This work was supported by a CIHR operating grant (MOP-102624) to Q. Y. We thank Dr Carolyn Harley for helpful discussions throughout the study, Dr. Qinlong Hou, Amin Shakhawat, and Andrea Darby-King for technical support.

Sprague Dawley rat (Charles River) pups of both genders are used. Litters are culled to 12 on PD1 (birth being PD0). The dams are maintained on a 12 hr light/dark cycle with ad libitum access to food and water. Experimental procedures have been approved by Memorial University&#8217;s Institutional Animal Care Committee.
1. Nose Plug Construction
NOTE: This procedure was adapted and modified from Cummings et al. (1997)35.
<ol>
	<li>Aquire polyethylene-20 tubing and 3-0 silk suture thread.</li>
	<li>Cut a small piece of polyethylene-20 tubing to approximately 0.8 mm.</li>
	<li>Thread silk suture through the prepared tubing such that there is thread on either side of the section of tubing.</li>
	<li>On one end of the thread outside of the plug, tie a knot in the thread.</li>
	<li>Pull the section of tubing down over the knot in the thread. The knot should lodge inside the tubing.</li>
	<li>Trim both ends of the thread such that ~2 mm of thread is protruding from one end of the tubing (see Figure 1A).</li>
</ol>
2. Naris Occlusion before Training
<ol>
	<li>Remove pup from dam and place in a secure dish covered with regular bedding.</li>
	<li>Use a cotton tip application to dab a local anesthetic jelly, 2% Xylocaine, on the naris to be occluded.</li>
	<li>Allow the pup to rest in the dish for ~3 min.</li>
	<li>Hold the pup gently but securely in the non-dominant hand.</li>
	<li>Using the dominant hand, pick up a nose plug and dab the same local anesthetic jelly around the tip from which the thread is not protruding. This will act as both an anesthetic for any minor pains associated with plug insertion and will act as a lubricant inside the naris. NOTE: The effect of Xylocaine starts within a couple of minutes and lasts 20-30 min. Pups in general show good tolerance for plug insertion after xylocaine jelly application (minimum struggling and vocalization).</li>
	<li>Gently insert the nose plug by firmly holding the pup and slowly rotating the plug with very gentle pushes until the plug is fully inserted and only the 2 mm thread is protruding from the naris (see Figure 1B). There should be no bleeding from either naris during this process. Pups with bleeding during nose plug insertion are excluded and returned to dams</li>
	<li>Allow the animal to rest in this dish for 5 min&#160;in order to habituate to the plug.</li>
	<li>Remove the pup from the habituation dish and begin the conditioning paradigm24.</li>
</ol>
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51808/51808fig1highres.jpg" width="500" /> 
Figure 1.&#160;Construction of a nose plug. A)&#160;Schematics showing the steps of making a nose plug. A thread is pulled through polyethylene tubing; a knot is made and pulled into the middle of the tubing to block it; two ends are cut with a 2 mm residue in one end out of the tubing. B)&#160;Front and lateral view of a rat with a nose plug in one naris.
3. Scented Bedding Preparation
<ol>
	<li>Wearing new gloves and in a fume hood to prevent odor contamination, place 500 ml of woodchip bedding into a plastic bag.</li>
	<li>Use a syringe to draw up 0.3 ml peppermint extract, and spray this over the bedding in the plastic bag.</li>
	<li>Tie the bag shut, shake the bag vigorously, and allow the bedding to rest in the bag for 5 min.</li>
	<li>Place the scented bedding in a clear, shallow, acrylic training box (20 x 20 x 5 cm3, Figure 2A) uncovered in a fume hood for 5 min&#160;before use. Once the bedding is prepared, discard these gloves, and do not allow these gloves to come in contact with the animals.</li>
	<li>Place the unscented bedding in an identical clear plastic box, and ensure that it does not come into contact with the scented bedding or used gloves.</li>
</ol>
4. Odor Conditioning Paradigm (See Picture in Figure 2A)
Pups undergo either a single conditioning session, on PD 6, or multiple trial sessions (one session per day, PD 3-6).
<ol>
	<li>Place the habituated pup on scented bedding. For control odor only (O/S-) pups, leave these pups on the bedding for 10 min, then skip to step 4.5. For experimental odor + stroke (O/S+) pups, continue to the following steps in this section.</li>
	<li>Stroke the pup for 30 sec&#160;using a small paintbrush. Use rapid circular motions primarily around the hind region of the pup.</li>
	<li>Allow the pup to rest for 30 sec.</li>
	<li>Repeat steps 4.2 &#38; 4.3 for a total of 10 min&#160;(i.e. 10 pairings of stroking + odor).</li>
	<li>Remove pup from the conditioning box, remove the nose plug and return the pup to the dam.</li>
</ol>
5. Lateralized Odor Preference Testing (See Picture in Figure 2B)
Testing occurs at various time points (e.g., 24 or 48 hr) following the final training session. Testing is carried out in a stainless steel testing chamber (30 x 20 x 18 cm3), which is placed on top of two training boxes (training box is described in 3.4), separated by a 2 cm neutral zone. One training box contains peppermint-scented bedding while the other box contains clean, unscented bedding. The floor of the testing chamber is a metal grid, which is then covered by a removable sheet of plastic mesh (Figure 2B).
<ol>
	<li>Prepare one peppermint and one unscented bedding as per Section 3, and place each box under opposite sides of the testing chamber, 2 cm apart. Place the plastic mesh on the metal-grid floor of the testing chamber.</li>
	<li>Remove the pup from the dam and place a firm dab of odorless silicone grease on the naris that is occluded during training. Re-apply the grease throughout the first testing procedure as needed. NOTE: Random naris occlusion during training and testing may be considered to avoid bias.</li>
	<li>Place pup in the neutral zone of the testing chamber.</li>
	<li>Allow the pup to explore the chamber for 1 min, recording how long the pup spent over the two sides of the chamber (i.e. over peppermint or neutrally-scented bedding).</li>
	<li>Allow the pup to rest for 1 min&#160;in a covered plastic holding chamber.</li>
	<li>Repeat steps 5.2 &#38; 5.3 for a total of 10 min&#160;(i.e. 5 test trials separated by 5 rest trials) switching the initial orientation of the pup in the chamber in order to control for direction preferences.</li>
	<li>Immediately following testing, wipe away the grease from the naris.</li>
	<li>Insert a polyethylene noseplug into the opposite naris as per section 2 and allow the animal to rest for 10 min.</li>
	<li>Test the pup once again as in 5.3 -5.6, remove the plug, and return the pup to the dam. Remove and clean the plastic mesh of the testing chamber with 95% ethanol and allow the liquid to evaporate. Place the mesh back before testing the next pup. 
	NOTE: Applying silicone grease at the first naris occlusion during testing prevents the chance of bleeding and stress associated with nose plug insertion.</li>
</ol>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51808/51808fig2highres.jpg" width="500" /> 
Figure 2. Early odor preference training and testing. A)&#160;Early odor preference training using odor + stroking paradigm. B)&#160;Two choice odor preference testing with peppermint bedding on one side, control unscented bedding on the opposite side. A 2 cm neutral zone is placed in between.
6. Testing the Effectiveness of Single Naris Occlusion
This experiment is performed to determine whether single naris occlusion leads to lateralized activation of the olfactory system.
<ol>
	<li>Perform unilateral naris occlusions on PD 6 or 7 pups as described in section 2.</li>
	<li>After ~5 min&#160;habituation, place the pup in a covered plastic container and expose it to 30 &#181;l pure peppermint oil soaked in a piece of tissue for 10 min.</li>
	<li>Immediately after the peppermint odor exposure, inject the pup intraperitoneally (i.p.) with chloral hydrate (400 mg/kg) as a general anesthetic,&#160;or pentobarbitol, (150 mg/kg).</li>
	<li>Once fully anesthetized (showing no response to tail or foot pinch), transcardially perfuse the pup with ice-cold solutions of saline (0.9%) for ~1 min, followed by paraformaldehyde (4%, dissolved in 0.1 M phosphate buffer solution, PBS).</li>
	<li>After 10 min&#160;of paraformaldehyde perfusion, collect the brain and place it in paraformaldehyde overnight at 4&#160;&#176;C, then transfer the brain to a sucrose solution (20% in PBS) for an additional 24 hr.</li>
	<li>Cut coronal brain slices at 30 &#956;m thickness with a cryostat. Collect OB and PC slices and mount onto gelatin-coated slides, followed by standard immunohistochemistry staining for pCREB antibody21,25,30.</li>
</ol>
7. Testing the Reversibility of Single Naris Occlusion
This experiment tests whether the blocking effect is reversible at 24 hr following the removal of the nose plug.
<ol>
	<li>Perform unilateral naris occlusions on PD 6 or 7 pups as described in section 2.</li>
	<li>After 15 min&#160;(equivalent to the duration of the naris occlusion during training &#8211; 5 min&#160;habituation + 10 min&#160;training), remove the nose plug, and return the pup to the dam.</li>
	<li>24 hr&#160;later, expose the pup to the peppermint odor in a covered plastic container for 10 min&#160;as described in 6.2.</li>
	<li>Follow same steps in sections 6.3-6.6.</li>
</ol>

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P. <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+the+neural+representation+of+odorants+after+olfactory+deprivation+in+the+adult+mouse+olfactory+bulb.">Changes in the neural representation of odorants after olfactory deprivation in the adult mouse olfactory bulb.</a> Chem Senses. 38, 77-89 (2013).</li><li>Kim, H. H., Puche, A. C., Margolis, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+deprivation+reversibly+modulates+transsynaptic+changes+in+the+NR2B-mediated+CREB+pathway+in+mouse+piriform+cortex.">Odorant deprivation reversibly modulates transsynaptic changes in the NR2B-mediated CREB pathway in mouse piriform cortex.</a> J Neurosci. 26, 9548-9559 (2006).</li><li>Korol, D. L., Brunjes, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+changes+in+2-deoxyglucose+uptake+and+amino+acid+incorporation+following+unilateral+odor+deprivation:+a+laminar+analysis.">Rapid changes in 2-deoxyglucose uptake and amino acid incorporation following unilateral odor deprivation: a laminar analysis.</a> Brain Res Dev Brain Res. 52, 75-84 (1990).</li><li>Leung, C. H., Wilson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-neuronal+regulation+of+cortical+apoptosis+in+the+adult+rat+olfactory+system.">Trans-neuronal regulation of cortical apoptosis in the adult rat olfactory system.</a> Brain Res. 984, 182-188 (2003).</li></ol>

The authors have nothing to disclose.

The lateralized odor learning and memory model in rat pups within a critical time window was first established by Hall and colleagues. In a series of studies33,34,36, they showed that an odor preference memory could be lateralized by odor + milk pairings to one naris at PD 6 in rat pups. Preference memory was robust when the same naris was open during training and testing, but not observed when the occluded naris was unblocked and tested. However, at PD 12, when anterior commissural connections from the anteri...

Olfaction is the primary sensory modality in rodents, without which they would not be able to successfully navigate or survive in their environment. It is especially critical for neonatal pups, which can neither see nor hear during the first post-natal week, to use olfaction in order to locate their mother to feed1. As a result, neonatal rat pups can be conditioned to prefer odors with simple experimental manipulations. A variety of stimuli have been used as the unconditioned stimulus (UCS) to induce conditioned responses to novel odors (conditioned stimulus, CS) in neonates, including the nesting environment2,3, milk suckling4-6, stroking or tactile stimulation7-12, tail pinch13, maternal saliva13, mild foot shock14-18, and intracranial brain stimulation19. The present study employs a well-established early odor preference paradigm wherein an odor, in this case peppermint, is combined with tactile stimulation in order to produce a preference for peppermint 24 hr&#160;later10,11,20. These odors memories are dependent on intact olfactory circuitry, primarily including the olfactory bulbs (OB)21-23 and the anterior piriform cortex (aPC)24,25.
Experimental investigations of the early odor preference learning have deepened and broadened our understanding of the molecular and physiological underpinnings of a mammalian memory. This mammalian model has several advantages in studying memory mechanisms. First, the neural sources of the UCS signal have been identified. Various stimuli as mentioned above stimulate locus coeruleus norepinephrine release26, which in turn activates multiple adrenoceptors in the OB and aPC, causing cellular and physiological effects that support learning22,27,28. Second, memory-supporting mechanisms take place in well-defined laminar neural structures. The simplicity of the olfactory circuitry in neonatal rats provides researchers with the ideal framework with which to uncover the intricate processes related to synaptic plasticity. Olfactory sensory neurons (OSN) in the olfactory epithelium project onto mitral/tufted cells in the OB and these mitral/tufted cells in turn project ipsilaterally to piriform cortex (PC) via the lateral olfactory tract (LOT), among other structures29. Both the OSN synapses in the OB30,31 and the LOT synapses24,25 in aPC have been identified as critical loci for synaptic changes that support learning and memory. Third, in an early age in rats, olfactory inputs can readily be lateralized. Each aPC has access to bilateral odor information via the anterior commissure once this white matter is fully formed at post-natal day 12 (PD12)32. Before PD 12, odor input can be isolated to ipisilateral OB and aPC through single naris occlusion24,25,31,33,34. Single naris occlusion permits the odor memory formation from the open naris, and prevents the same memory from the occluded naris prior to PD 1233. Odor memory is isolated to the ipsilateral hemisphere including both OB and aPC. Therefore, each rat pup can be its own control for learning and underpinning physiology.
In the present study, the lateralized early odor preference learning protocol is introduced. This method serves as a powerful tool for studying neural mechanisms underpinning odor learning by providing an intra-animal control24,25,31 , thereby reducing both the number of animals required and the general variation. Naris occlusion is reversible in that the grease or nose plug can be applied and removed with minimal stress or damage to the animal. Here, first, detailed procedures of early odor preference training and testing are described, with a focus on the lateralized protocol using single naris occlusion with a nose plug. Then results are presented to demonstrate the effectiveness of single naris occlusion in isolating odor input and producing lateralized odor memory. Finally, the potentials of using this lateralized learning model to study physiological changes in the olfactory system that both generate learning and support memory expression are discussed.

<table border="0" cellpadding="0" cellspacing="0" width="580">
	<tbody>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Polythylene 20 tubing</td>
			<td style="width:123px;">Intramedic</td>
			<td style="width:103px;">427406</td>
			<td style="width:205px;">Non radiopaque, Non toxic</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">3-0 silk suture thread</td>
			<td style="width:123px;">Syneture</td>
			<td style="width:103px;">Sofsilk</td>
			<td style="width:205px;">Non absorbant&#160;</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Silicone grease</td>
			<td style="width:123px;">Warner Instrument</td>
			<td style="width:103px;">64-0378</td>
			<td style="width:205px;">Odorless</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">2% xylocaine gel</td>
			<td style="width:123px;">AstraZeneca</td>
			<td style="width:103px;">Prod. No 061</td>
			<td style="width:205px;">Lidocaine hydrochloride jelly,&#160; purchased at local pharmacy</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Paint brush</td>
			<td style="width:123px;">Dynasty</td>
			<td style="width:103px;">206R</td>
			<td style="width:205px;">Similar size/other brands work too</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Peppermint extract</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:103px;">W284807</td>
			<td style="width:205px;">Other brand should be okay too</td>
		</tr>
		<tr height="171">
			<td height="171" style="height:171px;width:149px;">Training box</td>
			<td style="width:123px;">Custom-made</td>
			<td style="width:103px;">N/A</td>
			<td style="width:205px;">Acrylic box (20x20x5cm3), see Figure 2A. Parameters and material for the box are not critical and can be modified. Material used should be odorless and does not absorb odors</td>
		</tr>
		<tr height="182">
			<td height="182" style="height:182px;width:149px;">Testing chamber</td>
			<td style="width:123px;">Custom-made</td>
			<td style="width:103px;">N/A</td>
			<td style="width:205px;">Stainless steel (30x20x18cm3), see Figure 2B. Parameters and material for the chamber are not critical and can be modified. For example, an acrylic chamber instead of a stainless steel one can be used</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">pCREB antibody</td>
			<td style="width:123px;">Cell Signaling</td>
			<td style="width:103px;">9198</td>
			<td style="width:205px;">Ser 133 (87G3) Rabbit mAb</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Chloral hydrate</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:103px;">C8383</td>
			<td style="width:205px;">N/A</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Paraformaldehype</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:103px;">P6148</td>
			<td style="width:205px;">N/A</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Sucrose</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:103px;">S9378</td>
			<td style="width:205px;">N/A</td>
		</tr>
	</tbody>
</table>

a lateralized odor learning model in neonatal rats for dissecting neural circuitry underpinning memory formation

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.

Here we review some of the previously established results24 to demonstrate the effectiveness of the naris occlusion in isolating odor input and learning to one hemisphere, and the reversibility of this method.
Single naris occlusion during early odor preference training leads to a lateralized odor memory24. The memory is confined to the spared naris (Figure 3). When pups are tested for odor preference with the same naris occluded as during training, they ...

Watch this Scientific Journal Video about A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation at JoVE.com

A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal ...

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Neuroscience

8.9K Views.  Faculty of Medicine, Memorial University. This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.

Video: A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

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

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

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#039;s Disease Model Mice

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

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

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

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

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

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

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...