Juvenile stress potentiates aversive 22-kHz ultrasonic vocalizations and freezing during auditory fear conditioning in adult male rats

Figures & data

Figure 1.  (A) Spectrogram of an exemplary call with the three points where peak amplitudes and frequencies were measured. These parameters were also derived from the average spectrum of the entire call (not depicted). (B) Spectrogram of two successive bouts of calls. Shown are examples for calls starting a bout (Call 1), calls within a bout (Calls 2–4), bouts (Bout 1 and Bout 2) and an inter-bout interval (IBI).

Figure 2.  Time spent freezing (s/min) during fear conditioning and testing. (A) Freezing during 1–3 min (pre-shock period; left) and 4–11 min (tone+shock presentation; right) during fear conditioning (day 2). (B) Freezing during 1–3 min (context period; left) and 4–11 min (tone presentation+context phase; right) during testing (day 3). n = 21 control rats, 23 juvenile stress rats; **p < 0.01 compared with respective control group.

Figure 3.  USVs on the day of fear conditioning (day 2). (A) Total number of vocalizations. (B) Number of USVs emitted over time (per 20 s time bin) during the course of fear conditioning. (C) Total time spent vocalizing. (D) Time spent vocalizing during each 20-s time bin over the course of fear conditioning. Grey boxes denote occurrences of CS/US presentation. USVs, ultrasonic vocalizations; n = 21 control rats, 23 juvenile stress rats; *p < 0.05, **p < 0.01 compared with control group; statistical differences are not shown in (B) and (D) for the sake of clarity.

Table I.  Characteristics of USVs during fear conditioning.

	Control	Juvenile stress	p-value
Latency to first call (s)	418.3 ± 20.3	336.3 ± 14.6	0.002**
Call length (s)	1.26 ± 0.08	1.25 ± 0.07	0.908
Peak frequency (kHz)
Average spectrum	23.3 ± 0.2	23.0 ± 0.2	0.387
Start point	26.1 ± 0.8	24.8 ± 0.4	0.158
Centre	23.4 ± 0.2	23.0 ± 0.2	0.213
End point	23.0 ± 0.2	22.8 ± 0.2	0.403
Peak amplitude (dB)
Average spectrum	68.1 ± 1.5	69.9 ± 0.9	0.268
Start point	39.6 ± 1.0	41.1 ± 0.7	0.226
Centre	70.4 ± 1.2	71.7 ± 0.7	0.348
End point	34.9 ± 0.9	35.6 ± 0.7	0.499
Calls starting a bout
Number	44.6 ± 8.0	62.6 ± 6.1	0.075
Call length (s)	1.5 ± 0.1	1.4 ± 0.1	0.618
Peak frequency   (kHz; start)	27.8 ± 0.8	26.0 ± 0.4	0.035*
Calls within a bout
Number	34.2 ± 6.1	72.0 ± 12.7	0.015*
Call length (s)	1.0 ± 0.05	1.0 ± 0.05	0.897
Peak frequency   (kHz; start)	24.3 ± 0.8	23.5 ± 0.2	0.270

Note: USV parameters of vocalizing rats. n = 19 control, 22 Juvenile stress; data are given as means ± SEM; p-values are results of t-tests; *p < 0.05, **p < 0.01.

Table II.  Characteristics of USVs during fear conditioning according to their occurrence during CS/US presentation versus inter-stimulus interval phases.

	Control	Juvenile stress	p-value
Calls during CS/US
Number of calls (/min)	11.3 ± 1.5	18.7 ± 2.0	0.007**
Call length (s)	1.46 ± 0.10	1.32 ± 0.07	0.258
Peak frequency (kHz)
Average spectrum	23.0 ± 0.2	23.0 ± 0.1	0.991
Start point	25.6 ± 0.6	25.1 ± 0.5	0.519
Centre	23.1 ± 0.3	23.0 ± 0.2	0.715
End point	22.7 ± 0.3	22.5 ± 0.2	0.634
Peak amplitude (dB)
Average spectrum	66.7 ± 1.7	68.2 ± 1.1	0.463
Start point	39.6 ± 1.1	40.8 ± 0.8	0.345
Centre	68.9 ± 1.6	70.2 ± 0.9	0.477
End point	35.6 ± 0.9	35.5 ± 0.7	0.938
Calls during ISI
Number of calls (/min)	6.7 ± 0.9	11.7 ± 1.6	0.014*
Call length (s)	1.25 ± 0.07	1.24 ± 0.08	0.970
Peak frequency (kHz)
Average spectrum	23.3 ± 0.2	23.1 ± 0.2	0.368
Start point	26.0 ± 0.8	24.8 ± 0.4	0.161
Centre	23.4 ± 0.2	23.0 ± 0.2	0.200
End point	23.1 ± 0.2	22.9 ± 0.2	0.433
Peak amplitude (dB)
Average spectrum	68.4 ± 1.5	70.3 ± 0.8	0.274
Start point	39.8 ± 1.0	41.1 ± 0.7	0.263
Centre	70.7 ± 1.2	71.9 ± 0.7	0.355
End point	34.9 ± 0.9	35.7 ± 0.7	0.501

Note: USV parameters of vocalizing rats. n = 19 control, 22 Juvenile stress; CS/US, conditioned/unconditioned stimulus; ISI, inter-stimulus interval; data are given as means ± SEM; p-values are results of t-tests; *p < 0.05, **p < 0.01.

Figure 4.  Scatter plots showing individual relationships between USVs (total number of calls) and freezing (s) during 4–11 min on the day of fear conditioning (day 2). Regression lines: dotted line, control group; solid line, juvenile stress group. n = 21 control rats, 23 juvenile stress rats; r = correlation coefficient.