The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Kristen A McLaurin; Landhing M Moran; Hailong Li; Rosemarie M Booze; Charles F Mactutus

doi:10.3791/58659

JoVE Journal > Neuroscience

신경과학

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Published: April 19, 2019

doi:

10.3791/58659

Kristen A McLaurin¹, Landhing M Moran¹, Hailong Li¹, Rosemarie M Booze¹, Charles F Mactutus¹

¹Program in Behavioral Neuroscience, Department of Psychology,University of South Carolina

Summary

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Abstract

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Introduction

Temporal processing deficits have been implicated as a potential underlying neural mechanism for alterations in higher-level cognitive processes commonly observed in neurocognitive disorders. Prepulse inhibition (PPI) of the auditory startle response (ASR) is a translational experimental paradigm commonly used to examine temporal processing deficits, revealing profound alterations in neurocognitive disorders such as schizophrenia¹, attention deficit hyperactivity disorder² and HIV-1 associated neurocognitive disorders³^,⁴. Specifically, assessments of temporal processing in preclinical models of HIV-1 have revealed the generality, relative permanence, and suggested the diagnostic utility of PPI across the majority of the animals' functional lifespan³^,⁴^,⁵^,⁶.

Use of an approach varying interstimulus interval (ISI; i.e., the time between the prepulse and the startle stimulus) in the analysis of reflex modification dates back to Sechenov in 1863⁷. The seminal studies of reflex modification, a measure of sensorimotor gating, employed an approach varying ISI to assess flexor response and audition in frogs⁷^,⁸, as well as knee-jerk responses in humans⁹. The first clinical application of the reflex modification procedure assessed visual sensitivity in a man with hysterical blindness¹⁰. Over a century after the first reports of reflex modification, the approach of varying ISI was popularized across a series of seminal papers¹¹^,¹²^,¹³. Despite the inherent differences in the seminal studies on reflex modification (i.e., species, experimental procedures, reflexes), they established a temporal relationship that was strikingly similar between species.

Assessment of prepulse inhibition using an approach varying ISI, as detailed in the present protocol, has multiple advantages over the popularized percent of control approach. First, the approach affords an opportunity to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve)³^,¹⁵ in startle amplitude, as well as shifts in the peak point of response inhibition³^,⁵. Additionally, when an approach varying ISI is employed, startle response is a relatively stable phenomenon¹, suggesting the potential utility of the approach in longitudinal studies examining the progression of neurocognitive deficits⁵^,¹⁵. Finally, PPI provides a critical opportunity to understand the underlying neural circuitry involved in neurocognitive disorders¹⁶.

In our study, we employed two experimental paradigms (Figure 1), including cross-modal PPI and gap prepulse inhibition (gap-PPI), to evaluate the utility of an approach varying ISI to delineate effects of sensory modality, psychostimulant exposure, and age. The cross-modal PPI experimental paradigm utilizes the presentation of an added stimulus (e.g., tone, light, air puff) as a discrete prestimulus prior to an acoustic startling stimulus. In sharp contrast, in the gap-PPI experimental paradigm, the absence of a background (e.g., removal of background noise, light, or air puff) serves as a discrete prestimulus. Here, we describe both experimental paradigms for the assessment of temporal processing, as well as statistical approaches for the analysis of PPI and gap-PPI. Within the discussion, we compared the conclusions one would draw from the variable ISI approach and the popularized percent of control approach.

Protocol

All animal protocols were reviewed and approved by the Animal Care and Use Committee at the University of South Carolina (federal assurance number: D16-00028).

1. Defining Parameters and Calibration of the Startle Apparatus

Set up the startle response system (see Table of Materials) according to the manufacturer’s instructions.
1. Enclose the startle platform in a 10 cm-thick double-walled isolation cabinet.
Calibrate the response sensitivities using the startle calibration system.
Attach the high-frequency loudspeaker 30 cm above the animal holder.
1. Measure and calibrate the loudspeaker using a sound level meter by placing the microphone inside the animal holder.
Affix a white LED light (22 lux) on the wall in front of the animal holder.
1. Measure the lux presented as a visual prepulse using a light meter.
Connect a semi-rigid plastic tube (0.64 mm diameter) to a compressed air tank via an airline regulator.
1. Set the air tank to 16 psi for the presentation of tactile prestimuli.
2. Use a sound level meter to measure the amount of noise being emitted by the tactile stimulus inside the tube, 2.5 cm from the end of the animal holder. If using multiple chambers, ensure that all chambers are calibrated in the same manner.
  Note: To prevent the tactile stimulus from being perceived as an acoustic stimulus, the sound of the air puff prepulse must be less than or equal to the white noise background. In the present set-up, the air puff prepulse emitted 70 db(A) inside the tube while the background white noise was also set to 70 db(A).

2. Creation of Experimental Programs

Open the Startle Response System software (see Table of Materials).
Click Definitions and Select Define Trial.
Define a pulse-only ASR trial.
Note: The pulse-only ASR trial is run during the habituation session, and 6 times at the beginning of every cross-modal PPI and gap-PPI session for habituation.
1. Type a Trial Name. Hit Enter.
2. Record Data.
3. Set the Analog Level to 720.
4. Define the Wait Length as 20 ms.
5. Introduce Background.
6. End the Trial.
7. Hit Accept to save the trial.
Click Definitions and Select Define Trial.
Create six separate trial definitions for acoustic PPI, including one trial for each ISI (i.e., 0, 30, 50, 100, 200, 4000 ms).
1. Create a trial definition for the 0 ms ISI for acoustic PPI.
  1. Type a Trial Name. Hit Enter.
  2. Record Data.
  3. Set the Analog Level to 720.
  4. Assign the Wait Length to 20 ms.
  5. Introduce Background.
  6. End the Trial.
  7. Hit Accept to save the trial.
2. Create remaining trial definitions for ISIs with both a prestimulus and a stimulus (i.e., 30, 50, 100, 200, 4000 ms).
  1. Type a Trial Name. Hit Enter.
  2. Set the Analog Level to 600 at 0 ms to introduce the prestimulus.
  3. Assign the Wait Length to 20 ms to specify the length of the prestimulus.
  4. Set the Analog Level to 440 at 20 ms to remove the prestimulus.
  5. Define the Wait Length dependent upon ISI.
    Note: Define the wait length as: 10 ms for the 30 ms ISI, 30 ms for the 50 ms ISI, 80 ms for the 100 ms ISI, 180 ms for the 200 ms ISI, and 3980 ms for the 4000 ms ISI. Only one wait length is included for each ISI.
  6. Record Data.
  7. Set the Analog Level to 720.
  8. Assign the Wait Length to 20 ms.
  9. Introduce Background.
  10. End the Trial.
  11. Hit Accept to save the trial.
Click Definitions and Select Define Trial.
Create six separate trial definitions for visual or tactile PPI, including one trial for each ISI (i.e., 0, 30, 50, 100, 200, 4000 ms).
1. Create a trial definition for the 0 ms ISI for visual or tactile PPI.
  1. Type a Trial Name. Hit Enter.
  2. Record Data.
  3. Turn the Tactile on.
  4. Set the Analog Level to 720 and the Wait Length to 20 ms.
  5. Turn the Tactile off.
  6. Introduce Background.
  7. End the Trial.
  8. Hit Accept to save the trial.
2. Create remaining trial definitions for ISIs with both a prestimulus and a stimulus (i.e., 30, 50, 100, 200, 4000 ms).
  Note: Visual and tactile can not be run concurrently because of software and hardware limitations. The modality presented is dependent upon the input into the hardware (i.e., whether the light is connected or the air puff is connected).
  1. Type a Trial Name. Hit Enter.
  2. Turn the Tactile on to introduce the prestimulus.
    Note: In this instance, tactile refers to the modality (i.e., either visual or air puff) that is connected to the hardware.
  3. Set the Wait Length to 20 ms.
  4. Turn the Tactile off to remove the prestimulus.
  5. Set the Analog Level to 440 at 20 ms.
  6. Define the Wait Length dependent upon ISI.
    Note: Define the wait length as: 10 ms for the 30 ms ISI, 30 ms for the 50 ms ISI, 80 ms for the 100 ms ISI, 180 ms for the 200 ms ISI, and 3980 ms for the 4000 ms ISI.
  7. Record Data.
  8. Set the Analog Level to 720.
  9. Assign the Wait Length to 20 ms.
  10. Introduce Background.
  11. End the Trial.
  12. Hit Accept to save the trial.
Click Definitions and Select Define Trial.
Create six separate trial definitions for acoustic gap-PPI, including one trial for each ISI (i.e., 0, 30, 50, 100, 200, 4000 ms).
1. Create a trial definition for the 0 ms ISI for acoustic gap-PPI.
  1. Type a Trial Name. Hit Enter.
  2. Record Data.
  3. Set the Analog Level to 720 and the Wait Length to 20 ms.
  4. Introduce Background.
  5. End the Trial.
  6. Hit Accept to save the trial.
2. Create remaining trial definitions for ISIs with both a prestimulus and a stimulus (i.e., 30, 50, 100, 200, 4000 ms).
  1. Type a Trial Name. Hit Enter.
  2. Set the Analog Level to 0 at 0 ms to introduce the prestimulus.
  3. Assign the Wait Length to 20 ms to specify the length of the prestimulus.
  4. Set the Analog Level to 440 at 20 ms to remove the prestimulus.
  5. Define the Wait Length dependent upon ISI.
    Note: Define the wait length as: 10 ms for the 30 ms ISI, 30 ms for the 50 ms ISI, 80 ms for the 100 ms ISI, 180 ms for the 200 ms ISI, and 3980 ms for the 4000 ms ISI.
  6. Record Data.
  7. Set the Analog Level to 720.
  8. Assign the Wait Length to 20 ms.
  9. Introduce Background.
  10. End the Trial.
  11. Hit accept to save the trial.
Click Definitions and Select Define Trial.
Create six separate trial definitions for the visual or tactile gap-PPI, including one trial for each ISI (i.e., 0, 30, 50, 100, 200, 4000 ms).
1. Create a trial definition for the 0 ms ISI for visual or tactile gap-PPI.
  1. Type a Trial Name. Hit Enter.
  2. Turn the Tactile on.
  3. Record Data.
  4. Set the Analog Level to 720 and the Wait Length to 20 ms.
  5. Introduce Background.
  6. End the Trial.
  7. Hit Accept to save the trial.
2. Create remaining trial definitions for ISIs with both a prestimulus and a stimulus (i.e., 30, 50, 100, 200, 4000 ms).
  1. Type a Trial Name. Hit Enter.
  2. Turn the Tactile on.
  3. Set the Analog Level to 0 ms.
  4. Turn the Tactile off.
  5. Set the Wait Length to 20 ms.
  6. Turn the Tactile on.
  7. Set the Analog Level to 440.
  8. Define the Wait Length dependent upon ISI.
    Note: Define the wait length as: 10 ms for the 30 ms ISI, 30 ms for the 50 ms ISI, 80 ms for the 100 ms ISI, 180 ms for the 200 ms ISI, and 3980 ms for the 4000 ms ISI.
  9. Record Data.
  10. Set the Analog Level to 720.
  11. Assign the Wait Length to 20 ms.
  12. Introduce Background.
  13. End the Trial.
  14. Hit accept to save the trial.
Select Definitions and Define Session.
1. Create a habituation session.
  1. Set the Background Analog Level to 440, the Number of Record Samples to 200 the Samples per Second to 2000, the Acclimation Period to 5 min, and the Sequence Repetitions to 36.
  2. Type 10 into the intertrial interval (ITI) list box.
  3. Click Add and select the pulse-only ASR trial.
  4. Click Save to save the habituation session.
Select Definitions and Define Session.
Define the session for Cross-Modal PPI.
1. Set the Background Analog Level to 440, the Number of Record Samples to 200 the Samples per Second to 2000, the Acclimation Period to 5 min, and the Sequence Repetitions to 1.
2. Define the intertrial interval (ITI) list.
  1. Type 10 into the first 5 ITI list boxes.
  2. Type a variable ITI (15-25 s) into the next 72 ITI list boxes, representing trials with a prestimulus.
3. Click Add.
  1. Select the pulse-only ASR trial and load it 6 times for Trials 1-6.
  2. Create 6-trial blocks for each prestimulus modality using a Latin Square design (Table 1).
  3. Load the 6-trial blocks in an ABBA counterbalanced order of presentation (e.g., acoustic, visual, visual, acoustic, acoustic, etc.) for cross-modal PPI.
    Note: Each trial must be loaded individually.
    Note: Each cross-modal PPI session includes a total of 78 trials.
4. Click Save to save the session.
Select Definitions and Define Session.
1. Define the session for Gap-PPI.
  1. Set the Background Analog Level to 440, the Number of Record Samples to 200 the Samples per Second to 2000, the Acclimation Period to 5 min, and the Sequence Repetitions to 1.
  2. Define the intertrial interval (ITI) list.
    1. Type 10 into the first 5 ITI list boxes.
    2. Type a variable ITI (15-25 s) into the next 36 ITI list boxes, representing trials with a prestimulus.
  3. Click Load to load the trials.
    1. Select the pulse-only ASR trial and load it 6 times for Trials 1-6.
    2. Create 6-trial blocks for each prestimulus modality using a Latin Square design (Table 1).
  4. Click Save to save the session.
    Note: Each gap-PPI session includes a total of 42 trials. Each session assesses one sensory modality.

3. Protocol Structure

Use the F344/N rat strain, the most common inbred rat strain, for assessments.
Note: Cross-modal PPI and gap-PPI can be conducted in animals at a variety of ages, of both sexes, and regardless of hormonal status (i.e., ovariectomized, castrated, intact). Details regarding the animals used in the representative data are presented in the representative results.
Handle the animals to allow for acclimation across a series of days prior to beginning experimentation.
Randomize the order of animals for experimentation dependent upon the between-subjects’ factors of interest (e.g., biological sex, treatment).
Open the startle response system software. Click Run. Select the session of interest.
Note: Only one session is conducted per day and sessions need to be conducted in a sequential order (i.e., Habituation, Cross-Modal PPI, Gap-PPI)
Input an Output File Name and click OK.
Enter Subject, Group, and ID information and click 계속.
Place the animal into the startle apparatus using an animal enclosure that is most appropriate for the size of the animal. Click OK to begin the session.
Export data for analysis.
1. Click Reports | Concatenate Data. Load the data file and click Add. Click ASCII to save the data output.

4. Data Analysis

Calculate an adjusted V. Max for each trial by subtracting the V. Max from the Start value.
Note: The adjusted V. Max creates a measure of mean peak ASR amplitude.
Graphically visualize results for the habituation session.
1. Plot group means and standard errors of the mean for each trial. Regression analyses can be conducted and fit with 95% confidence intervals.
Graphically visualize results for cross-sectional cross-modal PPI and gap-PPI.
1. Calculate mean values for each ISI by averaging across the 6 trials individually for each animal.
2. Calculate and graph group means and standard errors of the mean for each ISI and sensory modality.
Statistically analyze cross-modal PPI and gap-PPI (Optional).
Note: Although the precise statistical approach will be dependent upon the experimental design and research question of interest, a mixed-design repeated measures ANOVA provides one appropriate approach.

Representative Results

A prominent non-monotonic ISI function was observed in cross-modal PPI (Figures 2A, 3A, 4A) and gap-PPI (Figures 2B, 3B, 4B). Baseline startle responses were observed at the 0 and 4000 ms ISIs, included as reference trials within a test session. The importance of the 4000 ms ISI cannot be understated, as it most closely resembles the PPI test trials (i.e., 30, 50, 100, 200 ms ISIs) in that the subject receives both the prepulse and startling stimuli. However, no significant decrease in ASR is observed at the 4000 ms ISI because of the large time interval between the prepulse and startling stimulus. Either the addition (i.e., cross-modal PPI) or removal (i.e., gap-PPI) of a discrete prestimulus produced robust inhibition at the 30, 50, 100, and 200 ms ISIs; inhibition that was dependent upon sensory modality, psychostimulant exposure or age. The power of the ISI approach is revealed by examining these effects on changes in the ISI function (i.e., sharper inflections of the ISI curve, flattening of the ISI curve, and shifts in the point of maximal inhibition).

The utility of an approach varying the ISI to delineate effects of sensory modality in cross-modal PPI are illustrated in Figure 2A (F344/N controls between 8 and 10 months of age, n=20). Due to hardware and software limitations, only two prestimulus modalities can be assessed at once. Following habituation, concurrent acoustic and visual prepulse stimuli were used to examine PPI. Next, concurrent acoustic and tactile prepulse stimuli were used to assess PPI. Data for acoustic PPI is presented from the experimental paradigm including the concurrent presentation of acoustic and visual prestimuli (i.e., visual context). A prominent shift in the point of maximal inhibition is dependent upon sensory modality, suggesting a differential sensitivity to the manipulation of ISI. Specifically, maximal inhibition is observed at the 30 ms ISI following the presentation of a discrete acoustic prestimulus, at the 50 ms ISI following the presentation of a discrete visual prestimulus, and at the 200 ms ISI following the presentation of a discrete tactile prestimulus. Additionally, a flatter ISI function, indicative of a relative insensitivity to the manipulation of ISI, is observed following the presentation of an acoustic prestimulus relative to a visual or tactile prestimulus. A repeated measures ANOVA was conducted to statistically analyze the data, confirming our observations and revealing a significant prestimulus modality x ISI interaction [F(10,190)=22.8, p_GG≤0.001, η_p²=0.546] with a prominent linear-linear component [F(1,19)=36.1, p≤0.001, η_p²=0.655]. Notably, the interaction accounted for a large proportion of variance within the model, evidenced via measures of η_p².

Following an animal's experience with each prestimulus in cross-modal PPI, the generalizability of sensory modality effects was assessed in gap-PPI. Acoustic gap-PPI, visual gap-PPI and tactile gap-PPI were each conducted separately. Figure 2B demonstrates the generalizability of varying the ISI to delineate effects of sensory modality. A prominent shift in the point of maximal inhibition, suggesting a differential sensitivity to the manipulation of ISI, was observed in tactile gap-PPI (i.e., 30 ms) relative to acoustic gap-PPI and visual gap-PPI (i.e., 50 ms). Additionally, a relative insensitivity to the manipulation of ISI, evidenced by a relatively flatter ISI function was observed in tactile gap-PPI and visual gap-PPI relative to acoustic gap-PPI. As in cross-modal PPI, a significant prestimulus modality x ISI interaction [F(10,190)=17.6, p_GG≤0.001, η_p²=0.481] with a prominent linear-quadratic component [F(1,19)=58.5, p≤0.001, η_p²=0.755] was revealed; an effect which again, accounts for a significant proportion of the variance.

After the completion of cross-modal PPI and gap-PPI, animals repeatedly orally self-administered methylphenidate (MPH). A post-test assessment of cross-modal PPI with concurrent acoustic and visual prestimuli and acoustic gap-PPI were conducted at approximately 14 months of age following 22-27 days of MPH exposure. The pre-test and post-test ISI functions for acoustic PPI are illustrated in Figure 3A. Most notably, at the post-test assessment, a relative flattening of the ISI function is observed, suggesting a relative insensitivity to the manipulation of ISI relative to the pre-test assessment. Additionally, a prominent shift in the point of maximal inhibition is revealed, with inhibition at the 30 ms ISI during the pre-test assessment and the 100 ms ISI at the post-test assessment, suggesting a differential sensitivity to the manipulation of ISI. A repeated-measures ANOVA confirmed these observations, revealing a significant test session x ISI interaction [F(5,95)=7.4, p_GG≤0.003, η_p²=0.280] with a prominent linear-quadratic component [F(1,19)=10.6, p≤0.004, η_p²=0.358].

Following the post-test cross-modal PPI assessment, acoustic gap-PPI was conducted to assess the generalizability of the effects of psychostimulant exposure on temporal processing. Figure 3B illustrates the generalizability of varying the ISI to delineate effects of psychostimulant exposure. The point of maximal inhibition was at the 50 ms ISI during both the pre-test and post-test assessment. However, a significantly flatter ISI function was observed following MPH exposure. A repeated-measures ANOVA confirmed these observations, revealing a significant test session x ISI interaction [F(5, 95)=3.6, p_GG≤0.013, η_p²=0.159] with a prominent linear-cubic component [F(1,19)=9.1, p≤0.007, η_p²=0.325].

The shape of the ISI function also affords an opportunity to assess the development of temporal processing across age. In a longitudinal study (F344/N controls, male: n=20, female: n=17), cross-modal PPI with concurrent acoustic and visual prestimuli was conducted every sixty days from postnatal day (PD) 30 to PD 150. The development of temporal processing in visual PPI is illustrated in Figure 4A. Within visual PPI, the point of maximal inhibition at all ages is at the 50 ms ISI. However, a sharper inflection of the ISI function is observed across age, suggesting a perceptual sharpening which occurs with development. A repeated-measures ANOVA, with sex as the between-subjects factor and age, ISI, and trial as within-subjects factors, confirmed these observations revealing a significant age x ISI interaction [F(10,350)=12.6, p_GG≤0.001, η_p²=0.265] with a prominent linear-quadratic component [F(1,35)=32.6, p≤0.001, η_p²=0.482] and a significant ISI x sex interaction [F(5,175)=4.0, p_GG≤0.014, η_p²=0.104] with a prominent quadratic component[F(1,35)=5.2, p≤0.028, η_p²=0.130].

At every age, acoustic gap-PPI was conducted following cross-modal PPI. The experiences an animal has had have a direct impact on its responses, necessitating the use of a sequential experimental design (i.e., always conducting cross-modal PPI prior to gap-PPI). Figure 4B illustrates the development of temporal processing, assessed using acoustic gap-PPI. At PD 30, a relative insensitivity to the manipulation of ISI was observed, evidenced by a flatter ISI function, relative to PD 90 or PD 150. Observations of the sharpest ISI function at PD 150 suggest a perceptual sharpening that occurs across development. Additionally, a prominent shift in the point of maximal inhibition is revealed, with maximal inhibition occurring at the 30 ms ISI at PD 30 and the 50 ms ISI at PD 90 and PD 150, suggesting a differential sensitivity to the manipulation of ISI. Statistically, the observation of a significant age x ISI interaction [F(10,350)=10.4, p_GG≤0.001, η_p²=0.230] with a prominent linear-quadratic component [F(1,35)=70.5, p≤0.001, η_p²=0.668] and an ISI x sex interaction [F(5,175)=3.8, p_GG≤0.010, η_p²=0.097] with a prominent quadratic component [F(1,35)=11.0, p≤0.002, η_p²=0.184240] confirms our observations.

Figure 1: Prepulse Inhibition Experimental Paradigms. A) Animals exhibit a baseline auditory startle response when an acoustic startle stimulus is presented. B) During cross-modal prepulse inhibition (PPI), the presentation of a discrete prestimulus (i.e., acoustic tone, light, air puff) 30 to 500 ms¹⁶ prior to an acoustic startle stimulus, produces robust inhibition. C) During gap prepulse inhibition (gap-PPI), the removal of a discrete prestimulus (gap in background noise, light, or air puff) 30 to 200 ms¹⁷ prior to an acoustic startle stimulus produces robust inhibition. The image is adapted from Maze Engineers¹⁸. Please click here to view a larger version of this figure.

Figure 2: Cross-Sectional Assessment of Temporal Processing: Sensory Modality. A) Representative analysis of the effect of sensory modality on the interstimulus interval (ISI) function in cross-modal prepulse inhibition (PPI). B) Representative analysis of the effect of sensory modality on ISI in gap prepulse inhibition (gap-PPI). Control results from McLaurin et al.⁶ are reanalyzed in a novel manner to assess the effect of sensory modality. Data are presented as mean ± standard error of the mean. Please click here to view a larger version of this figure.

Figure 3: Cross-Sectional Assessment of Temporal Processing: Psychostimulant Exposure. A) Representative analysis of the effect of psychostimulant exposure (pretest vs. posttest) on the interstimulus interval (ISI) function in acoustic prepulse inhibition (PPI). B) Representative analysis of the effect of psychostimulant exposure on ISI in acoustic gap prepulse inhibition (gap-PPI). Control results from McLaurin et al.⁶ are reanalyzed in a novel manner as the pretest component for psychostimulant exposure. Data are presented as mean ± standard error of the mean. Please click here to view a larger version of this figure.

Figure 4: Longitudinal Assessment of Temporal Processing. A) Representative analysis of the effect of age on the interstimulus interval (ISI) function in visual prepulse inhibition (PPI). B) Representative analysis of the effect of age on the ISI function in acoustic gap prepulse inhibition (gap-PPI). Data are presented as mean ± standard error of the mean. Please click here to view a larger version of this figure.

Trial Block	Interstimulus Interval
1	0	30	50	100	200	4000
2	30	50	100	200	4000	0
3	50	100	200	4000	0	30
4	100	200	4000	0	30	50
5	200	4000	0	30	50	100
6	4000	0	30	50	100	200

Table 1: Latin Square Experimental Design

Sensory Modality	Cross-Modal PPI	Gap-PPI
Auditory	85.7 (2.0)	25.0 (4.3)
Visual	72.6 (2.7)	52.8 (5.3)
Tactile	73.2 (3.0)	-3.6 (8.5)
Psychostimulant Exposure	Cross-Modal PPI	Gap-PPI
Pretest Assessment	85.7 (2.0)	25.0 (4.3)
Posttest Assessment	90.5 (1.3)	52.6 (4.5)
Age	Cross-Modal PPI	Gap-PPI
PD 30	51.3 (3.7)	29.7 (4.4)
PD 90	73.8 (2.2)	39.6 (5.7)
PD 150	66.3 (2.9)	45.0 (3.9)

Table 2: Percent of Control

Discussion

The present protocol describes the power of varying ISI for the assessment of temporal processing for studies employing either cross-sectional or longitudinal experimental designs. Examining the effects of sensory modality, psychostimulant exposure, or age on the shape of the ISI function demonstrated its utility in revealing a differential sensitivity to the manipulation of ISI (i.e., shifts in the point of maximal inhibition) or a relative insensitivity to the manipulation of ISI (i.e., sharper inflections of the ISI curve, flattening of the ISI curve). Use of two experimental paradigms, including cross-modal PPI and gap-PPI, demonstrates that the utility of ISI is independent of the addition (i.e., cross-modal PPI) or removal (i.e., gap-PPI) of a discrete prestimulus.

Critical experimental design considerations for the completion of cross-modal PPI and gap-PPI are included within the protocol. First, a Latin-Square experimental design is implemented for the presentation of ISIs within 6-trial blocks, controlling for variation due to the order of ISI presentation. Second, the use of two control trials, including both the 0 and 4000 ms ISIs, provides reference control trials within the test session. The use of the 4000 ms ISI is particularly critical, as it most appropriately resembles the other (i.e., 30, 50, 100, 200) prepulse + pulse trials, but without the expectation of significant inhibition. Third, a counterbalanced (i.e., ABBA) experimental design is employed within cross-modal PPI to account for the repeated measurement of sensory modalities within a test session. Finally, inclusion of a variable ITI during the prepulse + pulse trials prevents an animal from expecting, and thus preparing for, the start of a trial. Thus, the implementation of a comprehensive number of ISIs in accordance with an appropriate experimental design allows for the determination of relatively precise and defined response functions; functions which provide a critical opportunity to assess the construct of temporal processing.

Methodology described within the present protocol contrasts other contemporary protocols for the analysis of PPI, which have popularized an approach that commonly employs a single ISI¹⁹^,²⁰. The popularized approach is commonly analyzed using percent inhibition, calculated as follows: 100 x {[(startle response amplitude during control trials)-(startle response amplitude during prepulse + pulse trials)] / {startle response amplitude during control trials)}. Two major caveats of the contemporary protocols, including the preclusion of the assessment of temporal processing and inappropriate statistical analyses, are discussed in turn below.

Percent inhibition was calculated for the 100 ms ISI within the representative data to demonstrate the limitations of the popularized approach (Table 2). For example, results for the assessment of acoustic gap-PPI and tactile gap-PPI suggest that animals fail to display any significant inhibition. Examination of Figure 2, utilizing an approach varying ISI, however, reveals that the animals did not fail to inhibit, but displayed a significant shift in the point of maximal inhibition (i.e., 50 ms in acoustic gap-PPI, 30 ms in tactile gap-PPI). Most notably, however, use of percent inhibition precludes the use of longitudinal experimental designs for assessing the development of temporal processing as a function of age, a well-recognized phenomena¹⁴^,²¹. Thus, like any percent of control measure, percent inhibition fails to disambiguate changes in PPI from changes in baseline startle response precluding the assessment of temporal processing.

Despite the apparent simplicity of the popularized approach, inferences drawn from statistical analysis must be made with extreme caution. Assumptions of the analysis of variance (i.e., normality of sampling distribution of means, homogeneity of error variance, independence of errors, absence of outliers²²) may not be met with subjectively determined percentage data²³. Specifically, the error variance for percentage data are not normally distributed²⁴, but instead are more appropriately described by a Poisson or bimodal distribution²⁵. In sharp contrast, a repeated-measures ANOVA provides one valid and reliable method for the statistical analysis of the ISI function. However, it is vital to account for the potential violation of sphericity, an assumption only present in models involving repeated measures, either using planned orthogonal contrasts or the post hoc Greenhouse-Geisser df correction factor²⁶ (p_GG).

Utilization of an approach varying ISI, however, is not without limitation. First, hardware and software limitations allow only two prestimulus modalities to be assessed at once. Notably, a differential sensitivity to the manipulation of context (i.e., concurrent visual or tactile stimulus in acoustic PPI) was previously reported in Long-Evans rats¹⁴ and HIV-1 Tg animals⁶. Second, relative to the popularized approach, there is a greater experimental time for the ISI approach (i.e., ~30 minutes for cross-modal PPI; ~20 minutes for gap-PPI).

Thus, an approach varying ISI provides an experimental method for the assessment of temporal processing. In addition to the aforementioned strengths of the approach, the serial neural circuit mediating PPI has been well-established²⁷^,²⁸, allowing for the assessment of neural circuitry alterations in neurocognitive disorders. Additionally, cross-modal PPI and gap-PPI may serve as a diagnostic screening tool for neurocognitive disorders (e.g., HAND⁴^,⁵^,⁶). Utilization of the variable ISI approach, therefore, may potentially have translational clinical utility for neurocognitive disorders.

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported in part by grants from NIH (National Institute on Drug Abuse, DA013137; National Institute of Child Health and Human Development HD043680; National Institute of Mental Health, MH106392; National Institute of Neurological Diseases and Stroke, NS100624) and the interdisciplinary research training program supported by the University of South Carolina Behavioral-Biomedical Interface Program. Dr. Landhing Moran is currently a Scientific Officer at the NIDA Center for Clinical Trials Network.

Materials

SR-Lab Startle Response System	San Diego Instruments
Isolation Cabinet	Industrial Acoustic Company
SR-Lab Startle Calibration System	San Diego Instruments
High-Frequency Loudspeaker	Radio Shack	model #40-1278B
Sound Level Meter	Bruel & Kjaer	model #2203
Perspex Cylinder	San Diego Instruments		Included with the SR-Lab Startle Response System
SR-Lab Startle Response System Software	San Diego Instruments		Included with the SR-Lab Startle Response System
Light Meter	Sper Scientific, Ltd.	model #840006
Airline Regulator	Craftsman	model #16023
SPSS Statistics 24	IBM		Used for Statistical Analyses (Optional)

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McLaurin, K. A., Moran, L. M., Li, H., Booze, R. M., Mactutus, C. F. The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents. J. Vis. Exp. (146), e58659, doi:10.3791/58659 (2019).