Name: manipulation of epileptiform electrocorticograms (ecogs) and sleep in rats and mice by acupuncture
Uploaded: 2016-12-22T13:00:00
Description: Watch this Scientific Journal Video about Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture at JoVE.com

This work was supported by Ministry of Science and Technology (MOST) grants MOST104-2410-H-002-053 &amp; NSC99-2320-B-002-026-MY3. This manuscript was edited and proofread by Mr. Brian Chang, who has experience revising professional documents.

All experimental protocols are approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University.
1. Stereotaxic Surgery for Implanting ECoG Electrodes, EMG Electrodes, Brain Thermistor, and Injection Guide Cannula
<ol>
	<li>For rats (250 - 350 g, 6- to 8-week-old Sprague-Dawley rats)
	<ol>
		<li>Anesthetize the rats by IP injection with 50 mg/kg zoletil. Confirm the proper depth of anesthesia by observing a lack of response after pinching the...

<ol>
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Choosing a feasible epilepsy animal model is essential for each experimental purpose. One of our goals is to elucidate the effects of EA on epilepsy suppression. EA is an alternative medicine that may exhibit therapeutic effect in epilepsy and has been documented in ancient Chinese literature. However, there is a lack of scientific evidence to prove it. To determine the effects of EA on epilepsy, we primarily focused on the effects of EA on mild focal epilepsy, rather than on severe generalized seizure or SE. Our previou...

Epilepsy is a common neurological disorder in which recurrent seizures occur throughout a patient&#39;s lifespan. Most epileptic recurrences can be well-controlled by anti-epileptic drugs (AEDs). However, about 30% of epileptic patients develop refractory epilepsy1. Epilepsy causes sleep disturbances, which can further exacerbate recurrence. Evidence demonstrates that epilepsy may either disrupt sleep at night or may cause excessive daytime sleepiness2,3. Our previous studies further indicate that epilepsy occurring at zeitgeber time (ZT) 0, i.e., the beginning of the light period in the light:dark cycle, decreases sleep; this is mediated by corticotropin-releasing hormone (CRH), a homeostatic factor. Epilepsy at ZT13 (the beginning of the dark period) enhances the expression of another homeostatic factor, interleukin-1 (IL-1), which increases sleep. Sleep circadian rhythms are altered when epilepsy occurs at ZT6, the middle of the light period4,5. On the other hand, sleep problems further exacerbate the progression and recurrence of epilepsy6. Based on the aforementioned evidence, we try to reveal an optimal therapeutic method to simultaneously control epilepsy and prevent sleep disruptions in epilepsy patients. We previously found that electroacupuncture (EA) with a 10-Hz stimulation frequency, in which a certain amount of current is delivered into the acupoint through a stainless-steel needle, successfully suppresses electrocorticogram (ECoG) epileptic activities and epilepsy-induced sleep disturbances7. EA with a 100-Hz stimulation frequency further deteriorates epileptic activities and sleep disruptions in rats8,9. This successful experiment depends on three factors: firstly, a feasible epileptic animal model; secondly, a method for sleep recording and analysis in rodents; and thirdly, the accurate performance of acupuncture and the accuracy of the acupoint locations.
Epilepsy has been categorized into two major types: focal epilepsy and generalized epilepsy. We are interested in focal temporal lobe epilepsy (TLE), generalized epilepsy, status epilepticus (SE), and the recurrence of spontaneous generalized epilepsy. Therefore, different manipulations are applied to create suitable epileptic models for our experiments. To establish focal TLE, a low dose of pilocarpine is administered into the left central nucleus of the amygdala (CeA). To verify this model, six ECoG electrodes are implanted on the frontal (F1 &#38; F2), parietal (P1 &#38; P2), and occipital (O1 &#38; O2) lobes in both the left and right hemispheres, and another two reference electrodes (R1 &#38; R2) are placed over the cerebellum in both hemispheres. An additional microinjection guide cannula is surgically implanted into the left CeA (AP, 2.8 mm from bregma; ML, 4.2 mm; DV, 7.8 mm relative to bregma). The coordinates are adapted from the Paxinos and Watson rat atlas10. If the focal TLE is successfully induced, only the recording from the electrode on left parietal cortex (P1), which is near the left CeA, should acquire the dominant epileptiform ECoGs, with no significant epileptiform ECoGs recorded from the other ECoG electrodes. Intraperitoneal (IP) injections of pilocarpine into rats induce generalized epilepsy and SE, but this can be fatal. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induce spontaneous generalized epilepsy in mice and also ensure the mice&#39;s survival. Two wire ECoG electrodes are implanted into the frontal and parietal cortices in the mice to receive ECoG signals and to verify spontaneously recurrent epilepsy.
Polysomnography (PSG) is a comprehensive method to record physiological changes that occur during sleep, and it can objectively classify sleep into different stages of non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. PSG records parameters of body functions, including brain waves (electroencephalogram, EEG), eye movements (electrooculogram, EOG), skeletal muscle tones (electromyogram, EMG), heart rhythms (electrocardiogram, ECG), and blood oxygen levels and respiratory parameters. In rats, we record ECoGs, EMGs, cortical temperature, and locomotor activity to classify vigilance states into wakefulness, NREM sleep, and REM sleep. Sleep analysis in mice is conducted using ECoGs, EMGs, and locomotor activity results. Rats are surgically implanted with three ECoG screw electrodes at the frontal, parietal, and contralateral cerebellar cortices by stereotaxic surgery. Post-acquisition determination of the vigilance states (wakefulness, NREM sleep, and REM sleep) is conducted according to the parameters acquired from the ECoGs, EMG, brain temperature, and locomotor activity. Detailed criteria for categorizing the animal&#39;s behavior in both rats and mice are described in the protocol.
Both rats and mice need to be anesthetized with a low dose of zoletil (25 mg/kg), which is half the dosage of anesthetics normally administered during stereotaxic surgery, before performing manual acupuncture or EA. This dosage allows animals to wake up 30 to 35 min after the injection. Either manual acupuncture or EA is performed at the beginning of the dark period, with a constant time period of 30 min, and each animal is consecutively treated for two to three days. Stimulating EA currents are delivered into a particular acupoint through a stainless-steel needle that is inserted into the acupoint. The stimulus current is a train of biphasic square pulses, in which the pulse duration is 150 ms and the stimulation intensity is 1 mA. If a dry needle is used for manual acupuncture, the needle inserted into the acupoints is twitched 10 times every 5 min. The difficult part of manual acupuncture or EA is to localize the acupoints in rodents. The location of acupoints in rats or mice is similar to their anatomical location in humans. For example, the bilateral Fengchi acupoints are located 3 mm away from the posterior median line on the neck, between the two ears, which is similar to its anatomical location in humans11. Furthermore, the acupoints with low impedance on the skin can be further confirmed. Sham acupuncture or sham EA manipulation is necessary for acupuncture or EA experiments. Sham acupuncture or sham EA should be performed at a non-acupoint located close to the acupoint, such as near the axilla12.
To successfully investigate the effects of acupuncture or EA on epilepsy and epilepsy-induced sleep disruptions, the following factors must be in place: a feasible epileptic animal model, the precise analysis of epileptiform ECoGs and the recurrence of epilepsy, a method to classify vigilance states, and the accurate performance of acupuncture or EA in rodents.

<table border="0" cellpadding="0" cellspacing="0" width="751">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Drugs</td>
			<td style="width:277px;"></td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zoletil</td>
			<td>Virbac</td>
			<td></td>
			<td>50 mg/kg i.p.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pilocarpine</td>
			<td>Sigma-Aldrich</td>
			<td>P6503</td>
			<td>300 mg/kg i.p.; 1.2 mg microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PTZ</td>
			<td>Sigma-Aldrich</td>
			<td>P6500</td>
			<td>0.035 mg/mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polysporin</td>
			<td>Pfizer</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgery</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECoG electrode</td>
			<td>Plastics One</td>
			<td>E363/20</td>
			<td>screw electrode for rats</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pedestal</td>
			<td>Plastics One</td>
			<td>MS363</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cannula</td>
			<td>Plastics One</td>
			<td>C315G/spc</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermistor</td>
			<td>Omega Engineering</td>
			<td align="right">44008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dental acrylic</td>
			<td>Tempron</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereotaxic Instrument</td>
			<td>Stoelting</td>
			<td></td>
			<td>Dural arms</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recording equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECoG amplifier</td>
			<td>Colbourn Instruments</td>
			<td>V75-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A/D Board</td>
			<td>National Instruments</td>
			<td>NI PCI-6033E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Infrared-based motion detectors</td>
			<td>Biobserve GmbH</td>
			<td>custom-made</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ICELUS</td>
			<td>G-System</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AxoScope 10 Software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acupuncture needs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless needles</td>
			<td>Shanghai Yanglong Medical Articles Co.</td>
			<td>32 gauge x 1&#8221;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Functions Electrical Stimulator</td>
			<td>I.T.O., Japan</td>
			<td>Trio 300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AcuPen</td>
			<td>Lhasa OMS</td>
			<td>Pointer Excel II</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

There are different rat and mouse models to satisfy the needs of different epilepsy types. To induce focal TLE, 0.5 &#181;L of pilocarpine (2.4 mg/&#181;L) is administered into the left CeA. The predominant epileptiform ECoGs are acquired from the ECoG electrode on the parietal lobe of the left hemisphere (Figure 1A: b), and rare epileptic activities are picked up from the rest of the ECoG electrodes (Figure 1A: a, c, <st...

Watch this Scientific Journal Video about Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture at JoVE.com

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.

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 ...

The overall goal of this procedure is to assess epilepsy, sleep and the effects of acupuncture in an animal model. This method can help answer key questions in neuroscience field about how to establish epilepsy, analyze sleep and perform acupuncture in a small animal model. The main advantages of this technique are that the lateral cortical and the lateral myogram electrodes in that semester can be implanted quickly and easily by stereotaxic surgery.Demonstrating this procedure will be Yi-Jou Wu, a graduate student from my laboratories. After confirming the proper depth of sedation by a lack of response to a toe pinch, apply eye ointment to a six-to eight-week-old 250 to 350 gram Spregg Dolly rat from the first group. Use clippers to remove the hair from the top of the skull.Disinfect the skin with sequential applications of povidone iodine solution and 75 percent ethanol, followed by antibiotic injection to prevent infection. Next, mount the animal onto a stereotaxis machine and then place an ear bar into the ear canal. Using a scalpel, make an approximately two centimeter midline caudal incision along the skull between the ears.Then use hemostats to clip the skin flaps, exposing the skull, and use a scalpel to remove the tissue. When the skull is clean, use a rotary tool to drill eight approximately 0.7 millimeter diameter holes into the frontal, parietal, and occipital lobes and the cerebellum in both the left and right hemispheres of the exposed bone. Then screw eight electrocorticogram electrodes into the holes for focal epilepsy detection.In a separate group of rats, drill three holes and place two screw electroencephalogram electrodes over the right frontal and parietal lobes of the cortices with the same coordinates as in the first group. Next, place a third electroencephalogram electrode over the left cerebellum to ground the animal and reduce the signal artifacts. Separate the neck skin and muscle and insert two electromyogram electrodes into the neck muscle.Drill another hole on the skull and place a microinjection guide canula into the left central nucleus of the amygdala. Then, drill a 1.6 millimeter hole in the skull and insert a calibrated 30 kilovolt thermoster on the surface of the parietal cortex to monitor the cortical temperature. Route the insulated electrocortico and electromyogram electrode leads to a pedestal.Using dental acrylic to cement the pedestal, guide the canula to the skull and then connect the thermoster to the tether. Then, treat the incision topically with polysporin to prevent infection and give the animals water supplemented with ibuprofin and penicillin G for one week after surgery. To induce focal temporal lobe epilepsy, use a microinjection pump to administer 0.5 microliters of pilocarpine into the left central nucleus of the amygdala through the injection guide canula at 0.2 microliters per minute.To induce generalized epilepsy with recurrent status epilepticus, inject 300 milligrams per kilogram of pilocarpine IP.For acupuncture, first manually locate the acupoints. For example, the feng chi acupoints are located three millimeters away from the posterior median line between the ears on the neck. After confirming a low skin impedance at the acupoints of interest, insert stainless steel needles into the acupoints at a two millimeter depth.Then, using a functional electrical stimulator, deliver a 10 hertz train of 150 millisecond byphasic pulses with a one milliamp intensity to the acupoints through the needle. Here, a predominant epileptiform electrocorticogram acquired from the electrocorticogram electrode on the parietal lobe of the left hemisphere is shown. After pilocarpine administration, rare epileptic activities are picked up from the rest of the electrocorticogram electrodes, indicating a successful focal temporal lobe epilepsy induction.Within five minutes, severe cholinergic effects on behavior occur, gradually increasing in severity until an electrocorticogram generalized seizure occurs followed by a status epilepticus event. No epileptiform electrocorticograms are observed during the dark period after the first pentilene tetrasol injection, or during the second dark period on an off-day for injection until the fifth pentilene tetrasol injection, along with spontaneously and recurrently generalized epilepsy. Wakefulness exhibits a high amplitude electromyogram and an abundance of locomotor activity, with a gradual increase in the cortical temperature as the vigilant state transits from either non-REM or REM sleep to wakefulness.Non-REM sleep is characterized by synchronized electrocorticograms with a high amplitude and low frequency, with a gradual electromyogram amplitude decline and no locomotor activity, as well as a decrease in the cortical temperature as the vigilant state transits from wakefulness into NREM sleep. During REM sleep, the electrocorticogram wave is desynchronized with a reduced amplitude. The predominant electroencaphalogram power density occurs within the theta frequency.The electromyogram activity is the lowest, phasic body twitch is observed, and the cortical temperature rapidly increases. Administration of 100 hertz of electroacupuncture into the bilateral feng chi acupoints exacerbates the pilocarpine induced epileptiform electrocorticograms, while 10 hertz of electroacupuncture suppresses the epileptiform electrocorticograms. Further, pilocarpine administration into the left central nucleus of the amygdala also suppresses non-REM sleep during the light period of the 12-hour light/dark cycle.100 hertz of electroacupuncture further deteriorates the pilocarpine induced suppression of non-REM sleep. In contrast, a 10 hertz electroacupuncture into the bilateral feng chi acupoints increases non-REM sleep per se during the dark period, and blocks the pilocarpine-induced reduction of non-REM sleep during the light period. Once mastered, this technique can be completed in one and a half hours if it is performed properly.Before attempting this procedure, it is important to practice the skill of stereotaxic surgery until proficiency. After its development, this technique paves the way for the researchers in the field of neuroscience to explore the effects of acupuncture in epilepsy and sleep disturbance. After watching this video, you should have a good understanding of how to surgically implant electroencephalo and electromyogram electrodes, a guide canula, and a semester to assess sleep patterns and other effects of acupuncture on brain activity.

manipulation-epileptiform-electrocorticograms-ecogs-sleep-rats-mice

Research

JoVE Journal

Behavior

The Successive Alleys Test of Anxiety in Mice and Rats

The plus-maze was derived from the early work of Montgomery. He observed that rats tended to avoid the open arms of a maze, preferring the enclosed ones. Handley, Mithani and File et al. performed the first studies on the plus-maze design we use today, and in 1987 Lister published a design for use with mice. 
Time spent on, and entries into, the open arms are an index of anxiety; the lower these indices, the more anxious the mouse is. Alternatively, a mouse that spends most of its time in the closed arms is classed as anxious. 
One of the problems of the plus-maze is that, while time spent on, and entries into, the open arms is a fairly unambiguous measure of anxiety, time in the central area is more difficult to interpret, although time spent here has been classified as &#x201C;decision making&#x201D;. In many tests central area time is a considerable part of the total test time.
Shepherd et al. produced an ingenious design to eliminate the central area, which they called the &#x201C;zero maze&#x201D;. However, although used by several groups, it has never been as widely adopted as the plus-maze. 
In the present article I describe a modification of the plus-maze design that not only eliminates the central area but also incorporates elements from other anxiety tests, such as the light-dark box and emergence tests. It is a linear series of four alleys, each having increasing anxiogenic properties. It has given similar results to the plus-maze in general. Although it may not be more sensitive than the plus-maze (more data is needed before a firm conclusion can be reached on this point), it provides a useful confirmation of plus-maze results which would be useful when, for example, only a single example of a mutant mouse was available, as, for example, in ENU-based mutagenesis programs.

The plus-maze measures anxiety-like behaviour in rodents. There are two opposite closed and two opposite open arms; anxious rodents avoid the open arms. The central area is neither completely open nor closed, so time spent here is ambiguous and difficult to interpret. Here a modification of the plus-maze protocol eliminating this area is described.

The plus-maze was derived from the early work of Montgomery. He observed that rats tended to avoid the open arms of a maze, preferring the enclosed ones.  ...

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 ...

Visualizing Motion Patterns in Acupuncture Manipulation

Acupuncture manipulation varies widely among practitioners in clinical settings, and it is difficult to teach novice students how to perform acupuncture manipulation techniques skillfully. The Acupuncture Manipulation Education System (AMES) is an open source software system designed to enhance acupuncture manipulation skills using visual feedback. Using a phantom acupoint and motion sensor, our method for acupuncture manipulation training provides visual feedback regarding the actual movement of the student's acupuncture manipulation in addition to the optimal or intended movement, regardless of whether the manipulation skill is lifting, thrusting, or rotating. Our results show that students could enhance their manipulation skills by training using this method. This video shows the process of manufacturing phantom acupoints and discusses several issues that may require the attention of individuals interested in creating phantom acupoints or operating this system.

Here, we present a protocol for using the Acupuncture Manipulation Education System (AMES) in the training of acupuncture manipulation skills using phantom acupoints.

Acupuncture manipulation varies widely among practitioners in clinical settings, and it is difficult to teach novice students how to perform acupuncture ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

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 ...

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.

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 ...

Chronic Sleep Deprivation in Mouse Pups by Means of Gentle Handling

Sleep is critical for proper development and neural plasticity. Moreover, abnormal sleep patterns are characteristic of many neurodevelopmental disorders. Studying how chronic sleep restriction during development can affect adult behavior may add to our understanding of the emergence of behavioral symptoms of neurodevelopmental disorders. While there are many methods that can be used to restrict sleep in rodents including forced locomotion, constant disruption, presentation of an aversive stimulus, or electric shock, many of these methods are very stressful and cannot be used in neonatal mice. Here, we describe gentle handling, a sleep deprivation technique that can be used chronically throughout development and into adulthood to achieve sleep restriction. Gentle handling involves close observation of the mice throughout the sleep deprivation period and requires the researcher to gently prod the animals whenever they are inactive or display behaviors associated with sleep. Coupled with EEG recordings, gentle handling could be used to selectively disrupt a specific phase of sleep such as rapid eye movement (REM) sleep. The technique of gentle handling is a powerful tool for the study of the effects of chronic sleep restriction even in neonatal mice that circumvents many of the more stressful procedures used for sleep deprivation.

We describe a sleep deprivation technique known as gentle handling where investigators gently prod mice any time sleep behavior is observed. This method is a powerful tool that allows researchers to study the effects that chronic sleep restriction throughout development can have on future brain physiology and behavior.

Sleep is critical for proper development and neural plasticity. Moreover, abnormal sleep patterns are characteristic of many neurodevelopmental disorders. ...

Simultaneous Recordings of Cortical Local Field Potentials and Electrocorticograms in Response to Nociceptive Laser Stimuli from Freely Moving Rats

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and human studies to investigate the cortical processing of nociceptive information. These laser-evoked brain potentials (LEPs) consist of several transient responses that are time-locked to the onset of laser stimuli. However, the functional properties of the LEP responses are still largely unknown, due to the lack of a sampling technique that can simultaneously record neural activities at the surface of the cortex (i.e., electrocorticogram [ECoG] and scalp electroencephalogram [scalp EEG]) and inside the brain (i.e., local field potential [LFP]). To address this issue, we present here an animal protocol using freely moving rats. This protocol is composed of three main procedures: (1) animal preparation and surgical procedures, (2) a simultaneous recording of ECoG and LFP in response to nociceptive laser stimuli, and (3) data analysis and feature extraction. Specifically, with the help of a 3D-printed protective shell, both ECoG and LFP electrodes implanted on the rat&#39;s skull were securely held together. During data collection, laser pulses were delivered on the rat&#39;s forepaws through gaps in the bottom of the chamber when the animal was in spontaneous stillness. Ongoing white noise was played to avoid the activation of the auditory system by the laser-generated ultrasounds. As a consequence, only nociceptive responses were selectively recorded. Using the standard analytical procedures (e.g., band-pass filtering, epoch extraction, and baseline correction) to extract stimulus-related brain responses, we obtained results showing that LEPs with a high signal-to-noise ratio were simultaneously recorded from ECoG and LFP electrodes. This methodology makes the simultaneous recording of ECoG and LFP activities possible, which provides a bridge of electrocortical signals at the mesoscopic and macroscopic levels, thereby facilitating the investigation of nociceptive information processing in the brain.

We developed a technique that simultaneously records both electrocorticography and local field potentials in response to nociceptive laser stimuli from freely moving rats. This technique helps establish a direct relationship of electrocortical signals at the mesoscopic and macroscopic levels, which facilitates the investigation of nociceptive information processing in the brain.

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and ...

Automated Measurements of Sleep and Locomotor Activity in Mexican Cavefish

Across phyla, sleep is characterized by highly conserved behavioral characteristics that include elevated arousal threshold, rebound following sleep deprivation, and consolidated periods of behavioral immobility. The Mexican cavefish, Astyanax mexicanus (A. mexicanus), is a model for studying trait evolution in response to environmental perturbation. A. mexicanus exist as in eyed surface-dwelling forms and multiple blind cave-dwelling populations that have robust morphological and behavioral differences. Sleep loss has occurred in multiple, independently-evolved cavefish populations. This protocol describes a methodology for quantifying sleep and locomotor activity in A. mexicanus cave and surface fish. A cost-effective video monitoring system allows for behavioral imaging of individually-housed larval or adult fish for periods of a week or longer. The system can be applied to fish aged 4 days post fertilization through adulthood. The approach can also be adapted for measuring the effects of social interactions on sleep by recording multiple fish in a single arena. Following behavioral recordings, data is analyzed using automated tracking software and sleep analysis is processed using customized scripts that quantify multiple sleep variables including duration, bout length, and bout number. This system can be applied to measure sleep, circadian behavior, and locomotor activity in almost any fish species including zebrafish and sticklebacks.

This protocol details the methodology for quantifying locomotor behavior and sleep in the Mexican cavefish. Previous analyses are extended to measure these behaviors in socially-housed fish. This system can be widely applied to study sleep and activity in other fish species.

Across phyla, sleep is characterized by highly conserved behavioral characteristics that include elevated arousal threshold, rebound following sleep ...

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 ...