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Gap prepulse inhibition of acoustic startle (GPIAS) method has been used effectively for the objective assessment of tinnitus in animals. Among two types of enclosures for the GPIAS, the unconstrained type carries less risk of animal death due to the absence of binding stress in the enclosure, and lack of need for alteration to animal size variation as it grows. However, animals' voluntary movements, which have no relation to the startles evoked by acoustic stimuli, are problematic, as they cannot be excluded in the case of the unconstrained enclosure based GPIAS measurement system. In order to discount voluntary movements which are not associated with external acoustic stimuli, we propose the conditional random inter-stimulus interval (CR ISI) method for unconstrained enclosure based GPIAS measurement. With the proposed ISI method, the unconstrained enclosure based acoustic startle response measurement system has been implemented in this paper. As a result, the effectiveness of the proposed CR ISI method has been verified and compared with those of conventional ISI methods through animal experiments using SD-rats. The experimental results showed that abnormal startle responses and invalid GPIAS values caused by motion were prevented when our proposed CR ISI method was applied to our implemented system. It was also verified that our proposed CR ISI method is advantageous in reducing the total experimental time for acquiring normal startle responses and valid GPIAS values, compared to conventional ISI methods, since our proposed CR ISI can begin the acoustic stimulation only when the animal gets stable and motionless.

Proposal of conditional random inter-stimulus interval method for unconstrained enclosure based GPIAS measurement systems