The overall goal of this procedure is to assess sensory gating in rodents in the form of habituation and pre pulses. Inhibition of acoustic startle responses begin the experiment by allowing the animals to acclimate to their startle boxes. Once the animals are acclimated, the testing protocols are run and data is collected.
Ultimately, the results show the amount and course of habitation and the amount of pre pulses inhibition through changes in the amplitude of acoustic startle responses. This method assesses sensory filtering and rodents, but it can also be applied to humans and many other vertebrate and invertebrate animals. Before a set of experiments, use a sound meter to calibrate the amplifier input so the loudspeakers transduce the same volume as set by the controlling instruments.
Then calibrate the sensitivity of the transducer platform of the startle boxes. According to the supplier's manual, make sure that there are no ongoing experiments when calibrating the system and that all boxes are calibrated in the same way. Acclimate the mice to the experimenter, the procedure, and to the startle boxes in non restraining mouse holders for two to five minutes with background noise of 65 to 68 decibels.
Repeat this procedure three to five times, once or twice a day until defecation and urination during acclimation ceases or considerably decreases, always replace and clean the animal holder between subjects. Animals should be handled one to two times before being placed into non restraining animal holders and exposed to background noise. After removing an animal from a holder, reward it with sunflower seeds before the entire protocol is run.
Repeat this procedure two more times. Gradually expanding the acclimation time to 10 minutes when a new rat or mouse strain is measured. First, establish an input output function.
Subject the animals to an acclimation period of five to 10 minutes with a constant background white noise of 65 to 68 decibels. Following acclimation subject the animals to startle stimuli every 20 seconds. The startle stimuli begin around 70 to 75 decibels and increase in volume by two to five decibels between exposures.
Ultimately reaching 120 to 130 decibels. The result is 20 to 30 trials with increasing stimulus intensity Testing parameters such as startle, pulse and pre pulses, intensities, inter stimulus intervals, and inter trial intervals should be carefully chosen according to the hearing and motor abilities of the animal model used. Here we propose a general protocol that will provide comparability with many former studies.
This protocol is divided into the following blocks. First, an acclimation period. Second, a block allowing for startle habituation called block one and third, a block for pre pulses inhibition.
Measurement called block two. Block one is run so that startle attenuations due to habitation do not interfere with PPI. Measurements begin by loading an animal into each of the four testing chambers.
If there are different groups of animals being tested within an experiment, different genotypes or injections, for example, then pseudo randomize them in terms of chamber number and order of testing. If an animal is repeatedly tested, then it should be retested in the same startle box for repeated PPI testing in individual rats, but not mice run this entire testing protocol once before collecting data. Naive animals are allowed to experience the test once prior to data collection.
Because PPI data variability is often reduced between the first and second testing sessions. In addition, long-term habituation can be a confounding variable when measuring PPI and much of this long-term habituation is reduced between the first and second exposures of animals to the startle program. Begin the test by acclimating the animal to 65 to 68 decibels of white noise for five to 10 minutes.
Next, run a short term habituation block. This involves subjecting the rodents to 20 millisecond blasts of white noise at 22nd intervals. Random intervals times between 10 and 30 seconds can also be used.
The intensity of the startle stimulus is ideally where the input output function plateaued, which is usually between 105 and 115 decibels. Next measure PPI. During this block, pseudo randomized trials with a startle pulse alone and trials with a pre pulse are presented, keep the background and startle stimulus, volumes and inter trial interval the same as they were during the habituation phase.
A pre pulses stimulus is generated from white noise with the same steep rise as the habituation phase pulses, but is reduced to a four millisecond duration. During PPI measurement very two parameters. Interra stimulus interval is the time between the presentation of the pre pulses and startle pulse stimuli choose varying inter stimulus intervals in this example, 30 and 100 milliseconds were chosen.
Second, choose varying pre pulses intensities, for example, of 75 and 85 decibels. Next pseudo randomize the four different PrepU pulse trial types and the startle pulse alone trials and each trial type 10 times for a total of 50 trials. Sometimes it is beneficial to add a six trial type consisting of only a pre pulses in order to show that animals do not startle to the PrepU.
The MET associate equipment allows the presentation of this trial type to detect this type of error. Remove an animal from startle box and return it to its home cage. To measure long-term habituation or LTH run the entire protocol for at least five subsequent days.
Block two can be omitted if block one contains at least 100 startle stimuli on each day. Be sure to conduct the runs at the same time because startle response amplitudes fluctuate over the course of the diurnal cycle. Upon completing trials, take care to store, analyze and present the data appropriately.
Rodents typically begin to startle at a volume of 85 to 90 decibels and upwards. The startle response increases with increasing volume and normally reaches a maximum at 100 to 110 decibels. If animals deviate considerably from these values, animals may have hearing or motor disabilities, the strongest habituation effect occurs normally within the first several stimuli well-handled.
Rats normally habituate to around 60%of their initial startle response, whereas mice generally habituate to about 80%of baseline in either species. Individual differences and strain differences can be very large. Most rats show PPI at around 10%of baseline when subjected to an optimal pre pulses.
PPI is very robust and individual differences are relatively small with these experimental settings, lower volume, pre pulses yield less PPI and more variability even within an animal, but also seem to be more vulnerable to pharmacological or genetic manipulations. Long-term habituation can be observed over several testing sessions. LTH is very robust in rats to observe LTH in mice.
However, the presentation of a large number of startle stimuli in each session is usually required Following this standard procedure. Some parameters may be altered in order to address specific questions. This method can be combined with both systemic and stereotaxic injections in order to study the role of specific signaling molecules and brain structures in sensory filtering.
