We would like to dedicate this work to the memory of late Dr. William T. Greenough, a great mentor, a colleague and a friend. This work was supported by NIH/NIAAA grant number AA009838 and NIH/NIGMS COBRE: The Delaware Center for Neuroscience research grant 1P20GM103653 to AYK. We are grateful to the former and current members of Klintsova lab.

Ethics Statement: The following protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Delaware.
1. Developmental Exposure (or Model of Binge-like Ethanol Exposure)
<ol>
	<li>On PD3, determine the sex of each animal and cross-foster any animals if necessary to keep litter size (8 animals) and sex distribution (4 males : 4 females) consistent within each litter. 
	NOTE: It is important to keep litter size and sex distribution as consistent as possible to avoid experimental confounds. Although this protocol uses 8 pups (4 males and 4 females) per litter, alternative litter sizes or sex distributions may be tailored to the needs of the experimental design.</li>
	<li>Subcutaneously inject a small amount of black India ink into the paws to identify animals within each litter.</li>
	<li>Pseudo-randomly assign litters as experimental (containing 50% alcohol-exposed (AE) and 50% sham-intubated control (SI) pups) or suckle control (SC) (animals that do not undergo any intubation, tail clipping, or separation protocols from PD 4 - 9 except for daily weighing and ear-punching).
	<ol>
		<li>To retain consistent group size, assign twice as many experimental litters as SC litters.</li>
	</ol>
	</li>
	<li>Weigh each animal then return it to its home cage. Animal weighing should occur daily during the intubation period (PD 4 - 9).
	<ol>
		<li>Remove the whole litter from the dam.</li>
		<li>Place pups on heated pad.</li>
		<li>Record the weight of each individual pup.</li>
	</ol>
	</li>
	<li>On PD4, after weighing each animal calculate the necessary alcohol amount for a total of 5.25 g/kg/day per each animal (based on pup weight from step 1.4)8.
	<ol>
		<li>Administer alcohol as 11.9% ethanol-in-milk substitute (vol/vol).</li>
	</ol>
	</li>
	<li>Starting at 9 AM, remove one litter&#39;s pups from the mother at a time.</li>
	<li>Administer the ethanol-in-milk to each AE pup34.</li>
	<li>Sham-intubate each SI pup8.</li>
	<li>Repeat steps 1.5. through 1.8. for each experimental litter.</li>
	<li>Two hours following the first dose, repeat the dosing procedure (steps 1.5 through 1.8) for a second alcohol dose.</li>
	<li>One and a half hours after the second alcohol dose (the point at which peak daily blood alcohol content is achieved), collect and centrifuge blood from the AE and SI pups via tail clipping for future blood alcohol content analysis35.
	<ol>
		<li>Collect 60 &#181;L of blood.</li>
		<li>Place blood in a 1 mL microcentrifuge tube. Centrifuge blood at 1.5 x g for 25 min.</li>
		<li>Carefully collect the supernatant serum from the centrifuge tube and save for future blood alcohol content analysis.</li>
	</ol>
	</li>
	<li>Repeat the dosing procedure (steps 1.5 through 1.8) using milk instead of ethanol-in-milk to prevent nutritional deficits from nursing inability in AE pups.
	<ol>
		<li>Perform a total of 2 supplemental milk doses 2 h apart on PD 4.</li>
	</ol>
	</li>
	<li>Repeat steps 1.4 through 1.12 (except for step 1.11) on PD 5 &#8211; 9.</li>
	<li>Following the final supplemental milk dose on PD9, ear punch all pups for identification in the EC cage.
	<ol>
		<li>Coordinate punched ear with some measure of litter number or identifier (e.g., odd numbered litters within a cohort would get their left ear punched while animals from even numbered litters would get their right ears punched). This will make it easier to identify animals in the EC cage should multiple animals from different litters have the same pawmark pattern.</li>
	</ol>
	</li>
</ol>
2. Weaning
<ol>
	<li>On PD 23, house all animals in cages of 2 - 3.
	<ol>
		<li>Ensure that all animals housed in the same cage are the same sex.</li>
		<li>Include one SC, one SI, and one AE animal per cage when possible.</li>
		<li>Minimize the number of cage mates that are from the same litter.</li>
		<li>Make sure all animals are capable of accessing food and water.</li>
	</ol>
	</li>
</ol>
3. Wheel Running
<ol>
	<li>On PD30, allocate half of the cages with animals to WR. House these animals in cages with a free access to attached stainless steel running wheel.
	<ol>
		<li>Ensure that wheels have a counter to assess the total number of revolutions.</li>
	</ol>
	</li>
	<li>Weigh all animals on PD 30 and PD 36.</li>
	<li>Check the number of revolutions of each wheel at 9 AM every day.</li>
	<li>Leave animals in their respective housing condition for 12 days.</li>
</ol>
4. Environmental Complexity
<ol>
	<li>Prepare the EC cage before 9 AM on the day that corresponds to PD 42 for experimental animals.
	<ol>
		<li>Get a 30 &#34; x 18 &#34; x 36 &#34; galvanized steel cage. 
		NOTE: The cage should have multiple levels, be capable of supporting the weight of multiple rats, be filled with standard bedding, and have multiple locations to attach water bottles and food dispensers.</li>
		<li>Place novel, colorful objects of variable sizes and shapes in the cage.
		<ol>
			<li>Place 6 large toys in the EC cage. Ensure that each toy is big enough for 3 or more rats to interact with concurrently.</li>
			<li>Place 6 medium toys in the EC cage. Ensure that each toy is big enough for 3 - 4 rats to interact with concurrently.</li>
			<li>Place a lot (at least 20) of smaller toys in the EC cage.</li>
			<li>Use toys of varying colors, shapes, size, etc. Novelty is critical to this intervention (see discussion).</li>
		</ol>
		</li>
		<li>Place two dishes of food at opposite ends of the cage.</li>
		<li>Place two bottles of water at opposite ends of the cage.</li>
	</ol>
	</li>
	<li>At 9 AM on PD 42, weigh all animals and relocate the WR animals to the EC cage. Each EC cage should contain 9 - 12 animals.
	<ol>
		<li>Make sure that no animals have both the same pawmark and ear-punch patterns.</li>
	</ol>
	</li>
	<li>Check all food and water daily.</li>
	<li>Every two days, remove the toys from the EC cage and replace them (according to step 4.1.2.).</li>
	<li>Every three days, clean the EC cage.
	<ol>
		<li>Remove the animals from the EC cage and put them in temporary holding cages of 2 - 3 animals.</li>
		<li>Remove all of the bedding from the bottom of the cage.</li>
		<li>Return the same toys to the cage unless this day coincides with the toy replacement schedule (according to step 4.4.).</li>
		<li>Replace all of the food and water.</li>
		<li>Replace the rats into the EC cage.</li>
	</ol>
	</li>
</ol>
5. Collect Tissue
NOTE: Tissue collection (e.g., perfusion with paraformaldehyde) and storage (e.g., freezing, paraffin embedding) can be performed with a variety of methods. The following will explain the process of perfusion with 4% paraformaldehyde in 0.1 M phosphate buffered saline (4% paraformaldehyde in PBS) solution8.
Caution: Paraformaldehyde is carcinogenic and may also cause skin irritation, allergic skin reaction, or eye damage. Use appropriate eye/skin protection.
<ol>
	<li>Expose one rat at a time to isoflurane to lightly anesthetize the animal.</li>
	<li>Intraperitoneally inject the rat with 2 mL/kg of Ketamine/xylazine mixture (1.5 mL xylazine mixed with 10 mL of ketamine). 
	NOTE: Ketamine and xylazine are both at stock concentrations of 100 mg/mL before combining for injection mixture.</li>
	<li>Once rat is no longer responsive, perfuse the animal with 0.1 M phosphate buffered saline (PBS; pH = 7.2) followed by 4% paraformaldehyde in PBS (pH = 7.2).</li>
	<li>Remove brain and store in 4% paraformaldehyde in PBS at 4 &#176;C for 48 h.</li>
	<li>After 2 days, transfer to solution of 30% sucrose added to 4% paraformaldehyde in PBS at 4 &#176;C.</li>
</ol>

The authors have nothing to disclose.

In the above protocol, we demonstrated an expedient intervention to rescue neuroanatomical deficits following neonatal alcohol exposure. This intervention can be used as a therapeutic in other animal models due to the robustness of each of the components of the intervention. Voluntary cardiovascular activity in the form of WR has been shown to benefit several behavioral outcomes38,39 and induce functional plastic alterations in brain regions such as the hippocampus (reviewed in40). This is in part due to expression of growth factors and other neuroprotective mechanisms in the brain parenchyma in both rodents and humans21,41. Supplementing these effects, EC can induce beneficial cellular6,11,42,43, structural2 and pharmacological12,44 change in rodents.
In order for WR to be maximally effective in this particular model of human syndrome, it is critical for animals to have voluntary access to a functional running wheel; daily wheel access should last for an extended amount of time45 at least 10-12 h per day and preferably 24 h (some adverse effects of withdrawal from the running wheel were reported). This WR paradigm lasts for 12 days to allow for the combination of WR and EC to fit into adolescence and early adulthood. The duration, age at exposure, and modality of exercise (among other factors) can affect the efficacy of exercise as a therapeutic intervention46, and such critical factors should be considered when planning to implement this protocol or any other WREC paradigm. A key component of this EC paradigm is the novelty of the multiple objects in the environment and social interaction (reviewed in14,47). Therefore, it is critical for the items in this paradigm to be replaced every 48 h. Based on the need for multiple items, the interaction with the items and their exploration, and social interaction, we find that our number of unique items, frequency of item replacement, and number of cage mates is sufficient to induce therapeutic outcomes on the neuroanatomical measures that we assess. We found that continuous exposure for 30 days is more appropriate to overcome deficits induced by neonatal alcohol exposure than limited exposure interaction to a novel environment.
The goal of this protocol is to introduce a WREC paradigm that addresses both the cardiovascular exercise and environmental novelty components of plastic intervention. For this reason, we will address the modification that can be made to the paradigm but would caution use of modifications that may alter the ways that animals interact within the paradigm as well as the experimental conclusions that can be drawn. One possible alteration would be the introduction of running wheels to the EC environment. In doing so, it would be difficult to determine the relative contributions of each component. It would additionally be difficult to assure that all animals participate in both the WR components and EC components of the paradigm as housing of 8 - 10 animals together is required for EC. However, since long-term access to exercise is critical in the efficacy of this intervention45, further research may address the optimal ratio of WR access to EC access (although the methods in this protocol have shown robust neuroanatomical and behavioral implications8,33). Modifications to individual items used within the EC environment are acceptable, but it is critical for the items to be interesting, complex, novel, stimulating and frequently refreshed14.
This paradigm does contain several innate limitations in our hands, which should be considered when planning to implement this &#34;super intervention&#34;. One limitation to the WR component of the paradigm is the inability to assess the distance run by individual animals. One of the obvious and straightforward solutions would be an individual housing of the animals during WR component. However, it needs to be stressed that individual housing is widely accepted as detrimental to animals and can even directly counteract the beneficial effects of wheel running48. An additional alternative (although time-consuming and imperfect) would be to video record the running wheel at all times that the animals have access. This would require a unique identifier for each animal in a cage (e.g., painting unique colors or patterns on the fur of each animal)49. This technique would still be subject to confounds of multiple animals utilizing the wheel concurrently. A similar difficulty carries to EC where it becomes difficult to food restrict individual animals (without limiting the time period of food consumption). To reduce the impact of this, we would recommend housing in EC for a full 30 days followed by an immediate food restriction paradigm. Extended amounts of time out of EC could inhibit induced plasticity that occurs during this paradigm.
As mentioned previously, the importance of this article is to allow for consistent characterization of the EC paradigm and its implementation following cardiovascular exercise in the form of WR. Previous EC paradigms have exposed animals to EC housing without exposure to WR12,50, WR inside of the EC cage for a shorter amount of time51 or with less animals52, or the animals were exposed to an EC environment for a longer amount of time with less frequent changing of cage items13. It is likely that the beneficial effects of EC require the induced plasticity from WR in a temporally relevant time window to show long-term benefit. In this way, we believe that coupling WR and EC for 12 and 30 days respectively allows for a maximally beneficial and concise intervention.
At this point, the use of this model has been limited to the adolescent and early adult time periods. Further examination of the robustness of this intervention at different stages, and the ontogeny of neuroplastic benefit should be examined further in the future. Additionally, the use of different developmental deficits is greatly encouraged, as this will assist in developing effective therapeutic interventions for individuals afflicted by such disorders. Previous literature has demonstrated independent effects of WR or EC on adult neurogenesis, learning and memory, or anxiety-like behaviors in a genetic mouse model of anxiety53. The robustness of these two interventions and the synergistic effect of EC to sustain the short-term effects of increased WR-induced benefits (i.e., hippocampal cell proliferation and neurogenesis) makes it well poised for integration into a diverse range of research questions.

Rearing in different environments has long been known to cause changes in various measures of neurological wellness. Many studies look at the beneficial effects of rearing in a complex environment (EC) starting with groundbreaking research by Diamond and Rosenzweig (e.g., 1,2) and Greenough (e.g.,3,4). EC has been demonstrated to have an undeniable positive effects on synaptic and cellular changes in the brain5,6,7. EC can affect a multiplicity of brain regions including the hippocampus8,9 and visual cortex10,11, ventral striatum12,13, as well as brain-wide neuroimmune function (reviewed in14). Particular interest has developed from the studies on hippocampus when it was demonstrated that EC can increase the survival rate of adult-born granule cells of the dentate gyrus through dendritic plasticity 9,13. This last point has gathered much interest due to the growing body of literature indicating that cardiovascular exercise promotes adult neurogenesis in both the healthy and damaged brain15,16,17,18. Wheel running (WR) is an easy to implement form of voluntary cardiovascular activity that has been shown to be beneficial in rodent models of neurological disorders or aging 17,19,20. WR affects the expression of growth factors in both the central and peripheral nervous system 21,22,23.
Combining (subsequently) WR and EC into a &#34;superintervention&#34; (WR-EC) (i.e., 12 days of WR followed by 30 days in EC) provides a robust increase in hippocampal adult neurogenesis and increased survival of the newly proliferated cells8, the effect that in the animal model of FASD is not achieved by individual components (see below). Since both components of WR-EC affect a diverse array of structures within the brain13 (WR reviewed in22, EC reviewed in24), implementation of this intervention can easily be applied to rodent models of both developmental and later life onset models of neurological impairment (e.g., neonatal alcohol exposure, aging, early life stress).
Integration of WR-EC in the adolescent and early adult periods (i.e., postnatal days 30 - 72) can ameliorate some of the negative effects of a rat model of fetal alcohol spectrum disorders (FASDs)8. A collection of studies have demonstrated that rodents exposed to alcohol from postnatal day (PD) 4 through 9 display significant deficits in neuroanatomical measures such as dendritic complexity25, cerebellar development26,27 and neuroimmune responsiveness28 as well as manifestations of impaired learning and memory29,30,31. Even a reduced amount of alcohol exposure within this time window (i.e., PD 7 through 9) can lead to deficits in learning and memory in adolescent and adult rats32 while some structures no longer see significant neuroanatomical impairment27. Many of these deficits - in addition to behavioral impairments in hippocampus-dependent tasks - have been mitigated following exposure to this WR-EC paradigm8,33 or WR alone25,31. Although WR alone has been a widely used intervention, the combination of WR-EC has not yet been utilized in the literature despite its ability to sustain the relatively shorter-term benefits of WR8. This article will discuss the implementation of the WR-EC intervention during adolescence. Although this paradigm is used in the context of early postnatal alcohol exposure, it can be introduced to various rodent models to assess brain potential for neuroplasticity in the models of brain disorders.

<table border="0" cellpadding="0" cellspacing="0" width="698">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Female Time-pregnant Long Evans Rats</td>
			<td style="width:124px;">Envigo (Formerly: Harlan, Inc.)</td>
			<td style="width:136px;"></td>
			<td style="width:191px;">Average litter size is 8-10 pups</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Black India Ink</td>
			<td style="width:124px;">Higgins (Chartpak, Inc.)</td>
			<td style="width:136px;">44201</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:247px;">Syringes and Injection Needles</td>
			<td style="width:124px;">Becton, Dickinson and Company (BD)</td>
			<td style="width:136px;">Assorted</td>
			<td style="width:191px;">For injection of pawmarking ink, administration of milk-alcohol solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Ear Punch</td>
			<td style="width:124px;">Kent Scientific Corporation</td>
			<td style="width:136px;">INS750076</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:247px;">Running Wheels</td>
			<td style="width:124px;">Wahmann Labs</td>
			<td style="width:136px;"></td>
			<td style="width:191px;">Wahmann Running Wheel is discontinued. Substitute with&#160; One per cage</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">EC Cage</td>
			<td style="width:124px;">Martin&#39;s Cages, Inc.</td>
			<td style="width:136px;">R-695</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Small EC Toys</td>
			<td style="width:124px;">Assorted</td>
			<td style="width:136px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:247px;">Medium EC Toys</td>
			<td style="width:124px;">Assorted</td>
			<td style="width:136px;"></td>
			<td style="width:191px;">Should be able to fit 1-2 rats inside of/ on top of object</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:247px;">Large EC Toys</td>
			<td style="width:124px;">Assorted</td>
			<td style="width:136px;"></td>
			<td style="width:191px;">Should be able to fit 3 or more rats inside of/on top of object</td>
		</tr>
	</tbody>
</table>

wheel running and environmental complexity as a therapeutic intervention in an animal model of fasd

Aerobic exercise (e.g., wheel running (WR) extensively used in animal research) positively impacts many measures of neuroplastic potential in the brain, such as rates of adult neurogenesis, angiogenesis, and expression of neurotrophic factors in rodents. This intervention has also been shown to mitigate behavioral and neuroanatomical aspects of the negative impacts of teratogens (i.e., developmental exposure to alcohol) and age-related neurodegeneration in rodents. Environmental complexity (EC) has been shown to produce numerous neuroplastic benefits in cortical and subcortical structures and can be coupled with wheel running to increase the proliferation and survival of new cells in the adult hippocampus. The combination of these two interventions provides a robust &#34;superintervention&#34; (WR-EC) that can be implemented in a range of rodent models of neurological disorders. We will discuss the implementation of WR/EC and its constituent interventions for use as a more powerful therapeutic intervention in rats using the animal model of prenatal exposure to alcohol in humans. We will also discuss which elements of the procedures are absolutely necessary for the interventions and which ones may be altered depending on the experimenter&#39;s question or facilities.

In order to assess the effect of the super intervention, we must look at the effects of each of its constituent elements &#8212; WR and EC &#8212; on our measures of interest. Figures 1 through 3 (below) appeared in a previous publication utilizing this paradigm8. Figure 4 appeared in a doctoral dissertation36. These data illustrate the impact of WR-EC on hippocampal adult neurogenesis in the dentate gyrus. All graphs illustrate group means, with error bars indicating a single standard error from mean. Figure 1 demonstrates increases in cell proliferation following the WR portion of our intervention, indicating that the WR component is robustly capable of increasing cellular proliferation in the DG of the hippocampus in normally developing, early-life stressed, and alcohol-exposed animals. Figure 2 demonstrates the ability of EC to increase survival of adult generated cells in the DG in animals that were exposed to either stress or alcohol neonatally. Figure 3 demonstrates the increase in cells that differentiate into a neuronal phenotype, indicating that WR-EC can increase proliferation and survival of adult-born dentate gyrus granule cells in animals that undergo neonatal exposure to alcohol or intubation stress, implicating it as a therapeutic to rescue deficits in hippocampal adult neurogenesis. Finally, Figure 4 confirms the WR-EC effect on dendritic plasticity: the length of doublecortin-positive dendrites of dentate gyrus&#39; granule cells in AE rats is no longer different from control. Blood alcohol content (BAC) on PD 4 was 321.19 &#177; 14.03 mg/dl (mean &#177; SEM), comparable to other studies using this exposure paradigm28,37. Previous studies have demonstrated that animals across these treatment groups do not differ in distances run during WR15.
<img alt="Figure 1" src="/files/ftp_upload/54947/54947fig1.jpg" /> 
Figure 1. WR Robustly Increases Cell Proliferation in the Hippocampal DG. Photomicrographs illustrate differences in cell proliferation in the DG on PD42 (the cessation of WR) as labeled with Bromo-deoxyuridine (BrdU) in AE animals following WR (A) and social housing (B). WR robustly increases cell proliferations irrespective of neonatal treatment (C). A two-way ANOVA revealed a main effect of housing condition (WR vs. SH) (F1,40 = 19.703, p &#60; 0.001), while no significant main effect of postnatal treatment (SC vs. SI vs. AE) or interaction between the two factors were observed. Post hoc comparisons were performed as Tukey&#39;s tests. All values represent mean &#177; standard error of the mean (SEM). *p &#60; 0.05, #p &#60; 0.01. This figure has been reproduced from Hamilton et al., 20128. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54947/54947fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54947/54947fig2.jpg" /> 
Figure 2. WR Followed by EC Rescues Deficits in Cell Survival Following Neonatal Alcohol Exposure or Sham Stress. Photomicrographs illustrate differences in cells labeled with BrdU in AE animals from WR-EC (A) and social housed conditions (B) injected with BrdU on PD41. Socially housed animals displayed a decrease following alcohol exposure relative to suckle controls. Animals undergoing the WR-EC superintervention display increased survival rates of cells proliferating after PD41 in both SI and AE groups (C). A two-way ANOVA revealed a main effect of housing condition (WR vs. SH) (F1,29 = 11.402, p &#60; 0.01) and a significant interaction between postnatal treatment and housing condition (F1,29 = 3.870, p &#60; 0.05), while no significant main effect of postnatal treatment (SC vs. SI vs. AE) was observed. A one-way ANOVA within SH animals revealed a main effect of postnatal treatment (F1,19 = 3.727, p &#60; 0.05) whereas a one-way ANOVA within WREC animals revealed no significant differences between postnatal treatments. Post hoc comparisons were performed as Tukey&#39;s tests. All values represent mean &#177; SEM. *p &#60; 0.05, #p &#60; 0.01. This figure has been reproduced from Hamilton et al., 20128. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54947/54947fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54947/54947fig3.jpg" /> 
Figure 3. WR-EC Rescues Deficits in Neurogenesis Following Neonatal Alcohol Exposure or Sham Stress. Co-localization of BrdU (green) expression and NeuN (red) in hippocampal granule cells. Fluorescent confocal images were acquired following immunohistochemical procedures. BrdU was injected on PD41 tissue was collected on PD72. Both BrdU and NeuN were observed in the DG (A, B). Although SC animals did not show a significant increase in number of proliferating neurons, both AE and SI animals showed an increase in neurogenesis (as indicated by double labeling with BrdU and NeuN) following the WR-EC paradigm compared to socially housed animals (C). A two-way ANOVA revealed a main effect of housing condition (WR vs. SH) (F1,28 = 20.48, p &#60; 0.001), while no significant main effect of postnatal treatment (SC vs. SI vs. AE) or interaction between the two factors were observed. Post hoc comparisons were performed as Tukey&#39;s tests. All values represent mean &#177; SEM. *p &#60; 0.05, #p &#60; 0.01. This figure has been reproduced from Hamilton et al., 20128. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54947/54947fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54947/54947fig4.jpg" /> 
Figure 4. WR-EC Rescues Deficits in Dendritic Complexity of Hippocampal DG Granule Cells. Sholl analyses of dendritic intersections illustrate WR-EC&#39;s ameliorative effects on dendritic complexity in the dentate gyrus of adult rats following neonatal alcohol exposure. In social housing conditions, AE animals have a decreased number of DG granule cell dendrite intersections relative to control animals (a). Housing in WREC increases the number of intersections in AE animals relative to socially housed controls (b). AE animals reared in our WREC paradigm display similar numbers of intersections relative to control animals housed in WREC (c). Repeated measure ANOVAs were performed on the data in each graph. Panel a demonstrates a main effect of postnatal treatment (F1,11 = 6.265, p = 0.029). Panel b demonstrates a trend toward a main effect between housing conditions (F1,6 = 4.181, p = 0.087). Panel c demonstrates no significant difference between SC and AE animals within the WREC housing condition. All post hoc comparisons were performed as Tukey&#39;s tests. All values represent mean &#177; SEM. ^p &#60; 0.01, *p &#60; 0.05. This figure has been reproduced from Hamilton, 201236. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54947/54947fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Wheel Running and Environmental Complexity as a Therapeutic Intervention in an Animal Model of FASD at JoVE.com

Wheel Running and Environmental Complexity as a Therapeutic Intervention in an Animal Model of FASD

Cardiovascular exercise and stimulating experiences in a complex environment have positive benefits on multiple measures of neuroplasticity within the rodent brain. This article will discuss the implementation of these interventions as a &#34;superintervention&#34; which combines wheel running and environmental complexity and will address the limitations of these interventions.

Aerobic exercise (e.g., wheel running (WR) extensively used in animal research) positively impacts many measures of neuroplastic potential in the brain, ...

wheel-running-environmental-complexity-as-therapeutic-intervention-an

Nanyang Technological University

Research

JoVE Journal

Behavior

6.7K Views.  University of Delaware. Cardiovascular exercise and stimulating experiences in a complex environment have positive benefits on multiple measures of neuroplasticity within the rodent brain. This article will discuss the implementation of these interventions as a "superintervention" which combines wheel running and environmental complexity and will address the limitations of these interventions.

Video: Wheel Running and Environmental Complexity as a Therapeutic Intervention in an Animal Model of FASD

The Crossmodal Congruency Task as a Means to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden hand is touched. If the viewed and felt touches are given at the same time then this is sufficient to induce the compelling experience that the rubber hand is one's own hand. The RHI can be used to investigate exactly how the brain constructs distinct body representations for one's own body. Such representations are crucial for successful interactions with the external world. To obtain a subjective measure of the RHI, researchers typically ask participants to rate statements such as "I felt as if the rubber hand were my hand". Here we demonstrate how the crossmodal congruency task can be used to obtain an objective behavioral measure within this paradigm.
The variant of the crossmodal congruency task we employ involves the presentation of tactile targets and visual distractors. Targets and distractors are spatially congruent (i.e. same finger) on some trials and incongruent (i.e. different finger) on others. The difference in performance between incongruent and congruent trials - the crossmodal congruency effect (CCE) - indexes multisensory interactions. Importantly, the CCE is modulated both by viewing a hand as well as the synchrony of viewed and felt touch which are both crucial factors for the RHI.
The use of the crossmodal congruency task within the RHI paradigm has several advantages. It is a simple behavioral measure which can be repeated many times and which can be obtained during the illusion while participants view the artificial hand. Furthermore, this measure is not susceptible to observer and experimenter biases. The combination of the RHI paradigm with the crossmodal congruency task allows in particular for the investigation of multisensory processes which are critical for modulations of body representations as in the RHI.

We demonstrate how an objective measure can be employed in the widely employed rubber hand illusion paradigm. This measure is obtained by modifying the well-established crossmodal congruency task. This task allows the investigation of multisensory processes which are critical for modulations of body representations as in the rubber hand illusion.

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden ...

Nest Building as an Indicator of Health and Welfare in Laboratory Mice

The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.

We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

The minimization and alleviation of  suffering has moral and scientific implications. In order to mitigate this  negative experience one must be able to ...

The Use of the Puzzle Box as a Means of Assessing the Efficacy of Environmental Enrichment

Environmental enrichment can dramatically influence the development and function of neural circuits. Further, enrichment has been shown to successfully delay the onset of symptoms in models of Huntington&#8217;s disease 1-4, suggesting environmental factors can evoke a neuroprotective effect against the progressive, cellular level damage observed in neurodegenerative disorders. The ways in which an animal can be environmentally enriched, however, can vary considerably. Further, there is no straightforward manner in which the effects of environmental enrichment can be assessed: most methods require either fairly complicated behavioral paradigms and/or postmortem anatomical/physiological analyses. This protocol describes the use of a simple and inexpensive behavioral assay, the Puzzle Box 5-7 as a robust means of determining the efficacy of increased social, sensory and motor stimulation on mice compared to cohorts raised in standard laboratory conditions. This simple problem solving task takes advantage of a rodent&#8217;s innate desire to avoid open enclosures by seeking shelter. Cognitive ability is assessed by adding increasingly complex impediments to the shelter&#8217;s entrance. The time a given subject takes to successfully remove the obstructions and enter the shelter serves as the primary metric for task performance. This method could provide a reliable means of rapidly assessing the efficacy of different enrichment protocols on cognitive function, thus paving the way for systematically determining the role specific environmental factors play in delaying the onset of neurodevelopmental and neurodegenerative disease.

Environmental enrichment provides a potential protective effect against neurodegenerative disorders. Currently, however, there is no easy way of determining the efficacy of enrichment procedures. This protocol describes a simple &#8220;Puzzle Box&#8221; method for assessing an animal&#8217;s cognitive function, in order to reveal the effectiveness of environmental enrichment.

Environmental enrichment can dramatically influence the development and function of neural circuits.  Further, enrichment has been shown to successfully ...

The Forced Swim Test as a Model of Depressive-like Behavior

The goal of the present protocol is to describe the forced swim test (FST), which is one of the most commonly used assays for the study of depressive-like behavior in rodents. The FST is based on the assumption that when placing an animal in a container filled with water, it will first make efforts to escape but eventually will exhibit immobility that may be considered to reflect a measure of behavioral despair. This test has been extensively used because it involves the exposure of the animals to stress, which was shown to have a role in the tendency for major depression. Additionally, the FST has been shown to share some of the factors that are influenced or altered by depression in humans, including changes in food consumption, sleep abnormalities and drug-withdrawal-induced anhedonia. The main advantages of this procedure are that it is relatively easy to perform and that its results are easily and quickly analyzed. Moreover, its sensitivity to a broad range of antidepressant drugs that makes it a suitable screening test is one of the most important features leading to its high predictive validity. Despite its appeal, this model has a number of disadvantages. First, the issue of chronic augmentation is problematic in this test because in real life patients need to be treated for at least several weeks before they experience any relief from their symptoms. Last, due to the aversiveness of the FST, it is important to take into account possible influences it might have on brain structure/function if brain analyses are to be carried out following this procedure.

This protocol describes the forced swim test, which is used for the study of depressive-like behavior in rodents. This procedure involves placing an animal in a container filled with water that eventually will lead to the exhibition of immobility behavior, which is considered to reflect behavioral despair.

The goal of the present protocol is to describe the forced swim test (FST), which is one of the most commonly used assays for the study of depressive-like ...

An Innovative Running Wheel-based Mechanism for Improved Rat Training Performance

This study presents an animal mobility system, equipped with a positioning running wheel (PRW), as a way to quantify the efficacy of an exercise activity for reducing the severity of the effects of the stroke&#160;in rats. This system provides more effective animal exercise training than commercially available systems such as treadmills and motorized running wheels (MRWs). In contrast to an MRW that can only achieve speeds below 20 m/min, rats are permitted to run at a stable speed of 30 m/min on a more spacious and high-density rubber running track supported by a 15 cm wide acrylic wheel with a diameter of 55 cm in this work. Using a predefined adaptive acceleration curve, the system not only reduces the operator error but also trains the rats to run persistently until a specified intensity is reached. As a way to evaluate the exercise effectiveness, real-time position of a rat is detected by four pairs of infrared sensors deployed on the running wheel. Once an adaptive acceleration curve is initiated using a microcontroller, the data obtained by the infrared sensors are automatically recorded and analyzed in a computer. For comparison purposes, 3 week training is conducted on rats using a treadmill, an MRW and a PRW. After surgically inducing middle cerebral artery occlusion (MCAo), modified neurological severity scores (mNSS) and an inclined plane test were conducted to assess the neurological damages to the rats. PRW is experimentally validated as the most effective among such animal mobility systems. Furthermore, an exercise effectiveness measure, based on rat position analysis, showed that there is a high negative correlation between the effective exercise and the infarct volume, and can be employed to quantify a rat training in any type of brain damage reduction&#160;experiments.

This study presents an innovative running wheel-based animal mobility system to quantify an effective exercise activity in rats. A rat-friendly testbed is built, using a predefined adaptive acceleration curve, and a high correlation between the effective exercise rate and the infarct volume suggests the protocol&#39;s potential for stroke prevention experiments.

This study presents an animal mobility system, equipped with a positioning running wheel (PRW), as a way to quantify the efficacy of an exercise activity ...

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the "click" sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward. The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. The mice learned rather quickly when challenged with tasks of the clicker training protocol, and almost all trained mice overcame the challenges they were given (100% of female mice and 83% of male mice). This study has identified that clicker training for mice strongly correlates with reduced fear in the mice during human-mice interactions, as shown by reduced anxiety-related behaviors (e.g., defecation, vocalization, and urination) and fewer depression-like behaviors (e.g., floating). By developing a reliable protocol that can be easily integrated into the daily routine of the keeping of laboratory mice, the lifetime experience of welfare in the mice can be improved substantially.

The development of new refinement strategies for laboratory mice is a challenging task that contributes towards fulfilling the 3R principle. This protocol introduces clicker training as a cognitive enrichment program for laboratory mice.

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous ...

Establishment of a Valuable Mimic of Alzheimer's Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure change. With the increase of AD patients worldwide, the pathology and treatment of the disease has become a focus in the International Pharmaceutical Industry. Thus, the establishment of the animal model to mimic AD in the laboratory is of great importance.
Here, we describe a detailed protocol for establishing a mimic of AD in a rat animal model though intracerebroventricular injection of amyloid beta protein 25-35 (A&#946;25-35) combined with aluminum trichloride (AlCl3) and anterodorsal thalamic nucleus injection of recombinant human transforming growth factor-&#946;1 (RHTGF-&#946;1) to rats. The related markers of AD were measured, including: spatial memory, neuronal structure and substructure, neuronal A&#946;, and neurofibrillary tangles (NFT) production. This rat model demonstrates spatial memory impairment, neuronal structure and substructure pathological changes, neuron intracellular A&#946; burden, and NFT aggregation, and provides a close mimic of the neuronal structure and function disorder to that of clinical AD patients. Thus, the presented AD rat modelprovides a valuable in vivo tool for exploring neuronal function, neuronal pathology, and drug screening of AD.

This is a protocol to mimic Alzheimer&#39;s Disease in rats by evaluation of spatial memory impairment, neuronal pathological changes, neuronal amyloid beta protein (A&#946;) burden, and neurofibrillary tangles aggregation, induced by the injection of A&#946;25-35 combined with aluminum trichloride and recombinant human transforming growth factor-&#946;1.

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure ...

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

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

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

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

Nest Building Behavior as an Early Indicator of Behavioral Deficits in Mice

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents synthetic and/or natural materials (such as twine, tissue, cotton, paper, and hay) as a gauge of their overall well-being and as an ancillary assessment to predict the possible decline in cognition. Typically, changes in nesting behaviors, such as failure to create a nest, indicate a change in health or welfare. In addition, nesting behavior is sensitive to many environmental and physiological challenges, as well as many genetic mutations underlying pathological disease states. The following protocol describes a nesting behavior paradigm that explores the usage of four types of nesting material. In addition, the protocol utilizes intraclass correlations to demonstrate that inter-rater reliability is higher when nests are constructed out of shredded paper compared to other common nesting materials such as cotton squares, paper twists, and soft cob bedding. The chosen methodology and statistical considerations (i.e., intraclass correlation) for this assay may be of interest for those conducting experiments assessing the quality of living of mice.&#160;

Here, we present a protocol to quantify nest building behavior in mice, which is known to be impaired in several neurological disorders and diseases. This protocol examines the utility of four materials and offers the opportunity to quantify the rater agreement in scoring, improving the validity and reliability of the assay.

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents ...

The Use of Traditional Fear Tests to Evaluate Different Emotional Circuits in Cattle

Animal temperament is complex and has implications for productivity and economic profitability. Quantifying an animal&#39;s response to differing stimuli may facilitate breeding selections and identify animals that are better suited to specific management strategies. Multiple tests have been developed to evaluate cattle temperament (e.g., exit velocity, chute score, pen score, open field test, startle test, bovine zero maze), but each of these tests evaluates the animal&#39;s response to different stimuli (e.g., isolation, novel environment, startle, willingness to enter an enclosed area). Cattle temperament has been observed to be relatively stable over time. However, the evaluation of temperament has the potential to be influenced by current conditions, previous experiences, and observer bias. Many of these temperament tests have been improperly categorized as fear tests and have also been criticized for being subjective. This paper provides a framework for standardizing behavioral tests for cattle and suggests that these different evaluations assess different aspects of the animal&#39;s overall temperament.

Here, we present a protocol to conduct a variety of behavioral tests in cattle that have been designed to evaluate emotions. A battery of behavioral tests (open field test, startle test, bovine zero maze, exit velocity, pen score, and chute score) were conducted to evaluate different components of animal temperament.

Animal temperament is complex and has implications for productivity and economic profitability. Quantifying an animal&#39;s response to differing stimuli ...