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 e...

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 hippocamp...

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 th...

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, ...

The overall goal of this behavioral intervention is to enhance environmental stimulation of laboratory rodents via exposure to voluntary cardiovascular exercise and environmental complexity in a controlled laboratory setting. This technique can help answer key questions in the field of rehabilitation and neuroplasticity. It highlights the importance of social exercise and exploratory behaviors on neurological development in adolescence and health maintenance in adulthood.The main advantage of this procedure is that it combines the exercise and environmental complexity in a synergistic way, and it allows the interpretation of each of the components separately if need be. To begin the experiment, place weaned rat pups into an appropriate cage and make sure that they always have free access to food and water. A week later, on day 30 of their development, move half the rats into exercise cages with running wheels.Each running wheel should have a revolution counter and be freely accessible. On the same day, weigh all the rats. Every following day at the same hour, check the number of revolutions on the running wheels.On day 36, weigh the rats again. Then, leave them in their respective housing condition for 12 days until day 42. Early on developmental day 42, prepare the environmentally complex cages.The 30 by 18 by 36 inch galvanized steel cages should contain multiple levels capable of supporting the weight of multiple rats. These cages must also be capable of supporting standard bedding and multiple food and water dispensers. Place novel colorful objects of variable sizes and shapes in the cage.First, position six large toys in the cage that are large enough for three or more rats to interact with concurrently. Next, place six medium-sized toys in the cage each of which should also be large enough for three or more rats to use concurrently. Then, place at least 20 small toys in the cage that might only occupy one rat at a time.Select toys of different colors and shapes to enhance the complexity. The novelty of each toy is critical to this form of intervention. At the opposite ends of the cage, place food dispensers and attach water bottles so each cage has two of each.Once the cages are prepared, weigh all the animals and relocate them to the environmentally complex cages. Group nine to 12 animals per cage, making certain that they all have unique identifiers. Every two days, replace all the toys.Make sure to have enough toys such that animals rarely encounter the same toy twice during the paradigm. When replacing the toys, also refill the food and water. Every third day, clean the cages.Temporarily house the rats in groups of two or three and replace the bedding, food, and water. Return the same toys to the cage on these days unless cleaning coincides with the toy replacement schedule. The wheel running and complex environmental interventions impacted adult neurogenesis in the dentate gyrus as assessed by BrdU staining.Increases in cell proliferation were observed following the wheel running portion of the intervention in the normally developing early life stressed and alcohol-exposed animals. The complex environment intervention increased the survival of adult-generated cells in the dentate gyrus in animals that were exposed to either stress or alcohol neonatally. Furthermore, the interventions resulted in an increase in cells that differentiate into a neuronal phenotype as seen with increases in adult-born dentate gyrus granule cells despite neonatal exposure to alcohol or intubation stress.Investigating the length of double cort and positive dentrites of dentate gyrus granule cells showed that there was no difference between the alcohol-exposed rats and control rats after wheel running and environmental complexity intervention. After watching this video, you should have a really good understanding of how to appropriately lay out the environmental complexity cage as well as how to house animals in the wheel running cages and environmental complexity cages. When attempting this procedure, it's important to thoroughly clean all of your materials and check up on your animals daily.Following this procedure, the behavioral, neurophysiological, and neuroanatomical measures can be assessed in order to determine the consequences of exposure to wheel running and environmental complexity interventions. After its development, this technique paved the way for researchers in the field of neuroplasticity to explore experience-dependent changes in rodent models of neurological disease.

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Research

JoVE Journal

Behavior

6.7K Görüntüleme.  University of Delaware. The overall goal of this behavioral intervention is to enhance environmental stimulation of laboratory rodents via exposure to voluntary cardiovascular exercise and environmental complexity in a controlled laboratory setting. This technique can help answer key questions in the field of rehabilitation and neuroplasticity. It highlights the importance of social exercise and exploratory behaviors on neurological development in adolescence and health maintenance in adulthood.The main advan...