This review was not supported by grants. We thank Sanypet Forza 10 USA Corp. Orlando, FL, USA for kindly providing the ND used in this study.

The protocol was examined and approved by the Veterinary Ethical Review Committee prior to the beginning of the study. The recommendations of the ARRIVE guidelines in animal research were also consulted and considered21-25.
1. Dog Selection and Food Supplementation
<ol>
	<li>Select 24 dogs of different breeds (mean age and weight &#177; SEM: 2.9 &#177; 0.3 years and 32.01 &#177; 1.17 kg; 14 males, 10 females) with evident clinical symptoms of behavioral disturbances, such as anxiety, diffidence, irregular biorhythm, reactivity, activation, irritability, alertness, and constant environmental exploration.</li>
	<li>Randomly divide the animals into two groups and place each into a 215,278 sq ft single box. Following the manufacturer&#39;s instructions, give the appropriate dose of standard diet (SD, n = 12) or nutraceutical diet (ND, n = 12) for 10 days, according to the weights of the animals (Table 1).</li>
	<li>Complete two veterinary inspections of the dogs before (T0) and 10 days after (T1) the treatment.</li>
</ol>
2. Behavior Symptoms Acquiring and Scoring
<ol>
	<li>Have a certified veterinary behaviorist score the behavioral (marking, anxiety, diffidence, irregular biorhythm, reactivity, activation, irritability, alertness, environmental exploration and attention requirement) and clinical (dandruff, itchiness, flush, seborrhea, fur opacity, vomiting, diarrhea, flatulence, lachrymation and anal sac repletion) conditions of each dog.
	<ol>
		<li>For each dog, gather a score before and after the 10-days evaluation as follows: 1 = absence of symptom; 2 = moderate presence of symptoms; 3 = marked presence of symptoms.</li>
		<li>At the end of the evaluation summarize, for each symptom, the scores of the dogs of each group before and after 10 days. Plot the data on a statistical software.</li>
	</ol>
	</li>
</ol>
3. The Sensor
Note: The sensor has a 3-axis accelerometer, allowing it to gather movement in each direction (x, y, z). It weighs 8 g and is extremely small (41 x 28 x 11 mm). Moreover, it is compatible with every mobile device that features a global wireless communication. It has a rechargeable battery that can last for 14 days after charging. The data output is calculated in points, which are the units of time spent active and at rest by each dog during the day.
<ol>
	<li>Ensure that the collar is not wider than 30 mm for the optimal fit of the sensor.</li>
	<li>Using the procedure outlined in sections 3 and 4, evaluate the behavioral changes related to the time spent active and at rest before and after treatment with the specific diet.</li>
</ol>
4. Sensor Setting
<ol>
	<li>Open the micro USB cap on the bottom of the unit and use the cable provided to connect the sensor to the computer&#39;s USB 1x/2.0 port or to a Class 2/Limited power supply with a USB output. When an LED starts blinking, charge the sensor for at least 90 min.</li>
	<li>Download and install the dedicated free mobile app from the web store.</li>
</ol>
5. Activity Monitoring and Analysis
<ol>
	<li>Insert the plugs of a wireless router and the dedicated Wi-Fi base station into two different sockets. Wait until the router is ready and the Wi-Fi base station starts flashing.</li>
	<li>Enable Bluetooth on the mobile device and make sure that it is connected to the Internet.</li>
	<li>Open the app and sign up. Then, tap on &#34;Add new dog&#34; and follow the steps below.
	<ol>
		<li>Take a picture of the dog and provide its name.</li>
		<li>Appropriately set the gender, age, weight, neutering status, and location of the dog.</li>
		<li>Set the primary and the secondary breed of the dog.</li>
		<li>Select, if applicable, the presence of allergies (skin, ear, etc.), arthritis, brain aging, cancer, diabetes, heart disease, or excessive weight.</li>
		<li>Select one of the three available lifestyles, with their respective points, according to personal requirements (1. Average, 2. Active, or 3. Olympian). 
		NOTE: Depending on the age of the dog, the points of each lifestyle will change. Given that the aim of this evaluation was to decrease the hyperactivity and the stress of the dogs, the first lifestyle, &#34;Average,&#34; was set as the final goal to reach.</li>
		<li>Tap on &#34;Wi-Fi base station&#34; and then on &#34;Pair a base station&#34; to connect the sensor to the Wi-Fi base station. Wait until the word &#34;FitBark&#34; appears below &#34;Pair a base station.&#34;</li>
	</ol>
	</li>
	<li>Clip a charged sensor to the collar of the dog.</li>
	<li>Repeat steps 4.3 to 4.4 for each dog.</li>
</ol>
6. Bark Recording
<ol>
	<li>Fix a digital voice recorder to the wall of each box at the beginning of the study.</li>
	<li>Start recording the bark activity.
	<ol>
		<li>Every day, before acquiring new data, connect the digital voice recorder to a computer by means of a retractable USB connector provided along with the recorder.</li>
		<li>Drag the folder with voice data from the device to the computer and rename it with the current date.</li>
	</ol>
	</li>
	<li>Repeat steps 6.2.1 - 6.2.2 every day for 10 days.</li>
	<li>At the end of the evaluation transform (in s) the bark time recorded. Summarize the bark time for each group before and after 10 days. Plot the data on a statistical software.</li>
</ol>

The authors have nothing to disclose.

Both SD and ND were two commercially available diets that completely fulfill the recommendations for protein, carbohydrate, and fat content according to the nutritional guidelines for complete and complementary pet food. However, in the ND, nutraceutical substances, such as Punica granatum (0.0457%), Valeriana officinalis (0.026%), Rosmarinus officinalis (0.000044%), Tilia spp (0.0635%), tea extract (0.031%), and L-tryptophan (0.0329%), were added. It is noteworthy that this clinical evaluation was inspired by a previous trivial evaluation where 2 dogs with evident behavioral symptoms mainly ascribed to generalized anxiety showed significant improvements after 3 days of ND supplementation. Here, we successfully used the same ND with 24 dogs presenting behavioral symptoms mainly ascribed to generalized anxiety.
The only critical step that occurred in the protocol was related to the Wi-Fi connection. In some boxes, the sensor signal did not reach the Wi-Fi station and therefore did not provide any data on the activity of the dogs. Thus, a Wi-Fi range extender was used to completely cover the distance between those boxes and the Wi-Fi station. Many studies have been performed to validate the usefulness of small, lightweight, motion-sensing accelerometers for pets44 and humans46-54. The sensor used in this clinical evaluation presents some limitations with respect to the gold-standard method of using a video camera55,56, such as a lack of specificity in distinguishing rest activity from sleep and general motion from an anxiety-related one. On the other hand, the sensor allows for the easy and fast detection of movement, as well as the ability to monitor daily improvements by means of a mobile phone app. Moreover, with respect to the other commercially available devices, this new sensor has a lower weight and price, can be worn by a dog of any weight, and has a long-lasting (~ 14 days) battery life. Moreover, because of the Wi-Fi station, it does not require the owner to be close to the dog while it is registering the improvements57-59. In fact, after registration with the website of the product company, the station can gather and store information that can be seen either on a computer or on a mobile device, even at a long distance (i.e., beyond the Bluetooth and Wi-Fi range). Possible further applications of this sensor will be the monitoring of excessive movement in dogs affected by separation anxiety47-54, abnormal repetitive behaviors60, or narcolepsy61once left alone at home.
Our results pave the way for a different short-term approach to manage dogs with behavioral symptoms mainly ascribed to generalized anxiety disorders, allowing the owner to re-establish a mutual attachment relationship with the dog. In conclusion, a better understanding of dog behavior, both by pet owners and by behavioral experts able to recognize behavioral and clinical symptoms related to generalized anxiety, might be coupled with a specific diet in order to ensure a better quality of life for the animals.

Dogs are generally recognized as the most devoted domesticated animals that live with humans. They are often considered to be family members whose behavioral changes are regarded as serious concerns, especially when these changes threaten their physical integrity and general wellbeing1. Therefore, the development of adjuvant approaches to common therapies for relieving canine behavioral disturbances would help families to improve the quality of life of their dogs, avoiding unwanted phenomena such as dog abandonment and euthanasia2. Most of these behavioral disturbances are related to anxiety caused by stress, and the anxiety may become pathological without proper intervention1,3.
It has been hypothesized that anxiety disorders in dogs are caused not only by significant life changes, but also by chronic or post-traumatic stresses that can alter their homeostasis and can consequently lead to adaptation disorders1,3. This study is based on a clinical evaluation of behavioral disturbances mainly ascribed to generalized anxiety. The typical clinical symptoms of this disorder include constant or increasing reactivity, body and environmental exploration, activation, alertness, and excessive barking; it also often affects the social interactions between dog and owner1,4,5. The predisposing factors could be intrinsic, such as genetics, or extrinsic, such as environmental stimuli1,6. In fact, the aforementioned clinical symptoms can become frequent even without a triggering environmental stimulus. In this sense, the study of the origin of these factors becomes essential for proper diagnosis and subsequent therapy.
The most common therapies for generalized anxiety rely on counterconditioning and desensitization techniques, where the dog learns how to behave once faced with a stimulus that causes anxiety, or on a pharmacological approach based on anxiolytic drug administration7. Based on these considerations, 24 dogs affected by behavioral disturbances mainly ascribed to generalized anxiety received a counterconditioning and desensitization behavioral therapy combined with a nutraceutical diet for 10 days. The diet consisted of a mixed formula of fish proteins, rice carbohydrates, Punica granatum, Valeriana officinalis, Rosmarinus officinalis, Tilia spp, tea extract, and L-tryptophan, with an omega-3:-6 ratio of 1:0.8. Literature reports clearly evidenced that P. granatum is used to treat chronic anxiety and insomnia1,8, while Valeriana officinalis is used for mild sleep disorders and nervous tension9,10. Furthermore, anti-anxiety and anti-depressant effects have been observed after Rosmarinus officinalis11-14 and Tilia spp15,16 consumption. L-theanine, one of the tea constituents, has been shown to play a role in reducing stress and decreasing heart rate in chronic anxiety17,18. Conversely, many studies reported the onset of anxiety, mood, and depressive symptoms after L-tryptophan depletion and/or an omega-3 deficiency19,20.
Generalized anxiety behavior and clinical symptoms, including marking, anxiety, diffidence, irregular biorhythm, reactivity, activation, irritability, alertness, environmental exploration, body exploration, attention requirement, dandruff, itchiness, flush, seborrhea, fur opacity, vomiting, diarrhea, flatulence, lachrymation, and anal sac repletion, were also evaluated. Most of these symptoms were accompanied by signs that induced dogs to spend more time awake and active, rather than at rest or asleep. Therefore, activity and rest times spent by each dog before and after evaluation were assessed. To continuously monitor daily improvements of activity and rest time, a commercially available sensor, which was fixed to the collars of the dogs and connected to a mobile phone or to a Wi-Fi station, was used.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>FitBark Activity monitor</td>
			<td>FitBark Inc.</td>
			<td></td>
			<td>Sensor</td>
		</tr>
		<tr>
			<td>&#160;FitBark Wi-Fi Base Station</td>
			<td>FitBark Inc.</td>
			<td>7002</td>
			<td>Wi-Fi Base Station</td>
		</tr>
		<tr>
			<td>FORZA10 Behavioral Diet 6lbs</td>
			<td>Forza10 USA Corp</td>
			<td>E0030922007</td>
			<td>Nutraceutical diet</td>
		</tr>
		<tr>
			<td>M5, 3G Mobile Wi-Fi&#160;</td>
			<td>TP-LINK</td>
			<td>M5250</td>
			<td>Router</td>
		</tr>
		<tr>
			<td>SmartBox</td>
			<td>Laika</td>
			<td></td>
			<td>215,278 sq ft Dog box</td>
		</tr>
		<tr>
			<td>Recorder</td>
			<td>Olympus</td>
			<td>WS-831</td>
			<td>Voice recorder</td>
		</tr>
	</tbody>
</table>

behavioral disturbances: an innovative approach to monitor the modulatory effects of a nutraceutical diet

In dogs, diets are often used to modulate behavioral disturbances related to chronic anxiety and stress caused by intense and restless activity. However, the traditional ways to monitor behavioral changes in dogs are complicated and not efficient. In the current clinical evaluation, a new, simple monitoring system was used to assess the effectiveness of a specific diet in positively modulating the intense and restless activity of 24 dogs of different ages and breeds. This protocol describes how to easily and rapidly evaluate improvement in a set of symptoms related to generalized anxiety by using a specific sensor, a mobile phone app, a wireless router, and a computer. The results showed that dogs treated with specific diets showed significant improvement in the times spent active and at rest after 10 days (p &lt; 0.01 and p &lt; 0.05, respectively). These dogs also showed an overall significant improvement in clinical and behavioral symptoms. A specific sensor, along with its related hardware, was demonstrated to successfully monitor behavioral changes relating to movement in dogs.

Table 1 shows the daily amount of nutraceutical diet suggested by the manufacturer. In Figure 1, the daily mean activity and rest times spent by dogs belonging to the SD and ND groups during the evaluation period are shown. For instance, a significant decrease from a T0 value of 7,343 &#177; 611.7 to a T1 value of 5,093 &#177; 526.5 was observed in the ND group after 10 days, while no significant difference was observed in the SD group (Figure 1A, *p &#60; 0.05). Conversely, the daily mean rest time significantly increased from a T0 value of 7.6 &#177; 0.3 h to a T1 value of 9.5 &#177; 0.3 h in the ND group after 10 days (**p &#60; 0.01), while no significant difference was observed in the SD group (Figure 1B).
Regarding behavioral symptoms, a reduction of the mean intensity of marking, from 2.50 to 2.41, was observed in dogs belonging to ND group, while no difference was observed in those belonging to the SD group (Figure 2A). On the contrary, anxiety, diffidence, and irregular biorhythm showed a significant reduction in dogs belonging to the ND group, from a T0 value of 2.50 &#177; 0.19 to a T1 value of 1.16 &#177; 0.11 (***p &#60; 0.001, Figure 2B), from a T0 value of 2.08 &#177; 0.28 to a T1 value of 1.17 &#177; 0.12 (Figure 2C, *p &#60; 0.05), and from a T0 value of 2.08 &#177; 0.28 to a T1 value of 1.08 &#177; 0.08 (Figure 2D, **p &#60; 0.01), respectively. No significant difference was observed in the respective SD groups. Also, reactivity, activation, irritability, alertness, environmental exploration, and attention requirement mean intensities showed significant reductions after ND supplementation. In particular, reactivity decreased from a T0 value of 2.16 &#177; 0.27 to a T1 value of 1.25 &#177; 0.13 (Figure 2E, **p &#60; 0.01), activation decreased from a T0 value of 2.25 &#177; 0.25 to a T1 value of 1.33 &#177; 0.14 (Figure 2F, *p &#60; 0.05), irritability decreased from a T0 value of 2.66 &#177; 0.18 to a T1 value of 1.66 &#177; 0.22 (Figure 2G, **p &#60; 0.01), alertness decreased from a T0 value of 2.50 &#177; 0.19 to a T1 value of 1.66 &#177; 0.22 (Figure 2H, *p &#60; 0.05), environmental exploration decreased from a T0 value of 2.33 &#177; 0.18 to a T1 value of 1.66 &#177; 0.22 (Figure 2I, **p &#60; 0.01), and attention requirement decreased from a T0 value of 2.24 &#177; 0.15 to a T1 value of 1.55 &#177; 0.21 (Figure 2J, *p &#60; 0.05). No significant difference was observed in the respective SD group.
In Figure 3, the mean intensities of clinical symptoms in dogs belonging to the SD and ND groups, before (T0) and after the 10-day evaluation period (T1), are shown. Dandruff significantly decreased from a T0 value of 2.33 &#177; 0.14 to a T1 value of 1.08 &#177; 0.08 (Figure 3A, ***p &#60; 0.001). Also, itchiness, flush, seborrhea, and fur opacity significantly decreased from a T0 value of 2.08 &#177; 0.26 to a T1 value of 1.04 &#177; 0.05, from a T0 value of 2.11 &#177; 0.24 to a T1 value of 1.16 &#177; 0.11, from a T0 value of 2.35 &#177; 0.25 to a T1 value of 1.33 &#177; 0.14, and from a T0 value of 2.22 &#177; 0.13 to a T1 value of 1.41 &#177; 0.15, respectively (Figure 3B-E, *p &#60; 0.05). A similar trend was observed for vomiting, diarrhea, flatulence, lachrymation, and anal sac repletion scores, which significantly decreased from a T0 value of 2.75 &#177; 0.10 to a T1 value of 1.58 &#177; 0.17 (Figure 3F, *p &#60; 0.001), from a T0 value of 2.69 &#177; 0.12 to a T1 value of 2.06 &#177; 0.19 (Figure 3G, *p &#60; 0.01), from a T0 value of 1.75 &#177; 0.13 to a T1 value of 1.25 &#177; 0.13 (Figure 3H, *p &#60; 0.05), from a T0 value of 2.16 &#177; 0.11 to a T1 value of 1.32 &#177; 0.03 (Figure 3I, *p &#60; 0.05), and from a T0 value of 2.28 &#177; 0.12 to a T1 value of 1.30 &#177; 0.14 (Figure 3J, *p &#60; 0.01), respectively. No significant difference was observed in dogs belonging to the SD group.
Figure 4 shows the mean time spent in barking by dogs belonging to the SD and ND groups before (T0) and after the evaluation period (T1). A significant decrease from a T0 value of 180.21 &#177; 15.35 to a T1 value of 76.02 &#177; 7.22 was observed in the ND group after 10 days. No significant difference was observed in the SD group.
The data were analyzed using graphing and statistical software. All data are presented as &#177; the standard error of the mean and were first checked for normality using the D&#39;Agostino-Pearson normality test. Differences in activity, rest and barking time as well as symptoms during the evaluation period were analyzed using a two-way ANOVA test followed by Tukey&#39;s multiple comparisons test. p &#60; 0.05 was considered significant.
It is worth noting that, at the beginning of the study, each dog was automatically assigned by the mobile app software to achieve a desired daily activity according to weight, age, and breed. After treatment, all dogs belonging to the ND group showed a significant decrease in the mean daily activity (p &#60; 0.05) with respect to SD group, which was even lower than expected, and a consequent significant increase in daily mean rest time (p &#60; 0.01). These results were also well correlated with consequent clinical symptoms and signs (Figures 2 - 3), demonstrating significant improvement as well as a significant reduction in barking time (Figure 4, p &#60; 0.001). Taken together, all these considerations strengthened the efficacy of the ND in improving the results of common behavioral therapies for generalized anxiety. These results are also in agreement with our recent paper, which described the efficacy of a similar diet in relieving some of the clinical symptoms, such as dandruff, itchiness, flush, diarrhea, and flatulence, that have all been taken into account in this study26.
It is also worth noting that clinical symptoms might also be the manifestation of an overall inflammation status, with a consequent oxidative stress imbalance. Inflammation is also known to contribute to the etiology of anxiety disorders, depression27, and neurotransmitter activity28. In this sense, we recently identified a specific compound, oxytetracycline, as a possible agent able to trigger an inflammatory condition both in vitro29,30and in vivo31,32. Oxytetracycline belongs to the class of tetracyclines, which are the most widely and legally used antibiotics in intensive farming (e.g., poultry30, livestock33, and aquaculture34) due to their low cost and high efficacy35. Unfortunately, oxytetracycline also has a high affinity for calcium-rich tissues, such as bone and teeth36, and can remain fixed in treated animals for extended periods, even respecting withdrawal time30. Moreover, pet food production relies on meat (mainly poultry) by-products, which are mechanically separated37. This kind of separation generates a bone-based meal bearing oxytetracycline residues that are present in commercially available diets (canned, semi-moist, and dry) at 20 - 30% and can accumulate within a pet&#39;s body.
Concerning the ND group, it is reasonable to hypothesize that the mean score intensity reduction of all clinical symptoms was thus a consequence of the anti-inflammatory and antioxidant effect of the nutraceutical substances Punica granatum38, Valeriana officinalis39, Rosmarinus officinalis40, Tilia spp41, tea extract42, and polyunsaturated fatty acids (PUFA) present within the diet. For instance, PUFA have been show to modulate behavioral symptoms in attention-deficit hyperactivity disorder (ADHD) patients and in aggressive dogs43. These dogs had lower docosahexaenoic acid (DHA) levels than normal, as well as a higher omega-6:omega-3 ratio44,45.
<img alt="Figure 1" src="/files/ftp_upload/54878/54878fig1.jpg" /> 
Figure 1. ND Reduces Activity Time and Increases Rest Time in Dogs. Schematic representation of the daily mean activity (A) and rest time (B) of dogs before (T0) and 10 days after (T1) SD and ND supplementation (**p &#60; 0.01). The error bars are &#177; the standard error of mean. <a href="http://cloudflare.jove.com/files/ftp_upload/54878/54878fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54878/54878fig2.jpg" /> 
Figure 2. ND Improves the Behavioral Disturbances in Affected Dogs. Schematic representations of the mean score intensities of behavioral symptoms in dogs before (T0) and 10 days after (T1) SD and ND supplementation. (A) Marking. (B) Anxiety (***p &#60; 0.001). (C) Diffidence (*p &#60; 0.05). (D) Irregular biorhythm (**p &#60; 0.01). (E) Reactivity (**p &#60; 0.01). (F) Activation (*p &#60; 0.05). (G) Irritability (**p &#60; 0.01). (H) Alertness (*p &#60; 0.05). (I) Environmental exploration (**p &#60; 0.01). (J) Attention requirement (**p &#60; 0.01). The error bars are &#177; the standard error of mean. <a href="http://cloudflare.jove.com/files/ftp_upload/54878/54878fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54878/54878fig3.jpg" /> 
Figure 3. ND Improves the Clinical Signs in Affected Dogs. Schematic representation of the mean score intensities of clinical symptoms in dogs before (T0) and 10 days after (T1) SD and ND supplementation (A) Dandruff (***p &#60; 0.001). (B) Itch (*p &#60; 0.05). (C) Flush (*p &#60; 0.05). (D) Seborrhea (*p &#60; 0.05). (E) Fur opacity (*p &#60; 0.05). (F) Vomiting (***p &#60; 0.001). (G) Diarrhea (**p &#60; 0.01). (H) Flatulence (*p &#60; 0.05). (I) Lachrymation (*p &#60; 0.05). (J) Anal sac repletion (**p &#60; 0.01). The error bars are &#177; the standard error of mean. <a href="http://cloudflare.jove.com/files/ftp_upload/54878/54878fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54878/54878fig4.jpg" /> 
Figure 4. ND Reduces the Bark Time in Supplemented Dogs. Schematic representation of the mean time spent barking in dogs before (T0) and 10 days after (T1) SD and ND supplementation (***p &#60; 0.001). The error bars are &#177; the standard error of mean. <a href="http://cloudflare.jove.com/files/ftp_upload/54878/54878fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Daily Ratio</td>
		</tr>
		<tr>
			<td>Weight (kg)</td>
			<td>Amount (g)</td>
		</tr>
		<tr>
			<td>1 - 10</td>
			<td>30 - 180</td>
		</tr>
		<tr>
			<td>11 -20</td>
			<td>190 - 300</td>
		</tr>
		<tr>
			<td>21 - 35</td>
			<td>310 - 455</td>
		</tr>
		<tr>
			<td>36 - 50</td>
			<td>465 - 595</td>
		</tr>
	</tbody>
</table>
Table 1. Daily Amount of Food Provided to the Dogs.

Watch this Scientific Journal Video about Behavioral Disturbances: An Innovative Approach to Monitor the Modulatory Effects of a Nutraceutical Diet at JoVE.com

Behavioral Disturbances: An Innovative Approach to Monitor the Modulatory Effects of a Nutraceutical Diet

We report a simple approach to evaluate the effectiveness of a specific diet in positively modulating the daily activity and clinical and behavioral symptoms of dogs with evident behavioral disturbances.

In dogs, diets are often used to modulate behavioral disturbances related to chronic anxiety and stress caused by intense and restless activity. However, ...

behavioral-disturbances-an-innovative-approach-to-monitor-modulatory

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Behavior

8.8K Views.  “G. d’Annunzio” University. We report a simple approach to evaluate the effectiveness of a specific diet in positively modulating the daily activity and clinical and behavioral symptoms of dogs with evident behavioral disturbances.

Video: Behavioral Disturbances: An Innovative Approach to Monitor the Modulatory Effects of a Nutraceutical Diet

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 ...

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of chronic CNS disorders, there is an increasing demand for murine models with high validity at the behavioral level. However, multiple pathogenic mechanisms and complex functional deficits often impose challenges to reliably measure and interpret behavior of chronically sick mice. Therefore, the assessment of peripheral pathology and a behavioral profile at several time points using a battery of tests are required. Video-tracking, behavioral spectroscopy, and remote acquisition of physiological measures are emerging technologies that allow for comprehensive, accurate, and unbiased behavioral analysis in a home-base-like setting. This report describes a refined phenotyping protocol, which includes a custom-made monitoring apparatus (Integrated Behavioral Station, INBEST) that focuses on prolonged measurements of basic functional outputs, such as spontaneous activity, food/water intake and motivated behavior in a relatively stress-free environment. Technical and conceptual improvements in INBEST design may further promote reproducibility and standardization of behavioral studies.

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station INBEST

Prolonged and comprehensive monitoring of mice in a home-cage environment provides a deeper understanding of aberrant behavior in murine models of brain diseases. This paper describes the Integrated Behavioral Station (INBEST) as the key component of contemporary behavioral analysis.

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of ...

Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the test mouse is placed in a standardized apparatus and relevant behaviors are assessed in three different sessions (called session I, II, and III).
The apparatus has three compartments (see Figure 1), the left and right compartments contain an inverted cup which can house a mouse (called &#34;stimulus mouse&#34;).
In session I, the test mouse is placed in the cage and its mobility is characterized by the number of transitions made between compartments. In session II, a stimulus mouse is placed under one of the inverted cups and the sociability of the test mouse is quantified by the amounts of time it spends near the cup containing the enclosed stimulus mouse vs. the empty inverted cup. In session III, the inverted cups are removed and both mice interact freely. The sociability of the test mouse in session III is quantified by the number of social approaches it makes toward the stimulus mouse and by the number of times it avoids a social approach by the stimulus mouse.
The automated evaluation of the movie detects the nose of the test mouse, which allows the determination of all described sociability measures in session I and II (in session III, approaches are identified automatically but classified manually). To find the nose, the image of an empty cage is digitally subtracted from each frame of the movie and the resulting image is binarized to identify the mouse pixels. The mouse tail is automatically removed and the two most distant points of the remaining mouse are determined; these are close to nose and base of tail. By analyzing the motion of the mouse and using continuity arguments, the nose is identified.
<img alt="Figure 1" src="/files/ftp_upload/52508/52508fig1.jpg" /> 
Figure 1. Assessment of Sociability During 3 sessions. Session I (top): Acclimation of test mouse to the cage. Session II (middle): Test mouse moving freely in the cage while the stimulus mouse is enclosed in an inverted cup. Session III (bottom): Both test mouse and stimulus mouse are allowed to move freely and interact with each other.

This protocol describes a method to quantify mouse sociability. Mice are videotaped as they move and interact in a special cage. Movie processing allows for the automated quantification of sociability with excellent accuracy and reliability.

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the ...

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Using the Race Model Inequality to Quantify Behavioral Multisensory Integration Effects

Multisensory integration research investigates how the brain processes simultaneous sensory information. Research on animals (mainly cats and primates) and humans reveal that intact multisensory integration is crucial for functioning in the real world, including both cognitive and physical activities. Much of the research conducted over the past several decades documents multisensory integration effects using diverse psychophysical, electrophysiological, and neuroimaging techniques. While its presence has been reported, the methods used to determine the magnitude of multisensory integration effects varies and typically faces much criticism. In what follows, limitations of previous behavioral studies are outlined and a step-by-step tutorial for calculating the magnitude of multisensory integration effects using robust probability models is provided.

The current study aims to provide a step-by-step tutorial for calculating the magnitude of multisensory integration effects in an effort to facilitate the production of translational research studies across diverse clinical populations.

Multisensory integration research investigates how the brain processes simultaneous sensory information. Research on animals (mainly cats and primates) ...

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 ...

Flypub To Study Ethanol Induced Behavioral Disinhibition and Sensitization

Alcohol use disorder (AUD) remains a serious problem in our society. To develop effective interventions for addiction, it is important to understand the underlying neurobiological mechanisms, for which diverse experimental approaches and model systems are needed. The main ingredient of alcoholic beverages is ethanol, which causes adaptive changes in the central nervous system and behavior upon chronic intake. Behavioral sensitization (i.e., escalated responses) in particular represents a key adaptive change underlying addiction. Most ethanol-induced behavioral sensitization studies in animal models have been conducted on the locomotor activating effect of ethanol. A prominent effect of ethanol is behavioral disinhibition. Behavioral sensitization to the disinhibition effect of ethanol, however, is underrepresented. To address this issue, we developed the Flypub assay that allows measuring the escalated increase in disinhibited courtship activities upon recurring ethanol exposure in Drosophila melanogaster.&#160;Here, we report the step-by-step Flypub assay including assembly of ethanol exposure chambers, setup of the assay station, criteria for fly care and collection, ethanol delivery, quantification of disinhibited courtship activities, data processing and statistical analysis. Also provided are how to troubleshoot critical steps, overcome limitations and expand its utility to assess additional ethanol-induced behaviors. The Flypub assay in combination with powerful genetic tools in Drosophila melanogaster will facilitate the task of discovering the mechanism underlying ethanol-induced behavioral sensitization.

The Flypub assay measures the behaviors that the fruit fly Drosophila melanogaster displays under the influence of ethanol. The assay can be readily mastered by experimenters at all levels and applied to various vaporized stimuli, facilitating substance abuse and addiction studies.

Alcohol use disorder (AUD) remains a serious problem in our society. To develop effective interventions for addiction, it is important to understand the ...

Iterative Development of an Innovative Smartphone-Based Dietary Assessment Tool: Traqq

To collect dietary intake data in a fast and reliable manner, a flexible and innovative smartphone application (app) called Traqq was developed (iOS/Android). This app can be used as a food record and&#160;24-h recall (or shorter recall periods). Different sampling schemes can be created on either prespecified or random days/times within a predetermined period for both methods, with&#160;push notifications to urge the participants to register their food intake. In case of non-response, notifications are automatically rescheduled to ensure complete data collection. For use as a food record, respondents can access the app and log their food intake throughout the day. Food records close automatically at the end of the day; recalls close after submission of the consumed items. The recall as well as the food record module provide access to an extensive food list based on the Dutch food composition database (FCDB), which can be accustomed to fit different research purposes. When selecting a food item, respondents are simultaneously prompted to insert portion size, i.e., in household measures (e.g., cups, spoons, glasses), standard portion sizes (e.g., small, medium, large), or weight in grams, and eating occasion/time of consumption. Portion size options can be adjusted, e.g., only entry in grams in case of a weighed food record or time of consumption instead of eating occasion). The app also includes a My Dishes function, which allows the respondent to create their own recipes or product combinations (e.g., a daily breakfast) and only report the total quantity consumed. Subsequently, the app accounts for yield and retention factors. The data are stored on a secure server. If desired, additional questions, i.e., in general or those related to specific food items or eating occasions can be incorporated. This paper describes the development of the system (app and backend), including expert evaluations and usability testing.

This article describes the protocol for the development of an innovative smartphone-based dietary assessment application Traqq, including expert evaluations and usability testing.