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# Goal/Experiment:
**Goal:** Assessment of Prepulse Inhibition (PPI) of the Acoustic Startle Reflex in Rodents
## Assessment of Prepulse Inhibition (PPI) of the Acoustic Startle Reflex in Rodents
**Authors:** Franciele Kich Giongo1, Matheus Gallas-Lopes1, Ana P Herrmann1
**Institution:** Universidade Federal do Rio Grande do Sul
1 Universidade Federal do Rio Grande do Sul
### Abstract
The acoustic startle reflex is an automatic and involuntary response that occurs in humans and other animals when they are exposed to sudden and intense auditory stimuli, such as loud noises. This response can be influenced by several factors, including the intensity and timing of the stimulus, the individual's state of alertness, and contextual factors. It can also be modulated by prior exposure to a weak prepulse shortly before the startling stimulus, a phenomenon known as prepulse inhibition (PPI).
PPI reflects the brain's ability to filter out irrelevant or non-threatening sensory information and is an important indicator of sensorimotor gating. Here we describe a protocol to measure PPI in mice and rats using a startle chamber from Insight® (SP, Brazil).
## Materials
### Software
| Name | OS | Developer |
|---------------------------------|---------|--------------------------|
| Monitor de Sobressalto para Ratos e Camundongos | Windows | Insight Equipamentos Ltda |
### Equipment
| Name | Brand | SKU | Link |
|--------------------------|------------------------|-----|------------------------------------------------------|
| EP-175 Startle Sobressalto | Insight Equipamentos Ltda | N/A | [EP-175 Startle Sobressalto](https://www.insightltda.com.br/produto/ep-175-startle-sobressalto/) |
## Before Start Instructions
This is a step-by-step guide developed at PsychoLab to set up the software used to assess prepulse inhibition (PPI) of the acoustic startle reflex in mice or rats using the equipment acquired from Insight Equipamentos Ltda.
- Ensure the speakers and ventilation of the apparatus are turned on.
- Avoid using any light source inside the box.
- Read the entire protocol before starting.
## Setting up the Protocol
1. **To create a new protocol:** Click on the logo in the upper left corner and select "Edit" and then "Procedure".
2. **To create a new prepulse protocol:** Select "Create a new procedure file starting with a standard PPI block" and name the protocol.
3. **If you already have a saved protocol file:** Select "Open an existent procedure file" and then select the file (e.g., example.sbs).
4. **To calibrate the background noise:**
- Place the decibel meter inside the equipment, just above the accelerometer.
- Ensure the decibel meter’s battery is charged or new.
- Position the decibel meter on the accelerometer and adjust the detection range as necessary (range 30-80 dB for background noise; 80-130 dB for other measurements).
- To see the readings of the decibel meter, remove the lid of the equipment and close the door.
5. **Select the speaker:** The button to select the speaker is pointed by the red arrow. Speaker identification will be shown in the box "Confirmação de Ips" for selection.
6. **Adjust the intensity:** Adjust the intensity of the sound manually in the percentage bar and press play. Check the decibel meter to see if the desired range is shown. For other stimuli, repeat the process outlined in step 2.
7. **Note percentages:** Take notes of the percentages shown in each stimulus for easier stimulus calibration.
8. **Configure stimulus settings:** Set the duration of each period in the first line according to your protocol. Each stimulus will have its own configuration regarding background noise, white noise, pure tone, shock, and light.
9. **Save stimulus settings:** Once you have entered the stimulus settings for each block, click the checked box icon below the table, then save.
10. **To create new stimuli blocks:** Click on the "add PPI" icon. Set the desired percentage for pulse and prepulse. Adjust the decibel meter accordingly.
11. **Scale calibration:**
- First, remove the decibel meter from the scale.
- Click on the option "No, scale calibration presents considerable errors. Repeat scale calibration process now".
- Check whether the scale displays the weight of 50g correctly.
- Calibrate the scale at the beginning of the tests, if measuring is accurate, no need to recalibrate.
- Click on "Yes, current scale calibration is satisfactory. Execute the next step".
12. **Session configuration:** Create stimuli presentation order.
- Check the box for habituation with background noise if needed.
- Transfer blocks from procedure blocks to execution session.
- Transfer blocks in the same order, and randomly as needed.
- Fill in the session parameters (habituation period, inter-trial interval, etc.).
- Click on "Execute procedure file now".
13. **Adjust scale gain:**
- For mice, set scale gain to 1.
- For rats up to 200g, set scale gain to 2.
- For rats over 200g, set scale gain to 3.
## Running the Protocol
1. **To start the test:** Place the subject in the apparatus, close the lid, and click the play icon. The test will start automatically after selecting where to save the summary results.
- If there is a habituation period, a countdown will show.
- The session will end and a notification will pop up.
## Retrieving the Data
1. **Full reports:** Click on "Reports" on the main screen to export full reports. Use parameters set in step 8 as filters. Export data in `.xls`, `.doc`, or `.txt`.
## Data Analysis
1. **Reactivity Values:**
- Use the `mean value` of each stimulus for pulse and prepulse reactivity.
2. **Prepulse Inhibition Values:**
- Use the formula `(pulse alone - prepulse + pulse) / pulse alone * 100` for each combination.
3. **Mean PPI:**
- Use the mean value of all %PPI calculated for each combination of stimulus.
endofoutput
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