If responses are low, adjust the Response Adjust dial on the SR-Lab cabinet. In software, the Vavg values range for mice
Webinar Q&A Report: Employing Acoustic, Tactile and PPI Startle Response Procedures in Rodent Behavioral Research
Q: Can someone comment about ISI in PPI with mice? There seems to be a great deal of controversy among people I have spoken to Inter stimulus interval is set from onset of PPI to onset of Startle stimulus. There is a broad range of how many milliseconds to use but 60-120 is a common ISI. 70 millisecond example:
Onset time 0 and on for 20 milliseconds 50 seconds of no sound Startle stimulus on at 70.
Q: If ASR responses are very low (e.g. young mice), is enhanced sensitivity calibration a better option to use over switching to TSR? If responses are low, adjust the Response Adjust dial on the SR-Lab cabinet. In software, the Vavg values range for mice is 700 millivolts and 200 millivolts for rats. Also, make sure you are using the smallest animal holder available. If you are still getting weak responses after adjusting the sensitivity and using the smallest animal holder, then TSR would be a good option to use on young mice. If you generate a response while using TSR, this may determine hearing loss in your mice. Q: I am often told there are problems with running mice younger than 45 days due to weight. What is the lower limit for weight, or is there one? Can SR-Lab system be used with small mice (9 -10grams)? We recommend not going lower than 10 grams when running young mice. Some things to consider when running very young and lower limit weight mice;
use the smallest enclosure from San Diego Instruments adjust the sensitivity of the response dial to pick up the small movements from the smaller weighted mice. basic response dial is 700 for mice and 200 for rats but for young animals shift higher to be able to pick up the response of the small animals.
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Q: Can the presenters expand on how the accelerometer works to eliminate body mass effects? The accelerometer does not consider static weight. Only dynamic forces create a voltage output. The San Diego Instruments software accounts for an acclimation period which allows the animal to calm down before the actual recording of data begins. This will allow for the movement of the animal to not affect recorded data. Q: Do you have a rejection threshold for individual responses when an acoustic startle is close to baseline (due to animal's normal movement) Per Dr. Vorhees, one of the advantages of accelerometer detectors is that you don’t get much signal from small movements such as when an animal turns around inside the holder. But to answer your question, no, we don’t use a threshold. Rather we look at the data to see what the responses are like. In mice, if we see a lot of zero or very low values we may not use that subject’s data out of concern over its hearing. We don’t see these problems in Sprague-Dawley or Long-Evans rats. SR-LAB does provide a measure called VStart which is a reading taken 3 ms into the recording window. This shows any abrupt movement of the animal shortly before the pulse begins. Most of the time these readouts are very small but occasionally one sees a larger one. An option is to subtract VStart from Vmax to correct for this movement. In practice we find that for rats this adjustment makes little difference, i.e., if your treatment produces a significant startle effect it will be significant with or without adjustment using Vstart. However, in mice, especially with PPI, we don’t use the VStart adjustment because sometimes mice react to the prepulse and that can show up as a VStart signal and if you subtract it you won’t see a Vmax unless the Vmax is large. Q: During the acclimation, is it typical to generate background noise and how critical is this? Generating background noise and the need during acclimation will depend on your lab. If you are in a lab that is very quiet, the built-in fan in the SR-Lab cabinet may be sufficient for background noise. If you are in a lab with a lot of movement or extra noise then background noise during the acclimation period would be useful to eliminate the extra lab noise allowing the animal to acclimate before startle stimulus is applied. Q: How does the startle response correlate with other behavioral measures such as those on the elevated plus maze? Looking to see if startle will add in another component into a behavioral test battery for animals undergoing stress. Startle is quite a different measure than anxiety. Startle is a measurement of a reflex where the elevated plus maze (EPM) measures anxiety. As a general rule, acute stress will increase the startle response in animals whereas chronic stress may increase startle but depends on how long the delay between stressor and startle testing. It has been suggested that PPI can be thought of as a test of preattention or early attention since the prepulse acts as an orienting response whereas the startle signal itself is a defensive behavior. The prepulse orienting reaction has the effect of suppressing the defensive startle response so not only is startle different than EPM, PPI adds dimension to startle assessment beyond what ASR alone provides.
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Q: The software demonstrated seems to be a newer version of SR-Lab than we have... is this newer version available to existing users? Are there new features that current users should be aware of? Yes, the newer software is available to existing users for a nominal fee. There is also a new USB interface control box available. Please contact us at
[email protected] so we can look up your current software and let you know of the newest features. Q: Dr. Vorhees, in the PRM and DLM exposure data you shared, were the same subjects (Rats) used for each time point (2, 4, 6, 8 hours)? Yes, the same animals were used for each time point. The strength of the startle is you can test the reflex over and over again with very little loss of response amplitude. Q: What type of stimulation frequencies can be achieved with the air puff system? A typical range for stimulation frequency is 20-40 milliseconds. The air puff system has a range from 060 PSI allowing you to set the frequency for your particular study. Q: Some argue handling is NOT necessary before running mice in PPI- Thoughts? Per Dr. Vorhees, handling does have effects on startle although we’ve not done a formal experiment to see by how much. The effect is most noticeable on trial-1. Extensively handled animals from prior to weaning don’t show as large of a trial-1 response as naïve, unhandled rats newly arrived from the supplier, but after trial-1, handled and unhandled rats respond similarly. If you are concerned about this and want to test unhandled rats, I’d recommend testing for 51 or 101 trials and removing trial-1 and using the rest of the trials as your data. Q: Can the presenters explain what Acoustic startle rise time is? Why is this important? Acoustic startle rise time is a critical metric eliciting the startle response. Rise time is the time it takes for sound amplitude to change from 10% to 90%. San Diego Instruments SR-Labs acoustic startle rise time is approximately 1.5 milliseconds. Q: How do you calibrate sound levels between boxes (SR-Lab Cabinets) when running ASR? San Diego Instruments uses a third party sound level meter which is placed inside the SR-Lab cabinet and read with the door closed through the peep hole. Using the software, in the Sound Level Diagnostic window, displays the Analog to dB Conversion chart ranging from 0-750. Select an “analog level” that equals 120 dB to start. Hit the trigger button on the diagnostic screen and see if the meter reads 120 dB. If it does not, adjust your analog level and click the trigger again. While doing this make sure to have no extra noise in the lab. Continue to do this until your meter reads 120 dB with the door shut. Once you have this level save it in the User Lab dB Conversion List window in the sound level diagnostic screen. There is a hardware audio adjust dial that allows you to normalize sound levels between SR-Lab cabinets.The calibration needs to be completed periodically or at the beginning of a new experiment.
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Q: Is a decreased startle response measured in transgenic mice purely based on a misconnection in the startle circuitry system or may other factors be playing a role? One thing that leads to misconnect with the mice and acoustic startle is the decrease in hearing for mice. This is very common in C57 mice as they age. Look through your data for non-responders because this will show you which mice have hearing loss. But if your mutant mice are responding but the amplitude is just lower then it may not be hearing but an effect of the protein you disrupted/deleted by knocking out the gene. To really know you’d have to do further experiments; histology or brain-stem auditory evoked potentials, the latter to test hearing directly.
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