This work was funded by the National Institute of Health grant AA17991 (to J.L.), Carefree Biotechnology Foundation (to J.L.), University of Southern California (USC), USC Graduate School Travel/Research Award (to S.W.) Saudi Arabia Cultural Mission Scholarship (to A.A.O.), and Army Health Professions Scholarship Program (to A.S.S.).

All animal experiments are performed according to the protocols approved by the University of Southern California (USC) Institutional Animal Care and Use Committee (IACUC), and all methods are carried out in accordance with relevant guidelines, regulations, and recommendations.
1. Animals
<ol>
	<li>Obtain approval from appropriate animal care committees for the study.</li>
	<li>Set the vivarium to a dark-light 12 h cycle with controlled temperature and humidity between 24 &#177; 2 &#176;C and 50%-60%, respectively.</li>
	<li>Obtain male and/or female wild type C57BL/6 mice aged 6-8 weeks. After stratifying the animals by sex, randomly assign them to one of the following groups: 1) group house with vehicle treatment; 2) group house with drug treatment; 3) social isolation with vehicle treatment; or 4) social isolation with drug treatment. Aim for at least four mice per group per sex (ideally six mice per group).</li>
	<li>Upon arrival of the mice, acclimate them to the vivarium for at least 24 h. The mice should arrive singly housed.</li>
</ol>
2. Cage setup
<ol>
	<li>For social isolation animals, take a standard mouse cage (75 in2 floor space) and add half the amount of bedding and a 1 in2 piece of cotton (or equivalent) for nesting.</li>
	<li>Wrap the outside walls of the cages in opaque, black plastic bags (or equivalent) and secure using tape. Make sure the mice cannot see the outside environment or surrounding animals.
	<ol>
		<li>Leave the top and bottom of the cage unwrapped, unless the mice can see neighboring animals through them.</li>
		<li>When wrapping, ensure that no segment of the bag is accessible from the inside of the cage. This is to prevent the animal from tearing the bag apart.</li>
		<li>Do not include any form of environmental enrichment, such as toys or running wheels.</li>
	</ol>
	</li>
	<li>Carefully and gently place the mice in the prepared cages. Provide food and water ad libitum.</li>
	<li>House control mice in groups of two or three under normal caging conditions (i.e., in a standard mouse cage [75 in2 floor space], a full amount of bedding, a 2 in2 piece of cotton or equivalent for nesting, and no wrapping of opaque bags).
	<ol>
		<li>Ensure the group-housed mice are compatible with each other (i.e., there are no fighting/conflicts between them). If conflict occurs, remove the aggressor and exclude from the analysis.</li>
		<li>Separate male and female mice housing and keep distance between the males and females to avoid the possibility of affecting the endocrine level changes of female mice due to their ability to smell.</li>
	</ol>
	</li>
</ol>
3. Care and treatment during the social isolation period
<ol>
	<li>Disturb the mice as minimally as possible during the social isolation period. Perform any procedures and activities, such as cage changes and treatment administration, during their active period (i.e., during the dark cycle) and under minimal noise disturbances.</li>
	<li>Change the cages only once a week during the dark cycle. The same plastic bag can be removed and rewrapped to new cages, unless significant damage is present.
	<ol>
		<li>For control (group housed) mice, change the cages twice a week or more as necessary during the dark cycle.</li>
	</ol>
	</li>
	<li>Ensure the mice have plenty of water and food to last at least 1 week.</li>
	<li>Continue isolating (or group housing) the mice for at least 4 weeks to see optimal results.</li>
</ol>
4. Agar drug/treatment preparation-a noninvasive drug treatment
<ol>
	<li>If treatments (e.g., drugs under investigation) are involved in the study, ideally administer the treatment with as little handling as possible, by utilizing agar forms. Routes such as injection and oral gavage inflict additional stress on the mice that may become a confounding factor of anxiety.</li>
	<li>Adjust the timing and frequency of treatment based on the nature of the drug used. 
	NOTE: In this study, 2 mg/kg dihydromyricetin (DHM, [(2R,3R)-3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-2,3-dihydrochromen-4-one]) was used as the treatment. DHM was administered daily, in a single dose, during the dark phase of the last 2 weeks of the isolation (or group house) period.</li>
	<li>To prepare the treatment, add 3% (w/v) agar in deionized (DI) water and heat to ~90 &#176;C to dissolve. The solution will bubble. Prevent spillage or boiling over. 
	NOTE: Heat the solution in a glass flask via short, 10 s intervals of microwaving. 
	CAUTION: The glassware will be hot. Wear appropriate personal protective equipment (PPE) when handling the solution.</li>
	<li>Swirl the solution and visually ensure a homogenous solution. 
	NOTE: The solution should be translucent and light yellow to light brown in coloration.</li>
	<li>While the solution is still warm, add 5% (w/v) sucrose and the desired dose of treatment. Only add sucrose and do not add the treatment of interest to the vehicle control.</li>
	<li>Swirl the solution and visually ensure a homogenous solution. Then, pour the solution into a mold and let cool at room temperature to solidify. If the treatment is light-sensitive, make sure to protect it from light. 
	NOTE: The solution should be slightly viscous.</li>
	<li>Once solidified, cut the agar into cubes of 0.5 cm x 0.5 cm x 0.5 cm and store at 4 &#176;C until administration.</li>
	<li>To administer the treatment, place a single cube onto a small weigh boat. During the dark phase of the light-dark cycle, quietly and carefully place the agar-weigh boat into individual cages, without touching the mouse. Allow the mouse to consume the agar. 
	NOTE: Mice typically spend 15-45 min to completely consume the agar.</li>
	<li>Confirm complete consumption of the agar and then carefully remove the weigh boat from the cage. Repeat as necessary.</li>
	<li>Prepare agar cubes weekly to keep fresh and avoid any contamination.</li>
</ol>
5. Behavior analysis
<ol>
	<li>Perform behavioral tests 24 h after the last day of the 4 week (or more) isolation period. Conduct tests during the dark phase under indirect red lighting and record with a video camera.</li>
	<li>Arrange at least three individuals to conduct manual offline scoring in a double-blinded manner to minimize bias and error.</li>
	<li>Elevated plus maze (EPM)
	<ol>
		<li>Prepare the EPM apparatus. The apparatus used in this protocol was obtained commercially (see Table of Materials) and made of opaque plastic with two open arms and two closed arms (33 cm x 5 cm each, open arms perpendicular to the closed arms) with a center platform of 5 cm x 5 cm. Elevate the apparatus 50 cm above the floor.</li>
		<li>Place the animal on the center of the apparatus, facing an open arm. Allow the animal to explore for 5 min and record their activity using a video camera.
		<ol>
			<li>Clean the apparatus after each animal by thoroughly wiping all surfaces with disinfectant (70% ethyl alcohol). Ensure that all rodent droppings are wiped off.</li>
		</ol>
		</li>
		<li>Score the mice&#39;s behavior offline based on time spent in open arms, closed arms, and the center platform using a stopwatch. Start the stopwatch when the mouse places at least three paws in the respective arm or platform.</li>
	</ol>
	</li>
	<li>Open field (OF) test
	<ol>
		<li>Prepare the OF apparatus. The apparatus used in this protocol (see Table of Materials) was made of opaque plastic measuring 50 cm x 50 cm x 38 cm (length x width x height).</li>
		<li>Draw square grids (10 cm x 10 cm each) on the field for a total of 25 grids.</li>
		<li>Place the animal on the center of the field and allow to explore for 10 min. Record their activity on a video camera.
		<ol>
			<li>Clean the apparatus after each animal by thoroughly wiping all surface with disinfectant (70% ethyl alcohol). Ensure that all rodent droppings are wiped off.</li>
		</ol>
		</li>
		<li>Score the mice&#39;s behavior offline based on the time spent in the central zone, time spent in the corners, total distance traveled, and number of times the mouse reared.
		<ol>
			<li>Use a stopwatch to record the time spent in the center or corner. Start the stopwatch when the mouse places at least three paws in the respective area.</li>
			<li>Use a counter to record the distance traveled and frequency of rearing. Count the number of squares the mouse enters (when the mouse places at least three paws in the square). Count rearing when the mouse clearly stands up on its hind paws. Do not count when the mouse stands up and leans against the walls or when it stands up to groom.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Novel object recognition (NOR) test
	<ol>
		<li>Perform this test over 3 days. On day 1, prepare an open field apparatus of 50 cm x 50 cm x 38 cm (length x width x height). Place the animal in the center of the open field and allow to familiarize for 5 min. Then place the animal back in its home cage.
		<ol>
			<li>Clean the apparatus after each animal by thoroughly wiping all surfaces with disinfectant (70% ethyl alcohol). Ensure that all rodent droppings are wiped off.</li>
		</ol>
		</li>
		<li>On day 2, prepare the same open field apparatus and place two identical objects, such as a small cube. Place them symmetrically about 20 cm apart. Place the animal in the center of the apparatus and allow to explore for 5 min. Then place the animal back in its home cage.
		<ol>
			<li>Clean the apparatus after each animal by thoroughly wiping all surfaces with disinfectant (70% ethyl alcohol). Ensure that all rodent droppings are wiped off.</li>
		</ol>
		</li>
		<li>On day 3, prepare the same open field apparatus and one of the objects from day 2 (i.e., small cube), which will function as the familiar object. Place another, dissimilar novel object, such as a wooden pyramid, symmetrically from the familiar object about 20 cm apart. Allow the animal to explore for 3 min and record their activity on a video camera.
		<ol>
			<li>Clean the apparatus after each animal by thoroughly wiping all surfaces with disinfectant (70% ethyl alcohol). Ensure that all rodent droppings are wiped off.</li>
		</ol>
		</li>
		<li>Score the mice&#39;s behavior offline based on the time spent exploring the familiar object and the novel object. Calculate the object recognition index (ORI%), where <img alt="Equation 1" src="/files/ftp_upload/64567/64567eq01.jpg" style="margin: auto;" />; tf and tn represent the times of exploring the familiar and novel objects, respectively.</li>
	</ol>
	</li>
	<li>Novel context recognition (NCR) test
	<ol>
		<li>Perform this test over 2 days. Prepare two distinctly shaped open fields and two pairs of distinctly shaped objects. The OF apparatus can be used as one of the contexts (open field). The other context should be of similar size but different shape, such as a round open field.</li>
		<li>On day 1, place one pair of identical objects (i.e., two cubes) in the square context and the other pair of identical objects (i.e., two pyramids) in the round context. Objects should be placed symmetrically 15-20 cm apart.</li>
		<li>Place the animal in the center and allow to explore for 5 min in one context. Repeat in the other context. Then, place the animal back in its home cage.
		<ol>
			<li>Clean the apparatus after each animal by thoroughly wiping all surfaces with disinfectant (70% ethyl alcohol). Ensure that all rodent droppings are wiped off.</li>
		</ol>
		</li>
		<li>On day 2, swap one of the objects from one context with the other (i.e., place one cube and one pyramid in the square context, and one cube and one pyramid in the round context).</li>
		<li>Place the animal in the center and allow to explore for 3 min. Record their activity on a video camera. The animals do not need to be recorded in both contexts.
		<ol>
			<li>Clean the apparatus after each animal by thoroughly wiping all surfaces with disinfectant (70% ethyl alcohol). Ensure that all rodent droppings are wiped off.</li>
		</ol>
		</li>
		<li>Score the mice&#39;s behavior offline based on the time spent exploring the distinct objects. Calculate the recognition index (RI%) as the proportion of time spent investigating the novel &#34;out-of-context&#34; object (i.e., the pyramid in the square context) versus the familiar &#34;in-context&#34; object (i.e., the cube in the square context). <img alt="Equation 2" src="/files/ftp_upload/64567/64567eq02.jpg" style="margin: auto;" />.</li>
	</ol>
	</li>
</ol>

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The authors declare no conflicts of interest.

Critical steps in the protocol include properly setting up the social isolation cages (i.e., wrapping of opaque bags and reducing the amount of bedding), minimizing the handling and disturbance of mice throughout the isolation period, and making sure the mice obtain and consume the agar with drug completely. It is critical that the vivarium or housing condition is maintained at a constant temperature and humidity, as well as minimized external interferences. Significant effort should be placed toward reducing as...

Anxiety disorders represent the largest class and burden of mental diseases in the United States (US), with related annual costs exceeding US$42 billion1,2,3. In recent years, anxiety and stress have heightened the prevalence of suicide and suicide ideation by over 16%4. Patients with chronic diseases are especially vulnerable to unintended secondary effects of mental distress or reduced cognitive function5. Current treatments for anxiety include psychotherapy, medications, or a combination of both6. However, despite this crisis, less than 50% of patients achieve full remission6,7. Anxiolytics such as benzodiazepines (BZs) and selective serotonin reuptake inhibitors (SSRIs) have significant drawbacks or produce little to no immediate effects8. Moreover, there is a relative scarcity of novel anxiolytics under development, challenged by the costly and time-consuming process of drug development9,10.
A critical step in the drug development process is the establishment and utilization of an animal model, such as mice, to study pathological changes and test drug safety and efficacy11. Current approaches to establishing anxiety animal models include 1) genetic manipulation, such as knocking out serotonin receptors (5-HT1A) or &#947;-aminobutyric acid A receptor (GABAAR) &#945; subunits12; 2) chronically administering anxiety-inducers such as corticosterone or lipopolysaccharides (LPS)13,14; or 3) administering environmental stress including social defeat and maternal separation15. These methods, however, may not realistically reflect anxiety induced throughout daily life and therefore may not be suitable for investigating the underlying mechanism or testing novel drugs.
Like humans, mice and rats are highly social creatures16,17,18. Social contact and social interactions are essential for optimal brain health and are critical for proper neurodevelopment during the rearing period19. Thus, maternal separation or social isolation during the rearing period results in mice that show more anxiety, depression, and changes in neurotransmission20. Moreover, social grooming or allogrooming is a common form of bonding or comforting behavior among mice and rats that live together21. Thus, socialization is an integral part of rodent life, and isolation negatively impacts their health.
In this context, the present protocol describes a novel anxiety model to mimic the intentional or unintentional patterns of social isolation in modern life. This social isolation (SI) model minimizes perceived distractions and invasiveness and utilizes adult wild type C57BL/6 mice and Sprague-Dawley (SD) rats. The protocol presented here focuses on the anxiety mice model based on our published evidence, which showed increased anxiety-like behavior, aggression, decreased cognition, and increased neuroinflammation as a result of social isolation22,23,24. Anxiety-like behavior is confirmed by the elevated plus maze (EPM) and open field (OF) tests, while cognitive function is measured by novel object recognition (NOR) and novel context recognition (NCR) tests. This model is useful for investigating anxiety and related disorders but can also be adapted or modified to study the natural progression and development of&#160;mild cognitive impairment as well as metabolic changes due to stress.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Black Plastic Bags</td>
			<td>Office Depot</td>
			<td>791932</td>
			<td>24&#34; x 32&#34;</td>
		</tr>
		<tr>
			<td>Elevated Plus Maze</td>
			<td>SD Instruments</td>
			<td>NA</td>
			<td>Black color</td>
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			<td>Open Field enclosure</td>
			<td>SD Instruments</td>
			<td>NA</td>
			<td>White color</td>
		</tr>
		<tr>
			<td>Select Agar</td>
			<td>Invitrogen</td>
			<td>30391-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Square cotton for nesting (nestlet)</td>
			<td>Ancare Corporation</td>
			<td>NC9365966</td>
			<td>Divide a 2&#34; square piece into 4 pieces to create a 1&#34; square piece for isolation group</td>
		</tr>
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			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S1888-1KG</td>
			<td></td>
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			<td>Weigh boat</td>
			<td>SIgma</td>
			<td>HS1420A</td>
			<td>Small, square white polystyrene</td>
		</tr>
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social isolation model: a noninvasive rodent model of stress and anxiety

Anxiety disorders are one of the leading causes of disability in the United States (US). Current treatments are not always effective and less than 50% of patients achieve full remission. A critical step in developing a novel anxiolytic is to develop and utilize an animal model, such as mice, to study pathological changes and test drug target(s), efficacy, and safety. Current approaches include genetic manipulation, chronic administration of anxiety-inducing molecules, or the administration of environmental stress. These methods, however, may not realistically reflect anxiety induced throughout daily life. This protocol describes a novel anxiety model, which mimics the intentional or unintentional patterns of social isolation in modern life. The social isolation-induced anxiety model minimizes perceived distractions and invasiveness and utilizes wild type C57BL/6 mice. In this protocol, 6- to 8-week-old mice (male and female) are singly housed in opaque cages to visually block the external environment, such as neighboring mice, for 4 weeks. No environmental enrichments (such as toys) are provided, bedding material is reduced by 50%, any treatment of drug is administered as an agar form, and the exposure/handling of the mice is minimized. Socially isolated mice generated using this protocol exhibit greater anxiety-like behavior, aggression,&#160;as well as decreased cognition.

All representative results and figures were modified from our recent publications22,23. To evaluate the effects of social isolation on anxiety and exploratory behavior, EPM and OF tests were performed 24 h following the end date of the 4 week social isolation period. Socially isolated mice spent significantly less time in the open arm (1.28 &#177; 0.17 min) compared to the control (2.31 &#177; 0.27 min), and a significantly longer time in the closed arm (3.31 &#1...

Watch this Scientific Journal Video about Social Isolation Model: A Noninvasive Rodent Model of Stress and Anxiety at JoVE.com

Social Isolation Model: A Noninvasive Rodent Model of Stress and Anxiety

Presented here is a social isolation (SI)-induced anxiety mouse model that utilizes wild type C56BL/6J mice to induce stress and anxiety-like behavior with minimal handling and no invasive procedures. This model reflects modern life patterns of social isolation and is ideal for studying anxiety and related disorders.

Anxiety disorders are one of the leading causes of disability in the United States (US). Current treatments are not always effective and less than 50% of ...

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Research

JoVE Journal

Neuroscience

3.9K Views.  University of Southern California. Presented here is a social isolation (SI)-induced anxiety mouse model that utilizes wild type C56BL/6J mice to induce stress and anxiety-like behavior with minimal handling and no invasive procedures. This model reflects modern life patterns of social isolation and is ideal for studying anxiety and related disorders.

Video: Social Isolation Model: A Noninvasive Rodent Model of Stress and Anxiety

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Hyponeophagia: A Measure of Anxiety in the Mouse

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often hampered by a phenomenon known as "bait shyness". Mice and rats cannot vomit, due to the tightness of the cardiac sphincter of the stomach, so to overcome the problem of potential food toxicity they have evolved a strategy of first ingesting only very small amounts of novel substances. The amounts ingested then gradually increase until the animal has determined whether the substance is safe and nutritious. So the old rat-catchers would first put a palatable substance such as oatmeal, which was to be the vehicle for the toxin, in the infested area. Only when large amounts were being readily consumed would they then add the poison, in amounts calculated not to affect the taste of the vehicle. The poisoned bait, which the animals were now readily eating in large amounts, would then swiftly perform its function.
Bait shyness is now used in the behavioural laboratory as a way of measuring anxiety. A highly palatable but novel substance, such as sweet corn, nuts or sweetened condensed milk, is offered to the mice (or rats) in a novel situation, such as a new cage. The latency to consume a defined amount of the new food is then measured.
Robert M.J. Deacon can be reach at <a href="mailto:robert.deacon@psy.ox.ac.uk">robert.deacon@psy.ox.ac.uk</a>

Mice and rats, due to their innate cautiousness, are initially slow in consuming a novel food, particularly in a novel place. This hyponeophagia can readily be measured in the laboratory, even though laboratory animals are much less anxious than their wild counterparts

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often ...

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

High-density Electroencephalographic Acquisition in a Rodent Model Using Low-cost and Open-source Resources

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network connectivity, are applicable to an expanding variety of questions in neuroscience. Performing these kinds of analyses in a rodent model requires higher electrode density than traditional screw electrodes can accomplish. While higher-density electroencephalographic montages for rodents exist, they are of limited availability to most researchers, are not robust enough for repeated experiments over an extended period of time, or are limited to use in anesthetized rodents.1-3 A proposed low-cost method for constructing a durable, high-count, transcranial electrode array, consisting of bilaterally implantable headpieces is investigated as a means to perform advanced electroencephalogram analyses in mice or rats.

Procedures for headpiece fabrication and surgical implantation necessary to produce high signal to noise, low-impedance electroencephalographic and electromyographic signals are presented. While the methodology is useful in both rats and mice, this manuscript focuses on the more challenging implementation for the smaller mouse skull. Freely moving mice are only tethered to cables via a common adapter during recording. One version of this electrode system that includes 26 electroencephalographic channels and 4 electromyographic channels is described below.

Instructions for the low-cost construction and surgical implantation of a chronic transcranial high-density electroencephalographic montage into mice are provided. Signal recording, extraction, and processing techniques are also described.

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network ...

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during development, homeostasis, and disease. Although macrophages have been studied in detail for decades, specialized features of microglia, the tissue-resident macrophages of the CNS, have remained largely mysterious, in part due to limitations in the ability to recapitulate mature microglial properties in culture. Here, we illustrate a straightforward procedure for the rapid isolation of pure microglia from the mature rodent brain. We also describe serum-free culture conditions that support high levels of microglial viability over time. Microglia cultured under these defined-medium conditions exhibit elaborate ramified processes and dynamic surveillance behavior. We illustrate some effects of serum exposure on cultured microglia and discuss how these serum-free cultures compare to both serum-exposed cultures as well as microglia in vivo.

Efforts to understand microglial function in detail have been hindered by the lack of microglial culture models that recapitulate the properties of mature in vivo microglia. This protocol describes an isolation and culture approach designed to maintain robust survival of highly ramified mature rat microglia under defined-medium conditions.

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide range of neurological and psychiatric disorders, and memory enhancement in age-related cognitive decline and neurodegenerative diseases. We describe a laboratory protocol for transcranial photobiomodulation therapy (PBMT) in mice. Aged BALB/c mice (18 months old) are treated with a 660 nm laser transcranially, once daily for 2 weeks. Laser transmittance data shows that approximately 1% of the incident red light on the scalp reaches a 1 mm depth from the cortical surface, penetrating the dorsal hippocampus. Treatment outcomes are assessed by two methods: a Barnes maze test, which is a hippocampus-dependent spatial learning and memory task evaluation, and measuring hippocampal ATP levels, which is used as a bioenergetics index. The results from the Barnes task show an enhancement of the spatial memory in laser-treated aged mice when compared with age-matched controls. Biochemical analysis after laser treatment indicates increased hippocampal ATP levels. We postulate that the enhancement of memory performance is potentially due to an improvement in hippocampal energy metabolism induced by the red laser treatment. The observations in mice could be extended to other animal models since this protocol could potentially be adapted to other species frequently used in translational neuroscience, such as rabbit, cat, dog, or monkey. Transcranial photobiomodulation is a safe and cost-effective modality which may be a promising therapeutic approach in age-related cognitive impairment.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide ...

Implantation of Osmotic Pumps and Induction of Stress to Establish a Symptomatic, Pharmacological Mouse Model for DYT/PARK-ATP1A3 Dystonia

Genetically modified mouse models face limitations, especially when studying movement disorders, where most of the available transgenic rodent models do not present a motor phenotype resembling the clinical aspects of the human disease. Pharmacological mouse models allow for a more direct study of the pathomechanisms and their effect on the behavioral phenotype. Osmotic pumps connected to brain cannulas open up the possibility of creating pharmacological mouse models via local and chronic drug delivery. For the hereditary movement disorder of rapid-onset dystonia-parkinsonism, the loss-of-function mutation in the &#945;3-subunit of the Na+/K+-ATPase can be simulated by a highly specific blockade via the glycoside ouabain. In order to locally block the &#945;3-subunit in the basal ganglia and the cerebellum, which are the two brain structures believed to be heavily involved in the pathogenesis of rapid-onset dystonia-parkinsonism, a bilateral cannula is stereotaxically implanted into the striatum and an additional single cannula is introduced into the cerebellum. The cannulas are connected via vinyl tubing to two osmotic pumps, which are subcutaneously implanted on the back of the animals and allow for the chronic and precise delivery of ouabain. The pharmacological mouse model for rapid-onset dystonia-parkinsonism carries the additional advantage of recapitulating the clinical and pathological features of asymptomatic and symptomatic mutation carriers. Just like mutation carriers of rapid-onset dystonia parkinsonism, the ouabain-perfused mice develop dystonia-like movements only after additional exposure to stress. We demonstrate a mild stress paradigm and introduce two modified scoring systems for the assessment of a motor phenotype.

We provide a protocol to generate a pharmacological DYT/PARK-ATP1A3 dystonia mouse model via implantation of cannulas into basal ganglia and cerebellum connected to osmotic pumps. We describe the induction of dystonia-like movements via application of a motor challenge and the characterization of the phenotype via behavioral scoring systems.

Genetically modified mouse models face limitations, especially when studying movement disorders, where most of the available transgenic rodent models do ...