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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</ol>

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><tr><td>ReproSil-Pur C18 column (4 mm x 150 mm)</td><td>Dr. Maisch GmbH</td><td></td><td></td></tr><tr><td>EZ Clips</td><td>Stoelting Co.</td><td>59022</td><td></td></tr><tr><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&quot;, BOX of 12</td></tr><tr><td>Absorbable Sutures</td><td>Ethicon</td><td>J310H</td><td>4-0 Coated Vicryl Violet 1X27&#039;&#039; SH-1</td></tr><tr><td>Dental Cement</td><td>Stoelting Co.</td><td>51458</td><td></td></tr><tr><td>Drill Bit</td><td>Stoelting Co.</td><td>514551</td><td>0.45 mm</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Alliance HPLC system&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>E2695 separation module</td><td>Waters</td><td>176269503</td><td></td></tr><tr><td>2475 fluorescence detector</td><td>Waters</td><td>186247500</td><td></td></tr><tr><td>post-column reagent manager</td><td>Waters</td><td>725000556</td><td></td></tr><tr><td>Lenovo computer</td><td>Waters</td><td>668000249</td><td></td></tr><tr><td>Empower software</td><td>Waters</td><td>176706100</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Passive avoidance box for rat&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Extra tall MDF sound attenuating cubicle</td><td>MedAssociates</td><td>ENV-018MD</td><td>Interior: 22&quot; (W) x 22&quot; (H) x 16&quot; (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&quot; 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&#039; 3-Pin mini-molex extension</td><td>MedAssociates</td><td>SG-216A-2</td><td></td></tr><tr><td>10&#039; 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&#039;, 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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Sleep-Wake Monitoring Equipment&lt;/strong&gt;</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&quot; 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><td></td></tr><tr><td>Network switch</td><td>Data Sciences International (DSI)</td><td>SG200-08P</td><td></td></tr><tr><td>Neuroscore software</td><td>Data Sciences International (DSI)</td><td>271-0171-CFG</td><td></td></tr><tr><td>Two biopotential channels transmitter, model TL11M2-F40-EET</td><td>Data Sciences International (DSI)</td><td>270-0134-001</td><td></td></tr></tbody></table>

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

10.5K Views.  University of Maryland School of Medicine. Wijzigingen in de kynurenine traject (KP) neuroactive metabolieten zijn betrokken bij de psychiatrische ziekten. Onderzoek naar de functionele uitkomsten van een gewijzigde kynurenine traject metabolisme in vivo bij knaagdieren kan helpen nieuwe therapeutische benaderingen toelichten. Het huidige protocol combineert biochemische en gedragsmatige benaderingen voor het onderzoek naar de gevolgen van een acute kynurenine uitdaging bij ratten.

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

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 ...

Noninvasive, High-throughput Determination of Sleep Duration in Rodents

Traditionally, sleep is monitored by an electroencephalogram (EEG). EEG studies in rodents require surgical implantation of the electrodes followed by a long recovery period. To perform an EEG recording, the animal is connected to a receiver, creating an unnatural tether to the head-mount. EEG monitoring is time consuming, carries risk to the animal, and is not a completely natural setting for the measurement of sleep. Alternative methods to detect sleep, particularly in a high-throughput fashion, would greatly advance the field of sleep research. Here, we describe a validated method for detecting sleep via activity-based home-cage monitoring. Previous studies have shown that sleep assessed via this method has a high degree of agreement with sleep defined by traditional EEG-based measures. Whereas this method is validated for total sleep time, it is important to note that sleep bout duration should be assessed by an EEG which has better temporal resolution. The EEG can also differentiate rapid eye movement (REM) and non-REM sleep, giving more detail about the exact nature of sleep. Nevertheless, activity-based sleep determination can be used to analyze multiple days of undisturbed sleep and to assess sleep as a response to an acute event (like stress). Here, we show the power of this system to detect the response of mice to daily intraperitoneal injections.

We describe a high-throughput method of measuring sleep by means of activity-based home-cage monitoring. This method offers advantages over traditional EEG-based methods. It is well validated for the determination of total sleep duration and can be a powerful tool to monitor sleep in rodent models of human disease.

Traditionally, sleep is monitored by an electroencephalogram (EEG). EEG studies in rodents require surgical implantation of the electrodes followed by a ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Neuroscience

Collecting Sleep, Circadian, Fatigue, and Performance Data in Complex Operational Environments

Sleep loss and circadian misalignment contribute to a meaningful proportion of operational accidents and incidents. Countermeasures and work scheduling designs aimed at mitigating fatigue are typically evaluated in controlled laboratory environments, but the effectiveness of translating such strategies to operational environments can be challenging to assess. This manuscript summarizes an approach for collecting sleep, circadian, fatigue, and performance data in a complex operational environment. We studied 44 airline pilots over 34 days while they flew a fixed schedule, which included a baseline data collection with 5 days of mid-morning flights, four early flights, four high-workload mid-day flights, and four late flights that landed after midnight. Each work block was separated by 3&#8211;4 days of rest. To assess sleep, participants wore a wrist-worn research-validated activity monitor continuously and completed daily sleep diaries. To assess the circadian phase, pilots were asked to collect all urine produced in four or eight hourly bins during the 24 h after each duty block for the assessment of 6-sulfatoxymelatonin (aMT6s), which is a biomarker of the circadian rhythm. To assess subjective fatigue and objective performance, participants were provided with a touch-screen device used to complete the Samn-Perelli Fatigue Scale and Psychomotor Vigilance Task (PVT) during and after each flight, and at waketime, mid-day, and bedtime. Using these methods, it was found that sleep duration was reduced during early starts and late finishes relative to baseline. Circadian phase shifted according to duty schedule, but there was a wide range in the aMT6s peak between individuals on each schedule. PVT performance was worse on the early, high-workload, and late schedules relative to baseline. Overall, the combination of these methods was practical and effective for assessing the influence of sleep loss and circadian phase on fatigue and performance in a complex operational environment.

Sleep loss and circadian misalignment contribute to numerous operational accidents and incidents. The effectiveness of countermeasures and work scheduling designs aimed at mitigating fatigue can be challenging to evaluate in operational environments. This manuscript summarizes an approach for collecting sleep, circadian, fatigue, and performance data in complex operational environments.

Sleep loss and circadian misalignment contribute to a meaningful proportion of operational accidents and incidents. Countermeasures and work scheduling ...

Human Circadian Phenotyping and Diurnal Performance Testing in the Real World

In our continuously developing &#39;around the clock&#39; society, there is a need to increase our understanding of how changes in biology, physiology and psychology influence our health and performance. Embedded within this challenge, is the increasing need to account for individual differences in sleep and circadian rhythms, as well as to explore the impact of time of day on performance in the real world. There are a number of ways to measure sleep and circadian rhythms from subjective questionnaire-based methods to objective sleep/wake monitoring, actigraphy and analysis of biological samples. This paper proposes a protocol that combines multiple techniques to categorize individuals into Early, Intermediate or Late circadian phenotype groups (ECPs/ICPs/LCPs) and recommends how to conduct diurnal performance testing in the field. Representative results show large differences in rest-activity patterns derived from actigraphy, circadian phase (dim light melatonin onset and peak time of cortisol awakening response) between circadian phenotypes. In addition, significant differences in diurnal performance rhythms between ECPs and LCPs emphasizes the need to account for circadian phenotype. In summary, despite the difficulties in controlling influencing factors, this protocol allows a real-world assessment of the impact of circadian phenotype on performance. This paper presents a simple method to assess circadian phenotype in the field and supports the need to consider time of day when designing performance studies.

Here, we present a method to investigate diurnal rhythms in performance following accurate categorization of participants into circadian phenotype groups based on the Munich ChronoType Questionnaire, gold standard circadian phase biomarkers and actigraphic measures.

In our continuously developing &#39;around the clock&#39; society, there is a need to increase our understanding of how changes in biology, physiology and ...

Advancing Sleep Disorder Treatments: A Drosophila-Based Screening Approach

High-Throughput Small Molecule Drug Screening For Age-Related Sleep Disorders Using Drosophila melanogaster

Sleep, an essential component of health and overall well-being, often presents challenges for older individuals who frequently experience sleep disorders characterized by shortened sleep duration and fragmented patterns. These sleep disruptions also correlate with an increased risk of various illnesses in the elderly, including diabetes, cardiovascular diseases, and psychological disorders. Unfortunately, existing drugs for sleep disorders are associated with significant side effects such as cognitive impairment and addiction. Consequently, the development of new, safer, and more effective sleep disorder medications is urgently needed. However, the high cost and lengthy experimental duration of current drug screening methods remain limiting factors.
This protocol describes a cost-effective and high-throughput screening method that utilizes Drosophila melanogaster, a species with a highly conserved sleep regulation mechanism compared to mammals, making it an ideal model for studying sleep disorders in the elderly. By administering various small compounds to aged flies, we can assess their effects on sleep disorders. The sleep behaviors of these flies are recorded using an infrared monitoring device and analyzed with the open-source data package Sleep and Circadian Analysis MATLAB Program 2020 (SCAMP2020). This protocol offers a low-cost, reproducible, and efficient screening approach for sleep regulation. Fruit flies, due to their short life cycle, low husbandry cost, and ease of handling, serve as excellent subjects for this method. As an illustration, Reserpine, one of the tested drugs, demonstrated the ability to promote sleep duration in elderly flies, highlighting the effectiveness of this protocol.

Author Spotlight: Overcoming Challenges in Drosophila Sleep Measurement Using DAM System 

Presented is a protocol for high-throughput drug screening to improve sleep by monitoring the sleep behavior of fruit flies in an elderly Drosophila model.

Sleep, an essential component of health and overall well-being, often presents challenges for older individuals who frequently experience sleep disorders ...

Polygraphic Recording Procedure for Measuring Sleep in Mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Bioengineering

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

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

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