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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The authors have nothing to disclose.

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 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 &#945;7nACh 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.

<table>
	<tbody>
		<tr>
			<td>Wistar rats</td>
			<td>Charles River Laboratories</td>
			<td></td>
			<td>adult male, 250-350 g</td>
		</tr>
		<tr>
			<td>L-kynurenine sulfate</td>
			<td>Sai Advantium</td>
			<td></td>
			<td></td>
		</tr>
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			<td>ReproSil-Pur C18 column (4 x 150 mm)</td>
			<td>Dr. Maisch GmbH</td>
			<td></td>
			<td></td>
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			<td>EZ Clips</td>
			<td>Stoelting Co.</td>
			<td>59022</td>
			<td></td>
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			<td>Mounting materials screws</td>
			<td>PlasticsOne</td>
			<td>00-96 X 1/16</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonabsorbable Sutures</td>
			<td>MedRep Express</td>
			<td>699B</td>
			<td>CP Medical Monomid Black Nylon Sutures, 4-0, P-3, 18&#34;, BOX of 12</td>
		</tr>
		<tr>
			<td>Absorbable Sutures</td>
			<td>Ethicon</td>
			<td>J310H</td>
			<td>4-0 Coated Vicryl Violet 1X27&#39;&#39; SH-1</td>
		</tr>
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			<td>Dental Cement</td>
			<td>Stoelting Co.</td>
			<td>51458</td>
			<td></td>
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			<td>Drill Bit</td>
			<td>Stoelting Co.</td>
			<td>514551</td>
			<td>0.45 mm</td>
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			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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			<td>Alliance HPLC system</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>E2695 separation module</td>
			<td>Waters</td>
			<td>176269503</td>
			<td></td>
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			<td>2475 fluorescence detector</td>
			<td>Waters</td>
			<td>186247500</td>
			<td></td>
		</tr>
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			<td>post-column reagent manager</td>
			<td>Waters</td>
			<td>725000556</td>
			<td></td>
		</tr>
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			<td>Lenovo computer</td>
			<td>Waters</td>
			<td>668000249</td>
			<td></td>
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			<td>Empower software</td>
			<td>Waters</td>
			<td>176706100</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
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			<td>Passive avoidance box for rat</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Extra tall MDF sound attenuating cubicle</td>
			<td>MedAssociates</td>
			<td>ENV-018MD</td>
			<td>Interior: 22&#34;W x 22&#34;H x 16&#34;D</td>
		</tr>
		<tr>
			<td>Center channel modulator shuttle box chamber</td>
			<td>MedAssociates</td>
			<td>ENV-010MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel grid floor for rat</td>
			<td>MedAssociates</td>
			<td>ENV-010MB-GF</td>
			<td></td>
		</tr>
		<tr>
			<td>Auto guillotine door</td>
			<td>MedAssociates</td>
			<td>ENV-010B-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick disconnect shuttle grid floor harness for rat</td>
			<td>MedAssociates</td>
			<td>ENV-010MB-QD</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulus light, 1&#34; white lens, mounted on modular panel</td>
			<td>MedAssociates</td>
			<td>ENV-221M</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonalert module with volume control for rat chamber</td>
			<td>MedAssociates</td>
			<td>ENV-223AM</td>
			<td></td>
		</tr>
		<tr>
			<td>SmartCtrl 8 input/16 output package</td>
			<td>MedAssociates</td>
			<td>DIG-716P2</td>
			<td></td>
		</tr>
		<tr>
			<td>8 Channel IR control for shuttle boxes</td>
			<td>MedAssociates</td>
			<td>ENV-253C</td>
			<td></td>
		</tr>
		<tr>
			<td>Infrared source and dectector array strips</td>
			<td>MedAssociates</td>
			<td>ENV-256</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop interface cabinet, 120 V 60 Hz</td>
			<td>MedAssociates</td>
			<td>SG-6080C</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual range constant current aversive stimulation module</td>
			<td>MedAssociates</td>
			<td>ENV-410B</td>
			<td></td>
		</tr>
		<tr>
			<td>Solid state grid floor scrambler module</td>
			<td>MedAssociates</td>
			<td>ENV-412</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual A/B shock control module</td>
			<td>MedAssociates</td>
			<td>ENV-415</td>
			<td></td>
		</tr>
		<tr>
			<td>2&#39; 3-Pin mini-molex extension</td>
			<td>MedAssociates</td>
			<td>SG-216A-2</td>
			<td></td>
		</tr>
		<tr>
			<td>10&#39; Shock output cable, DB-9 M/F</td>
			<td>MedAssociates</td>
			<td>SG-219G-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Shuttle shock control cable 15&#39;, 6</td>
			<td>MedAssociates</td>
			<td>SG-219SA</td>
			<td></td>
		</tr>
		<tr>
			<td>Small tabletop cabinet and power supply, 120 V 60 Hz</td>
			<td>MedAssociates</td>
			<td>SG-6080D</td>
			<td></td>
		</tr>
		<tr>
			<td>PCI interface package</td>
			<td>MedAssociates</td>
			<td>DIG-700P2-R2</td>
			<td></td>
		</tr>
		<tr>
			<td>Shuttle box avoidance utility package</td>
			<td>MedAssociates</td>
			<td>SOF-700RA-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Sleep-Wake Monitoring Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ponehmah software</td>
			<td>Data Sciences International (DSI)</td>
			<td>PNP-P3P-610</td>
			<td></td>
		</tr>
		<tr>
			<td>MX2 8 Source Acquisition interface</td>
			<td>Data Sciences International (DSI)</td>
			<td>PNM-P3P-MX204</td>
			<td></td>
		</tr>
		<tr>
			<td>Dell computer, Optiplex 7020, Windows 7, 64 bit</td>
			<td>Data Sciences International (DSI)</td>
			<td>271-0112-013</td>
			<td></td>
		</tr>
		<tr>
			<td>Dell 19&#34; computer monitor</td>
			<td>Data Sciences International (DSI)</td>
			<td>271-0113-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Receivers for plastic cages, 8x</td>
			<td>Data Sciences International (DSI)</td>
			<td>272-6001-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cisco RV130 VPN router</td>
			<td>Data Sciences International (DSI)</td>
			<td>RV130</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix 2.0</td>
			<td>Data Sciences International (DSI)</td>
			<td>271-0119-001</td>
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			<td>Network switch</td>
			<td>Data Sciences International (DSI)</td>
			<td>SG200-08P</td>
			<td></td>
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			<td>Neuroscore software</td>
			<td>Data Sciences International (DSI)</td>
			<td>271-0171-CFG</td>
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			<td>Two biopotential channels transmitter, model TL11M2-F40-EET</td>
			<td>Data Sciences International (DSI)</td>
			<td>270-0134-001</td>
			<td></td>
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a high-performance liquid chromatography measurement of kynurenine and kynurenic acid: relating biochemistry to cognition and sleep 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 &#945;7 nicotinic acetylcholine (&#945;7nACh) 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.

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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58129/58129fig1.jpg" /> 
Figure 1: Standard curves for a kynurenine and KYNA injection. 20 &#181;L of each standard was injected to create the standard curve for (A) kynurenine and (B) KYNA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58129/58129fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58129/58129fig2.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58129/58129fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58129/58129fig3.jpg" /> 
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 &#60;0.001 indicates the significance by a two-way analysis of variance (ANOVA) followed by a Bonferroni t-test correction. N = 4&#8211;5 per group. All data are mean &#177; SEM. This figure has been modified from Pocivavsek et al.11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58129/58129fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58129/58129fig4.jpg" /> 
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 &#60;0.01, *** P &#60;0.001 indicate the significance by a two-way analysis of variance (ANOVA) followed by a Bonferroni t-test correction. N = 8&#8211;14 per group. All data are mean &#177; SEM. This figure has been modified from Pocivavsek et al.11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58129/58129fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58129/58129fig5.jpg" /> 
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 &#60;0.05 indicates the significance by a two-way analysis of variance (ANOVA) followed by a Bonferroni t-test correction. N = 6&#8211;8 per group. All data are mean &#177; SEM. This figure has been modified from Pocivavsek et al.11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58129/58129fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Mobile phase composition</td>
			<td>50 mM sodium acetate, pH 6.2</td>
		</tr>
		<tr>
			<td>5% acetonitrile</td>
		</tr>
		<tr>
			<td>Mobile phase flow rate</td>
			<td>0.5 mL/min</td>
		</tr>
		<tr>
			<td>Post-column derivatization reagent</td>
			<td>500 mM zinc acetate</td>
		</tr>
		<tr>
			<td>Post-column derivatization flow rate</td>
			<td>0.1 ml/min</td>
		</tr>
		<tr>
			<td>Column type</td>
			<td>ReproSil-Pur C18</td>
		</tr>
		<tr>
			<td>Column dimensions</td>
			<td>4 x 150 mm2</td>
		</tr>
		<tr>
			<td>Detector temperature</td>
			<td>4.0 &#186;C</td>
		</tr>
		<tr>
			<td>Kynurenine wavelength</td>
			<td>Ex: 365, Em: 480</td>
		</tr>
		<tr>
			<td>Kynurenine retention time</td>
			<td>~6 min</td>
		</tr>
		<tr>
			<td>KYNA wavelength</td>
			<td>Ex: 344, Em: 398</td>
		</tr>
		<tr>
			<td>KYNA retention time</td>
			<td>~11 min</td>
		</tr>
	</tbody>
</table>
Table 1: Parameters for an HPLC determination of kynurenine pathway metabolites.

Watch this Scientific Journal Video about A High-performance Liquid Chromatography Measurement of Kynurenine and Kynurenic Acid: Relating Biochemistry to Cognition and Sleep in Rats at JoVE.com

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

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 ...

a-high-performance-liquid-chromatography-measurement-kynurenine

Nanyang Technological University

Research

JoVE Journal

Behavior

10.4K Views.  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.

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

The Crossmodal Congruency Task as a Means to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden hand is touched. If the viewed and felt touches are given at the same time then this is sufficient to induce the compelling experience that the rubber hand is one's own hand. The RHI can be used to investigate exactly how the brain constructs distinct body representations for one's own body. Such representations are crucial for successful interactions with the external world. To obtain a subjective measure of the RHI, researchers typically ask participants to rate statements such as "I felt as if the rubber hand were my hand". Here we demonstrate how the crossmodal congruency task can be used to obtain an objective behavioral measure within this paradigm.
The variant of the crossmodal congruency task we employ involves the presentation of tactile targets and visual distractors. Targets and distractors are spatially congruent (i.e. same finger) on some trials and incongruent (i.e. different finger) on others. The difference in performance between incongruent and congruent trials - the crossmodal congruency effect (CCE) - indexes multisensory interactions. Importantly, the CCE is modulated both by viewing a hand as well as the synchrony of viewed and felt touch which are both crucial factors for the RHI.
The use of the crossmodal congruency task within the RHI paradigm has several advantages. It is a simple behavioral measure which can be repeated many times and which can be obtained during the illusion while participants view the artificial hand. Furthermore, this measure is not susceptible to observer and experimenter biases. The combination of the RHI paradigm with the crossmodal congruency task allows in particular for the investigation of multisensory processes which are critical for modulations of body representations as in the RHI.

We demonstrate how an objective measure can be employed in the widely employed rubber hand illusion paradigm. This measure is obtained by modifying the well-established crossmodal congruency task. This task allows the investigation of multisensory processes which are critical for modulations of body representations as in the rubber hand illusion.

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden ...

Eye Tracking, Cortisol, and a Sleep vs. Wake Consolidation Delay: Combining Methods to Uncover an Interactive Effect of Sleep and Cortisol on Memory

Although rises in cortisol can benefit memory consolidation, as can sleep soon after encoding, there is currently a paucity of literature as to how these two factors may interact to influence consolidation. Here we present a protocol to examine the interactive influence of cortisol and sleep on memory consolidation, by combining three methods: eye tracking, salivary cortisol analysis, and behavioral memory testing across sleep and wake delays. To assess resting cortisol levels, participants gave a saliva sample before viewing negative and neutral objects within scenes. To measure overt attention, participants&#8217; eye gaze was tracked during encoding. To manipulate whether sleep occurred during the consolidation window, participants either encoded scenes in the evening, slept overnight, and took a recognition test the next morning, or encoded scenes in the morning and remained awake during a comparably long retention interval. Additional control groups were tested after a 20&#160;min&#160;delay in the morning or evening, to control for time-of-day effects. Together, results showed that there is a direct relation between resting cortisol at encoding and subsequent memory, only following a period of sleep. Through eye tracking, it was further determined that for negative stimuli, this beneficial effect of cortisol on subsequent memory may be due to cortisol strengthening the relation between where participants look during encoding and what they are later able to remember. Overall, results obtained by a combination of these methods uncovered an interactive effect of sleep and cortisol on memory consolidation.

We present a protocol used to discover an interactive effect between sleep and cortisol on memory consolidation, particularly for negative arousing images. Specifically, the experimental design utilizes eye tracking, salivary cortisol analysis, and behavioral memory testing &#8211; methods that can be used with both healthy and clinical participants.

Although rises in cortisol can benefit memory consolidation, as can sleep soon after encoding, there is currently a paucity of literature as to how these ...

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here with the goal of uncovering rhythm disorders, such as beat deafness. Beat deafness is characterized by poor performance in perceiving durations in auditory rhythmic patterns or poor synchronization of movement with auditory rhythms (e.g., with musical beats). These tasks include the synchronization of finger tapping to the beat of simple and complex auditory stimuli and the detection of rhythmic irregularities (anisochrony detection task) embedded in the same stimuli. These tests, which are easy to administer, include an assessment of both perceptual and sensorimotor timing abilities under different conditions (e.g., beat rates and types of auditory material) and are based on the same auditory stimuli, ranging from a simple metronome to a complex musical excerpt. The analysis of synchronized tapping data is performed with circular statistics, which provide reliable measures of synchronization accuracy (e.g., the difference between the timing of the taps and the timing of the pacing stimuli) and consistency. Circular statistics on tapping data are particularly well-suited for detecting individual differences in the general population. Synchronized tapping and anisochrony detection are sensitive measures for identifying profiles of rhythm disorders and have been used with success to uncover cases of poor synchronization with spared perceptual timing. This systematic assessment of perceptual and sensorimotor timing can be extended to populations of patients with brain damage, neurodegenerative diseases (e.g., Parkinson&#8217;s disease), and developmental disorders (e.g., Attention Deficit Hyperactivity Disorder).

Behavioral tasks that allow for the assessment of perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) are presented. Synchronization of finger tapping to the beat of an auditory stimuli and detecting rhythmic irregularities provides a means of uncovering rhythm disorders.

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

Modulating Cognition Using Transcranial Direct Current Stimulation of the Cerebellum

Numerous studies have emerged recently that demonstrate the possibility of modulating, and in some cases enhancing, cognitive processes by exciting brain regions involved in working memory and attention using transcranial electrical brain stimulation. Some researchers now believe the cerebellum supports cognition, possibly via a remote neuromodulatory effect on the prefrontal cortex. This paper describes a procedure for investigating a role for the cerebellum in cognition using transcranial direct current stimulation (tDCS), and a selection of information-processing tasks of varying task difficulty, which have previously been shown to involve working memory, attention and cerebellar functioning. One task is called the Paced Auditory Serial Addition Task (PASAT) and the other a novel variant of this task called the Paced Auditory Serial Subtraction Task (PASST). A verb generation task and its two controls (noun and verb reading) were also investigated. All five tasks were performed by three separate groups of participants, before and after the modulation of cortico-cerebellar connectivity using anodal, cathodal or sham tDCS over the right cerebellar cortex. The procedure demonstrates how performance (accuracy, verbal response latency and variability) could be selectively improved after cathodal stimulation, but only during tasks that the participants rated as difficult, and not easy. Performance was unchanged by anodal or sham stimulation. These findings demonstrate a role for the cerebellum in cognition, whereby activity in the left prefrontal cortex is likely dis-inhibited by cathodal tDCS over the right cerebellar cortex. Transcranial brain stimulation is growing in popularity in various labs and clinics. However, the after-effects of tDCS are inconsistent between individuals and not always polarity-specific, and may even be task- or load-specific, all of which requires further study. Future efforts might also be guided towards neuro-enhancement in cerebellar patients presenting with cognitive impairment once a better understanding of brain stimulation mechanisms has emerged.

Transcranial direct current stimulation (tDCS) over the cerebellum exerts a remote effect on the prefrontal cortex, which can modulate cognition and performance. This was demonstrated using two information-processing tasks of varying complexity, whereby only cathodal tDCS improved performance when the task was difficult, but not easy.

Numerous studies have emerged recently that demonstrate the possibility of modulating, and in some cases enhancing, cognitive processes by exciting brain ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture

This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little ...

A New Method for Inducing a Depression-Like Behavior in Rats

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is no existing format for studying the mechanism of action, prevention, containment, and treatment of contagious depression. The purpose of this study, therefore, was to establish the first animal model of contagious depression.
Healthy rats can contract depressive behaviors if exposed to depressed rats. Depression is induced in rats by subjecting them to several manipulations of chronic unpredictable stress (CUS) over 5 weeks, as described in the protocol. A successful sucrose preference test confirmed the development of depression in the rats. The CUS-exposed rats were then caged with na&#239;ve rats from the contagion group (1 na&#239;ve rat/2 depressed rats in a cage) for an additional 5 weeks. 30 social groups were created from the combination of CUS-exposed rats and na&#239;ve rats.
This proposed depression-contagion protocol in animals consists mainly of cohabiting CUS-exposed and healthy rats for 5 weeks. To ensure that this method works, a series of tests are carried out - first, the sucrose preference test upon inducing depression to rats, then, the sucrose preference test, alongside the open field and forced-swim tests at the end of the cohabitation period. Throughout the experiment, rats are given tags and are always returned to their cages after each test.
A few limitations to this method are the weak differences recorded between the experimental and control groups in the sucrose preference test and the irreversible traumatic outcome of the forced swim test. These may be worth considering for suitability before any future application of the protocol. Nonetheless, following the experiment, na&#239;ve rats developed contagion depression after 5 weeks of sharing the same cage with the CUS-exposed rats.

This protocol describes a new model by which healthy rats could contract depression over a given time periodthrough contagion by exposure to chronic unpredictable stressed (CUS) rats.

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is ...

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...

A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.

A comprehensive behavioral test battery of motor skills, mood&#8212;including social interaction, depression, and anxiety&#8212;and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.