We thank Professor Olena Severynovska; Maryna Kuscheriava M.Sc; Maksym Kryvonosov M.Sc; Daryna Yakumenko M.Sc; Evgenia Goncharyk M.Sc; and Olha Shapoval, PhD candidate at the Department of Physiology, Faculty of Biology, Ecology, and Medicine, Oles Honchar Dnipro University, Dnipro, Ukraine for their supportive and useful contributions. The data was obtained as part of Dmitry Frank&#39;s PhD dissertation.

The experiments were performed following the recommendations of the Declarations of Helsinki and Tokyo and the Guidelines for the Use of Experimental Animals of the European Community. The experiments were approved by the Animal Care Committee of Ben-Gurion University of the Negev. A protocol timeline is illustrated in Figure 1.
1. Surgical procedures and fluid percussion TBI
<ol>
	<li>Select male and female adult Sprague-Dawley rats, housed at a room temperature of 22 &#177; 1 &#176;C, and humidity of 40%-60%, with 12-12 h light-dark cycles.</li>
	<li>Provide food as chow and water ad libitum. Perform experiments between morning hours, i.e., 6:00 a.m. and 12:00 p.m.</li>
	<li>Perform a baseline neurological assessment for both the control and TBI groups prior to the start of the experiment (see section 2 below).</li>
	<li>Anesthetize the rats with inhaled 4% isoflurane for induction and 1.5% for maintenance of anesthesia. Ensure that rat is immobilized by testing pedal reflex or movement in response to an irritant. 
	&#8203;NOTE: Use a continuous isoflurane administration system for anesthesia. Perform all procedures in aseptic conditions.</li>
	<li>Perform parasagittal fluid-percussion injury as previously described21,22.</li>
	<li>Subcutaneously inject 0.2 mL of 0.5% bupivacaine along the prospective incision site, prior to incision. ransfer the rat to the recovery room and continue monitoring the neurological (e.g., paralysis), respiratory (e.g., respiratory arrest) and cardiovascular state (e.g., decreases in soft tissue perfusion, changes in color of pupils, and bradycardia) for 24 h. Prior to emergence from anesthesia, administer 0.01 - 0.05 mg/kg intramuscular buprenorphine as postoperative analgesia. Repeat doses every 6 - 12 h for at least 48 h.</li>
</ol>
2. Evaluation of Neurological Severity Score (NSS)
NOTE: Assessment of neurological deficit was performed and graded using an NSS, as previously described23,24. The maximum score of alteration in motor function and behavior is 24 points. A score of 0 indicates an intact neurological status and 24 indicates severe neurological dysfunction, as previously described24.
<ol>
	<li>Test the rat&#39;s inability to leave a circle (50 cm in diameter) when placed in its center. Perform this task three times, with each session lasting 30 min, 60 min, and more than 60 min each. 
	NOTE: If picking up rats by the tail, hold the base of the tail.</li>
	<li>Test the rat for a loss of righting reflex.
	<ol>
		<li>Place the animal on its back in the palm of the researcher&#39;s hand. Give a score of 1 if the animal is able to right itself25 (standing on all four paws).</li>
	</ol>
	</li>
	<li>Test the rat for hemiplegia, the inability of the rat to resist forced positioning.</li>
	<li>Raise the rat by its tail to test the reflexive bending of the hindlimb.</li>
	<li>Put the rat on the floor to test its ability to walk straight.</li>
	<li>Perform testing for three reflexive behaviors: the pinna reflex, the corneal reflex, and the startle reflex.
	<ol>
		<li>For the pinna reflex, perform light tactile stimulation to test ear retraction as previously described25.</li>
		<li>To test the corneal reflex, monitor blink response when applying a soft stick lightly to the eye and measure on a scale of 0 (no response) to triple eye blink (3), as previously described25.</li>
		<li>For the startle reflex, drag a pen across the top of the wire cage and record response with a scale from 0 (no response) to 3 (1 cm jump or more), as previously described25.</li>
	</ol>
	</li>
	<li>Grade the rat based on loss of seeking behavior and prostration (not moving their whiskers, sniffing, or running after being transferred to a new environment)24.</li>
	<li>Test limb reflexes for the placement on the left and right forelimbs, and then the left and right hindlimbs.</li>
	<li>Analyze functionality via the beam balancing task with a beam that is 1.5 cm wide. Perform the test for sessions lasting 20 seconds, 40 seconds, and more than 60 seconds.</li>
	<li>Run the beam walking test with three different beams: 8.5 cm wide, 5 cm wide, and 2.5 cm wide.</li>
</ol>
3. Preparing for the metric task
<ol>
	<li>Equipment
	<ol>
		<li>Place a black circular platform 200 cm in diameter and 1 cm thick on a table. The height of the table should be 80 cm above the floor.</li>
		<li>Establish two different objects in the center of circular platform 68 cm away from each other. 
		NOTE: In this experiment, two glass bottles were used for objects, one round bottle with a height of 13.5 cm and another faceted bottle with a height of 20 cm. Fill bottles with water to ensure stability.</li>
		<li>Prepare a camera and install the required computer software for capturing, saving, and processing data. Install the camera at a height of 290 cm from the floor. 
		NOTE: The distance between the platform and camera depends on the camera specifications. The camera frame should cover the entire area of the arena in which the test is being conducted. The distance for our experiment between the platform and the camera was 210 cm.</li>
	</ol>
	</li>
	<li>Habituation
	<ol>
		<li>On the day before the task, habituate the rat to the new environment for 10 min by placing on the arena without video recording. 
		NOTE: Do not perform the neurological tasks and the metric task on the same day.&#160; 
		NOTE: Perform metric tests in a red light area.</li>
	</ol>
	</li>
</ol>
4. Performing the metric task
NOTE: Performing the metric task consists of two periods: 1) habituation (15 min) and 2) test (5 min) period.
<ol>
	<li>Habituation period
	<ol>
		<li>Establish two different objects in the center of the circular platform 68 cm away from each other.</li>
		<li>Place the rat on the end of the platform equidistant from the objects for a 15 min period, and record the video.</li>
		<li>Remove the rat from the platform and place in an individual cage for 5 min.</li>
		<li>Clean the platform with 5%-10% alcohol. 
		&#8203;NOTE: Up to 70% alcohol may be used to clean the platform in well-ventilated areas.</li>
	</ol>
	</li>
	<li>Test period
	<ol>
		<li>Reduce the distance between objects to 34 cm.</li>
		<li>Place the rat on the platform for 5 min and record the rat&#39;s exploration activity on video.</li>
		<li>Clean the platform with 5%-10% alcohol.</li>
	</ol>
	</li>
</ol>
5. Data analysis
NOTE: Data analysis is performed by video tracking software specifically designed for animal behavior studies that automatically records animal activity and movement (see Table of Materials). This software automates a range of behavioral variables, including mobility, activity, and explorative behavior.
<ol>
	<li>Prior to analyzing the video files, insert the software hardware key. Start the video tracking software and open preset Template.</li>
	<li>In the Setup section, verify settings as follows: Arena, Trial Control, and Detecting Settings (see Figure 2a) . 
	NOTE: For this experiment, parameters for the exploration area are defined as 6 cm around the object of interest. The time the rat entered into this area was measured.</li>
	<li>After verifying the settings, duplicate and rename them.</li>
	<li>On the general screen of the program, Grab Background by right clicking on the mouse.</li>
	<li>Select a video file for the background image. In the Browse menu, select the location of the video file.</li>
	<li>Capture the image and mark the investigated areas and zones, calibrating the image for analysis. Perform the same steps for Trial Control and Detecting Settings.</li>
	<li>In the general menu, select Trial List and download the list of video files for analysis.</li>
	<li>Add the videos and indicate the location with the required settings.</li>
	<li>Select acquisition and Start Trial (see Figure 2b,c). Export all data as Excel files (see Figure 2d). 
	NOTE: Perform all calculations for the habituation and test periods. Metric task assessment is prepared with an advanced template.</li>
</ol>

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The authors have nothing to disclose.

By specifically targeting the metric spatial information process, this metric test provides a necessary tool toward understanding memory deficiency after TBI. The protocol presented in this paper is a modification of previously described behavioral tasks11. One previously described metric task used two different paradigms, each consisting of three habituation sessions and one testing session. The first paradigm consisted of moving the familiar objects closer together after habituation and the seco...

The prevalence of neurological deficits such as attention, executive function, and certain memory deficits after moderate traumatic brain injury (TBI) is more than 50 percent1,2,3,4,5,6,7,8. TBI can lead to severe impairments in spatial short-term, long-term, and working memory9. These memory impairments have been observed in rodent models of TBI. Rodent models have enabled the development of techniques to test memory, allowing for deeper examinations into the effect of TBI on memory processing in neural memory systems.
Two tests, related to topological and metric spatial information processing respectively, assist with measuring spatial working memory (SWM). The topological test depends on changing the size of environmental space or related spaces of connection or enclosure around an object, while the metric test assesses changes in angles or distance between objects10,11. Goodrich-Hunsaker et al. first adapted the human topological test for rats10 and applied the metric task to dissociate the roles of the parietal cortex (PC) and dorsal hippocampus in spatial information processing11. Similarly, Gurkoff and colleagues evaluated metric, topological, and temporal ordering memory tasks after lateral fluid percussion injury9. There is a correlation between damage to certain regions of the brain and impairment of metric or topological memory. It has been suggested that metric memory impairment is related to lesions in bilateral dorsal dentate gyrus and cornu ammonis (CA) sub-region CA3 of the hippocampus, and that topological memory impairment is related to bilateral parietal cortex lesions10,12.
The purpose of this protocol is to assess spatial memory deficit in a rat population via a metric task. This method is a suitable alternative to investigate mechanisms of SWM after brain injury, and its advantages include the relative ease of implementation, high sensitivity, low cost of reproducibility, the possibility of dynamic observation, and a low stress environment. Compared to other behavioral tasks such as the Barnes maze13,14, Morris water navigation task15,16,17, or spatial maze tasks18,19, this metric test is less complicated. Due to its ease of implementation, the metric test requires a shorter and less stressful training period and takes place over only 2 days9: 1 day for habituation and 1 day for the task. Moreover, our proposed test is easier to perform than other low stress tests, such as the novel object recognition (NOR) task, and does not require the extra day of habituation20.
This paper provides a straightforward model for evaluating SWM after brain injury. This assessment of post-TBI SWM may assist in a more comprehensive investigation of its pathophysiology.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2% chlorhexidine in 70% alcohol solution</td>
			<td>SIGMA - ALDRICH</td>
			<td>500 cc</td>
			<td>For general antisepsis of the skin in the operatory field</td>
		</tr>
		<tr>
			<td>&#160;Bupivacaine 0.1 %</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">4 boards of different thicknesses (1.5cm, 2.5cm, 5cm and 8.5cm)</td>
			<td>This is to evaluate neurological defect</td>
		</tr>
		<tr>
			<td>4-0 Nylon suture</td>
			<td>4-00</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bottles</td>
			<td>Techniplast</td>
			<td>ACBT0262SU</td>
			<td>150 ml bottles filled with 100 ml of water and 100 ml 1%(w/v) sucrose solution</td>
		</tr>
		<tr>
			<td>Bottlses (four) for topological an metric tasks</td>
			<td></td>
			<td></td>
			<td>For objects used two little bottles, first round (height 13.5 cm) and second faceted (height 20 cm) shape and two big faceted bottles, first 9x6 cm (height 21 cm) and second 7x7 cm (height 21 cm).</td>
		</tr>
		<tr>
			<td>Diamond Hole Saw Drill 3mm diameter</td>
			<td></td>
			<td>Glass Hole Saw Kit</td>
			<td>Optional.&#160;</td>
		</tr>
		<tr>
			<td>Digital Weighing Scale</td>
			<td>SIGMA - ALDRICH</td>
			<td>Rs 4,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>SIGMA - ALDRICH</td>
			<td>Z265969</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 99.9 %</td>
			<td>&#160;Pharmacy</td>
			<td></td>
			<td>5%-10% solution used to clean equipment and remove odors</td>
		</tr>
		<tr>
			<td>EthoVision XT (Video software)</td>
			<td>Noldus, Wageningen, Netherlands</td>
			<td></td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Fluid-percussion device</td>
			<td>custom-made at the university workshop</td>
			<td></td>
			<td>&#160;&#160; No specific brand is recommended.</td>
		</tr>
		<tr>
			<td>Gauze Sponges</td>
			<td>Fisher</td>
			<td>22-362-178</td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves (thin laboratory gloves)</td>
			<td></td>
			<td></td>
			<td>Optional.</td>
		</tr>
		<tr>
			<td>Heater with thermometer</td>
			<td>Heatingpad-1</td>
			<td>Model: HEATINGPAD-1/2</td>
			<td>&#160;&#160; No specific brand is recommended.</td>
		</tr>
		<tr>
			<td>Horizon-XL</td>
			<td>Mennen Medical Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluran, USP 100%</td>
			<td>Piramamal Critical Care, Inc</td>
			<td>NDC 66794-017</td>
			<td>Anesthetic liquid for inhalation</td>
		</tr>
		<tr>
			<td>Office 365 ProPlus</td>
			<td>Microsoft</td>
			<td>-</td>
			<td>Microsoft Office Excel</td>
		</tr>
		<tr>
			<td>Olympus BX 40 microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating&#160; forceps</td>
			<td>SIGMA - ALDRICH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating&#160; Scissors</td>
			<td>SIGMA - ALDRICH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PC Computer for USV recording and data analyses</td>
			<td>Intel</td>
			<td></td>
			<td>Intel&#174; core i5-6500 CPU @ 3.2GHz, 16 GB RAM, 64-bit operating system</td>
		</tr>
		<tr>
			<td>Plexiglass boxes linked by a narrow passage</td>
			<td></td>
			<td></td>
			<td>Two transparent 30 cm &#215; 20 cm &#215; 20 cm plexiglass boxes linked by a narrow 15 cm &#215; 15 cm &#215; 60 cm passage</td>
		</tr>
		<tr>
			<td>Purina Chow</td>
			<td>Purina</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Rat cages&#160; (rat home cage or another enclosure)</td>
			<td>Techniplast</td>
			<td>2000P</td>
			<td>No specific brand is recommended</td>
		</tr>
		<tr>
			<td>Scalpel blades 11</td>
			<td>SIGMA - ALDRICH</td>
			<td>S2771</td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS</td>
			<td>SPSS Inc., Chicago, IL, USA</td>
			<td>&#160;20 package</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic Instrument</td>
			<td>custom-made at the university workshop</td>
			<td></td>
			<td>&#160;&#160; No specific brand is recommended</td>
		</tr>
		<tr>
			<td>Timing device</td>
			<td>Interval Timer:Timing for recording USV&#39;s</td>
			<td></td>
			<td>Optional. Any timer will do, although it is convenient to use an interval timer if you are tickling multiple rats</td>
		</tr>
		<tr>
			<td>Topological and metric tasks device</td>
			<td>Self made in Ben Gurion University of Negev</td>
			<td></td>
			<td>White circular platform 200 cm in diameter and 1 cm thick on table</td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td>Logitech</td>
			<td>C920 HD PRO WEBCAM</td>
			<td>Digital video camera for high definition recording of rat behavior under plus maze test</td>
		</tr>
		<tr>
			<td>Windows 10</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a metric test for assessing spatial working memory in adult rats following traumatic brain injury

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of human studies, animal models provide suitable alternatives to test treatment methods, and to study the mechanisms and related complications of the condition. Experimental rodent models have historically been the most widely used due to their accessibility, low cost, reproducibility, and validated approaches. A metric test, which tests the ability to recall the placement of two objects at various distances and angles from one another, is a technique to study impairment in spatial working memory (SWM) after TBI. The significant advantages of metric tasks include the possibility of dynamic observation, low cost, reproducibility, relative ease of implementation, and low stress environment. Here, we present a metric test protocol to measure impairment of SWM in adult rats after TBI. This test provides a feasible way to evaluate physiology and pathophysiology of brain function more effectively.

The significance of comparisons between groups was determined using the Mann-Whitney test. Statistical significance of results was considered at P &#60; 0.05, while statistically high relevance was measured at P &#60; 0.01.
The results showed no differences in NSS between all groups before intervention and 28 days after TBI. Each group consisted of 12 female or 12 male rats. The NSS scores obtained 48 h after TBI are presented in Table 1. Rats from the TBI group that showed si...

Watch this Scientific Journal Video about A Metric Test for Assessing Spatial Working Memory in Adult Rats Following Traumatic Brain Injury at JoVE.com

A Metric Test for Assessing Spatial Working Memory in Adult Rats Following Traumatic Brain Injury

Traumatic brain injury (TBI) is commonly associated with memory impairment. Here, we present a protocol to assess spatial working memory after TBI via a metric task. A metric test is a useful tool to study spatial working memory impairment after TBI.

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of ...

a-metric-test-for-assessing-spatial-working-memory-adult-rats

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

3.2K Views.  Soroka Medical Center, Ben-Gurion University of the Negev. Traumatic brain injury (TBI) is commonly associated with memory impairment. Here, we present a protocol to assess spatial working memory after TBI via a metric task. A metric test is a useful tool to study spatial working memory impairment after TBI.

Video: A Metric Test for Assessing Spatial Working Memory in Adult Rats Following Traumatic Brain Injury

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, interest in the cognitive processes of nonmodel species, typically within an ecological context, has also become an emerging field of study. In particular, interest in the cognitive processes in reptiles is growing although experimental studies on reptilian cognition are sparse. The few reptilian studies that have experimentally tested for spatial learning and memory have used rodent paradigms modified for use in reptiles. However, ecologically important aspects of the physiology and behavior of this taxonomic group must be taken into account when testing for spatially based cognition. Here, we describe modifications of the dry land Barnes maze and associated testing protocol that can improve performance when probing for spatial learning and memory ability in small squamate reptiles. The described paradigm and procedures were successfully used with male side-blotched lizards (Uta stansburiana), demonstrating that spatial learning and memory can be assessed in this taxonomic group with an ecologically relevant apparatus and protocol.

This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use in small squamate reptiles.

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

A Metric Test for Assessing Spatial Working Memory in Adult Rats Following Traumatic Brain Injury

Summary

Explore More Videos

Chapters in this video

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Assessing Spatial Learning and Memory in Small Squamate Reptiles

A Novel In Vitro Model of Blast Traumatic Brain Injury

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries