Relationships between the acoustic startle response and prepulse inhibition in C57BL/6J mice: a large-scale meta-analytic study
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Prepulse inhibition (PPI) is the suppression of a startle reflex response to a startle stimulus that occurs when a weak prepulse stimulus precedes the startle stimulus. PPI is measured to assess sensorimotor gating across species, including humans and rodents. Reduced PPI, which is thought to reflect dysfunction of sensorimotor gating, is reported in patients with psychiatric disorders, such as schizophrenia, bipolar disorder, and post-traumatic stress disorder (PTSD), and in animal models of these disorders. Individual differences in basal startle reactivity occur even in a genetically homogenous group of animals; however, there is limited information regarding whether basal levels of the startle response are associated with variations in PPI levels. Here, to explore the relationship between an acoustic startle response (ASR) and PPI, we performed a meta-analysis of data obtained from more than 1300 C57BL/6J male mice on the influence of an ASR to 110- and 120-dB startle stimuli on the PPI levels of the ASR at 74- and 78-dB prepulse intensities. Examination of scatter plots of the ASR amplitudes and PPI levels followed by correlation analyses indicated that there is no simple linear relationship between the two measures; when mice were divided into three groups on the basis of their startle amplitudes, there were positive correlations between the amplitude of the ASR to the 110-dB stimulus and PPI levels in a group of mice that showed lower ASR amplitudes among the genetically homogenous group, whereas no significant correlations were identified in groups of mice that showed intermediate and higher ASR amplitudes. As indicated by the correlation analysis, the lowest responders to the 110-dB stimulus exhibited lower levels of PPI than the intermediate or higher responders. In contrast, for the 120-dB stimulus, a negative correlation was identified between the amplitude of the ASR to the 120-dB stimulus and the PPI levels in the groups of mice that showed intermediate or higher ASR amplitudes. Lower and intermediate responders showed higher levels of PPI than higher responders to the 120-dB stimulus. These findings suggest that basal startle reactivity may affect PPI levels in male C57BL/6J mice, thus representing one potential confounding factor that may confuse the interpretation of PPI results. These findings emphasize the importance of careful examination of startle reactivity to ensure a reliable assessment of PPI.
KeywordsAcoustic startle response Prepulse inhibition Meta-analysis Behavioral test battery Mouse phenotype database
Acoustic startle response
Prepulse inhibition (PPI) is a phenomenon in which a weak sensory stimulus suppresses a startle response caused by a sudden intense stimulus . PPI is thought to reflect sensorimotor gating, a form of central nervous system inhibition in which irrelevant sensory information is filtered out during the early stages of processing so that attention may be focused on more salient features of the environment [2, 3]. Reduced PPI of the startle response has been extensively demonstrated in patients with schizophrenia [1, 2, 4, 5, 6] and potentially in patients with other psychiatric and neurological disorders, such as obsessive compulsive disorder (OCD) , Huntington’s disease , Tourette syndrome , panic disorder , and post-traumatic stress disorder (PTSD) , which suggests sensorimotor gating deficits in these patients.
A PPI test is a widely used paradigm that enables the assessment of sensorimotor gating across species, including humans and rodents, in a similar fashion . In rats and mice, the startle reflex response is measured as a whole-body flinch that may be elicited by an acoustic or tactile (air puff) startle-eliciting stimulus similar to stimuli used in humans. The PPI of the startle response occurs when a weak prepulse stimulus is presented 30–500 ms prior to the startling stimulus. In general, 100- to 120-dB sound stimuli are used as the startling stimulus to measure the acoustic startle response (ASR), and an acoustic prepulse is presented at an intensity of 4–16 dB above a continuous background noise, e.g., 70-dB white noise, to evaluate PPI of the ASR. For the analysis of each animal, the percent PPI is calculated as the percent decrease in the startle amplitude at each prepulse intensity relative to the startle-alone trial. Animal studies have indicated that genetic and pharmacological manipulations may alter the ASR and PPI, which demonstrates that the PPI test is a useful tool for investigating the underlying mechanisms of sensory inhibitory processing in the brain, as well as translational research of neuropsychiatric disorders [6, 13].
The ASR and PPI may be affected by genetic, biological, and environmental factors, such as strain [14, 15], age [16, 17], sex [18, 19], and housing conditions [20, 21]. Various experimental factors and other unidentified factors potentially lead to individual or group differences in the startle response in animals. In these cases, the interpretation of PPI differences between groups may be complicated by the differences in the basal level of the startle response. Therefore, researchers have investigated whether baseline ASR magnitudes affect PPI levels. Ison et al.  reported that although the percent PPI of the startle reflex did not differ between CBA/J mice with high-startle baselines and low-startle baselines, there was a weak positive correlation between the basal startle response and percent PPI. Similar results were obtained in C57BL/6J mice that exhibited low or intermediate startle amplitudes in a group under testing conditions, whereas the startle reactivity was not correlated with the percent PPI in mice with high startle amplitudes . Although the previous studies, based on a small number of animals, suggest that the association of basal startle reactivity with behavioral responses reflects an inhibitory mechanism of sensory information processing, a paucity of information remains regarding the relationship between ASR and PPI.
In our laboratory, we have analyzed various types of behavior in more than 180 strains of genetically engineered mice in a battery of behavioral tests with our standardized procedure [17, 24, 25]. In the PPI test, we used several different combinations of a startle stimulus and a prepulse stimulus to identify potential phenotypes in ASR and PPI. The mice were exposed to two different intensities of startle stimulus (i.e., 110- or 120-dB stimulus) with 74-dB, 78-dB, or no prepulse stimulus in a test session. In this study, we used the behavioral data of more than 1300 wild-type C57BL/6J male mice subjected to the PPI test with data stored in our database and examined the relationships between ASR amplitudes and PPI levels using this substantial amount of data from a genetically homogeneous group of mice.
Animals and experimental design
Genetically engineered mice and their wild-type control mice were transported from the animal facilities of other laboratories or vendors to the facilities of our laboratory (for detailed information on individual mice included in the studies previously published , refer to the Mouse Phenotype Database, URL: http://www.mouse-phenotype.org). The mice were subjected to a battery of behavioral tests with our standardized protocol (for details, refer to Shoji et al. ). We used behavioral data of the startle response/PPI test that were obtained from 1363 wild-type control male mice 2–12 months old (2–3 months old, n = 757; 4–5 months old, n = 389; 6–7 months old, n = 167; 8–12 months old, n = 50). The wild-type mice were derived from a C57BL/6J strain and its substrains (6JJcl or 6JJmsSlc) maintained in Japan, and more than 90% of the mice were backcrossed at least six times (and more than 95% of the mice used were backcrossed at least five times) with C57BL/6J mice. Thus, the genetic backgrounds of the mice were regarded as “C57BL/6J”. They were housed in plastic cages with paper bedding (Paper Clean; Japan SLC, Inc., Shizuoka, Japan) under a 12-h light/dark cycle (lights on at 7:00 a.m.) with access to food (CRF-1; Oriental Yeast Co., Ltd.) and water ad libitum. Behavioral testing was performed between 9:00 a.m. and 6:00 p.m. The testing apparatuses were cleaned with super hypochlorous water and 70% ethanol to remove olfactory stimuli after each test. All behavioral testing procedures were approved by the Animal Care and Use Committee of Kyoto University Graduate School of Medicine and the National Institute for Physiological Sciences in Japan.
Startle response/PPI test
A startle reflex measurement system (O’Hara & Co., Tokyo, Japan) was used to measure the ASR to a loud noise and PPI of the startle response, as previously described . A 20-min test session was initiated by placing a mouse in a plastic cylinder in a sound-attenuating chamber, in which the mouse was left undisturbed for the first 10-min period and was subsequently subjected to startle-stimulus-only trials and PPI trials for 10 min. The last 10-min session consisted of six trial types, i.e., two types of startle-stimulus-only trials (110 or 120 dB auditory stimulus) and four types of PPI trials (74 dB prepulse + 110 dB stimulus, 74–110 dB; 78 dB prepulse + 110 dB stimulus, 78–110 dB; 74 dB prepulse + 120 dB stimulus, 74–120 dB; and 78 dB prepulse + 120 dB stimulus, 78–120 dB). White noise (40 ms) was used as the startle stimulus for all trial types. A background white noise was presented at a level of 70 dB during the test. The prepulse sound was presented for 20 ms at an intensity of 74 or 78 dB 100 ms prior to the presentation of the startle stimulus. The startle response was recorded for 400 ms starting with the onset of the startle stimulus using the startle reflex measurement system with an accelerometer mounted below the cylinder. Six blocks of the six trial types were presented to the mice in a pseudo-random order, and each trial type was presented once within a block. The average inter-trial interval was 15 s with a range of 10–20 s. The peak startle amplitude was recorded for use as a dependent variable. The percent PPI was calculated for analysis for each mouse according to the following formula: the percentage of PPI = 100 × [1 - (startle response for prepulse + startle trial)/(startle response for startle stimulus alone trial)].
Statistical analysis was conducted with statistical software (SAS University Edition; SAS Institute, Cary, NC, USA). Behavioral data were analyzed using a Spearman rank correlation test and Kruskal-Wallis test followed by post hoc comparisons with the Dwass-Steel-Critchlow-Fligner test to avoid potential statistical problems with non-normal distributions. The statistical significance level was set at p < 0.05.
Groups of mice used for correlation analyses between the ASR amplitudes and percent PPI
110-dB startle-stimulus only trial
120-dB startle-stimulus only trial
ASR < 0.26 (12.5 percentile)
ASR < 0.61 (12.5 percentile)
0.26 (12.5 percentile) ≤ ASR < 0.45 (25 percentile)
0.61 (12.5 percentile) ≤ ASR < 0.9 (25 percentile)
0.45 (25 percentile) ≤ ASR < 0.62 (37.5 percentile)
0.9 (25 percentile) ≤ ASR < 1.16 (37.5 percentile)
0.62 (37.5 percentile) ≤ ASR < 0.8 (50 percentile)
1.16 (37.5 percentile) ≤ ASR < 1.44 (50 percentile)
0.8 (50 percentile) ≤ ASR < 0.99 (62.5 percentile)
1.44 (50 percentile) ≤ ASR < 1.76 (62.5 percentile)
0.99 (62.5 percentile) ≤ ASR ≤ 1.25 (75 percentile)
1.76 (62.5 percentile) ≤ ASR ≤ 2.24 (75 percentile)
1.25 (75 percentile) < ASR ≤ 1.725 (87.5 percentile)
2.24 (75 percentile) < ASR ≤ 2.93 (87.5 percentile)
1.725 (87.5 percentile) < ASR
2.93 (87.5 percentile) < ASR
Significant differences in PPI among the eight subgroups of responders to the 120-dB stimulus were identified for each trial type (for 74–120 dB, χ2 = 26.3291, p = 0.0004; for 78–120 dB, χ2 = 97.5232, p < 0.0001) (Fig. 3b and Additional file 1). The post hoc analyses indicated that Groups 7′ and 8′, the high startle responder groups, exhibited significantly lower percentages of PPI than the intermediate responder mice in Groups 3′, 4′ or 5′ in the 74–120 dB trial (Group 8′ < Groups 3′, 4′, and 5′; Group 7′ < Group 3′, all p < 0.05). In addition, in the 78–120 dB trial, the percentages of PPI in Groups ‘7 and 8’ were significantly lower than the percentages in Groups 1′, 2′, 3′, 4′, 5′, or 6′ (Groups 8′ < Groups 1′, 2′, 3′, 4′, 5′, and 6′; Group 7′ < Groups 1′, 2′, 3′, 4′, and 5′, all p < 0.05), and the percentage of PPI in Group 6′ was lower than the percentages in Groups 2′ and 3′ (Group 6′ < Groups 2′ and 3′, all p < 0.05). In general, mice that showed a low startle response to the 110-dB stimulus exhibited the lowest levels of PPI, and mice that showed a high startle response to the 120-dB startle stimulus displayed lower levels of PPI than mice with low or intermediate ASR amplitudes.
Latency to peak startle response is a relevant variable affecting ASR amplitudes and PPI levels. To further examine the relationships between ASR latency and ASR/PPI amplitudes, we performed correlation analyses with Spearman’s method between the latencies to the peak ASR with 110- or 120-dB stimulus and the percentages of PPI using data of 721 C57BL/6J mice that are available from the Mouse Phenotype Database (Additional file 2: Figure S1). The ASR latency to 110-dB stimulus was negatively correlated with ASR amplitude at 110-dB stimulus (Additional file 2: Figure S1A; Rho = − 0.2287, p < 0.0001) and PPI levels (Additional file 2: Figure S1B,C; for 74–110 dB, Rho = − 0.0742, p = 0.0463; for 78–110 dB, Rho = − 0.0885, p = 0.0175). Similarly, the ASR latency to 120-dB stimulus showed slight but significant negative correlation with PPI levels at 74–120 dB (Additional file 2: Figure S1E; Rho = − 0.0940, p = 0.0115), although the latency was not significantly correlated with ASR amplitudes at 120-dB stimulus (Additional file 2: Figure S1D; Rho = − 0.0220, p = 0.5537) nor PPI levels at 78–120 dB (Additional file 2: Figure S1F; Rho = − 0.0196, p = 0.5983). The inspection of the scatter plots in the ASR latency and PPI levels and correlation analyses indicate that mice showing a longer latency to respond startle stimuli tend to exhibit lower ASR amplitudes and PPI levels in most conditions that were used in our experiments.
This study aimed to investigate the relationships between an ASR to loud noise and the PPI of the startle response in adult C57BL/6J male mice. We performed a large-scale meta-analysis of the association of ASR amplitudes with percentages of PPI at different startle stimuli, with a focus on individual differences in the ASR amplitude in a genetically homogeneous group of 1363 C57BL/6J mice. Our study, which included a correlation analysis of ASR amplitudes and PPI levels, demonstrated that there was no simple linear relationship between ASR amplitudes and PPI levels, as indicated by weak positive or negative correlations between the two measures in the subgroups of mice with different ASR amplitudes in response to 110-dB and 120-dB stimuli. Moreover, positive correlations between the amplitude of the ASR to the 110-dB stimulus and PPI levels were identified in the low-responder group, whereas no significant correlations were identified in the intermediate- and high-responder groups at the 110-dB stimulus. At the higher intensity acoustic stimulus or 120-dB stimulus, there were no significant correlations between the ASR amplitudes and PPI levels in the low-responder group, and a significant negative correlation was identified in the intermediate- and high-responder groups. Further analysis of group comparisons indicated that the lowest responder group (Group 1) showed the lowest levels of PPI at the 110 dB startle stimulus compared with the other responder groups (Groups 2–8), and at the 120 dB startle stimulus, the higher responder groups (Groups 7′ and 8′) exhibited lower levels of PPI than the other groups (Groups 1′-6′), as expected from the results of the correlation analysis. Collectively, these results indicate that basal startle reactivity is associated with PPI levels in C57BL/6J mice.
The preceding study by Yee et al. , in which a cohort of 102 C57BL/6J mice was subdivided into three groups on the basis of ASR amplitudes, showed that there were significant positive correlations between ASR amplitudes and PPI levels in the groups of mice that exhibited lower and intermediate ASR levels at 100-dB and 110-dB stimuli, whereas no significant correlation was identified in the group of mice with higher ASR levels. Their findings of a positive correlation between the two measures in the lower responders to the smaller acoustic startle stimulus appear to be consistent with our results. Yee et al.  also reported that at a 120-dB stimulus, there was a significant positive correlation between ASR amplitudes and PPI levels in the low-responder group, whereas a negative but non-significant correlation between the two measures was identified in the intermediate-responder groups; no correlation was identified in the high-responder group. Similar results were obtained in our study, in which small positive but non-significant correlations between the amplitude of the ASR to the 120-dB stimulus and PPI levels were identified in the lower responders, and significant negative correlations between the two measures were identified in the intermediate or higher responders to 120-dB. Overall, our meta-analytic study of a large number of C57BL/6J mice, in which the correlation patterns identified are generally similar to the patterns obtained in the previous findings by Yee et al. , strengthens the reliability of the findings regarding the relationships between basal startle reactivity and percentages of PPI in C57BL/6J mice.
Variations in PPI may be caused by interactions of multiple genetic and environmental factors, and the precise causes of the variations remain unclear. In this study, the large-scale meta-analysis of data from more than 1300 C57BL/6J male mice indicates that basal acoustic startle reactivity, which is associated with variations in PPI levels, may be a confounding factor for PPI results. Furthermore, additional correlation analyses of ASR latency and PPI revealed that mice showing a longer ASR latency tend to exhibit lower ASR amplitudes and lower PPI levels, indicating that differences in latency to ASR can also be a potential confounding factor in evaluating PPI. The analyses shown in scatter plots of basal ASR measures and PPI levels may facilitate an understanding of the relationships between the two behavioral measures in mice. Thus, our study indicates the importance of describing the details of behavioral characteristics in each group examined in this test paradigm to draw a convincing conclusion when interpreting PPI results.
We thank our many collaborators and the members of our laboratory for the animal husbandry and behavioral analysis of mice. We also thank Dr. Akito Nakao for reviewing the manuscript.
This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas “Systems Genomics” (20016013), “Pathomechanisms of Brain Disorders” (20023017), Young Scientists A (16680015), Exploratory Research (19653081), Scientific Research (B) (21300121), Integrative Brain Research (IBR-shien), and Comprehensive Brain Science Network from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), Neuroinformatics Japan Center (NIJC), and grants from CREST and BIRD of the Japan Science and Technology Agency (JST).
Availability of data and materials
All data are available from the Mouse Phenotype Database (http://www.mouse-phenotype.org).
HS performed data analysis and wrote manuscript. TM designed the study and wrote manuscript. Both authors read and approved the final manuscript.
All experimental procedures were approved by the Animal Care and Use Committee of Kyoto University Graduate School of Medicine and the National Institute for Physiological Sciences in Japan.
Consent for publication
The authors declare that they have no competing interests.
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