Male rats emit aversive 44-kHz ultrasonic vocalizations during prolonged Pavlovian fear conditioning
- Behavior and Metabolism Research Laboratory, Mossakowski Medical Research Institute, Polish Academy of Sciences, Poland
- Bioinformatics Laboratory, Mossakowski Medical Research Institute, Polish Academy of Sciences, Poland
- Department of Renal and Body Fluid Physiology, Mossakowski Medical Research Institute, Polish Academy of Sciences, Poland
Figures
Figure 1 with 4 supplements
Characteristics of vocalizations emitted by Wistar rats during fear conditioning training with 10 aversive foot-shocks (Table 1/Exp. 1–3/#2,4,8,13; n = 46).
(A) Some rats produced aversive 22-kHz vocalizations with typical features, that is, constant-frequency of <32 kHz, >300 ms duration – both values marked as dotted lines); example emission from one rat. (B) Some rats produced 44-kHz vocalizations with constant frequency of >32 kHz and long duration (>150 ms); example emission from one rat. (C) Rats that emitted aversive vocalizations during fear session produced 50-kHz vocalizations during appetitive playback session the following day (full data published in Olszyński et al., 2021); representative data from same rat in (A). (D) The onset of long 22-kHz alarm calls typically occurred after first shock stimulus (vertical dotted lines mark time of shock deliveries in D, E); note the gradual rise in peak frequency, not exceeding 32-kHz (horizontal dotted line in D, E); data from the same rat as (A, C). (E) In rats that emitted 44-kHz calls, the onset was usually delayed to after several foot-shocks; note the gradual rise in peak frequency of both long 22-kHz and 44-kHz vocalizations throughout training (comp. Figure 1—figure supplement 2C and D); data from same rat in (B). (F) Call rate of long 22-kHz calls was higher than 44-kHz calls (*p<0.05, **p<0.01, ***p<0.001) and with different time course – maximum number of 22-kHz calls at inter-trial interval (ITI)-3 (higher than ITI-1, 2, 5–10; <0.0001–0.0005 p levels); and higher number of 44-kHz calls at ITI-5–10, that is, 6.6 ± 2.3 vs. ITI-1–4, that is, 0.4 ± 0.2; p<0.0001; all Wilcoxon; numbers of ITI correspond to the numbers of previous foot-shocks, values are means ± SEM. (G) Long 44-kHz vocalizations had a higher incidence rate (15.5%) than short 22-kHz (8.8%) and 50-kHz calls (5.6%); values are calculated for the sum of all vocalizations obtained during the entire training sessions (there were fewer 50-kHz calls, i.e., 3.7%, when vocalizations prior to the first shock were not included). (A–E) Dots reflect specified single rat values. (F, G) n = 46, other results from these rats are previously published (Olszyński et al., 2021; Olszyński et al., 2023).
Figure 1—figure supplement 1
Variations of call frequency shown in relation to call duration in Wistar rats that had undergone 6 or 10 trials of delay fear conditioning training (n = 16, selected from Table 1/Exp. 2–3/#7,8,13).
Vocalizations plotted in relation to peak frequency (x-axis) and duration (y-axis). Each point corresponds to one vocalization. Vertical dotted line marks threshold value (32-kHz) between 22-kHz and 50-kHz calls. Horizontal dotted line marks threshold value (300 ms) between short and long 22-kHz calls (Brudzynski et al., 1993). Rat identifier is given in the lower right corner; the number after dash indicates the number of conditioning trials. (A) Examples from four rats that emitted typical long 22-kHz calls (no 44-kHz calls). (B) Four typical long 22-kHz vocalizations with few long 22-kHz calls crossing the 32-kHz threshold. (C) Eight sample rats that emitted typical long 22-kHz vocalizations and atypical high-frequency aversive calls forming a separate 44-kHz group.
Figure 1—figure supplement 2
Changes in distribution (A, B), frequency (C), duration (D), and mean power (E, F) of long aversive vocalizations throughout fear conditioning training session.
Data were acquired from all Wistar rats subjected to a 10-trial fear conditioning training procedure (Table 1/Exp. 1–3/#2,4,8,13; n = 46). X-axes represent subsequent inter-trial intervals (ITI) numbered after the preceding conditioned stimulus. (A, B) Number or percentage of rats emitting long vocalizations. Bubbles represent long 22-kHz calls (white) or 44-kHz calls (red); bubble size scales with the amount of vocalizations. Emission of 44-kHz calls and the number of animals emitting them increases in the latter half of the session. Data are absolute values (A) or percentages (B); 'mean’, average value from all ITI, ‘max’, maximum values from each rat. (C) Frequencies of 22-kHz and 44-kHz vocalizations. Horizontal dotted line marks the threshold value (32 kHz) between 22-kHz and 50-kHz/44-kHz calls. Peak frequency of long vocalizations rose gradually in all rats. (D) Duration of 22-kHz and 44-kHz vocalizations. The duration of 22-kHz calls gradually declined. The duration of 44-kHz calls peaked after the fourth ITI. (E) Mean power of 22-kHz and 44-kHz vocalizations. Mean power spectral density (loudness, amplitude) of 22-kHz calls, n = 14–17 per ITI; and 44-kHz calls, n = 5–17 per ITI. (F) Results for 44-kHz vocalizations (from E) were adjusted for angular attenuation, that is, +10 dB. Before the adjustment: during the first half of the session, 22-kHz calls appeared louder than 44-kHz calls, in the second half of the session the difference dissipated. After the adjustment: both types of calls started on a comparable amplitude level, but in the 6th–10th ITI, 22-kHz calls became quieter than 44-kHz calls. Values are means ± SEM (C–F). Graphs show either all rats (A–D, n = 46) or rats that met the criteria of emitting >20 of 44-kHz calls (E, F, n = 17 selected from n = 46); *p<0.05, **p<0.01, ***p<0.001.
Figure 1—figure supplement 3
Percentage of animals emitting 44-kHz calls (A, B) and percentage of 44-kHz calls in all vocalizations (C, D) emitted by Wistar rats and spontaneously hypertensive rats (SHR).
Results from three main fear conditioning experiments are shown (comp. Table 1), that is, Exp. 1 (light gray bars), Exp. 2 (dark gray bars), and Exp. 3 (black bars), which all were performed with Wistar rats or SHR (when specified in the x-axis labels). The labels denote different experimental groups used across the experiments (see Table 1 for the number of animals in each group). Results were obtained during fear conditioning training (A, C) and testing sessions (B, D). Rats subjected to trace fear conditioning were tested in safe and unsafe contexts, while in delay fear conditioning, the rats were tested only in an unsafe context (see ‘Materials and methods’). 44-kHz calls appeared most often in Wistar rats that had undergone 10-trial fear conditioning procedures. Please note that the experiments were not performed in parallel.
Figure 1—video 1
Rat transitioning from emitting long 22-kHz calls to emitting 44-kHz calls.
Included below the video of the fear conditioning training session is a synchronized recording of a spectrogram generated using SASlab Pro (version 5.2.xx, Avisoft Bioacoustics, Germany). Audio was added post-recording after synchronizing the video and audio files and converting the frequency of the sounds to audible range using ‘change pitch’ function in Audacity 2.4.2 (Muse Group, Cyprus).
Figure 2 with 1 supplement
Five subtypes (B–F) of high-frequency 44-kHz aversive vocalizations.
(A) Standard aversive 22-kHz vocalization with peak frequency <32 kHz (peak frequency = 24.4 kHz). Five 44-kHz aversive vocalization subtypes: (B) flat (constant frequency call; peak frequency = 42.4 kHz); (C) step up (peak frequency = 39.5 kHz); (D) step down (peak frequency = 52.2 kHz); (E) insert (peak frequency = 38.5 kHz); and (F) complex (peak frequency = 46.3 kHz). (G) Percentage share of 44-kHz call subtypes in all cases of detected 44-kHz vocalizations.
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Figure 2—source data 1
Example of a step up 44-kHz ultrasonic vocalization.
Ultrasonic vocalization emitted during fear conditioning training session. Sound transformed to audible frequency using ‘change pitch’ function in Audacity 2.4.2 (Muse Group, Cyprus).
- https://cdn.elifesciences.org/articles/88810/elife-88810-fig2-data1-v1.zip
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Figure 2—source data 2
Example of a step down 44-kHz ultrasonic vocalization.
Ultrasonic vocalization emitted during fear conditioning training session. Sound transformed to audible frequency using ‘change pitch’ function in Audacity 2.4.2 (Muse Group, Cyprus).
- https://cdn.elifesciences.org/articles/88810/elife-88810-fig2-data2-v1.zip
Figure 2—figure supplement 1
Non-typical 44-kHz aversive vocalizations.
(A, B) Constant frequency calls with very high peak frequency (A, peak frequency = 62.9 kHz; B, peak frequency = 65.9 kHz, start peak frequency = 78.1 kHz). (C, D) Harmonic aversive vocalizations, where element with fundamental frequency (F0, lowest frequency of the vocalization) is not with maximum amplitude, that is, peak frequency is determined from the higher call component (C, F0 = 27.8 kHz, peak frequency = 55.6 kHz; D, F0 = 40 kHz, peak frequency = 81.5 kHz). (E, F) Vocalizations with prominent duration but with modulated frequency (E, peak frequency = 69.3 kHz; F, peak frequency = 39.0 kHz). (A, G) Constant frequency calls from SHR (G, flat 44-kHz call, peak frequency = 42.4 kHz).
Figure 3 with 2 supplements
Clustering of ultrasonic vocalizations from fear conditioning training sessions using two independent methods.
(A) DBSCAN algorithm (ε = 0.14) clustering of vocalizations from all fear conditioning experiments (Table 1/Exp. 1–3/#1–13, n = 218), silhouette coefficient = 0.198, two clusters emerge, cluster of green dots n = 77,243 (due to high generality of cluster average peak frequency and duration deemed redundant), cluster of red dots n = 5,646 (average peak frequency = 43,826.6 Hz, average duration = 0.524 s), some calls were not assigned to any cluster, that is, outlier vocalizations, black dots, n = 4,139. (B, C) Clustering by k-means algorithm and visualization of calls emitted by selected rats, that is, with >30 of 44-kHz vocalizations, during trace and delay fear conditioning training (n = 26, selected from Table 1/Exp. 1–3/#2,4,7,8,11–13), total number of calls n = 40,084. (B) Topological plot of ultrasonic calls using UMAP embedding, particular agglomerations of calls labeled with their type or subtype. (C) Spectrogram images from DeepSqueak software superposed over plot B, colors denote clusters from unsupervised clustering, number of clusters set using elbow optimization (max number = 4), two clusters emerge; see also Figure 3—figure supplement 1.
Figure 3—figure supplement 1
Clustering of ultrasonic vocalizations from rats emitting 44-kHz calls using UMAP and k-means.
(A) Topological plot of ultrasonic calls using UMAP embedding from selected rats emitting 44-kHz vocalizations during trace and delay fear conditioning training (n = 26, selected from Table 1/Exp. 1–3/#2,4,7,8,11–13), total number of calls n = 40,084, with spectrogram miniatures pointing to the general location from which they originated. (B) Comparison of unsupervised k-means clustering with different maximum possible number of clusters using elbow optimization (different clusters denoted by colors) done by DeepSqueak software, superposed over UMAP topological plot, number on the bottom left of the miniature denotes the maximum possible number of clusters set for elbow optimization, and number on the bottom right denotes the resulting number of clusters after elbow optimization.
Figure 3—figure supplement 2
Examples of parts of ultrasonic bouts comprised of long 22-kHz vocalizations (A) and 44-kHz vocalizations (B) emitted by Wistar rats during fear conditioning training.
The average frequencies of these particular calls are 23.4 kHz (A) and 44.5 kHz (B); each vertical scale is 125 kHz.
Figure 4 with 1 supplement
Physiological and behavioral response to playback of 44-kHz calls (vs. 50-kHz and 22-kHz calls) presented from a speaker to naïve Wistar rats.
(A) Heart rate (HR). (B) The number of emitted vocalizations. (A, B) Gray sections correspond to the 10-s-long ultrasonic playback. Each point is a mean for a 10-s-long time-interval with SEM. (C, D) Properties of 50-kHz vocalizations emitted in response to ultrasonic playback, that is, number of calls (C) and duration (D) calculated from the 0-120 s range. (A) 50-kHz playback resulted in HR increase (playback time interval vs. 10–30 s time interval, p=0.0007), while the presentation of the aversive playbacks resulted in HR decrease, both in case of 22-kHz (p<0.0001) and 44-kHz (p=0.0014, average from –30 to –10 time intervals [i.e., ‘before’] vs. playback interval, all Wilcoxon), which resulted in different HR values following different playbacks, especially at +10 s (p=0.0097 for 50-kHz vs. 22-kHz playback; p=0.0275 for 50-kHz vs. 44-kHz playback) and +20 s time intervals (p=0.0068, p=0.0097, respectively, all Mann–Whitney). (B) 50-kHz playback resulted also in a rise of evoked vocalizations (before vs. 10–30 s time interval, p=0.0002, Wilcoxon) as was the case with 44-kHz playback (p=0.0176 in respective comparison), while no rise was observed following 22-kHz playback (p=0.1777). However, since the increase in vocalization was robust in case of 50-kHz playback, the number of emitted vocalizations was higher than after 22-kHz playback (e.g., p<0.0001 during 0–30 time intervals) as well as after 44-kHz playback (e.g., p<0.0001 during 0–10 time intervals, both Mann–Whitney). Finally, when the increases in the number of emitted ultrasonic calls in comparison with before intervals were analyzed, there was a difference following 44-kHz vs. 22-kHz playbacks during 30 s and 40 s time intervals (p=0.0420 and 0.0430, respectively, Wilcoxon). (C) During the 2 min following the onset of the playbacks, rats emitted more ultrasonic calls during and after 50-kHz playback in comparison with 22-kHz (p<0.0001) and 44-kHz (p=0.0011) playbacks. The difference between the effects of 22-kHz and 44-kHz playbacks was not significant (p=0.2725, comp. Figure 4—figure supplement 1F; all Mann–Whitney). (D) Ultrasonic 50-kHz calls emitted in response to playback differed in their duration, that is, they were longer to 50-kHz (p=0.0004) and 44-kHz (p=0.0273, both Mann–Whitney) playbacks than to 22-kHz playback. * 50-kHz vs. 44-kHz, $ 50-kHz vs. 22-kHz, # 22-kHz vs. 44-kHz; one character (*, $, or #), p<0.05; two, p<0.01; three, p<0.001; Mann–Whitney (A, B) or Wilcoxon (C, D). Values are means ± SEM, n = 13–16.
Figure 4—figure supplement 1
Behavioral response to playback of 44-kHz calls (vs. 50-kHz and 22-kHz calls).
(A, B) Rats with implanted heart rate transmitters (comp. Figure 4), Wistar, n = 13–16. (C–G) Rats without transmitters, Sprague–Dawley, n = 15. (A, C) Distance traveled. (B, D) Time spent in the speaker’s half of the cage; the dotted horizontal line marks a 50% chance value for time in a side of the cage. (E) Number of emitted vocalizations. (A–E) Gray sections correspond to the 10-s-long ultrasonic presentation, each point is a mean for a 10-s-long time-interval with SEM. (F, G) Properties of 50-kHz vocalizations emitted in response to ultrasonic playback, that is, number of calls (F) and duration (G) in 0–120 s range. (A–D) Playback presentation resulted in increased motor activity in case of, especially, 50-kHz playback and 44-kHz playback. Also, all kinds of playback resulted in increased time spent in the half of the cage next to the speaker. (E) 50-kHz playback resulted in a rise of the number of evoked vocalizations (average from –30 to –10 time intervals aka before vs. 10–30 s time interval, p=0.0010) as was the case with 44-kHz playback (p=0.0142), respectively, while no rise was observed following 22-kHz playback (p=0.2271, all Wilcoxon). However, since the increase in vocalization was robust in case of 50-kHz playback, the number of emitted vocalizations was higher than both after 22-kHz playback (e.g., p<0.01 during 0–20 time intervals) and after 44-kHz playback (p=0.0172, 0 s time interval, all Mann–Whitney). Finally, when the increases in the number of emitted ultrasonic calls in comparison with before intervals were analyzed, there was a difference following 44-kHz vs. 22-kHz playbacks during the 40 s time interval (p=0.0017, Wilcoxon, comp. Figure 4B). (F) During the 2 min following the onset of the playbacks, the rats emitted more ultrasonic calls during and after 50-kHz playback in comparison with 22-kHz (p=0.0002) and 44-kHz (p=0.0067) playbacks; also, the rats emitted more ultrasonic calls during and after 44-kHz playback in comparison with 22-kHz playback (p=0.0369), comp. Figure 4C; all Wilcoxon. (G) Ultrasonic 50-kHz calls emitted in response differed also in their duration, that is, they were shorter to 22-kHz (p=0.0195) and 44-kHz (p=0.0039) playbacks than to 50-kHz playback. The difference between the effects of 22-kHz and 44-kHz playbacks was not significant (p=0.5469) comp. Figure 4D; all Wilcoxon. * 50-kHz vs. 44-kHz, $ 50-kHz vs. 22-kHz, # 22-kHz vs. 44-kHz; one character (*, $, or #), p<0.05; two, p<0.01; three, p<0.001; Mann–Whitney (A, B) or Wilcoxon (C, D). Values are means ± SEM.
Tables
Table 1
All fear conditioning (FC) experiments described in the text.
| # | Exp. | FC type | Strain | # of trials (shocks) | # of rats (n) | Housing | Transmitters | Age in weeks | Description/history |
|---|---|---|---|---|---|---|---|---|---|
| 1 | 1 | Trace | Wistar | 0* | 7 | Single | + | 13 | Rats that had undergone FC after playback experiments (published in Olszyński et al., 2020) |
| 2 | 10 | 7 | |||||||
| 3 | 0* | 10 | Paired | ||||||
| 4 | 10 | 10 | |||||||
| 5 | 2 | Delay | Wistar | 0* | 37 | Paired | + | 12 | FC experiments which were described before in detail (Olszyński et al., 2021; Olszyński et al., 2023) |
| 6 | 1 | 16 | |||||||
| 7 | 6 | 22 | |||||||
| 8 | 10 | 19 | |||||||
| 9 | SHR | 0* | 31 | ||||||
| 10 | 1 | 17 | |||||||
| 11 | 6 | 17 | |||||||
| 12 | 10 | 15 | |||||||
| 13 | 3 | Delay | Wistar | 10 | 10 | Paired | - | 12 | New FC experiment |
-
*
Control groups.
Table 2
Freezing associated with emission of long, monotonous vocalizations.
All Wistar rats that had undergone 10 trials of fear conditioning training were analyzed (Table 1/Exp. 1–3/#2,4,8,13; n = 46). (A) Freezing (%) in 10-s-long bins where rats emitted exclusively long 22-kHz vocalizations vs. exclusively 44-kHz vocalizations. Results were compared to baseline freezing levels before conditioning training (‘First 5 min’) and during 10-s-long periods with no vocalizations (‘no calls’). More information in the text. (B) Freezing during the emission episodes of long 22-kHz and 44-kHz calls. Pairs of 44-kHz and long 22-kHz vocalizations were randomly selected from each animal. Freezing levels (%) did not differ between 22-kHz vs. 44-kHz calls (0.2054–0.7776 p levels, Wilcoxon). Minimum freezing duration used: 30 frames (A), 3 frames (for pairs of ≥150 ms vocalizations), or 5, 10, and 15 frames for ≥500 ms vocalizations (B).
| (A) Freezing behavior during 10-s-long time intervals; analyzed with 30 frames | ||||
| Group of rats analyzed | Freezing (%) | |||
| First 5 min | 10-s-long bins with | |||
| no calls | 22-kHz calls only | 44-kHz calls only | ||
| Rats with long 22-kHz calls (n = 41) | 6.3 ± 2.3 | 39.7 ± 3.5 | 47.8 ± 3.3***,# | NA |
| Rats with long 22-kHz calls and 44-kHz calls (n = 21) | 9.1 ± 4.2 | 33.7 ± 4.9 | 40.4 ± 4.3*** | 50.3±7.0***,# |
| (B) Freezing behavior during selected calls; analyzed with reduced number of frames | ||||
| Rats with long 22-kHz calls and 44-kHz calls | No. of frames | Freezing (%) during emission of | ||
| 22-kHz calls | 44-kHz calls | |||
| with calls of ≥150 ms duration (n = 32) | 3 | 49.5 ± 7.6 | 58.8 ± 8.0 | |
| with calls of ≥500 ms duration (n = 28) | 5 | 67.3 ± 7.3 | 54.1 ± 9.1 | |
| 10 | 61.9 ± 8.1 | 53.3 ± 9.1 | ||
| 15 | 52.6 ± 9.0 | 48.4 ± 9.4 | ||
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NA, not analyzed.
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*** vs. ‘First 5 min’, p<0.001; # vs. ‘no calls’, p<0.05; both Wilcoxon.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Wistar rat, Rattus norvegicus) | Wistar rat | The Center for Experimental Medicine of the Medical University of Białystok | CMDB:WI | 7 weeks old upon arrival at our lab |
| Strain, strain background (SHR, Rattus norvegicus) | SHR | Mossakowski Medical Research Institute | SHR/NHsd/Cmd | 7 weeks old upon arrival at our lab |
| Strain, strain background (Sprague–Dawley Rat, R. norvegicus) | SD rat | Mossakowski Medical Research Institute | Tac:Cmd:SD | 7 weeks old upon arrival at our lab |
| Software, algorithm | SASLab Pro | Avisoft Bioacoustics | RRID:SCR_014438 | version 5.2.xx |
| Software, algorithm | Avisoft Recorder | Avisoft Bioacoustics | RRID:SCR_014436 | version 4.2.28 |
| Software, algorithm | Deep Squeak | https://github.com/DrCoffey/DeepSqueak; Coffey and Marx, 2022 | RRID:SCR_021524 | version 3.0.4 |
| Software, algorithm | sklearn | PMID:24600388 | RRID:SCR_002577 | |
| Other, hardware | Ultrasonic Microphone | Avisoft Bioacoustics | CM16/CMPA | |
| Other, hardware | Radiotelemetric transmitters | Data Sciences International | HD-S10 |
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Male rats emit aversive 44-kHz ultrasonic vocalizations during prolonged Pavlovian fear conditioning
eLife 12:RP88810.
https://doi.org/10.7554/eLife.88810.4
