The authors wish to thank our funding agencies, which include NeuroDevNet (a National Centres of Excellence), the ALVA Foundation, the Women's and Children's Health Research Institute, and the University of Alberta.

The Animal Care and Use Committee, Health Sciences at the University of Alberta approved all animal studies.
Note: While this protocol may be adapted to other species and strains, this protocol is written for Long-Evans rats. These rats have been shown to have superior motor performances and visual acuity compared to other rodent strains. 25,26 The protocol for timed pregnancies, dietary supplementation, maternal inflammatio...

<ol>
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Neurodevelopmental reflex testing is a predictive measure of abnormal cortical development and maturation, which may be of significance under circumstances where overt neuropathology is not evident. During neurodevelopmental testing, it is critical to ensure that pups are examined at the same time daily. Rats are nocturnal and therefore, their circadian rhythm may alter performance if testing is performed at different times during the day.34 Testing should be completed in a quiet room as loud nois...

Neurodevelopmental reflexes, or developmental milestones, are one of the earliest assessments used on human newborns and infants. Neurological reflexes are involuntary and repetitive movements that demonstrate brain stem and spinal cord reflexes. Maturation of higher cortical networks characterized by evolving migration, myelination, and synaptogenesis promote voluntary control and cortical inhibition. Alterations in the normal progression of the central nervous system evolution can disrupt brain development, resulting in abnormal cortical wiring, functioning, and myelination, causing neurodevelopmental reflex delays or absences. Human infants at high risk for neurodevelopmental disability often display abnormal early reflexes. Abnormal reflexes can present as a delay in acquisition, absence, prolonged presence, or reappearance later in life, and are predictive of developmental disabilities.1,2 Therefore, it is important to mimic reflex delays in experimental models of neurodevelopmental disabilities.
Rodents are commonly employed as experimental models. Rat pups are altricial when born, and therefore too immature to undertake specific or complex motor, sensory and/or cognitive behavioral tasks. In this regard, their developmental immaturity relates to both their physical and organ development. Rats are born hairless with an inability to thermoregulate, are blind, and unable to walk. With reference to brain development, substantial cortical maturation occurs postnatally. Newborn rat pups (day of birth referred to as postnatal day 1; PD1) have been suggested to reach a brain maturation level that is similar to a preterm human brain of 23 - 28 weeks gestation, whereas PD7-10 pups are equivalent to near-term human brain. 3,4,5,6 This correlation is based on gross anatomical analyses, however, other measures of brain maturation such as myelination and amplitude integrated electroencephalograms have also been described. 5,7 For example, pre-oligodendrocytes are the predominant cells in the developing human fetal brain from 23 - 32 weeks in utero, and this maturational stage corresponds to a PD1-3 rodent. 5,8,9,10 Moreover, myelination begins in utero in humans whereas in rat pups it appears in the forebrain around PD7-10; the newborn rodent brain remains largely un-myelinated. 11,12 Tucker et al. found that the amplitude integrated electroencephalogram pattern of a P1 rat to be similar to a 23-week gestation human fetus, whereas a PD7 and PD10 pup is akin to a 30 - 32 week and term infant, respectively.7 For these reasons, newborn reflex testing in neonatal rat pups provides an opportunity for capturing the ontogeny and/or disruption of brain development.
The battery of reflexes described below are adapted from studies by W.M. Fox and A. Lubics13,14 W.M. Fox was one of the earliest investigators with respect to the ontogeny of reflexes in the mouse. 13 These reflexes include, but are not limited to, limb grasping and placing, cliff avoidance, righting, accelerated righting, gait, auditory startle, posture, and eye opening. Both forelimb and hindlimb grasp (referred to as palmar and plantar grasp in humans, respectively) are facilitated by spinal reflexes and corticospinal inhibition from non-primary motor areas. 15,16 Hindlimb placing (plantar reflex) reflects maturation of the corticospinal tract. 16,17,18 Cliff avoidance (protective responses), righting (labyrinth), and accelerated righting involve integration and communication between sensory input and motor output (such as those involved with the vibrissae and vestibular systems). 19,20,21 Gait reflects locomotion.14 Auditory startle assesses acoustic stimulation and synaptic connections of giant neurons in the nucleus reticularis pontis caudalis.21 Posture involves appropriate cortical-spinal/spinal-cortical projections, muscle strength, and neuromuscular innervation. 22,23 Maturation of the gamma-aminobutyric acid receptors may correlate with eye opening.24 It is important to keep in mind that the reflexes reflect a much more complicated network and provided here is a general correlation. Moreover, these reflexes provide a quick and easy method of assessing neurological development at very young ages where more complex behavioral testing is not feasible.
The objective of this paper is to provide a general guideline for neurodevelopmental reflex testing that can be easily incorporated into experimental neonatal rat studies. The methodology described was carried out in Long-Evans neonatal rat pups and quantification of the results was based on first day of appearance. The day that reflex testing is initiated and the equipment utilized may be modified to better suit a different experimental model (such as for different strains and species). By establishing the normal physiological progression of reflex maturation in a specific animal model, investigators can evaluate the effects of external stressors, endogenous manipulations, and/or therapeutic interventions on neurodevelopment in neonatal rat models. Overall, the use of reflexes as a determination of brain maturity is advantageous in predicting perinatal brain injury, and is reflective of later neurodevelopmental outcomes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Breeding</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Fisherbrand</td>
			<td>12-711-9AM</td>
			<td>Used for vaginal flushes.</td>
		</tr>
		<tr>
			<td>Sterile Saline</td>
			<td>Hospira</td>
			<td>7983254</td>
			<td>The solution used to collect cells during vaginal flushes.</td>
		</tr>
		<tr>
			<td>400 &#181;l Microcentrifuge tubes</td>
			<td>Fisherbrand</td>
			<td>05-408-120</td>
			<td>Used to hold the saline solution.</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Leica</td>
			<td>Leica ATC 2000</td>
			<td>For observation of the saline solution. Can be any light microscope used in the lab.</td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>Fisherbrand</td>
			<td>12-552-5</td>
			<td>The saline solution is placed on the slide. Can be any slides used in the lab.</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisherbrand</td>
			<td>12-545-F</td>
			<td>To coverslip the slides. Can use any coverslips used in the lab.</td>
		</tr>
		<tr>
			<td>Dietary&#160; Supplementation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Broccoli Sprouts seeds</td>
			<td>Mumm&#39;s Sprouting Seeds</td>
			<td></td>
			<td>Broccoli sprouts seeds are ordered and grown in the lab.</td>
		</tr>
		<tr>
			<td>Countertop Seed Sprouter Box</td>
			<td>Mumm&#39;s Sprouting Seeds</td>
			<td></td>
			<td>A box is used to germinate and grow the seeds prior to harvest.</td>
		</tr>
		<tr>
			<td>250 mL beaker</td>
			<td></td>
			<td></td>
			<td>The beaker is used to soak the seed. Any size beaker that would fit can be used.</td>
		</tr>
		<tr>
			<td>Maternal Inflammation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipoplysaccharide (LPS)</td>
			<td>Sigma</td>
			<td>L3129</td>
			<td>The endotoxin used to mimic maternal inflammation.</td>
		</tr>
		<tr>
			<td>1 mL Syringe</td>
			<td>BD Syringe</td>
			<td>309659</td>
			<td>Used to inject the pregnant rat.</td>
		</tr>
		<tr>
			<td>Gauge (30G X 1/2)</td>
			<td>BD PrecisionGlide Needle</td>
			<td>305106</td>
			<td>Use the smallest needle to avoid pain and discomfort.</td>
		</tr>
		<tr>
			<td>Sterile Saline (0.9% Sodium Chloride, USP)</td>
			<td>Hospira</td>
			<td></td>
			<td>Saline is used to dissolve LPS.</td>
		</tr>
		<tr>
			<td>Weights</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Denver Instrument</td>
			<td></td>
			<td>For recording the weights. Can be any scale with 2 decimal places used in the lab.</td>
		</tr>
		<tr>
			<td>Neurodevelopmental Reflexes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thin blunt rod</td>
			<td></td>
			<td></td>
			<td>Can be a paperclip or toothpick. This is for forelimb and hindlimb grasping.</td>
		</tr>
		<tr>
			<td>Round filter paper</td>
			<td>Whatman</td>
			<td>1001 150</td>
			<td>15 cm diameter paper used for gait analysis.</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>Fisher Scientific</td>
			<td>06-662-51</td>
			<td>For timing the time allocated to righting and gait.</td>
		</tr>
		<tr>
			<td>Blunt surface</td>
			<td></td>
			<td></td>
			<td>Can be an edge of a table. This is for hindlimb placing and cliff avoidance.</td>
		</tr>
		<tr>
			<td>Foam landing</td>
			<td></td>
			<td></td>
			<td>For when the pups perform accelerated righting.</td>
		</tr>
		<tr>
			<td>Video recorder</td>
			<td>Sony</td>
			<td>VCT-D580RM</td>
			<td>To record all reflexes tested. Must be able to record at 1/1000 fps</td>
		</tr>
		<tr>
			<td>Bell</td>
			<td></td>
			<td></td>
			<td>For auditory startle.&#160;</td>
		</tr>
		<tr>
			<td>Heat lamp or pad</td>
			<td></td>
			<td></td>
			<td>To maintain the body temperature of the pups underoing examination.</td>
		</tr>
	</tbody>
</table>

neurodevelopmental reflex testing in neonatal rat pups

Neurodevelopmental reflex testing is commonly used in clinical practice to assess the maturation of the nervous system. Neurodevelopmental reflexes are also referred to as primitive reflexes. They are sensitive and consistent with later outcomes. Abnormal reflexes are described as an absence, persistence, reappearance, or latency of reflexes, which are predictive indices of infants that are at high risk for neurodevelopmental disorders. Animal models of neurodevelopmental disabilities, such as cerebral palsy, often display aberrant developmental reflexes, as would be observed in human infants. The techniques described assess a variety of neurodevelopmental reflexes in neonatal rats. Neurodevelopmental reflex testing offers the investigator a testing method that is not otherwise available in such young animals. The methodology presented here aims to assist investigators in examining developmental milestones in neonatal rats as a method of detecting early-onset brain injury and/or determining the effectiveness of therapeutic interventions. The methodology presented here aims to provide a general guideline for investigators.

The timeline of this experimental design is presented in Figure 2. 30 The methods and results have previously been published. 30 The objective of the study was to assess whether dietary supplementation with broccoli sprouts during gestation and the preweaning period protected the offspring from neurodevelopmental delay induced by in utero exposure to LPS. Timed pregnant rats were given intraper...

Watch this Scientific Journal Video about Neurodevelopmental Reflex Testing in Neonatal Rat Pups at JoVE.com

Neurodevelopmental Reflex Testing in Neonatal Rat Pups

Behavioral testing is the gold standard for determining outcomes following brain injury, and can identify the presence of developmental disabilities in infants and children. Neurodevelopmental reflexes are an early indicator of these abnormalities. A host of easily accomplished developmental reflex tests in the neonatal rodent were developed and described here.

Neurodevelopmental reflex testing is commonly used in clinical practice to assess the maturation of the nervous system. Neurodevelopmental reflexes are ...

The overall goal of this neonatal rat reflex methodology is to learn how to perform these tasks quickly, accurately, and comparably. This method can answer key questions in the field of nerve developmental disabilities such as cerebral palsy. The main advantage of this technique is that it offers a way to test early developmental reflexes in newborn rat pups.Like humans, newborn rodents have early developmental reflexes that offer a window into later development. Individuals new to this method will struggle as the reflexes occur quickly and can be difficult to score. It is important to be as consistent as possible.The implications of this technique extend toward the therapy and diagnosis of cerebral palsy and developmental disabilities. Determination of reflex behaviors is the gold standard for the ascertainment and the efficacy of therapeutic interventions. Visual demonstration of this method is critical as the steps are difficult to learn through written words.To begin, acclimate the rats to the environment and to human handling as described in the accompanying text protocol. Once acclimated, mate the rats to produce a litter of pups for testing. Beginning on post-natal day three, transfer the pups to a quiet room at a consistent time each day.Let them acclimate for a minimum of one hour prior to performing an array of neurodevelopmental reflex tests. In the room, set up a heating lamp or heat pad to maintain the pup's body temperature at 36.5 degrees Celsius. Also, record the pup's weight each day.Next, set up a video camera with a shutter speed of at least 1/1000th of a second to analyze the reflex tests frame by frame. Position the camera so that it will be directly above or beside the rat pup and the testing materials during each test. For each of the following tests, perform the test each day until the pup is able to perform the task on two consecutive days.The date of the first occurrence is the date which should be recorded for data quantification. Beginning on postnatal day three, perform the forelimb grasp reflex test. For this test, place a blunt rod against the palm of each of the pup's forepaws, one at a time, and manually apply light pressure.The pressure should slightly displace the forepaw to ensure that contact is made, and the pups can feel the rod. Grasping will appear as flexion of all digits around the rod. Successful acquisition of this reflex occurs when both forepaws grasp the rod for two days in a row.Next, place a blunt rod against the sole of each hindpaw, and apply light pressure manually to conduct the hindlimb grasp reflex test. The light pressure should slightly displace the hindpaw to ensure that contact is made and the pups can feel the rod. Successful grasping of the rod appears as flexion of the digits around the rod.Acquisition of this reflex occurs when both hindpaws grasp the rod for two days in a row. Also beginning on postnatal day three, conduct the righting reflex test. For this test, firmly hold the pup in a supine position, so that all four paws are upright.Let go of the pup and immediately start the timer. Righting is achieved when the pup is able to flip or roll over on to all four paws and each paw is perpendicular to the body. Give each pup a maximum of 15 seconds to achieve this goal.Beginning on postnatal day four, perform the hindlimb placing reflex test. For this test, hold the pup vertically in the air by the torso. Gently stroke the dorsum of the hindpaw with a blunt surface, such as the edge of a table.A correct placing reflex is when the rat pup withdraws the stimulated hindlimb, followed by placement of the hindlimb down on that surface. Also beginning on postnatal day four, conduct the cliff avoidance test. For this test, place the rat pup at the edge of a flat surface, such that the forepaws and snout of the pup are over the edge.The correct outcome is a protective response where the rat pup turns away from the edge of the cliff. Begin the evaluation of gait on postnatal day six. For this test, place pups in the center of a 15 centimeter diameter circle and allow the pup 30 seconds to move off the circle.A successful gait is performed when the rat pup is able to move both forepaws outside the circle in less than 30 seconds. Begin the analysis of auditory startle on postnatal day 10. Here, something, such as a bell, that is capable of producing a loud noise, is required.Position the bell directly over the pup. Ring the bell, and then assess whether or not a startle response is present. A positive startle response is observed when the pup displays a jerking movement away from the sound.Starting on postnatal day 12, place the pups on a non-slippery open surface, and observe the posture the pup has when moving. An immature posture is reflected by dragging of the abdomen when moving, and perpendicular pointing of both forepaws and hindpaws relative to the body. A mature posture is acquired when the pup can lift the abdomen from the surface and both forepaws and hindpaws are pointed straight or parallel to the body when moving.Also starting on postnatal day 12, begin observing the pup's eyelids. Record the day both eyelids open. On postnatal day 14, begin the accelerated righting testing.For this test, place the pup in a supine position 30.48 centimeters above a foam landing pad. Drop the pup and observe its ability to right itself. Proper righting refers to when the pup is able to turn over to the prone position and land on its paws.Newborn pups exposed to in utero inflammation were born significantly smaller than controls. Dietary supplementation with broccoli sprouts was able to increase the birth weights of these pups. Along with birth weight, neurodevelopmental reflex testing is a predictive measure of abnormal cortical development and maturation, which may be of significance under circumstances where overt neuropathology is not evident.Following birth, examination of the neonatal reflex hindlimb placing in these groups showed a significant delay in the acquisition of this reflex for pups exposed to in utero inflammation compared to controls. Dietary supplementation with broccoli sprouts prevented the delay. The results of the neonatal reflex cliff avoidance test revealed that pups exposed to in utero inflammation are significantly delayed in acquiring a protective response.Again, broccoli sprouts consumption prevented significant delay. This same result was seen following examination of the neonatal reflex gait. During this procedure, other methods like daily weight recordings can be performed in order to answer additional questions like daily growth rate, catch-up growth, the severity of injury, and the effect of growth restriction.After its development, this technique paved the way for researchers in the field of neuroscience to explore developmental reflex testing in neonatal rodents. After watching this video, you should have a good understanding of how to perform early developmental reflex testing in newborn rat pups. Moreover, you should understand the significance of early behavioral reflexes as they provide an early biomarker of injury and the effect of therapeutic intervention.

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Research

JoVE Journal

Behavior

19.4K ビュー.  University of Alberta. The overall goal of this neonatal rat reflex methodology is to learn how to perform these tasks quickly, accurately, and comparably. This method can answer key questions in the field of nerve developmental disabilities such as cerebral palsy. The main advantage of this technique is that it offers a way to test early developmental reflexes in newborn rat pups.Like humans, newborn rodents have early developmental reflexes that offer a window into later development. Individuals new to thi...

ビデオ: Neurodevelopmental Reflex Testing in Neonatal Rat Pups

Behavioral testing is the gold standard for determining outcomes following brain injury, and can identify the presence of developmental disabilities in infants and children. Neurodevelopmental reflexes are an early indicator of these abnormalities. A host of easily accomplished developmental reflex tests in the neonatal rodent were developed and described here.

recording of induced seizures through electroencephalography in freely moving rat pups

The video demonstrates the recording of electroencephalogram (EEG) signals from seizure-induced rat pups. Take a freely moving pup implanted with electrodes connected to a data acquisition system. A neurotoxin is administered to induce epileptic seizures, and the resulting electrical bursting activity is monitored using the EEG...

Recording of Induced Seizures through Electroencephalography in Freely Moving Rat Pups

Next, on the data acquisition unit, select a sampling rate of at least 10,000 hertz for recording, and check for proper data transmission. Collect a baseline recording. After taking a baseline recording, inject the pup intraperitoneally with kainic acid to induce epileptic seizures. 15 minutes after the injection, observe and record the epileptic...

non-invasive model of neuropathogenic escherichia coli infection in the neonatal rat

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

isolation and cryopreservation of neonatal rat cardiomyocytes

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure.  This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each...

methods for in vivo biomechanical testing on brachial plexus in neonatal piglets

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Presented here are methods to perform in vivo biomechanical testing on brachial plexus in a neonatal piglet...

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

chronic sleep deprivation in mouse pups by means of gentle handling

Chronic Sleep Deprivation in Mouse Pups by Means of Gentle Handling

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...

three dimensional vestibular ocular reflex testing using a six degrees of freedom motion platform

Three Dimensional Vestibular Ocular Reflex Testing Using a Six Degrees of Freedom Motion Platform

A method is described to measure three-dimensional vestibulo ocular reflexes (3D VOR) in humans using a six degrees of freedom (6DF) motion simulator. The gain and misalignment of the 3D angular VOR provide a direct measure of the quality of vestibular function. Representative data on healthy subjects are...

terminal h-reflex measurements in mice

Terminal H-reflex Measurements in Mice

The clinical evaluation of spasticity based on the Hoffmann reflex (H-reflex) and using electrical stimulation of peripheral nerves is an established method. Here, we provide a protocol for a terminal and direct nerve stimulation for H-reflex quantification in the mouse...

rapid detection of neurodevelopmental phenotypes in human neural precursor cells (npcs)

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC...

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

probiotic studies in neonatal mice using gavage

Probiotic Studies in Neonatal Mice Using Gavage

This study details the process of gavaging precise amounts of probiotics to neonatal mice. The experimental set-up was optimized to include but is not limited to probiotic dosing, methods of administration, and quantification of bacteria in the intestines.

primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions.  This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream...

early pathological and magnetic resonance detection of cerebral injury using a rat model of neonatal hypoxic ischemic encephalopathy

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.

assessing cellular stress and inflammation in discrete oxytocin-secreting brain nuclei in the neonatal rat before and after first colostrum feeding

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte...

a neonatal heterotopic rat heart transplantation model for the study of endothelial-to-mesenchymal transition

Author Spotlight: A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition  

This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.

In Vivo Heart Transplantation to Study Endothelial-to-Mesenchymal Transition

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in vivo electrophysiological measurement of the rat ulnar nerve with axonal excitability testing

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized...

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

anatomically realistic neonatal heart model for use in neonatal patient simulators

Anatomically Realistic Neonatal Heart Model for Use in Neonatal Patient Simulators

This protocol describes a procedure for creating functional artificial neonatal heart models by utilizing a combination of magnetic resonance imaging, 3D printing, and injection molding. The purpose of these models is for integration into the next generation of neonatal patient simulators and as a tool for physiological and anatomical...

a battery of motor tests in a neonatal mouse model of cerebral palsy

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between...

intravenous injections in neonatal mice

Intravenous Injections in Neonatal Mice

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

a new optokinetic testing method to measure rat vision

A New Optokinetic Testing Method to Measure Rat Vision

The Optokinetic Nystagmus (OKN) behavioral testing method is used for the assessment of visual acuity in rodents. Here a simple method is demonstrated that can be easily set up in research laboratories for a reliable assessment of visual function in both normal and experimental...

Testing for Odor Discrimination and Habituation in Mice

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the mouse can discriminate between non-social odors and social odors, whether the mouse habituates to a repeatedly presented odor, and whether the mouse demonstrates dishabituation when presented with a novel odor. Since many social behavior tests measure the experimental animal&#8217;s response to a familiar or novel mouse, false positives can be avoided by establishing that the animals can detect and discriminate between social odors. There are similar considerations in learning tests such as fear conditioning that use odor to create a novel environment or olfactory cues as an associative stimulus. Deficits in the olfactory system would impair the ability to distinguish between contexts and to form an association with an olfactory cue during fear conditioning. 
In the odor habitation/dishabituation test, the mouse is repeatedly presented with several odors. Each odor is presented three times for two minutes. The investigator records the sniffing time directed towards the odor as the measurement of olfactory responsiveness. A typical mouse shows a decrease in response to the odor over repeated presentations (habituation). The experimenter then presents a novel odor that elicits increased sniffing towards the new odor (dishabituation). After repeated presentation of the novel odor the animal again shows habituation. This protocol involves the presentation of water, two or more non-social odors, and two social odors. In addition to reducing experimental confounds, this test can provide information on the function of the olfactory systems of new knockout, knock-in, and conditional knockout mouse lines.

This manuscript describes a protocol to examine the olfactory system of rodents. The olfactory habituation/dishabituation test will allow investigators to determine whether a mouse habituates to a repeatedly presented odor and whether the mouse demonstrates dishabituation when presented a novel odor.

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the ...

Testing Sensory and Multisensory Function in Children with Autism Spectrum Disorder

In addition to impairments in social communication and the presence of restricted interests and repetitive behaviors, deficits in sensory processing are now recognized as a core symptom in autism spectrum disorder (ASD). Our ability to perceive and interact with the external world is rooted in sensory processing. For example, listening to a conversation entails processing the auditory cues coming from the speaker (speech content, prosody, syntax) as well as the associated visual information (facial expressions, gestures). Collectively, the &#8220;integration&#8221; of these multisensory (i.e., combined audiovisual) pieces of information results in better comprehension. Such multisensory integration has been shown to be strongly dependent upon the temporal relationship of the paired stimuli. Thus, stimuli that occur in close temporal proximity are highly likely to result in behavioral and perceptual benefits &#8211; gains believed to be reflective of the perceptual system&#39;s judgment of the likelihood that these two stimuli came from the same source. Changes in this temporal integration are expected to strongly alter perceptual processes, and are likely to diminish the ability to accurately perceive and interact with our world. Here, a battery of tasks designed to characterize various aspects of sensory and multisensory temporal processing in children with ASD is described. In addition to its utility in autism, this battery has great potential for characterizing changes in sensory function in other clinical populations, as well as being used to examine changes in these processes across the lifespan.

We describe how to implement a battery of behavioral tasks to examine the processing and integration of sensory stimuli in children with ASD. The goal is to characterize individual differences in temporal processing of simple auditory and visual stimuli and relate these to higher order perceptual skills like speech perception.

In addition to impairments in social communication and the presence of restricted interests and repetitive behaviors, deficits in sensory processing are ...

Simple and Computer-assisted Olfactory Testing for Mice

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected with aging and is a strong predictor of neurodegenerative diseases. Thus, olfaction testing is used as a non-invasive diagnostic method to detect neurological deficits early on. In order to understand the mechanisms underlying olfactory network susceptibility, olfactory research in rodents has gained momentum in the past decade.
Here, we present a very simple, time efficient and reproducible olfactory testing method of innate odor perception and sensitivity in mice without the need of any prior food or water restriction. The tests are performed in a familiar environment to the mice, require only the scents and a 2 min session of odorant exposure. The analysis is performed, post-hoc, using computer-assisted commands on ImageJ and can be, therefore, carried out from start to end by one researcher.
This protocol does not require any special hardware or setup and is indicated for any laboratory interested in testing olfactory perception and sensitivity.

We present a simple and unbiased olfactory test in mice. With this protocol olfactory discrimination, preference, avoidance and sensitivity to a novel odor as compared to water can be assessed in single behavioral sessions. This method is indicated for a single experimenter and analysis is based on computer-assisted video processing.

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected ...

Testing Tactile Masking between the Forearms

Masking, in which one stimulus affects the detection of another, is a classic technique that has been used in visual, auditory, and tactile research, usually using stimuli that are close together to reveal local interactions. Masking effects have also been demonstrated in which a tactile stimulus alters the perception of a touch at a distant location. Such effects can provide insight into how components of the body's representations in the brain may be linked. Occasional reports have indicated that touches on one hand or forearm can affect tactile sensitivity at corresponding contralateral locations. To explore the matching of corresponding points across the body, we can measure the spatial tuning and effect of posture on contralateral masking. Careful controls are required to rule out direct effects of the remote stimulus, for example by mechanical transmission, and also attention effects in which thresholds may be altered by the participant's attention being drawn away from the stimulus of interest. The use of this technique is beneficial as a behavioural measure for exploring which parts of the body are functionally connected and whether the two sides of the body interact in a somatotopic representation. This manuscript describes a behavioural protocol that can be used for studying contralateral tactile masking.

Here we explore contralateral tactile masking between the forearms in which tactile detection thresholds are modulated by vibration applied to a remote site. The details of which remote sites have an effect can tell us about how the body is represented in the brain.

Masking, in which one stimulus affects the detection of another, is a classic technique that has been used in visual, auditory, and tactile research, ...

New Variations for Strategy Set-shifting in the Rat

Behavioral flexibility is crucial for survival in changing environments. Broadly defined, behavioral flexibility requires a shift of behavioral strategy based on a change in governing rules. We describe a strategy set-shifting task that requires an attentional shift from one stimulus dimension to another. The paradigm is often used for testing cognitive flexibility in primates. However, the rodent version has not been as extensively developed. We have recently extended an established set-shifting task in the rat1 by requiring attention to different stimuli according to context. All the experimental conditions required animals to choose either a left or right lever. Initially, all animals had to choose on the basis of the location of the lever. Subsequently, a change in the rule occurred, which required a shift in set from location-based rule to a rule in which the correct lever was indicated by a light cue. We compared performance on three different versions of the task, in which the light stimulus was either novel, previously relevant, or previously irrelevant. We found that specific neurochemical lesions selectively impaired the ability to make particular types of set shift as measured by the performance on the different versions of the task.

Set-shifting, a form of behavioral flexibility, requires an attentional shift from one stimulus dimension to another. We extended an established rodent set-shifting task1 by requiring attention to different stimuli according to context. The task was combined with specific lesions to identify neuron subtypes underlying a successful shift.

Behavioral flexibility is crucial for survival in changing environments. Broadly defined, behavioral flexibility requires a shift of behavioral strategy ...

Testing Animal Anxiety in Rats: Effects of Open Arm Ledges and Closed Arm Wall Transparency in Elevated Plus Maze Test

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral science, conflicting outcomes are often provided from different laboratories. To identify reasons for the different outcomes, we previously focused on arm features, which differ between laboratories, most notably the presence/absence of ledges at the sides of open arms and the transparency/opaqueness of closed arm walls. In a previous report, we used a custom designed container to compare rat behavior on different combinations of open and closed arm designs under otherwise identical experimental conditions, and showed that differences in arm features interfere with experimental outcomes. In brief, open arm ledges significantly increased anxiety-like behavior in rats, while transparent arms may also have decreased this behavior. Furthermore, we verified a higher detection sensitivity of the effect of an anxiolytic drug in a combination of no-ledged open arm + opaque walled closed arm compared with a combination of ledged open arm + transparent walled closed arm. In this report, we introduce our protocol for the elevated plus maze test, together with discussion of the key results from the previous report and our experimental experience. We believe this report will provide useful information for researchers who have employed or who plan to use the elevated plus maze in their studies.

Rats' anxiety-like behavior tested by the elevated plus maze varies highly depending on the testing environment. Here we show how apparatus settings, especially arm structure, affect behavior on the maze, and we explain a reliable protocol to detect effects of anxiolytic drugs. This report will contribute to anxiety research.

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral ...

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, ...

Computerized Adaptive Testing System of Functional Assessment of Stroke

The computerized adaptive testing system of the functional assessment of stroke (CAT-FAS) can simultaneously assess four functions (motor functions of the upper and lower extremities, postural control, and basic activities of daily living) with sufficient reliability and administrative efficiency. CAT, a modern measurement method, aims to provide a reliable estimate of the examinee's level of function rapidly. CAT administers only a few items whose item difficulties match an examinee's level of function and, thus, the administered items of CAT can provide sufficient information to reliably estimate the examinee's level of function in a short time. The CAT-FAS was developed through four steps: (1) determining the item bank, (2) determining the stopping rules, (3) validating the CAT-FAS, and (4) establishing a platform of online administration. The results of this study indicate that the CAT-FAS has sufficient administrative efficiency (average number of items = 8.5) and reliability (group-level Rasch reliability: 0.88 - 0.93; individual-level Rasch reliability: &#8805;70% of patients had Rasch reliability score &#8805;0.90) to simultaneously assess four functions in patients with stroke. In addition, because the CAT-FAS is a computer-based test, the CAT-FAS has three additional advantages: the automatic calculation of scores, the immediate storage of data, and the easy exporting of data. These advantages of the CAT-FAS will be beneficial to data management for clinicians and researchers.

Here, we present a protocol to develop the computerized adaptive testing system of the functional assessment of stroke (CAT-FAS). The CAT-FAS can simultaneously assess four functions (two motor functions [upper and lower extremities], postural control, and basic activities of daily living) with sufficient reliability and administrative efficiency.

The computerized adaptive testing system of the functional assessment of stroke (CAT-FAS) can simultaneously assess four functions (motor functions of the ...

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. ...

Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various physiological or pathological changes. The object location task (OLT) and the novel object recognition task (NORT) are two effective behavioral tasks commonly used to reveal the function and relative health of specific brain regions involved in memory. While both of these tests exploit the inherent preference of mice for the novelty to reveal memory for previously encountered objects, the OLT primarily evaluates spatial learning, which relies heavily on hippocampal activity. The NORT, in contrast, evaluates non-spatial learning of object identity, which relies on multiple brain regions. Both tasks require an open-field-testing arena, objects with equivalent intrinsic value to mice, appropriate environmental cues, and video recording equipment and the software. Commercially available systems, while convenient, can be costly. This manuscript details a simple, cost-effective method for building the arenas and setting up the equipment necessary to perform the OLT and NORT. Furthermore, the manuscript describes an efficient testing protocol that incorporates both OLT and NORT and provides typical methods for data acquisition and analysis, as well as representative results. Successful completion of these tests can provide valuable insight into the memory function of various mouse model systems and appraise the underlying neural regions that support these functions.

Here we provide a protocol which includes comprehensive instructions for the economical establishment of murine object location and novel object recognition behavioral testing, including the design, cost, and construction of required equipment as well as execution of behavioral testing, data collection, and analysis.