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 inflammation, and neurodevelopmental reflexes is as follows.
1. Experimental Animals
<ol>
	<li>When rats arrive to the animal housing facility, refrain from any interaction for a day or two to allow them to acclimate to their new environment. Following acclimatization, handle the rats every day for approximately five days, or when the rats are no longer showing signs of stress. Human-induced stress can alter test results and lead to pregnancy loss.</li>
	<li>Once the rats are comfortable, breeding begins. Place two females and one male rat in a double decker breeding cage (h= 38 cm, 1,800 cm2 floor area) overnight (3 PM - 8 AM). These cages prevent stress and overcrowding. In the morning, using a transfer pipette, flush the vagina of the female rats with approximately 0.25 mL of saline. Transfer the solution into a microcentrifuge tube.</li>
	<li>Place the two females and male back into their respective conventional cages.</li>
	<li>View the solution collected in section 1.2 under a light microscope (100X) to determine which stage of the estrous cycle the rat is experiencing and whether or not sperm are present. If there are sperm, record the date as embryonic day (E1) (Figure 1). If pregnant, give the female her own cage on E14. 
	NOTE: The cells in the vaginal smear are clear, therefore use a low light setting. Ensure that the condenser is adjusted to its lowest setting. Adjust the brightness control to dim the light. If a brightness dial is available, set it to 1.</li>
</ol>
2. Dietary Supplementation
NOTE: This protocol is intended to assess the therapeutic effects of dietary broccoli sprout consumption during the last week of pregnancy. Grow the sprouts according to Wu et al. 27.
<ol>
	<li>Soak the broccoli sprouts seeds for 2 - 3 h. Spread the seeds out on a countertop seed sprouter box and set on a warm surface for germination.</li>
	<li>Water the seeds twice daily, once in the AM (8 - 9 AM) and once in the PM (3 - 4 PM).</li>
	<li>On day five, place the sprouts by a window for the day (sprouts should have two leaves each and will become green over the day).</li>
	<li>At the end of the day, harvest the sprouts by gently pulling the broccoli sprouts out of the sifted box and lay on a flat surface to dry.</li>
	<li>Once the sprouts are completely dried, weigh out 200 mg and place in a sealed plastic bag.</li>
	<li>Beginning on E14, feed each pregnant rat 200 mg of dried broccoli sprouts daily until PD21 (the day pups are weaned).</li>
</ol>
3. Inflammation
<ol>
	<li>To induce inflammation and reproduce white matter injury in offspring, inject pregnant rats with lipopolysaccharide (LPS) every 12 h on E19 and E20. On the day of injection, bring the cage into the lab from the housing facility. Allow the pregnant rat to acclimate and become calm for at least 1 h.&#160;If a rat shows clinical signs of permanent systemic or kidney damage, it will be euthanized immediately and removed from the study.&#160;</li>
	<li>Weigh the pregnant dam.</li>
	<li>Dissolve 200 &#181;g/kg of LPS (serotype 0127:B8) in 100 &#181;L of saline. Aspirate the solution into a 1 mL syringe equipped with a 30 G &#189; needle.
	<ol>
		<li>Use a new needle for each injection. Allow the solution to warm up for a few min to minimize discomfort.</li>
	</ol>
	</li>
	<li>Record the time and inject the pregnant dam intraperitoneally. Alternate injections between sides to ensure equal distribution. 
	NOTE: Machholz et al. describes proper restraint and injection procedures. 28</li>
</ol>
4. Developmental Reflex Tests
<ol>
	<li>On PD1 (day of birth), remove pups from the dam and record birth weights. Place the pups back into their respective cages and move them to the animal facility until PD3. This ensures maternal bonding between dam and pup.
	<ol>
		<li>Record the reflex testing for reliable assessment. Adjust the video camera so that it is at maximum shutter speed for frame-by-frame analyses (1/1,000 s). Position the video camera so that it is directly above or beside the rat pup and all materials used for each reflex. For example, for hindlimb placing, place the camera beside the rat pup to ensure the lifting and placing of the hindlimb is captured.</li>
		<li>For accelerated righting, place the camera above the rat pup and landing pad in order to record the ability of the pup to right in midair.</li>
	</ol>
	</li>
	<li>Record weights daily until the end of the experiment. On PD3, begin neurodevelopmental reflex testing (Figure 4). Move cages into a quiet room for a minimum of 1 h prior to testing to allow for acclimatization to the environment.</li>
	<li>Perform neurodevelopmental reflex testing at a consistent time each day as rats are nocturnal. For example, test between 9 AM - 12 PM daily. Keep the pups directly under a heating lamp or on a heat pad at all times, to maintain a stable body temperature of 36.5 &#176;C; rat pups of this age lose heat easily. Measure with a rectal temperature probe.&#160;If done efficiently, pups body temperatures do not change because they are not removed from the dam and litter long enough. NOTE: Monitor the temperature of heat lamp to ensure it does not exceed 37&#160;&#176;C.
	<ol>
		<li>Record the score of each reflex each day until a positive response is observed. A positive reflex response occurs when the pup is able to perform a task on two consecutive days. The date of the first occurrence of a positive response is used for quantification. No further testing is required following the positive response. Hence the end date for each reflex is variable.</li>
	</ol>
	</li>
	<li>Forelimb Grasping
	<ol>
		<li>Beginning on PD3, conduct the forelimb grasp reflex test. Place a blunt rod against the palm of each forepaw and apply light pressure manually. The light 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.</li>
		<li>Score the forelimb grasp: 
		0 for no grasping 
		1 for successful grasping by one forepaw (left or right forepaw can be specified here) 
		2 for successful grasping by both forepaws</li>
	</ol>
	</li>
	<li>Hindlimb Grasping
	<ol>
		<li>Beginning on PD3, conduct the hindlimb grasp reflex test. Place a blunt rod against the sole of each hindpaw and apply light pressure manually. 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. Successful acquisition of this reflex occurs when both hindpaws grasp the rod for two days in a row.</li>
		<li>Score the hindlimb grasp: 
		0 for no grasping 
		1 for successful grasping by one hindpaw (left or right hindpaw can be specified here) 
		2 for successful grasping by both hindpaws</li>
	</ol>
	</li>
	<li>Righting
	<ol>
		<li>Beginning on PD3, begin the righting reflex test. Firmly hold the pup in a supine position, with all four paws upright. Let go of the pup and immediately start the timer. Righting is achieved when the pup is able to flip/roll over onto all four paws, and each paw is perpendicular to the body. Give each pup a maximum of 15 s to achieve this goal.</li>
		<li>Score the righting: 
		0 for lying on the back (or 15 s for the maximum allocated time) 
		1 for lying on the side (left or right side can be specified here) or the ability of the pup to right but in the wrong posture (or 15 s for the maximum allocated time) 
		2 for successful righting and appropriate posture</li>
	</ol>
	</li>
	<li>Hindlimb placing
	<ol>
		<li>Beginning on PD4, begin the hindlimb placing reflex 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.</li>
		<li>Score the hindlimb placing reflex: 
		0 for no successes 
		1 for placing of one hindpaw (left or right hindpaw can be specified here) 
		2 for successful hindlimb placing for both hindpaws</li>
	</ol>
	</li>
	<li>Cliff Avoidance
	<ol>
		<li>Beginning on PD4, begin the cliff avoidance testing. 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.&#160;The experimenter&#8217;s hand or a foam landing is placed beneath the cliff to catch the pup from the fall. &#160;&#160;</li>
		<li>Score the cliff avoidance: 
		0 for no movement or falling off the edge 
		1 for attempts to move away from the cliff but with hanging limbs 
		2 for successful movement away from the cliff</li>
	</ol>
	</li>
	<li>Gait
	<ol>
		<li>Begin the evaluation of gait on PD6. Place pups in the center of a 15 cm diameter circle and allow the pup 30 s to complete the task.14 A successful gait is performed when the rat pup is able to move both forepaws outside the circle in less than 30 s.</li>
		<li>Score the gait as the time in seconds it takes the rat pup to move outside the circle. Record the time it takes the pup to move both forepaws outside the circle. Record 30 s if the pup is unable to complete the task within the given time</li>
	</ol>
	</li>
	<li>Auditory Startle
	<ol>
		<li>Begin the analysis of auditory startle on PD10. Present a loud noise (bell) directly over the pup to assess whether or not a startle response is present. A positive startle response is observed when the pup displays a &#39;jerking&#39; movement, away from the sound.</li>
		<li>Score the auditory startle: 
		(or no) for no startle response 
		(or yes) for a positive startle response</li>
	</ol>
	</li>
	<li>Posture
	<ol>
		<li>Begin posture analysis on PD12. Place 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.</li>
		<li>Score the posture: 
		0 for no movement 
		1 for immature posture when moving 
		2 for mature posture while moving</li>
	</ol>
	</li>
	<li>Eye Opening
	<ol>
		<li>Begin eye-opening analysis on PD12. Record the day both eyelids open: 
		0 for no visible eyes 
		1 for one visible eye (left or right side can be specified here) 
		2 for two visible eyes</li>
	</ol>
	</li>
	<li>Accelerated Righting
	<ol>
		<li>Beginning on PD14, begin accelerated righting testing. Place the pup in a supine position 30.48 centimeters above a foam landing. Drop the pup and observe its ability to right itself. Righting refers to when the pup is able to turn over to the prone position and land on its paws.</li>
		<li>Score the accelerated righting: 
		0 for falling on its back 
		1 for falling on its side (left or right side can be specified here) 
		2 for landing on its paws</li>
	</ol>
	</li>
</ol>
Note: Different types of quantification of the reflexes are available depending on the question of the investigators. Quantification of the reflexes can be done as initial observation of a reflex, first day of appearance up until the disappearance of the reflex, time it takes to successfully perform the task, speed of performance, and/or improvement of performance over time. 13, 14, 29,30,31 For the current study, the first day of appearance was used as a score. When using multiparous models, pups from the same litter typically behave similarly due to their genetic makeup, in utero and postpartum environment, and nutritional availability.32 Therefore, a single pup cannot be accounted for as an n = 1 due to litter bias. Pups examined within a litter can be averaged so that each litter represents an n = 1. 32, 33 Alternatively, several pups within the same litter can be analyzed using the mixed effects model, which takes into account pups within the same litter. 32, 33

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Brain Res. 291, 289-298 (2015).</li><li>Wainwright, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Issues+of+design+and+analysis+relating+to+the+use+of+multiparous+species+in+developmental+nutritional+studies.">Issues of design and analysis relating to the use of multiparous species in developmental nutritional studies.</a> J. Nutr. 128 (3), 661-663 (1998).</li><li>Lazic, S. E., Essioux, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+basic+and+translational+science+by+accounting+for+litter-to-litter+variation+in+animal+models.">Improving basic and translational science by accounting for litter-to-litter variation in animal models.</a> BMC Neurosci. 14, 14-37 (2013).</li><li>Sergio, D. P., Sanchez, S., Ruben, V. R., Ana, B. R., Barriga, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+behaviour+and+in+the+circadian+rhythms+of+melatonin+and+corticosterone+in+rats+subjected+to+a+forced-swimming+test.">Changes in behaviour and in the circadian rhythms of melatonin and corticosterone in rats subjected to a forced-swimming test.</a> J Appl Biomed. 1 (47), (2005).</li><li>Castelhano-Carlos, M., Baumans, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+light,+noise,+cage+cleaning+and+in-house+transport+on+welfare+and+stress+of+laboratory+rats.">The impact of light, noise, cage cleaning and in-house transport on welfare and stress of laboratory rats.</a> Lab. Anim. 43 (4), 311-327 (2009).</li><li>Zimmerberg, B., Ballard, G. A., Riley, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+thermoregulation+after+prenatal+exposure+to+alcohol+in+rats.">The development of thermoregulation after prenatal exposure to alcohol in rats.</a> Psychopharmacology (Berl. 91 (4), 479-484 (1987).</li><li>Fan, L. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-ischemia+induced+neurological+dysfunction+and+brain+injury+in+the+neonatal+rat).">Hypoxia-ischemia induced neurological dysfunction and brain injury in the neonatal rat).</a> Behav. Brain Res. 165 (1), 80-90 (2005).</li><li>Smart, J. L., Dobbing, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vulnerability+of+developing+brain.+VI.+Relative+effects+of+foetal+and+early+postnatal+undernutrition+on+reflex+ontogeny+and+development+of+behaviour+in+the+rat.">Vulnerability of developing brain. VI. Relative effects of foetal and early postnatal undernutrition on reflex ontogeny and development of behaviour in the rat.</a> Brain Res. 33 (2), 303-314 (1971).</li><li>Fox, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuro-Behavioral+ontogeny:+A+synthesis+of+ethological+and+neurophysiological+concepts.">Neuro-Behavioral ontogeny: A synthesis of ethological and neurophysiological concepts.</a> Brain Res. 2 (1), 3-20 (1966).</li><li>Piper, M. C., Mazer, B., Silver, K. M., Ramsay, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+neurological+symptoms+in+high-risk+infants+during+the+first+two+years+of+life.">Resolution of neurological symptoms in high-risk infants during the first two years of life.</a> Dev. Med. Child Neurol. 30 (1), 26-35 (1988).</li><li>Shiotsuki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rotarod+test+for+evaluation+of+motor+skill+learning.">A rotarod test for evaluation of motor skill learning.</a> J. Neurosci. Methods. 189 (2), 180-185 (2010).</li><li>Vorhees, C. V., Williams, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morris+water+maze:+procedures+for+assessing+spatial+and+related+forms+of+learning+and+memory.">Morris water maze: procedures for assessing spatial and related forms of learning and memory.</a> Nat. Protoc. 1 (2), 848-858 (2006).</li><li>Walf, A. A., Frye, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+elevated+plus+maze+as+an+assay+of+anxiety-related+behavior+in+rodents.">The use of the elevated plus maze as an assay of anxiety-related behavior in rodents.</a> Nat. Protoc. 2 (2), 322-328 (2007).</li></ol>

The authors have nothing to disclose.

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 ...

neurodevelopmental-reflex-testing-in-neonatal-rat-pups

Research

JoVE Journal

Behavior

19.7K Views.  University of Alberta. 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. 

Video: Neurodevelopmental Reflex Testing in Neonatal Rat Pups

Barnes Maze Testing Strategies with Small and Large Rodent Models

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.&#160;bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.&#160;distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.&#160;random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.&#160;shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.

The dry-land Barnes maze is widely used to measure spatial navigation ability in response to mildly aversive stimuli. Over consecutive days, performance (e.g.&#160;latency to locate escape cage) of control subjects improves, indicative of normal learning and memory. Differences between rats and mice necessitate apparatus and methodology changes that are detailed here.

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land ...

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 ...

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer&#8217;s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer&#8217;s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

 The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, ...

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 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 ...

Chronic Sleep Deprivation in Mouse Pups by Means of Gentle Handling

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

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

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