We thank Professor Olena Severynovska of the Department of Physiology, Faculty of Biology, Ecology, and Medicine, Oles Honchar Dnipro University, Dnipro, Ukraine for her support and helpful contributions to our discussions. The data obtained are part of R.K.&#8217;s PhD thesis.

The Animal Care Committee of the Ben-Gurion University of the Negev, Israel approved all treatment and testing procedures used in this protocol.
1. Preparation of Rats for the Experimental Procedure
NOTE: Select adult male Sprague-Dawley rats weighing 300-350 g.
<ol>
	<li>House the rats, four per cage, in a vivarium at 22 &#176;C and 40% humidity, with a reversed 12 h light/dark cycle (lights off at 8:00 a.m.) and unlimited access to food and water.</li>
	<li>Randomly assign the rats to two groups, namely an MCAO group (N = 24) and a Sham group (N = 19).</li>
</ol>
2. Preparation of Rats for Surgery
<ol>
	<li>To prepare the rats for the modified MCAO procedure, anesthetize each rat for 30 min with a mixture of isoflurane (4% for induction, 2% for surgery, and 1.3% for maintenance) in 24% oxygen (2 L/min) in an induction chamber and allow it to breathe spontaneously29,30. 
	NOTE:&#160;Stimulate pedal withdrawal reflex by pinching the skin between the toes and/or toe pads using&#160;blunt forceps. Consider the depth of anesthesia adequate when the reflex disappears.</li>
	<li>Maintain the core body temperature at 37 &#176;C with a heating plate.</li>
	<li>Measure the body temperature of all rats through a probe placed in the rat&#8217;s rectum before beginning the surgical procedure.</li>
	<li>Keep the body temperature constant (37 &#176;C) for all rats to minimize any hypothermic effect on the neurological outcome and any neurological injury.</li>
	<li>Apply artificial tears ointment to both of each animal&#8217;s eyes while they lie on the prep table.</li>
	<li>Shave the neck of each rat and disinfect the skin with 70% alcohol chlorhexidine (70% alcohol and 0.5% chlorhexidine gluconate). Repeat the disinfection step two more times.&#160;</li>
	<li>Cover the rats with sterilized surgical drapes.</li>
</ol>
3. Surgery (the MCAO Technique)
NOTE: Perform surgery as described by Boyko et al.28 and use the instruments provided by McGarry et al.29 and Ulu&#231; et al.30.
<ol>
	<li>Perform a ventral midline incision and dissect the superficial fascia.</li>
	<li>Carefully perform a sharp and blunt dissection within the three triangle-shaped muscle (the sternohyoid, the digastric, and the sternomastoid muscle) to identify the carotid arteries (the ECA, the ICA, and the common carotid artery [CCA]).</li>
	<li>Carefully dissect and expose the right CCA and the ICA.</li>
	<li>Separate the right CCA and ICA from the vagus nerve. 
	NOTE: Upon identifying the carotid arteries as stipulated in step 3.2, the ICA would be recognized as one-half of the CCA branches alongside the ECA.</li>
	<li>Insert a catheter (heat-blunted or silicone-coated 4-0 nylon) monofilament directly through the ICA, ~18.5-19 mm from the bifurcation point of the right CCA into the circle of Willis until reaching a mild resistance, to occlude MCA. 
	NOTE: The heat-blunted filament and the silicone-coated 4-0 nylon play the same role and are the more preferred monofilaments in recent times, given that they provide better occlusion than the plain nylon thread28,30.</li>
	<li>Tie a 4-0 silk suture around the ICA just above the right CCA (pterygopalatine artery) bifurcation to block the ICA proximal to the filament insertion point permanently and distal to it temporarily.</li>
	<li>Tie the ICA around the intraluminal thread to fasten the silk suture to prevent bleeding.</li>
	<li>Subject the sham-operated rats to the same surgical procedure as the MCAO rats but insert a nylon thread instead28. 
	NOTE: The nylon thread, like the silicon-coated nylon, occludes the ICA, but it is not as effective as the latter.</li>
	<li>Close the wound on the rat&#8217;s neck after occluding the MCA, turn off the anesthesia, and place the rat in an incubator under observation until it wakes up. 
	NOTE: The need for the proximal ligation is to occlude the ICA, and that of the additional distal ligation is to reduce the bleeding around the filament and secure it in place. Also, the rat should wake up a few minutes after its wound has been closed.</li>
</ol>
4. Post-surgical Recovery
<ol>
	<li>Administer 5 mL of 0.9% saline solution to each rat intraperitoneally, immediately after surgery, to prevent dehydration.</li>
	<li>Administer analgesia unconditionally on the first day post-operation; use meloxicam 1 mg/kg SQ q24 h.&#160;Give diluted dipyrone (0.5 g dissolved in 400 mL of drinking water) to rats showing symptoms of pain during the first 3 days.</li>
	<li>Sacrifice any rats with seizures (seizures are caused by the increased intracranial pressure from cerebral edema or cerebral hemorrhage28).</li>
</ol>
5. Neurological Severity Score31
NOTE: This procedure is performed by two observers who do not take part in the surgical proceedings; they test the neurological deficits and grade motor deficits on a cumulative score of 0-432. The evaluation of this score may be performed at different time intervals; in this investigation, it was performed 50 min, 24 h, 7, 15, and 30 days post-surgery. Find below the steps for evaluating the NSS. Although not a necessity in this situation, this score is required to ascertain stroke in rodents in order to administer treatment.
<ol>
	<li>Place the rat on a ceramic floor and allow it to move about freely for 1 min.</li>
	<li>Gently pull the rat backward by the tail and grade as follows.
	<ol>
		<li>Grade the rat with score 0 for no neurological deficit.</li>
		<li>Grade the rat with score 1 for forelimb flexion.</li>
		<li>Grade the rat with score 2 for contralateral weak forelimb grip.</li>
		<li>Grade the rat with score 3 for circling to the paretic side when pulled by the tail.</li>
		<li>Grade the rat with score 4 for spontaneous circling32. 
		NOTE: If more than one of the reactions are observed, preference is given to the action with a higher score.</li>
	</ol>
	</li>
</ol>
6. Determination of the Infarct Volume (Hhistologic Examination)
<ol>
	<li>Measurement of the infarct volume 
	NOTE: Perform this procedure as described previously33,34. Measure brain infarct volume using 2,3,5-triphenyltetrazolium chloride (TTC) staining 24 h after reperfusion.
	<ol>
		<li>Euthanize five rats from each group, 24 h after the last NSS, by exposing them to an isoflurane overdose in an induction chamber.</li>
		<li>Decapitate the rats and quickly isolate their brains using small scissors and forceps.</li>
		<li>Wash the isolated brains in 0.9% saline.</li>
		<li>Examine bleeding points on the brains to exclude the mice that underwent subarachnoid hemorrhage at the circle of Willis.</li>
		<li>Place each brain on a clean glass slide on a -20 &#176;C ice pack and then place them in a -20 &#176;C fridge for 5 min to make the brain easier to slice.</li>
		<li>Take the glass slide with the brain on it out of the -20 &#176;C fridge, put it back on the -20 &#176;C ice pack, and dissect the frontal pole and the cerebellum with blade and forceps.</li>
		<li>Slice the brain sections horizontally into 2 mm thickness with a blade to produce six slices.</li>
		<li>Prepare a 0.05% TTC solution by adding 1.25 g of TTC powder to 500 mL of normal saline solution prior to sacrifice, transfer the solution to a 24-well plate (1 mL per well) covered in foil, and store it at 4 &#176;C. 
		NOTE: TTC and tissue stained with TTC are light sensitive.</li>
		<li>Using forceps, transfer the brain slices to the 24-well plate containing the TTC solution (one slice per well) and stretch out the slices in the solution.</li>
		<li>Incubate the plate content at 37 &#176;C in a shallow water bath for 30 min.</li>
		<li>Aspirate the TTC solution from the plate with a pipette, wash them with a brainwashing fluid, and incubate at room temperature for 30 min.</li>
		<li>Place the slices in the order in which they were cut on a laboratory glass and examine the segments with a scanner.</li>
		<li>Analyze the infarct size as a percentage of the whole brain slice, using ImageJ analysis software, based on visual identification.</li>
	</ol>
	</li>
	<li>Analysis of the infarct volume35
	<ol>
		<li>Quantify the infarct volume by using a standard image analysis software (ImageJ) and analyze the infarct brain volume as a percentage of the whole brain size33.</li>
		<li>Place the brain slices on glass microscope slides and scan them with an optical scanner at a high resolution (1,600 x 1,600 dpi) for an adequate analysis.</li>
		<li>Crop the images and standardize the scale for all images, using the metric ruler included in the scanned image.</li>
		<li>Measure the area of marked pallor in six consecutive 2 mm coronal sections using a Wand (tracing) tool and freehand selection on the ImageJ 1.37v software36.</li>
		<li>Calculate the indirect infarct volume using the following formula37.</li>
	</ol>
	</li>
</ol>
7. Measurement of Brain Edema38
NOTE: Brain edema was measured 24 h after the last MCAO.&#160;&#160;Euthanize animals with severe neurological deficit interfering with eating and/or drinking for at least three days, more than 20% weight loss, hemiplegia or seizures.
<ol>
	<li>Assess the magnitude of the edema of the right hemisphere by using the summation of coronally sliced areas to calculate the volumes of the right and left hemispheres in arbitrary units (pixels). 
	NOTE: Use the ImageJ 1.37v software for this calculation, after optical scanning (resolution: 1,600 x 1,600 dpi). Select the area of interest and use the Measure function from the Analysis menu. Macros were used in our investigation.</li>
	<li>Express the brain edema area as a percentage of the standard areas in the unaffected contralateral hemisphere.</li>
	<li>Calculate the degree of swelling using the equation developed previously39.</li>
</ol>
8. Behavioral Paradigms
<ol>
	<li>Perform behavioral tests between days 30 and 33 after surgery in a closed, quiet, and light-controlled room.</li>
	<li>Assign investigators blinded to all experimental procedures to videotape all behavioral tests with a commercially available program.</li>
	<li>Sucrose preference test40,41
	<ol>
		<li>Place the rats in individual cages in the same room as where they are housed during the dark cycle.</li>
		<li>For the next 24 h, place one bottle of 100 mL of 1% (w/v) sucrose solution in each cage with the rat to be tested and allow for the adaptation of the rat.</li>
		<li>After 24 h, deprive the rats of food and water for 12 h by removing the bottles.</li>
		<li>Then after 12 h, place two bottles in each cage for 4 h, one containing 100 mL of tap water and another containing 100 mL of sucrose solution (1% [w/v]).</li>
		<li>Record the amount of sucrose solution and water consumed by rats in milliliters. Calculate the affinity to sucrose preference as follows.</li>
	</ol>
	</li>
	<li>Porsolt forced swim test42 
	NOTE: The Porsolt forced swim test was carried out as described in a previous protocol published by Zeldetz et al.40 and Boyko et al.42. The principle of this test states that, when rats are forced to swim in a restricted area from whence, they cannot escape, they eventually become immobile, ceasing any attempts to escape the water43,44. This test was carried out in a different room during the dark cycle.
	<ol>
		<li>Place each rat in a vertical plexiglass cylinder (height: 100 cm; diameter: 40 cm) containing 80 cm of water at 25 &#176;C for 15 min for habituation.</li>
		<li>Take the rat out and allow it to dry for 15 min in a heated enclosure (32 &#176;C).</li>
		<li>Return the rat to its home (original) cage.</li>
		<li>Repeat step 8.4.1 24 h later, this time for the investigation, for 5 min.</li>
		<li>Videotape the 5 min test and calculate the total duration of immobility during that period.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

One of the ways in which the MCAO technique presented here could be deemed safer than the original MCAO model is illustrated by the fact that the ECA and its branches, including the occipital artery, the terminal lingual, and the maxillary artery, are not compromised when occluding the MCA via the ICA. The original MCAO model&#39;s offset of the ECA (and its branches), by distally dissecting and coagulating them46, causes impaired mastication, owing to a compromise to the vascular supply to mastic...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/58875">A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats</a>. The Authors section was updated.
One of the author names was updated from:
Dmitri Frank
to
Dmitry Frank

An erratum was issued for:&#160;<a href="https://www.jove.com/t/58875">A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats</a>. The Authors section was updated.

Erratum: A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

Stroke is fourth on the list of death-perpetrating diseases in the United States1,2,3, while it causes the majority of disabilities in adults in developed countries4; this makes stroke a leading contender among the world&#39;s most significant health issues. Normalcy in stroke-surviving patients is rare, with about 15%-40% of survival victims suffering permanent disability, 20% requiring institutional care 3 months after stroke onset5, and about a third of 6-month survivals needing others to help them live through each day6. Stroke reportedly also accounts for the rising national health expenditures7. Estimates from the American Heart Association has stroke-related costs in the United States at over $50 billion in 20108.
Not only does stroke cause individuals&#39; long-term damages, but some survivors tend to suffer emotional and behavioral disorders, such as dementia, fatigue, anxiety, depression, delirium, and aggression9,10,11,12,13,14. The most recurrent psychological sequel after a stroke is post-stroke depression (PSD), diagnosed in about 40%-50% of survivals15,16,17. Stroke-induced depression results in increased morbidity and mortality18,19,20,21,22. The pathophysiology of PSD is not known completely, but it is apparently caused by multiple factors and is linked to disability, cognitive impairment, and lesion site23.
The rat model of focal brain ischemia, created by MCAO, is the most widespread animal model of stroke24,25,26,27. In demonstrating the induction of PSD in rats by occluding the MCA via the ICA, techniques that minimize mortality and variability in the MCAO model are employed28.
The primary objective of this protocol is to outline the steps for inducing PSD in rats by occluding the MCA via the ICA, a modified model of MCAO, which reduces mortality and the outcome of variability28. Specific aims include performing neurological and histological examinations (determining the neurological severity score [NSS], the volume of the infarction zone, and brain edema) to verify the efficacy of MCAO and using behavioral tests to examine the influence of this MCAO procedure on the development of emotional disorders, mainly PSD.

<table border="0" cellpadding="0" cellspacing="0" style="width:572px;" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Absorbent pad</td>
			<td style="width:71px;">-</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Black lusterless perspex box</td>
			<td style="width:71px;">-</td>
			<td style="width:119px;">-</td>
			<td>(120 cm &#215; 60 cm &#215; 60 cm), divided into a 25% central zone and the surrounding border zone</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bottles</td>
			<td style="width:71px;">Techniplast</td>
			<td style="width:119px;">ACBT0262SU</td>
			<td>150 ml bottles filled with 100 ml of water and 100 ml 1%(w/v) sucrose solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electric Shock</td>
			<td>Heat System Ultasonic Inc.</td>
			<td style="width:119px;">-</td>
			<td>-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Horizon-XL</td>
			<td colspan="2">Mennen Medical Ltd</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Imaging System</td>
			<td style="width:71px;">Kodak</td>
			<td style="width:119px;">-</td>
			<td>For imaging and quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Monofilament</td>
			<td style="width:71px;">-</td>
			<td style="width:119px;">-</td>
			<td>-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paper towels</td>
			<td style="width:71px;">Pharmacy</td>
			<td style="width:119px;">-</td>
			<td>Dry towels used for keeping rats dry after immersing them in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pexiglass cylinder</td>
			<td style="width:71px;">-</td>
			<td style="width:119px;">-</td>
			<td>a 100 cm tall and 40 cm in diameter cylinder used for carrying out the forced swim test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Purina Chow</td>
			<td style="width:71px;">Purina</td>
			<td align="right" style="width:119px;">5001</td>
			<td>Rodent laboratory chow given to rats, mice and hamster is a life-cycle nutrition that has been used in biomedical researc for over 5 decades. Provided to rats ad libitum in this experiment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rat Cages</td>
			<td style="width:71px;">Techniplast</td>
			<td style="width:119px;">2000P</td>
			<td>Conventional housing for rodents. Was used for housing rats throughout the experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scanner&#160;</td>
			<td>Canon CanoScan</td>
			<td style="width:119px;">4200F</td>
			<td>-</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Video Camera</td>
			<td style="width:71px;">ETHO-VISION (Noldus)</td>
			<td style="width:119px;">-</td>
			<td>Digital video camera for high definition recording of rat behavior under open field test</td>
		</tr>
	</tbody>
</table>

a middle cerebral artery occlusion technique for inducing post-stroke depression in rats

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of all ischemic stroke patients suffer from PSD, a disorder considered to be an ischemic stroke-related precursor for increased death and degradation in health. The pathophysiology of PSD is still obscure. To study the mechanism of development and occurrence of PSD further, and to find out a therapy, we attempted to develop a new protocol that requires occluding the middle cerebral artery (MCA) via the internal carotid artery (ICA) in rats. This protocol describes a model of PSD induced in rats through the middle cerebral artery occlusion (MCAO). Also used in the experiment are the Porsolt forced swim test and the sucrose preference test to confirm and evaluate the depressive mood of the rats under investigation. Rather than inserting the catheter through the external carotid artery (ECA), as stipulated for the original procedure, this MCAO technique has the monofilament passing directly through the ICA. This MCAO technique was developed a few years ago and leads to a reduction in mortality and variability. It is generally accepted that the criteria used are preferred in the selection of biological models. The data obtained with this protocol show that this model of MCAO could be a way of inducing PSD in rats and could potentially lead to the understanding of the pathophysiology and the future development of new drugs and other neuroprotective agents.

Histological findings (Table 1) revealed a statistically significant infarct volume as a percentage of total brain (p &#60; 0.0001) post-MCAO when compared to animals in the sham-control group. Also reported was a statistically significant brain edema when the evaluation from the experimental group (p &#60; 0.0003) was put side by side with that of the sham-control group.
The NSS scores obtaine...

Watch this Scientific Journal Video about A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats at JoVE.com

A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

Here we present a protocol to induce post-stroke depression in rats by occluding the middle cerebral artery via the internal carotid artery. We use the Porsolt forced swim test and the sucrose preference test to confirm and evaluate induced depressive moods.

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of ...

a-middle-cerebral-artery-occlusion-technique-for-inducing-post-stroke

Research

JoVE Journal

Behavior

10.1K Views.  Ben-Gurion University of the Negev. Here we present a protocol to induce post-stroke depression in rats by occluding the middle cerebral artery via the internal carotid artery. We use the Porsolt forced swim test and the sucrose preference test to confirm and evaluate induced depressive moods.

Video: A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals and humans. However, many animal models of depression were developed in males and are not applicable or effective in studies of postpartum females. Recent studies have reported significant effects of chronic social stress during lactation, an ethologically relevant and effective stressor, on maternal behavior, growth, and behavioral neuroendocrinology. This manuscript will describe this chronic social stress paradigm using repeated exposure of a lactating dam to a novel male intruder, and the assessment of the behavioral, physiological, and neuroendocrine effects of this model. Chronic social stress (CSS) is a valuable model for studying the effects of stress on the behavior and physiology of the dam as well as her offspring and future generations. The exposure of pups to CSS can also be used as an early life stress that has long term effects on behavior, physiology, and neuroendocrinology.

This article describes the use of chronic resident intruder social stress as an ethologically relevant paradigm to model postpartum depression and anxiety in lactating rodents.

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals ...

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

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

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

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

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

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 ...

Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, few studies have investigated the long-term effects of injury in rodent models of TBI. Shown here are behavioral measures that are well-established in TBI research for times early after injury, such as two weeks, until two months. Some of these methods have previously been used at later times after injury, up to one year, but by very few laboratories. The methods demonstrated here are a short neurological assessment to test reflexes, a Beam-Balance to test balance, a Beam-Walk to test balance and motor coordination, and a working memory version of the Morris water maze that can be sensitive to deficits in reference memory. Male rats were handled and pre-trained to neurological, balance, and motor coordination tests prior to receiving parasagittal fluid percussion injury (FPI) or sham injury. Rats can be tested on the short neurological assessment (neuroscore), the beam-balance, and the Beam-Walk multiple times, while testing on the water maze can only be done once. This difference is because rats can remember the task, thus confounding the results if repeated testing is attempted in the same animal. When testing from one to three days after injury, significant differences are detected in all three non-cognitive tasks. However, differences in the Beam-Walk task were not detectable at later time points (after 3 months). Deficits were detected at 3 months in the Beam-Balance and at 6 months in the neuroscore. Deficits in working memory were detected out to 12 months after injury, and a deficit in a reference memory first appeared at 12 months. Thus, standard behavioral tests can be useful measures of persistent behavioral deficits after FPI.

The goal of the behavioral tests presented here is to detect functional deficits in rats after traumatic brain injury. Four specific tests are presented that detect deficits in behaviors to reflect the damage to specific brain areas at times extending to one year after injury.

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, ...

Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For example, in the cat, visual information about an impending obstacle can modulate stepping for avoidance. Locomotor adaptation can also occur independent of vision, as sudden tactile inputs to the leg by an expected obstacle can modify the stepping of all four legs for avoidance. Such complex locomotor coordination involves supraspinal structures, such as the parietal cortex. This protocol describes the use of reversible, cooling-induced cortical deactivation to assess parietal cortex contributions to memory-guided obstacle locomotion in the cat. Small cooling loops, known as cryoloops, are specially shaped to deactivate discrete regions of interest to assess their contributions to an overt behavior. Such methods have been used to elucidate the role of parietal area 5 in memory-guided obstacle avoidance in the cat.

Complex locomotion in naturalistic environments requiring careful coordination of the limbs involves regions of the parietal cortex. The following protocol describes the use of reversible cooling-induced deactivation to demonstrate the role of parietal area 5 in memory-guided obstacle avoidance in the walking cat.

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For ...

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

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

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

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

Tickling, a Technique for Inducing Positive Affect When Handling Rats

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk for both the animals and the hands of their handlers, decreased animal welfare, and less valid research data. To minimize negative effects on experimental results and human-animal relationships, research animals are often habituated to being handled. However, the methods of habituation are highly variable and often of limited effectiveness. More potently, it is possible for humans to mimic aspects of the animals&#39; playful rough-and-tumble behavior during handling. When applied to laboratory rats in a systematic manner, this playful handling, referred to as tickling, consistently gives rise to positive behavioral responses. This article provides a detailed description of a standardized rat tickling technique. This method can contribute to future investigations into positive affective states in animals, make it easier to handle rats for common husbandry activities such as cage changing or medical/research procedures such as injection, and be implemented as a source of social enrichment. It is concluded that this method can be used to efficiently and practicably reduce rats&#39; fearfulness of humans and improve their welfare, as well as reliably model positive affective states.

This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection.

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk ...

Testing for Metacognitive Responding Using an Odor-based Delayed Match-to-Sample Test in Rats

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, numerous behavioral tasks have been created, many of which utilize an option to decline memory tests. To assess metamemory in rats, we utilized this decline-test option paradigm by adapting previous visual delayed-match-to-sample tests (DMTS)1,2 developed for primate species to an odor-based test suitable for rodents. First,&#160;rats are given a sample to remember by digging in a cup of scented sand. After a delay, the rat is presented with four distinctly scented cups, one of which contains the identical scent experienced during the sample; if this matching cup is selected, then the rat obtains a preferred, larger reward. Selection of any of the other three non-matching sand-filled scented cups results in no reward. Retention intervals are individually titrated such that subjects perform between 40 and 70% correct, therefore ensuring rats sometimes remember and sometimes forget the sample. Here, the operational definition of metamemory is the ability to distinguish between the presence and absence of memory through behavioral responding. Towards this end, on two-thirds of trials, a decline option is presented in addition to the four choice cups (choice trials). If the decline-test option- an unscented colored sand cup, is selected, the subject receives a smaller less-preferred reward and avoids the memory test. On the remaining third of trials, the decline-test option is not available (forced trials), causing subjects to guess the correct cup when the sample is forgotten. On choice tests, subjects that know when they remember should select the decline option when memory is weak rather than take the test and choose incorrectly. Therefore, significantly higher performance on chosen tests as compared to forced memory tests is indicative of the adaptive use of the decline-test response and metacognitive responding.

This protocol describes a method for investigating the possibility of metamemory, or memory awareness, in rodents. The odor-based delayed-matching-to-sample paradigm is a novel, ecologically-relevant behavioral test useful for determining the extent to which rodents can adaptively respond based on cognitively monitoring the strength of their memory states.

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, ...