The Acoustic Startle Response Chamber is utilized in the assessment of underlying mechanisms of sensorimotor integration. The acoustic startle response is a protective reflex to a sudden or a threatening auditory stimulus often characterized by rapid closing of the eyes and muscle contractions. In animals, the startle response may be observed during a predator confrontation, and thus is also known as the escape response (Fetcho & McLean, 2009). The short pathways of the startle circuitry have relatively fast conducting neurons that are receptive to different sensory modalities. These pathways are, in turn, connected to the motor neurons, often the largest ones, which undergo strong excitation in response to the startle stimuli (Fetcho, & McLean, 2009). The startle response, hence, is useful in understanding cellular functions and synaptic connections.

While reflexive, the startle response may also be elicited by other sensory stimuli such as sensory gating (Fendt, Li, & Yeomans, 2001). Impairments of sensory gating, measured using pre-pulse inhibition, are often biomarkers of many neuropsychiatric disorders such as schizophrenia (Mena et al., 2016) and obsessive-compulsive disorder (Ahmari, Risbrough, Geyer, & Simpson, 2016). Impaired pre-pulse inhibition is also a characteristic of neurological disorders such as Huntington’s disease (Swerdlow et al., 1995) and Tourette syndrome (Zebardast et al., 2013). Thus, the acoustic startle response is helpful in assessing sensory gating impairments and development of the associated treatments. Additionally, the startle response can also be used to evaluate learning and emotions.

The Acoustic Startle Response Chamber consists of a sound-attenuated isolation chamber. The chamber comes equipped with an animal holder having a grid floor and transducer. The stimulus presentation can be done via speakers, visible light bulb, and shock grid attachment. The chamber can be calibrated using the Conductor Software. Other apparatuses used in the assessment of reflex and similar behaviors include the Step Down Avoidance, the Geotaxis Test, the Electrical von Frey Filament, and Predator Odor Exposure Test.

The Acoustic Startle Response Chamber consists of a sound-attenuated isolation chamber with provisions for the animal holder and startle-inducing cues. The animal holder is available in a range of sizes to accommodate animals ranging in weight from 0 to 500 grams. The holder is constructed using metal rods that have a diameter of 0.47 cm (flooring) and 0.318 cm (sides) that are spaced according to the weight of the animal. The animal holder can also be fitted with a transducer to convert vertical movements of the platform into voltage signals. In addition to auditory stimuli, electrical as well as light stimuli can be presented in the Acoustic Startle Response Chamber. Auditory stimuli can be presented through the two speakers present in the chamber. The speakers allow two independent channels, each having a frequency range of 100 to 40,000 Hz and an intensity range of 1 to 150 dB. The chamber is also equipped with a sound detector to assist volume control. Light cues can be produced using the dual visible light and infrared bulbs. A removable DC current shock grid can also be used to provide electrical stimuli. The shock intensity can be raised in 0.1 mA steps between 0.1 to 4.0 mA.

Clean the Acoustic Startle Response Chamber and its components between trials and subjects to prevent the influence of any lingering stimuli on the performances. Calibrate the speaker volumes as well as the transducer sensitivity in the absence of any external disturbances before commencing with the protocol.

The general protocol can be divided into the acclimation phase, the habituation phase, and the pre-pulse inhibition phase. Throughout the session, white background noise is maintained.

Acclimation Phase

Acclimatize the subject to handling at least one week prior to beginning the sessions. Subjects that have undergone surgery should be allowed at least 1 week of rest to prevent surgery stress from interfering with the task.

Allow the subject to acclimatize to the testing room for at least 30 minutes without any stimulations. Weigh the subject before placing it in the apparatus and allow it to acclimatize to the apparatus with the background noise (65 to 68 dB white noise) on for 5 to 15 minutes.

Habituation Phase (Acoustic Startle Response Trials)

Following the acclimation phase, begin the habituation phase using only startle stimuli. Present the subject with 30 to 100 startle stimuli in the range of 105 to 120 dB having a duration between 20 to 40 msec. Though debated, it is recommended that the intertrial interval between the startle stimuli be randomized between 10 to 30 sec to prevent the possible prediction of the timing of the stimuli by the subject. However, fixed intervals may also be used.

Pre-pulse Inhibition Phase

Allow the subject 5 to 10 minutes acclimation period before beginning the pre-pulse inhibition phase. Begin the session with 4 to 5 successive bursts of startle pulse (120 dB for 40 msec) to normalize the subject response. Follow this trial with a pseudorandomized presentation of startle pulse, no stimulation and three distinct pre-pulse (for example 70, 75, and 80 dB for 20 msec) followed by startle pulse (after 30 or 100 msec). Perform at least 10 trials under each of the three conditions with inter-trials interval randomized between 10 to 30 sec (a fixed interval of 20 sec may also be used). A pre-pulse alone condition may also be included in the pre-pulse inhibition block.

Startle Threshold Trial

The startle threshold trial allows the establishment of the appropriate startle pulse and is usually performed following the habituation phase. Present each decibel in a randomized order for a 40 msec duration. Present at least 3 to 5 instances of each decibel and average it.

Long-Term Habituation Protocol

For long term habituation protocol, the general protocol (acclimation phase, habituation phase, and pre-pulse inhibition phase) are to be repeated over a course of at least 5 consequent days. It must be ensured that all conditions, including housing and timing of the experiment, are maintained across all days with very little variability.

The long-term habituation assessment can also be performed only with the acclimation phase and the habituation phase, with the habituation phase having at least 100 startle pulse used.

Investigation of the effect of predator odor exposure in early adolescence

Wah, Ossenkopp, Bishnoi, and Kavaliers (2018) investigated the effects of repeated adolescent stress as well as propionic acid on the behaviors and the response to sensory stimuli of Long-Evans rats. Based on the drug; sodium propionate (PPA, 500 mg/kg) and phosphate-buffered saline (PBS) male subjects (post-natal day 27) were divided into 4 treatment groups: cat-odor PPA, cat-odor PBS, no odor PPA and no odor PBS. The subjects were exposed to cat odors obtained from worn collars (male and female cats) for 30 minutes across 2 weeks (post-natal days 28, 30, 32, and 34) in a Predator Odor Exposure Test chamber. Controls were exposed to the odor of unworn collars in the same set-up. On post-natal day 40, the subjects were intraperitoneally administered the treatment drugs at 2.0 ml/kg body weight. Fifteen minutes following the administration, behavioral alterations were assessed in the Light-Dark Box. On post-natal day 43, subjects were once again treated with the drugs, 10 minutes following which they were evaluated in the Acoustic Startle Response Chamber (acoustic startle response, 120 dB; pre-pulse inhibition, 76 and 82 dB). Treatment procedures and behavioral assays were repeated once again on post-natal day 74 (Light-Dark Box) and 77 (Acoustic Startle Response Chamber) to evaluate the changes in adulthood behaviors. While no significant changes in the duration of time spent in the light chamber were found between the cat-odor exposed groups, the groups did show decreased nose pokes into the light area compared to their controls. In comparison to PBS treated groups, PPA treatment decreased the number of transitions between compartments, decreased corrected total distance traveled, and decreased corrected vertical time in both adolescents and adults.  No effect on the magnitude of the response could be observed between the cat odor groups, or the PPA treated groups. Pre-pulse inhibition testing at 76 dB revealed a decreased percentage of pre-pulse inhibition in PPA treated groups in comparison to the PBS groups. However, no significant differences between groups were observed at 82 dB.

Investigation of startle latency in the 5xFAD mouse model of Alzheimer’s disease

Story, Chan, Munro, Rossignol, and Dunbar (2018) observed that the 5xFAD mice model displayed similar startle response as Alzheimer’s disease sufferers.  Twelve 5xFAD mice and 9 wild types consisting of both male and females (8 months in age) were evaluated for their startle reflex in the Acoustic Startle Response Chamber. Acclimation was carried out twice a day for 3 to 5 days using 65 dB white noise for 5-minute intervals. Following this period subjects received 30 pseudorandomized trials that included 10 startle trials (120 dB, 50 msec) and 20 (10 + 10 trials) pre-pulse trials. Pre-pulse (30 msec) was generated as an 8 or 16 dB increase in white noise which was followed 70 msec later by 120 dB startle pulse. The average inter-trial interval was approximately 24 seconds, and the startle response was recorded in the 250 msec window following the startle pulse. In comparison to their wild type counterparts, 5xFAD mice displayed a significant reduction in peak startle response and a significant increase in the latency to peak startle response (mean latency 83.9 msec). Pre-pulse inhibition trials revealed that the 5xFAD maintained the baseline startle response while the wild types displayed significantly lower pre-pulse inhibition at both pre-pulse increments (70% at 8 dB increment and 51% at 16 dB increment). Subjects were also tested in the Open-Field. However, no significant differences in performances could be observed.

Investigation of deep brain stimulation in a schizophrenic rodent model

Subjects were offspring of Wistar rats that had received a single intravenous injection of either poly I:C (4 mg/kg) or saline on day 15 of gestation. The subjects (aged more than 90 post-natal days) underwent surgery for placement of electrodes in the mPFC (ventral prelimbic cortex; anteroposterior (AP) +3.5, mediolateral (ML) 0.6, dorsoventral (DV) -3.4) or the Nacc shell. Following surgery, the subjects were allowed to recover for 1 to 2 weeks and received habituation to handling, deep brain stimulation procedure as well as the test chamber during the last 5 days. The pre-pulse inhibition trials were carried out in three sessions: session 1 served as the baseline performance and did not involve deep brain stimulations, session 2 included deep brain stimulation of the mPFC or the Nacc, and session 3 was used to evaluate if session 2 effects continued and thus did not include brain stimulation. The sessions were spaced 24 hours apart. Subjects were placed in the Acoustic Startle Response Chamber and underwent acclimation (5 min) with 60 dB background noise and 10 startle stimuli (20 msec) following which test session commenced. Test session trials included 10 presentation of each 1 pulse alone (100dB, 20 msec), 1 no stimulus, 2 pre-pulse alone (72 dB or 68 dB, pure tone, 10 Hz, 20 msec), 3 pre-pulse (72 dB, 68 dB, or 64 dB) followed by pulse after 100 msec trials. A significant decrease in the pre-pulse inhibition was observed in the poly I:C offspring group, relative to saline offspring group, in sessions 1 and 3, which was normalized by deep brain stimulation (both mPFC and Nacc group) in session 2. On the other hand, Nacc deep brain stimulation-induced pre-pulse inhibition deficits in the saline-treated dams’ offspring. (Bikovsky et al., 2016)

Investigation of the behavioral effects of methoxetamine

Halberstadt, Slepak, Hyun, Buell, and Powell (2016) investigated the “k” analog, methoxetamine, for causing behavioral changes similar to PCP and “k”. Male Sprague-Dawley rats were housed in 12-hour reverse light/dark cycle to allow testing in the awake phase. Subjects were administered 1 ml/kg injections of either “p” hydrochloride (PCP) (0.8, 1, or 1.2 mg/kg); (±)-N-allylnormetazocine hydrochloride (NANM) (2.5, 5, or 10 mg/kg); S-(+)-“k” hydrochloride (1.5, 3, 6, or 12 ,g/kg); R-(−)-“k” hydrochloride (1.5, 3, 6, or 12 ,g/kg); and methoxetamine hydrochloride (MXE) (1, 3, or 10 mg/kg). The effects of each drug were evaluated in the Acoustic Startle Response Chamber 15 min after treatment with NANM, 5 min after treatment with MXE or PCP, and immediately after treatment with “k”. Startle sessions included pseudorandomized trials of 12 pulse-alone (120 dB, 40 msec) and 36 pre-pulse (12 trials each at 68, 71, and 77 dB) + pulse trials. Additionally, subjects were also presented with 6 pulse alone trials before and after testing. At doses 3 and 10 mg/kg of MXE, resulted in a decrease of pre-pulse inhibition at all 3 pre-pulse intensities. Startle response observation revealed a 3-fold increase in the magnitude of response at a dose of 1 mg/kg. While PCP did not affect the amplitude of the response, it resulted in a significant decrease in pre-pulse inhibition at all intensities. Similarly, “k” isomers (S-(+)-“k” hydrochloride and R-(−)-“k” hydrochloride) did not have any effect on the magnitude of the response. Though both resulted in reduced pre-pulse inhibition, in comparison to the R-(−)-“k”, S-(+)-“k” proved to be more potent. Further, only S-(+)-“k” showed a drug and pre-pulse intensity interaction with no reduction in the pre-pulse intensity at 68 dB. NANM reduced pre-pulse inhibition with 10 mg/kg reducing pre-pulse inhibition at all intensities while 5 mg/kg showing a significant reduction only at 68 and 71 dB. NANM also did not affect the startle amplitude. Overall, MXE was observed to have resulted in behavioral alterations similar to PCP.

In addition to the observation of behavioral reactions, the following data can be recorded in the Acoustic Startle Inhibition Chamber,

• Peak startle response amplitudes
• Average startle response amplitudes
• Percentage of pre-pulse inhibition

%PPI = [pulse-alone – (pre-pulse + pulse score)]/pulse-alone score

• Reflex latency

Strength

The Acoustic Startle Response Chamber allows the automation of the entire process. The apparatus allows control over the intensity and combinations of cues used. The all-in-one nature of the apparatus permits the application of various protocols such as fear-potentiated startle. The system can also be used to induce stress in subjects as part of the protocol of other behavioral assays.

Limitations

Behavioral performances of the subjects may be affected by changes in illumination and noise levels in the holding area from the housing conditions. Any variations in the isolation chamber conditions, such as background noise, from trial to trial may result in erroneous data. Improper calibration of the components of the apparatus may affect the quality of the data. Lingering olfactory cues or other cues from previous trials may influence performances. Subjects age, gender, weight, species, and sensitivity to the cues used can impact performances.

• The Acoustic Startle Response Chamber is used in the assessment of sensorimotor gating, learning, and emotions.
• The sound-attenuated chamber minimizes the influence of external auditory stimuli on the performances.
• The chamber accommodates multiple animal holders, thus allowing simultaneous assessments in groups of animals.
• The Acoustic Startle Response Chamber can be used for other protocols such as fear-potentiated startle, emotions assessment, learning assessment, stress induction, and hearing assessment.

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