Name: Developing a Behavioral Box for Assessing Prepulse Inhibition and Neural Activity in Psychiatric Animal Models (Video) | JoVE Journal
Uploaded: 2025-07-22T12:30:00.000+00:00
Duration: 6 min 55 s
Description: Learn to assess prepulse inhibition in socially isolated rats using a cost-effective behavioral box with synchronized accelerometer and neuronal data. This method advances sensory gating research in psychiatric disorders.

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Edith Granados, Cinvestav Guadalajara, for kindly supporting this project with PPI scripts and data synchronization. This work was funded by the Santos Dumont Institute, Ministry of Education, CNPq, CAPES, and FINEP.

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<li>Chen, X., Li, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ArControl%3A+An+Arduino-based+comprehensive+behavioral+platform+with+real-time+performance%5BTitle%5D)+AND+%22Front+Behav+Neurosci%22%5BJournal%5D)">ArControl: An Arduino-based comprehensive behavioral platform with real-time performance.</a> Front Behav Neurosci. 11, 244(2017).</li>
<li>Oliveira Silva, J. W., Lima, R. A., Morya, E., Brasil, F. L., Gonçalves, L. M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+behavioral+box+and+sensing+techniques+for+analysis+of+tactile+discrimination+tasks+in+rodents%5BTitle%5D)+AND+%22Sensors%22%5BJournal%5D)">Improved behavioral box and sensing techniques for analysis of tactile discrimination tasks in rodents.</a> Sensors. 23 (1), 288(2022).</li>
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</ol>

The authors declare that they have no competing financial interests.

This protocol presented a DIY behavioral box to study prepulse inhibition tasks in freely behaving Wistar rats while recording brain data (in this example, local field potential and spiking activity). The box can be built and customized with components commonly found in neuroscience laboratories, thus being accessible and cheap. Such DIY solutions also allow for more flexibility in designing and personalizing experiments.
Many benefits can be observed with the use of a DIY system, including lo...

Individuals diagnosed with schizophrenia (SCZ) often present sensorimotor deficits. Decreased response to sensory stimulation and difficulty in discerning relevant information from diverse sensory inputs is often reported in those patients1,2. For healthy individuals, delivering a weaker stimulus before a stronger one diminishes the usual startle reflex to the stronger stimulus3. This phenomenon is known as prepulse inhibition (PPI) and has been used to investigate sensory gating deficits in patients with SCZ and animal models of SCZ4.
In an acoustic PPI task, a startle response to a single strong acoustic pulse (e.g., ~120 dB) is compared to the startle response when the same stimulus is preceded in a short interval (30 to 500) by a weaker stimulus (e.g., ~65 dB)3,4. In healthy individuals, the startle response to the pulse is diminished when the weaker prepulse is presented (when compared to the startle response to the single strong pulse alone). This phenomenon is disrupted in individuals with SCZ, who exhibit less suppression of the startle response even when the weaker prepulse is presented preceding the strong pulse1,5,6,7,8.
Similar to SCZ patients, rats with mesolimbic dopamine overactivity also present diminished acoustic PPI9, making this paradigm a useful tool to investigate behavioral and physiological alterations in animal models of schizophrenia. Generally, the commercial apparatus for PPI consists of an acoustic chamber containing sensors, speakers, and software for setting the experimental parameters (e.g., inter-pulse interval and intensity of stimuli), recording rodents&#39; startle response, and processing the data. However, commercial systems often offer limited customization options and are costly. Do-it-yourself (DIY) approaches to building flexible research apparatus are increasing access to equipment and transferring knowledge10. DIY solutions to study sensorimotor gating mechanisms like PPI must consider several key factors to ensure effectiveness and reliability. Some essential considerations include design specifications, environment and light control, sound attenuation, stimulus delivery systems, behavioral measurement sensors, and data acquisition and analysis.
DIY solutions for behavioral boxes have been developed for different functions, such as operant licking experiments11, auditory discrimination tasks12, and forelimb function13. Although several DIY approaches focus on video tracking14, this type of data requires high computational resources to be analyzed. An open-source hardware and software platform can be used to build a low-cost PPI behavioral box with the flexibility to test customized experiments15,16,17, and synchronize neuronal recordings of freely behaving animals.
This article introduces a method for developing a behavioral box to assess PPI with a low-cost platform to investigate the post-weaning isolation rat model, an early-life stress contributing to the development of SCZ-like symptoms, that can also be used with simultaneous electrophysiological brain recordings.

<table><tbody><tr><td>Acrylic box</td><td>Custom made</td><td></td><td>5mm thick acrylic sheet, 20 cm x 25 cm x 25 cm</td></tr><tr><td>Adhesive rubber bumper pads</td><td>Gorilla grit</td><td>MPU6050&amp;nbsp;</td><td>Diameter 0.8&quot; and height 0.38&quot;. Rubber pads to stabilize the acrylic box and isolate from external vibration. The height should be more that 5mm due to MPU6050 underneath the box.&amp;nbsp;</td></tr><tr><td>Arduino Uno</td><td>Arduino</td><td>ATmega328P&amp;nbsp;</td><td>Arduino microcontroller board based on the ATmega328P with 14 digital input/output pins, 6 analog inputs, and clock speed of 16 MHz.</td></tr><tr><td>Cedrus stim tracker (audio detector)</td><td>Cedrus</td><td>https://cedrus.com/stimtracker/index.htm</td><td>Cedrus stim tracker detects acoustic stimuli and sends TTL markers to accelerometer data.</td></tr><tr><td>Cineplex Research System</td><td>Plexon</td><td>https://plexon.com/plexon-systems/cineplex-behavioral-research-system/</td><td>Recording video of the animal synchornized with electrophysiological data.</td></tr><tr><td>Cyanoacrilate glue</td><td></td><td>MPU6050</td><td>Super glue to attach firmly the MPU6050 underneath the box.</td></tr><tr><td>Digital sound lever meter</td><td></td><td></td><td>Decibel meter to calibrate the stimuli set before the experiment.</td></tr><tr><td>IBM SPSS</td><td>SPSS (Statistical Package for the Social Sciences, USA)</td><td></td><td>IBM SPSS Statistics software platform.</td></tr><tr><td>Matlab</td><td>MathWorks</td><td>https://www.mathworks.com/products/matlab.html</td><td>MATLAB platform for data analysis, algorithm development, and model creation.</td></tr><tr><td>Micro stainless steel screw</td><td></td><td></td><td>Glasses screws with 1.2mm diameter and 4 mm length.</td></tr><tr><td>Microeletrode array</td><td>In house custom made</td><td></td><td>It uses omnetics connector and tungsten 50micron wires coated with parylene of California Fine Wire.</td></tr><tr><td>Microphone</td><td></td><td></td><td>Microphone to acquire acoustic stimulation synchronized with electrophysiological data.</td></tr><tr><td>MPU-6050</td><td>InvenSense</td><td>https://invensense.tdk.com/products/motion-tracking/6-axis/mpu-6050/</td><td>MPU-6050 is a 3-axis gyroscope and a 3-axis accelerometer&amp;nbsp;</td></tr><tr><td>Multichannel acquisition processor&amp;nbsp;</td><td>Plexon</td><td>SPIKE/LFP</td><td>MAP Plexon to acquire neuronal electrophyiological data SPIKE/LFP and auxiliar channels (microphone, TTL markers, accelerometer).</td></tr><tr><td>NeuroExplorer</td><td>Nex&amp;nbsp;</td><td>https://www.neuroexplorer.com/</td><td>NeuroExplorer data analysis program of continuously recorded signals and timestamps (spike trains, behavioral events)</td></tr><tr><td>Neuroexplorer</td><td>Nex Technolgie</td><td>https://www.neuroexplorer.com/</td><td>Software for SPIKE and LFP neurophysiological data analysis.</td></tr><tr><td>Offline Sorter</td><td>Plexon</td><td>https://plexon.com/products/offline-sorter/#38957t34842</td><td>Software for offline spike sorting.</td></tr><tr><td>OffLineSorter</td><td>Offline Sorter</td><td>https://plexon.com/products/offline-sorter/</td><td>Offline Sorter is a offline spike sorting software</td></tr><tr><td>Rectal veterinary SpO2 sensor</td><td>KTMED</td><td>FS-03</td><td>SpO2 sensor for small animals</td></tr><tr><td>Ribbon cable</td><td></td><td></td><td>Thin ribbon cable to connect MPU6050 to Arduino.</td></tr><tr><td>Roboti drill</td><td>Neurostar</td><td>https://robot-stereotaxic.com/drill-injection-robot/</td><td>Robotic drill used with Neurostar stereotaxic. It can be replaced by manual drill.</td></tr><tr><td>Speaker</td><td></td><td></td><td>Speaker has range of 100 to 40 kHz and intensity of up to 150 dB.</td></tr><tr><td>Stereotaxic apparatus</td><td>Neurostar</td><td></td><td>Stereotaxic used for neurosurgery in rodents</td></tr></tbody></table>

developing a behavioral box for assessing prepulse inhibition and neural activity in psychiatric animal models

Developing a Behavioral Box for Assessing Prepulse Inhibition and Neural Activity in Psychiatric Animal Models

Early-life distress is recognized as a potential precursor to neurodevelopmental disorders. One approach to investigating this condition in animal models is post-weaning social isolation. This early-life distress in rodents has been shown to lead to neural changes similar to those observed in patients with schizophrenia, including diminished prefrontal cortex volume, decreased dendritic spine density, and increased dopaminergic activity in the ventral tegmental area. The prepulse inhibition (PPI) paradigm is an established method for assessing sensory gating deficits in both human and animal models. PPI refers to the suppression of the startle reflex to a stronger acoustic stimulus (e.g., 120 dB pulse) when it is preceded by a weaker acoustic pre-stimulus (e.g., 65 dB pulse). This phenomenon is often reduced in psychiatric patients and animal models of psychiatric disorders. This article introduces a protocol to build a behavioral box for PPI studies, enabling the use of an accessible, cost-effective animal model of schizophrenia and the synchronization of the behavioral box with neuronal recordings from the model (e.g., electrophysiological recordings). This customized behavioral box effectively detected startle responses and facilitated PPI assessment with simultaneous neuronal recording in freely behaving rodents subjected to post-weaning isolation.

Prepulse inhibition showed no difference for pulse 1 and 3 compared to pulse 5 (120dB only), but showed a statistical difference for pulse 2 and 4 (p &lt; 0.05) (Figure 6).
<img alt="PPI percentage bar chart, statistical significance indicated in experimental data analysis." class="xfigimg" src="/files/ftp_upload/67005/67005fig06.jpg" data-altupdatedat="1762425657000" data-alt="Figure 6"> 
<st...

This protocol presents a microcontroller-based behavioral box to assess prepulse inhibition (PPI) by collecting acceleration data from a sensor beneath the box. This data evaluates sensory gating deficits in socially isolated rats, with an added method for synchronizing data with neuronal activity to advance neurophysiology studies on behavioral disorders.

Early-life distress is recognized as a potential precursor to neurodevelopmental disorders. One approach to investigating this condition in animal models ...

This protocol outlines a straightforward method for evaluating animal models of schizophrenia designed for behavioral researchers without programming or electronics expertise. We created a cost-effective behavioral box to assess prepulse inhibition in rats affected by neurodevelopmental issues stemming from early life distress. Recent advancements in psychiatric animal models using the prepulse inhibition test focus on understanding sensory gating deficits seen in disorders like schizophrenia.This includes genetic modifications, neuroimaging, and pharmacological interventions to investigate prepulse inhibition disruptions, ultimately improving diagnostics and therapies. We hope that our protocol will popularize the study of prepulse inhibition in animal models of schizophrenia, and labs that are studying molecular, behavioral, and physiological aspects of the disease can use it to contribute to the understanding of this complex psychiatric disorder. To begin, make an acrylic transparent box to accommodate a freely-behaving rat equipped with a wired headstage.Use self-adhesive rubber pads to isolate the acrylic box from vibrations and secure the accelerometer below without contact with other parts. Using a soldering iron, solder a ribbon cable directly to the four pinholes on the accelerometer board. VCC, GND, SCL, and SDA.Clean the center of the acrylic and the flat face board of the accelerometer. Then, connect the opposite end of the ribbon cable to the microcontroller by connecting VCC to 3.3 volts, GND to GND, SCL to SCL, and SDA to SDA. Next, construct a soundproof chamber with a frontal door using 80 centimeter edges of 15 millimeters thick medium-density fiberboard.Position a speaker 20 centimeters above the acrylic box, ensuring that the headstage and cable have a full range of motion. Connect the audio out from the computer to the microcontroller board. Using a universal serial bus cable, connect the microcontroller board to the computer.Then, open the integrated development environment software for the microcontroller board on the computer. After selecting the corresponding microcontroller board, COM port and baud rate, open the file sketch, compile, and upload the code to the microcontroller board. Click on the monitor's serial port to check for incoming data.Then, close the development environment software. Now, position a digital sound level meter within the acrylic box where the animal will be placed. Play the acoustic stimuli and adjust the detection range on the sound level meter to calibrate the stimulus levels being sent by the computer.Then, open the preferred open-source software for acquiring and saving data from the serial port. Configure the software settings, including the COM port, microcontroller board, and baud rate. Click Connect to read raw data or the chart view.Then, click Save to start recording the experiments data. After implanting the electrode array in the rat's brain, carefully connect the headstage to the connector implant on the rat's skull. Then, power on the neuronal data acquisition system and confirm that it receives data from the acoustic stimuli through either the microcontroller or a TTL generator connected to the audio detector or microphone.Turn on the open-source serial port recorder set up earlier. Now, use electrophysiology acquisition software to collect electrophysiological and accelerometer data. Expose the animal to white background noise for seven minutes, with random presentation of all types of pulses, repeating each pulse type 10 times.At the end of the recording session, turn off the acquisition software. Then, carefully disconnect the headstage connector from the implant on the animal's skull. To perform electrophysiological and behavioral analysis, import the raw data containing brain activity, acceleration, and acoustic stimuli into a signal processing platform.Extract EPICS centered on each acoustic stimulus. Then, normalize the means to enable comparison between animals. Set the response to a single pulse as the maximum response and normalize the reflex responses to other pulses relative to this value.Finally, define the startle amplitude for pulse 5 as 100%and represent responses to other pulses as a function of the pulse 5 amplitude. Local field potential analysis indicated no significant power modulation in the prelimbic and infralimbic cortices post-stimulation, while areas like the ventral tegmental area, amygdala, nucleus accumbens, and hippocampus showed decreased power spectrum density in delta, theta, and alpha bands, with a notable beta modulation. Both commercial and DIY accelerometers demonstrated consistent amplitude and time-based data, with baseline readings and startle response amplitudes showing close alignment across devices.

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Neuroscience

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The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Learn to assess prepulse inhibition in socially isolated rats using a cost-effective behavioral box with synchronized accelerometer and neuronal data. This method advances sensory gating research in psychiatric disorders.

Developing a Behavioral Box for Assessing Prepulse Inhibition and Neural Activity in Psychiatric Animal Models

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