration when using the telemetric system is the limited ability to recording from multiples channels simultaneous. The current paradigm pairs one EEG channel and one EMG channel, and allows for the scoring of REM, NREM, and wake behavior.
The combination of methods described presently provides insight into behavioral and biochemical assays to elucidate the functional outcomes of a kynurenine elevation. These protocols demonstrate a relationship between the KP, cognition, and sleep, a previously unexplored dynamic. Utilizing the detailed experimental methods provided here will enhance the scientific understanding of the functional impacts of kynurenine and KYNA neurobiology in animals. Ultimately, continued work in this field may lead to new therapeutic approaches to alleviate outcomes in individuals combatting psychiatric disturbances and disruptions in sleep.
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.
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 |