- Email: [email protected]
Power spectrum analysis of ultrasonic vocalization elicited by maternal separation in rat pups
BR A IN RE S EA RCH 1 2 83 ( 20 0 9 ) 5 8 –64
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Power spectrum analysis of ultrasonic vocalization elicited by maternal separation in rat pups Satoko Ise⁎, Hisashi Ohta Pharmacology, Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., 3 Okubo, Tsukuba, Ibaraki 300-2611, Japan
A R T I C LE I N FO
AB S T R A C T
Article history:
The present study investigated how frequency distribution of maternal separation-induced
Accepted 3 June 2009
ultrasonic vocalization was altered by environmental stimuli and pharmacological agents.
Available online 11 June 2009
Sprague–Dawley rat pups at 10 days were used to measure numbers and frequencies of the ultrasonic vocalizations under various ambient temperatures and with pharmacological
Keywords:
manipulations of the corticotropin-releasing factor (CRF) and GABAergic systems. The
Rat
ultrasonic vocalization consisted of two distinct peaks in the frequency range of 30 kHz to
Ultrasonic vocalization
50 kHz. The area under the curve (AUC) in the high-frequency range and the number of the
Maternal separation
ultrasonic vocalizations increased when ambient temperature was lowered. Systemic
Frequency
administration of a selective CRF1 receptor antagonist, NBI27914, and a typical anxiolytic,
Power spectrum
diazepam, decreased the AUC in the high-frequency range and the number of the ultrasonic vocalizations in a dose-dependent manner at an ambient temperature of 24 °C. The AUC in the low-frequency range did not change with an alteration in ambient temperature or treatment with NBI27914 and diazepam except a high dose (1 mg/kg) of diazepam. These results demonstrated that emotional levels of isolated pups reflected not only the number but also the frequency distribution of maternal separation-induced ultrasonic vocalizations. High-frequency components, but not low-frequency components, of the ultrasonic vocalization were sensitive to changes in negative affective state of isolated pups. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
An infant rat emits ultrasonic vocalizations when it is isolated from its dam and littermates. The number of ultrasonic vocalizations was shown to be modulated by changes in environmental conditions, such as ambient temperature, tactile stimuli, and odors normally provided by the dam (Allin and Banks, 1971; Shair et al., 1999) and was often reduced by anxiolytics and antidepressants (Olivier et al., 1998a, 1998b; Branchi et al., 2001). Our previous report demonstrated that the number of ultrasonic vocalizations reflected the level of environmental stress in the isolated rat
pup (Ise et al., 2008). The administration of corticotropinreleasing factor (CRF), which played a pivotal role in stressinduced endocrine and behavioral responses (Vale et al., 1981; Heinrichs and Koob, 2004), dose-dependently increased the number of ultrasonic vocalizations at 37 °C (low stress condition). The increases in the number of ultrasonic vocalizations elicited by CRF were blocked by a CRF1 receptor antagonist, NBI27914, but not a CRF2 receptor antagonist, K41498. Our results indicated that the CRF–CRF1 receptor system was involved in modulating the number of the ultrasonic vocalizations by rat pups isolated from their dam.
⁎ Corresponding author. E-mail address: [email protected] (S. Ise). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.06.003
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Fig. 1 – Schematic drawing of the power spectrum in maternal separation-induced ultrasonic vocalization emitted by an isolated pup for 5 min. Ordinate represented the liner scale power (Vrms); abscissa represents the frequency (kHz). The horizontal-striped area indicated the low-frequency AUC (AUCL) from 30 kHz to the frequency at the nadir. The hatched area indicated the high-frequency AUC (AUCH) from the frequency at the nadir to 50 kHz. The highest peaks in both areas were called PeakL and PeakH, respectively.
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Although most investigators indicated that the major frequency of the ultrasonic vocalization was distributed around 40 kHz, as detected by the pre-turned bat detector (Noirot, 1986; Hofer and Shair, 1978), one study reported on the frequency distribution of ultrasonic vocalization during maternal separation (Brudzynski et al., 1999). The frequency of the sound pressure peak shifted from low to high frequency with age. However, no information was available on whether changes in emotional level in isolated pup affected the frequency distribution of the ultrasonic vocalization. Although we have demonstrated that the number of ultrasonic vocalizations caused by the maternal separation was affected by external stimuli such as environmental changes as well as pharmacological manipulation, no attempt was made to examine if the frequency distribution of the ultrasonic vocalization was modulated by the environmental stimuli or pharmacological treatment. In the present study, we performed spectrum analysis in the ultrasonic vocalizations under various ambient temperature conditions of 37 °C, 24 °C, and 15 °C and under pharmacological treatment with agents that affected the CRF and GABAergic systems.
Fig. 2 – Effect of ambient temperature on the number and power spectrum variables of maternal separation-induced ultrasonic vocalizations at 37 °C, 24 °C, and 15 °C. A and B were the number and power spectrum of the ultrasonic vocalizations at three different ambient temperatures, respectively. C and D were the AUCL and AUCH, respectively. Detailed explanations of power spectrum variables were described in Fig. 1 legend. **P < 0.01 compared with the number at 37 °C. #P < 0.05, ##P < 0.01 compared with the AUCL or AUCH at 37 °C (Williams test; n = 5–6).
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2.
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Results
2.1. Effect of ambient temperature on the number and power spectrum variables of ultrasonic vocalizations elicited by maternal separation in rat pups We evaluated the number and power spectrum variables in maternal separation-induced ultrasonic vocalizations obtained from isolated pup under various ambient temperatures (37 °C, 24 °C, and 15 °C) that were considered to represent different levels of environmental stress. The detailed definitions of power spectrum variables were described in Fig. 1. Briefly, the power spectrum in the frequency range between 30 kHz and 50 kHz had two peaks. The area under the curve (AUC) was calculated based on these two peaks. The lowfrequency AUC (AUCL) was defined from 30 kHz to the nadir frequency, where the lowest power between the highest peak (PeakL) in low-frequency range and the highest peak (PeakH) in high-frequency range. The high-frequency AUC (AUCH) was defined from the nadir frequency to 50 kHz. Statistical analysis indicated that lowering the ambient temperature from 37 °C to 24 °C to 15 °C increased both the number and AUCH of ultrasonic vocalizations (number, 24 °C, P = 0.0019, 15 °C, P = 0.0005; AUCH, 24 °C, P = 0.02, 15 °C, P = 0.0001, Figs. 2A and D). In contrast, the AUCL (Fig. 2C) and frequencies of PeakL, nadir, and PeakH in the ultrasonic vocalizations (Table 1) did not differ at any ambient temperature condition.
2.2. Effect of NBI27914 on the number and power spectrum variables of ultrasonic vocalizations elicited by maternal separation in rat pups at 24 °C Administration of a CRF 1 receptor antagonist, NBI27914, significantly reduced the number and AUCH of the ultrasonic vocalizations at 24 °C in a dose-dependent manner (number, 10 mg/kg, P = 0.004, 30 mg/kg, P < 0.0001, Fig. 3A; AUCH, 30 mg/kg, P = 0.035, Fig. 3D). In contrast, the AUCL (Fig. 3C) and frequencies of PeakL, nadir, and PeakH in the ultrasonic vocalizations (Table 1) were not altered at any dose of NBI27914 at 24 °C.
2.3. Effect of diazepam on the number and power spectrum variables of ultrasonic vocalizations elicited by maternal separation in rat pups at 24 °C Administration of diazepam significantly reduced the number, AUCL, and AUCH of the ultrasonic vocalizations at 24 °C in a dose-dependent manner (number, 0.3 mg/kg, P = 0.0002, 1 mg/kg, P < 0.0001, Fig. 4A; AUCL, 1 mg/kg, P = 0.0109, Fig. 4C; AUCH, 1 mg/kg, P = 0.0046, Fig. 4D). The frequencies of PeakL, nadir, and PeakH in the ultrasonic vocalizations shifted to low frequency at 1 mg/kg of diazepam (Table 1). Both doses of diazepam did not affect behaviors of an isolated pup during the measurement.
3.
Discussion
The present study addressed if frequency distribution as well as the number of maternal separation-induced ultrasonic vocalizations changed under various stressful conditions induced by ambient temperatures and examined if such frequency distribution was modified by pharmacological treatments. In order to study the frequency distribution in the ultrasonic vocalization, we used various environmental ambient temperatures (37 °C, 24 °C and 15 °C) as environmental stimuli. The present results revealed that the frequency distribution of ultrasonic vocalization consisted of two distinct peaks between 30 kHz and 50 kHz. The height of PeakH, around 40 kHz, became higher, the AUCH increased, and the frequency of the highest peak shifted from PeakL to PeakH when the ambient temperature was decreased from 37 °C to 24 °C and 15 °C. It synchronized to the increases of the number of the ultrasonic vocalizations. These findings suggested that the AUCH, as well as the number of the ultrasonic vocalizations, were more sensitive to the level of emotional state in isolated pup than AUCL. Until now, no report has addressed if the alteration of environmental stimuli affects the frequency distribution of the ultrasonic vocalization elicited by maternal separation. The present study was the first report to examine the relationship between the
Table 1 – Power spectrum variables during maternal separation-induced ultrasonic vocalizations under various stressrelated conditions. Treatment
n
PeakL frequency (kHz)
Ambient temperatures 37 °C 5 24 °C 6 15 °C 5 NBI27914 at 24 °C Control 12 3 mg/kg 5 10 mg/kg 6 30 mg/kg 11 Diazepam at 24 °C Control 12 0.1 mg/kg 8 0.3 mg/kg 9 1 mg/kg 7 a
Nadir frequency (kHz)
PeakH frequency (kHz)
AUCL (V·Hz)
AUCH (V·Hz)
37.3 ± 1.0 37.4 ± 0.3 37.6 ± 0.3
39.9 ± 0.9 39.0 ± 0.4 39.2 ± 0.3
40.7 ± 0.9 41.5 ± 0.4 42.3 ± 0.4
5.2 ± 1.5 7.0 ± 1.7 4.6 ± 1.1
1.9 ± 0.7 8.7 ± 2.1 a 18.4 ± 2.3 a
36.3 ± 0.3 35.8 ± 0.4 36.0 ± 0.3 36.1 ± 0.2
38.7 ± 0.3 37.9 ± 0.4 37.9 ± 0.3 38.7 ± 0.2
40.9 ± 0.3 40.5 ± 0.2 39.9 ± 0.6 40.1 ± 0.3
10.4 ± 1.6 9.6 ± 2.6 7.9 ± 2.3 8.8 ± 1.3
8.7 ± 3.0 6.6 ± 1.0 4.2 ± 0.8 2.9 ± 1.3 a
36.5 ± 0.3 36.4 ± 0.3 35.6 ± 0.4 34.0 ± 0.7 a
39.0 ± 0.3 38.9 ± 0.4 38.3 ± 0.3 36.7 ± 1.0 a
42.1 ± 0.5 41.5 ± 0.4 40.7 ± 0.4 38.3 ± 1.4 a
P < 0.05 compared with the corresponding 37 °C-group or control group. (Williams' test, n = 5–21 per group).
8.4 ± 2.0 9.8 ± 1.2 6.9 ± 1.3 2.1 ± 0.8 a
9.3 ± 1.7 6.2 ± 2.0 7.2 ± 2.1 1.1 ± 0.2 a
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Fig. 3 – Effects of a CRF1 receptor antagonist, NBI27914, on the number and power spectrum variables of maternal separation-induced ultrasonic vocalizations at 24 °C. NBI27914 (3, 10, and 30 mg/kg, i.p.) was administered 30 min before the test. A and B were the number and power spectrum of ultrasonic vocalizations at 24 °C, respectively. C and D represented the AUCL and AUCH, respectively. Detailed explanations of power spectrum variables were described in Fig. 1 legend. **P < 0.01 compared with the number in the control group. #P < 0.05 compared with the AUCL or AUCH in the control group (Williams test; n = 5–12).
magnitude of external stimuli and distribution of frequency in the maternal separation-induced ultrasonic vocalization. The number of ultrasonic vocalizations was pharmacologically inhibited by not only CRF-related compounds but also anxiolytics and antidepressants (Kehne et al., 2000; Iijima and Chaki, 2005; Olivier et al., 1998a, 1998b). In a previous report, we demonstrated that the endogenous CRF–CRF1 receptor system was involved in the generation of the ultrasonic vocalization elicited by maternal separation under environmental stress conditions, but not the CRF–CRF2 receptor system (Ise et al., 2008). Contribution of the CRF–CRF1 receptor system on the ultrasonic vocalization was clear at 24 °C. In this study, we assessed the effects of a CRF1 receptor antagonist, NBI27914, and a typical anxyolytic drug, diazepam, on the frequency distribution as well as the number of the ultrasonic vocalizations at 24 °C. The number and AUCH of the ultrasonic vocalizations were decreased dose-dependently by administration of both drugs at 24 °C. The frequency distribution after
administration of both drugs at the highest dose became similar to that at 37 °C. Namely, AUCH in the ultrasonic vocalization was less prominent at 37 °C as well as with treatment with NBI27914 and diazepam. These data supported that the highfrequency component was sensitive to environmental stimuli modified by ambient temperatures and, in addition to the number of the ultrasonic vocalizations, might be a useful variable for evaluating anxiety- and stress-related agents. In contrast, AUCL did not change in any case except with a high dose of diazepam (1 mg/kg). AUCL would be an essential component for the ultrasonic vocalization. Although the AUCL decreased and the PeakL shifted to a low-frequency side by diazepam at a dose of 1 mg/kg, these effects might be due to the low number of the ultrasonic vocalizations at a dose of 1 mg/kg diazepam. Alternatively, it might be hypothesized that diazepam, which activates the GABAergic system, had a distinct effect on the frequency distribution of the ultrasonic vocalizations, unlike activating the CRF–CRF1 receptor mechanisms.
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Fig. 4 – Effects of an anxiolytic agent, diazepam, on the number and power spectrum variables of maternal separation-induced ultrasonic vocalizations at 24 °C. Diazepam (0.1, 0.3, and 1 mg/kg, s.c.) was administered 30 min before the test. A and B were the number and power spectrum of ultrasonic vocalizations at 24 °C, respectively. C and D represented the AUCL and AUCH, respectively. Detailed explanations of power spectrum variables were described in Fig. 1 legend. **P < 0.01 compared with the number in the control group. #P < 0.05, ##P < 0.01 compared with the AUCL or AUCH in the control group (Williams test; n = 7–12).
Although distribution of CRF1 receptor and benzodiazepine receptor in brain are broad (Rybnikova et al., 2003; FernandezLopez et al., 1997), the CRF–CRF1 receptor and GABAergic system might modulate vocal output at a different level of the brain. Since a CRF1 receptor antagonist significantly attenuated AUCH and the number of ultrasonic vocalizations without affecting AUCL which would be an essential component for the output of ultrasonic vocalization, the CRF–CRF1 receptor system might regulate a receptive system at the level of higher brain such as amygdala where one of the key areas to respond to aversive input is. Meanwhile benzodiazepine attenuated all AUCH, AUCL and the number of ultrasonic vocalizations, suggesting that benzodiazepine might regulate an efferent system of the vocalization at the level of periaqueductal grey on brain stem motor neuron as well as higher brain areas. Changes in frequency distribution caused by centrally acting agents might reflect mechanisms of action for each agent. Further studies will be necessary to clarify if various agents that affect the number of ultrasonic vocalizations alter the frequency distribution in different ways.
In conclusion, our results demonstrated that the emotional state of an isolated pup was reflected not only in the number but also in the frequency distribution of maternal separationinduced ultrasonic vocalizations. In particular, we revealed that the high-frequency component was sensitive to stress stimuli based on responses to lowering the ambient temperature, as well as activation of the CRF–CRF1 receptor and GABAergic systems.
4.
Experimental procedures
4.1.
Animals
Male and female 10-day-old Sprague–Dawley rat pups (Charles River Inc., Yokohama, Japan) were used. Newborn rats were culled to maintain less than 12 pups per dam on the day after birth (postnatal day 1). No sex differences in the number of vocalizations were observed in 10-day-old rat pups at various ambient temperatures in our preliminary study. The 10-day-old rat pups were used because the number of the ultrasonic
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vocalizations reached a peak at postnatal day 10 during 3 weeks of postnatal periods in our preliminary study. Pups were housed with the dam in their home cage in a controlled animal room (room temperature: 23.0 ± 2.0 °C, humidity: 55.0 ± 15.0%) on a 12 h light–dark cycle (lights on at 07:00). All animal procedures were approved by the Banyu Institutional Animal Care and Use Committee.
4.2. Measurement of maternal separation-induced ultrasonic vocalizations On the day of the study, the rat pups and their dam were moved to the experimental room from the animal housing room and left undisturbed for at least 1 h before initiation of the study. In order to minimize maternal and temporal effects, pups from one mother were randomly assigned to treatments. That is, for testing an effect of ambient temperatures, pups from two mothers were used and litters from each mother were randomly divided into three groups corresponding to three different ambient temperatures, another one was the same. Ambient temperature was set at 37 °C, 24 °C and then 15 °C to expose pups. We avoided reintroducing pups exposed to a lower temperature at first into the nest because pups exposed to lower ambient temperature might maintain more physical and psychological abnormalities than pups exposed to 37 °C when returned to the nest. In the case of pharmacological treatments, pups from three mothers were used for each drug. To minimize order effects and intra-mother's effects, both vehicle and one drug treated pups were at least assigned from each mother and the order of administration of vehicle and drugs were psudo-randomly conducted. After habituation, each pup received an intraperitoneal (i.p.) or subcutaneous (s.c.) injection of the vehicle and test drugs, and then was returned to the dam and littermates in the case of pharmacological manipulation. Thirty minutes after the administration, each pup was placed in a stainless-steel chamber (10.5 cm diameter × 16 cm height) on a Cool Plate® (NCP-2215; Nisshin Rika, Tokyo, Japan), which maintained the chamber at 37 °C or 24 °C or 15 °C in a soundproof room (AT-81; RION, Tokyo, Japan). Ultrasonic vocalizations were measured for 5 min. Individual calls made by each pup during this period were detected using an ultrasound-sensitive microphone (Inazu keisoku, Saitama, Japan) and amplified by a dedicated preamplifier. Analog signals were converted to digital signals using an A/D converter (CH-3150; Exacq Technologies, Indiana, USA) and stored on a personal computer (PRECISION 470®; Dell, Texas, USA). Analog signals were filtered (NF Corporation, Yokohama, Japan; settings: high-pass 30 kHz) before being digitized. Transferred digital data were automatically counted using recording software (Dasy Lab® 7.0; measX GmbH, Moenchengladbach, Germany). The threshold value was set at a signal amplitude of 0.1 V to exclude background noise. Each pup was immediately returned to its dam in the home cage after measurements. The digital data were transformed into the power spectrum using fast Fourier transformation and then converted into liner scale power spectrum in order to directly express the relationship between sound pressure and frequency (Dasy Lab® 7.0). In our preliminary study, ultrasonic vocalizations emitted by 10day-old rat pups were monitored up to 100 kHz. Power
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spectrum of full range showed that there was no clear peak fewer than 30 kHz and that large portions of power were found from 30 kHz to 50 kHz. Thus, analog signals were filtered by high-pass filter setting over 30 kHz and the power spectrum was analyzed ranging from 30 kHz to 50 kHz. In addition to these main portions at a range of 30 kHz to 50 kHz, power spectrum showed small portions at a ranging from 60 kHz to 100 kHz due to a harmonic overtone of the main portions. To assess frequency properties in the ultrasonic vocalization, we identified the predominant frequency peak between 30 kHz and 50 kHz and calculated the area under the curve (see detailed explanation in Fig. 1 legend).
4.3.
Drugs
A selective CRF1 receptor antagonist, NBI27914 was obtained from Tocris Bioscience (Bristol, UK) and dissolved in 100% dimethyl sulfoxide. NBI27914 was intraperitoneally injected in a volume of 1 ml/kg of body weight following previous report (Baram et al., 1997). Control group was received vehicle intraperitoneally at 1 ml/kg of body weight. A benzodiazepine receptor agonist, diazepam, was obtained from Wako Pure Chemicals (Osaka, Japan) and suspended in 0.5% methylcellulose solution. Diazepam was administered subcutaneously in a volume of 5 ml/kg of body weight. Control group received vehicle subcutaneously at 5 ml/kg of body weight.
4.4.
Data analysis
All data are expressed as mean ± standard error of the mean. Statistical analysis consisted of one-way analysis of variance followed by post-hoc multiple comparison tests and Williams test for ambient temperature- and dose-dependency. P < 0.05 was considered statistically significant.
Acknowledgments This research was supported by Dr. Norihiro Nagano (Pharmacology, Tsukuba Research Institute Banyu Pharmaceutical Co., Ltd.). He provided software for measuring the ultrasonic vocalization and the fast Fourier transform and calculating the power spectrum variables. We also thank him for his technical assistance.
REFERENCES
Allin, J.T., Banks, E.M., 1971. Effects of temperature on ultrasound production by infant albino rats. Dev. Psychobiol. 4, 149–156. Baram, T.Z., Chalmers, D.T., Chen, C., Koutsoukos, Y., De Souza, E.B., 1997. The CRF1 receptor mediates the excitatory actions of corticotropin releasing factor (CRF) in the developing rat brain: in vivo evidence using a novel, selective, non-peptide CRF receptor antagonist. Brain Res. 770, 89–95. Branchi, I., Santucci, D., Alleva, E., 2001. Ultrasonic vocalisation emitted by infant rodents: a tool for assessment of neurobehavioural development. Behav. Brain Res. 125, 49–56. Brudzynski, S.M., Kehoe, P., Callahan, M., 1999. Sonographic structure of isolation-induced ultrasonic calls of rat pups. Dev. Psychobiol. 34, 195–204.
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Fernandez-Lopez, A., Chinchetru, M.A., Calvo, Fernandez, P., 1997. The autoradiographic perspective of central benzodiazepine receptors; a short review. Gen. Pharmacol. 29, 173–180. Heinrichs, S.C., Koob, G.F., 2004. Corticotropin-releasing factor in brain: a role in activation, arousal, and affect regulation. J. Pharmacol. Exp. Ther. 311, 427–440. Hofer, M.A., Shair, H., 1978. Ultrasonic vocalization during social interaction and isolation in 2-weeek-old rats. Dev. Psychobiol. 11, 495–504. Iijima, M., Chaki, S., 2005. Separation-induced ultrasonic vocalization in rat pups: further pharmacological characterization. Pharmacol. Biochem. Behav. 82, 652–657. Ise, S., Nagano, N., Okuda, S., Ohta, H., 2008. Corticotropin-releasing factor modulates maternal separation-induced ultrasonic vocalization in rat pups via activation of CRF1 receptor. Brain Res. 1234, 59–65. Kehne, J.H., Coverdale, S., McCloskey, T.C., Hoffman, D.C., Cassella, J.V., 2000. Effects of the CRF(1) receptor antagonist, CP 154,526, in the separation-induced vocalization anxiolytic test in rat pups. Neuropharmacology 39, 1357–1367.
Noirot, E., 1968. Ultrasounds in young rodents, II: changes with age in albino rats. Anim. Behav. 16, 129–134. Olivier, B., Molewijk, H.E., van der Heyden, J.A., van Oorschot, R., Ronken, E., Mos, J., Miczek, K.A., 1998a. Ultrasonic vocalizations in rat pups: effects of serotonergic ligands. Neurosci. Biobehav. Rev. 23, 215–227. Olivier, B., Molewijk, H.E., van Oorschot, R., van der Heyden, J., Ronken, E., Mos, J., 1998b. Rat pup ultrasonic vocalization: effects of benzodiazepine receptor ligands. Eur. J. Pharmacol. 358, 117–128. Rybnikova, E.A., Pelto-Huikko, M., Rakitskova, V.V., Shalvapina, V.G., 2003. Localization of corticoliberin receptors in the rat brain. Neurosci. Behav. Physiol. 33, 399–404. Shair, H.N., Masmela, J.R., Hofer, M.A., 1999. The influence of olfaction on potentiation and inhibition of ultrasonic vocalization of rat pups. Physiol. Behav. 65, 769–772. Vale, W., Spiess, J., Rivier, C., Rivier, J., 1981. Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science 213, 1394–1397.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Power spectrum analysis of ultrasonic vocalization elicited by maternal separation in rat pups Satoko Ise⁎, Hisashi Ohta Pharmacology, Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., 3 Okubo, Tsukuba, Ibaraki 300-2611, Japan
A R T I C LE I N FO
AB S T R A C T
Article history:
The present study investigated how frequency distribution of maternal separation-induced
Accepted 3 June 2009
ultrasonic vocalization was altered by environmental stimuli and pharmacological agents.
Available online 11 June 2009
Sprague–Dawley rat pups at 10 days were used to measure numbers and frequencies of the ultrasonic vocalizations under various ambient temperatures and with pharmacological
Keywords:
manipulations of the corticotropin-releasing factor (CRF) and GABAergic systems. The
Rat
ultrasonic vocalization consisted of two distinct peaks in the frequency range of 30 kHz to
Ultrasonic vocalization
50 kHz. The area under the curve (AUC) in the high-frequency range and the number of the
Maternal separation
ultrasonic vocalizations increased when ambient temperature was lowered. Systemic
Frequency
administration of a selective CRF1 receptor antagonist, NBI27914, and a typical anxiolytic,
Power spectrum
diazepam, decreased the AUC in the high-frequency range and the number of the ultrasonic vocalizations in a dose-dependent manner at an ambient temperature of 24 °C. The AUC in the low-frequency range did not change with an alteration in ambient temperature or treatment with NBI27914 and diazepam except a high dose (1 mg/kg) of diazepam. These results demonstrated that emotional levels of isolated pups reflected not only the number but also the frequency distribution of maternal separation-induced ultrasonic vocalizations. High-frequency components, but not low-frequency components, of the ultrasonic vocalization were sensitive to changes in negative affective state of isolated pups. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
An infant rat emits ultrasonic vocalizations when it is isolated from its dam and littermates. The number of ultrasonic vocalizations was shown to be modulated by changes in environmental conditions, such as ambient temperature, tactile stimuli, and odors normally provided by the dam (Allin and Banks, 1971; Shair et al., 1999) and was often reduced by anxiolytics and antidepressants (Olivier et al., 1998a, 1998b; Branchi et al., 2001). Our previous report demonstrated that the number of ultrasonic vocalizations reflected the level of environmental stress in the isolated rat
pup (Ise et al., 2008). The administration of corticotropinreleasing factor (CRF), which played a pivotal role in stressinduced endocrine and behavioral responses (Vale et al., 1981; Heinrichs and Koob, 2004), dose-dependently increased the number of ultrasonic vocalizations at 37 °C (low stress condition). The increases in the number of ultrasonic vocalizations elicited by CRF were blocked by a CRF1 receptor antagonist, NBI27914, but not a CRF2 receptor antagonist, K41498. Our results indicated that the CRF–CRF1 receptor system was involved in modulating the number of the ultrasonic vocalizations by rat pups isolated from their dam.
⁎ Corresponding author. E-mail address: [email protected] (S. Ise). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.06.003
BR A IN RE S E A RCH 1 2 83 ( 20 0 9 ) 5 8 –6 4
Fig. 1 – Schematic drawing of the power spectrum in maternal separation-induced ultrasonic vocalization emitted by an isolated pup for 5 min. Ordinate represented the liner scale power (Vrms); abscissa represents the frequency (kHz). The horizontal-striped area indicated the low-frequency AUC (AUCL) from 30 kHz to the frequency at the nadir. The hatched area indicated the high-frequency AUC (AUCH) from the frequency at the nadir to 50 kHz. The highest peaks in both areas were called PeakL and PeakH, respectively.
59
Although most investigators indicated that the major frequency of the ultrasonic vocalization was distributed around 40 kHz, as detected by the pre-turned bat detector (Noirot, 1986; Hofer and Shair, 1978), one study reported on the frequency distribution of ultrasonic vocalization during maternal separation (Brudzynski et al., 1999). The frequency of the sound pressure peak shifted from low to high frequency with age. However, no information was available on whether changes in emotional level in isolated pup affected the frequency distribution of the ultrasonic vocalization. Although we have demonstrated that the number of ultrasonic vocalizations caused by the maternal separation was affected by external stimuli such as environmental changes as well as pharmacological manipulation, no attempt was made to examine if the frequency distribution of the ultrasonic vocalization was modulated by the environmental stimuli or pharmacological treatment. In the present study, we performed spectrum analysis in the ultrasonic vocalizations under various ambient temperature conditions of 37 °C, 24 °C, and 15 °C and under pharmacological treatment with agents that affected the CRF and GABAergic systems.
Fig. 2 – Effect of ambient temperature on the number and power spectrum variables of maternal separation-induced ultrasonic vocalizations at 37 °C, 24 °C, and 15 °C. A and B were the number and power spectrum of the ultrasonic vocalizations at three different ambient temperatures, respectively. C and D were the AUCL and AUCH, respectively. Detailed explanations of power spectrum variables were described in Fig. 1 legend. **P < 0.01 compared with the number at 37 °C. #P < 0.05, ##P < 0.01 compared with the AUCL or AUCH at 37 °C (Williams test; n = 5–6).
60
2.
BR A IN RE S EA RCH 1 2 83 ( 20 0 9 ) 5 8 –64
Results
2.1. Effect of ambient temperature on the number and power spectrum variables of ultrasonic vocalizations elicited by maternal separation in rat pups We evaluated the number and power spectrum variables in maternal separation-induced ultrasonic vocalizations obtained from isolated pup under various ambient temperatures (37 °C, 24 °C, and 15 °C) that were considered to represent different levels of environmental stress. The detailed definitions of power spectrum variables were described in Fig. 1. Briefly, the power spectrum in the frequency range between 30 kHz and 50 kHz had two peaks. The area under the curve (AUC) was calculated based on these two peaks. The lowfrequency AUC (AUCL) was defined from 30 kHz to the nadir frequency, where the lowest power between the highest peak (PeakL) in low-frequency range and the highest peak (PeakH) in high-frequency range. The high-frequency AUC (AUCH) was defined from the nadir frequency to 50 kHz. Statistical analysis indicated that lowering the ambient temperature from 37 °C to 24 °C to 15 °C increased both the number and AUCH of ultrasonic vocalizations (number, 24 °C, P = 0.0019, 15 °C, P = 0.0005; AUCH, 24 °C, P = 0.02, 15 °C, P = 0.0001, Figs. 2A and D). In contrast, the AUCL (Fig. 2C) and frequencies of PeakL, nadir, and PeakH in the ultrasonic vocalizations (Table 1) did not differ at any ambient temperature condition.
2.2. Effect of NBI27914 on the number and power spectrum variables of ultrasonic vocalizations elicited by maternal separation in rat pups at 24 °C Administration of a CRF 1 receptor antagonist, NBI27914, significantly reduced the number and AUCH of the ultrasonic vocalizations at 24 °C in a dose-dependent manner (number, 10 mg/kg, P = 0.004, 30 mg/kg, P < 0.0001, Fig. 3A; AUCH, 30 mg/kg, P = 0.035, Fig. 3D). In contrast, the AUCL (Fig. 3C) and frequencies of PeakL, nadir, and PeakH in the ultrasonic vocalizations (Table 1) were not altered at any dose of NBI27914 at 24 °C.
2.3. Effect of diazepam on the number and power spectrum variables of ultrasonic vocalizations elicited by maternal separation in rat pups at 24 °C Administration of diazepam significantly reduced the number, AUCL, and AUCH of the ultrasonic vocalizations at 24 °C in a dose-dependent manner (number, 0.3 mg/kg, P = 0.0002, 1 mg/kg, P < 0.0001, Fig. 4A; AUCL, 1 mg/kg, P = 0.0109, Fig. 4C; AUCH, 1 mg/kg, P = 0.0046, Fig. 4D). The frequencies of PeakL, nadir, and PeakH in the ultrasonic vocalizations shifted to low frequency at 1 mg/kg of diazepam (Table 1). Both doses of diazepam did not affect behaviors of an isolated pup during the measurement.
3.
Discussion
The present study addressed if frequency distribution as well as the number of maternal separation-induced ultrasonic vocalizations changed under various stressful conditions induced by ambient temperatures and examined if such frequency distribution was modified by pharmacological treatments. In order to study the frequency distribution in the ultrasonic vocalization, we used various environmental ambient temperatures (37 °C, 24 °C and 15 °C) as environmental stimuli. The present results revealed that the frequency distribution of ultrasonic vocalization consisted of two distinct peaks between 30 kHz and 50 kHz. The height of PeakH, around 40 kHz, became higher, the AUCH increased, and the frequency of the highest peak shifted from PeakL to PeakH when the ambient temperature was decreased from 37 °C to 24 °C and 15 °C. It synchronized to the increases of the number of the ultrasonic vocalizations. These findings suggested that the AUCH, as well as the number of the ultrasonic vocalizations, were more sensitive to the level of emotional state in isolated pup than AUCL. Until now, no report has addressed if the alteration of environmental stimuli affects the frequency distribution of the ultrasonic vocalization elicited by maternal separation. The present study was the first report to examine the relationship between the
Table 1 – Power spectrum variables during maternal separation-induced ultrasonic vocalizations under various stressrelated conditions. Treatment
n
PeakL frequency (kHz)
Ambient temperatures 37 °C 5 24 °C 6 15 °C 5 NBI27914 at 24 °C Control 12 3 mg/kg 5 10 mg/kg 6 30 mg/kg 11 Diazepam at 24 °C Control 12 0.1 mg/kg 8 0.3 mg/kg 9 1 mg/kg 7 a
Nadir frequency (kHz)
PeakH frequency (kHz)
AUCL (V·Hz)
AUCH (V·Hz)
37.3 ± 1.0 37.4 ± 0.3 37.6 ± 0.3
39.9 ± 0.9 39.0 ± 0.4 39.2 ± 0.3
40.7 ± 0.9 41.5 ± 0.4 42.3 ± 0.4
5.2 ± 1.5 7.0 ± 1.7 4.6 ± 1.1
1.9 ± 0.7 8.7 ± 2.1 a 18.4 ± 2.3 a
36.3 ± 0.3 35.8 ± 0.4 36.0 ± 0.3 36.1 ± 0.2
38.7 ± 0.3 37.9 ± 0.4 37.9 ± 0.3 38.7 ± 0.2
40.9 ± 0.3 40.5 ± 0.2 39.9 ± 0.6 40.1 ± 0.3
10.4 ± 1.6 9.6 ± 2.6 7.9 ± 2.3 8.8 ± 1.3
8.7 ± 3.0 6.6 ± 1.0 4.2 ± 0.8 2.9 ± 1.3 a
36.5 ± 0.3 36.4 ± 0.3 35.6 ± 0.4 34.0 ± 0.7 a
39.0 ± 0.3 38.9 ± 0.4 38.3 ± 0.3 36.7 ± 1.0 a
42.1 ± 0.5 41.5 ± 0.4 40.7 ± 0.4 38.3 ± 1.4 a
P < 0.05 compared with the corresponding 37 °C-group or control group. (Williams' test, n = 5–21 per group).
8.4 ± 2.0 9.8 ± 1.2 6.9 ± 1.3 2.1 ± 0.8 a
9.3 ± 1.7 6.2 ± 2.0 7.2 ± 2.1 1.1 ± 0.2 a
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Fig. 3 – Effects of a CRF1 receptor antagonist, NBI27914, on the number and power spectrum variables of maternal separation-induced ultrasonic vocalizations at 24 °C. NBI27914 (3, 10, and 30 mg/kg, i.p.) was administered 30 min before the test. A and B were the number and power spectrum of ultrasonic vocalizations at 24 °C, respectively. C and D represented the AUCL and AUCH, respectively. Detailed explanations of power spectrum variables were described in Fig. 1 legend. **P < 0.01 compared with the number in the control group. #P < 0.05 compared with the AUCL or AUCH in the control group (Williams test; n = 5–12).
magnitude of external stimuli and distribution of frequency in the maternal separation-induced ultrasonic vocalization. The number of ultrasonic vocalizations was pharmacologically inhibited by not only CRF-related compounds but also anxiolytics and antidepressants (Kehne et al., 2000; Iijima and Chaki, 2005; Olivier et al., 1998a, 1998b). In a previous report, we demonstrated that the endogenous CRF–CRF1 receptor system was involved in the generation of the ultrasonic vocalization elicited by maternal separation under environmental stress conditions, but not the CRF–CRF2 receptor system (Ise et al., 2008). Contribution of the CRF–CRF1 receptor system on the ultrasonic vocalization was clear at 24 °C. In this study, we assessed the effects of a CRF1 receptor antagonist, NBI27914, and a typical anxyolytic drug, diazepam, on the frequency distribution as well as the number of the ultrasonic vocalizations at 24 °C. The number and AUCH of the ultrasonic vocalizations were decreased dose-dependently by administration of both drugs at 24 °C. The frequency distribution after
administration of both drugs at the highest dose became similar to that at 37 °C. Namely, AUCH in the ultrasonic vocalization was less prominent at 37 °C as well as with treatment with NBI27914 and diazepam. These data supported that the highfrequency component was sensitive to environmental stimuli modified by ambient temperatures and, in addition to the number of the ultrasonic vocalizations, might be a useful variable for evaluating anxiety- and stress-related agents. In contrast, AUCL did not change in any case except with a high dose of diazepam (1 mg/kg). AUCL would be an essential component for the ultrasonic vocalization. Although the AUCL decreased and the PeakL shifted to a low-frequency side by diazepam at a dose of 1 mg/kg, these effects might be due to the low number of the ultrasonic vocalizations at a dose of 1 mg/kg diazepam. Alternatively, it might be hypothesized that diazepam, which activates the GABAergic system, had a distinct effect on the frequency distribution of the ultrasonic vocalizations, unlike activating the CRF–CRF1 receptor mechanisms.
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Fig. 4 – Effects of an anxiolytic agent, diazepam, on the number and power spectrum variables of maternal separation-induced ultrasonic vocalizations at 24 °C. Diazepam (0.1, 0.3, and 1 mg/kg, s.c.) was administered 30 min before the test. A and B were the number and power spectrum of ultrasonic vocalizations at 24 °C, respectively. C and D represented the AUCL and AUCH, respectively. Detailed explanations of power spectrum variables were described in Fig. 1 legend. **P < 0.01 compared with the number in the control group. #P < 0.05, ##P < 0.01 compared with the AUCL or AUCH in the control group (Williams test; n = 7–12).
Although distribution of CRF1 receptor and benzodiazepine receptor in brain are broad (Rybnikova et al., 2003; FernandezLopez et al., 1997), the CRF–CRF1 receptor and GABAergic system might modulate vocal output at a different level of the brain. Since a CRF1 receptor antagonist significantly attenuated AUCH and the number of ultrasonic vocalizations without affecting AUCL which would be an essential component for the output of ultrasonic vocalization, the CRF–CRF1 receptor system might regulate a receptive system at the level of higher brain such as amygdala where one of the key areas to respond to aversive input is. Meanwhile benzodiazepine attenuated all AUCH, AUCL and the number of ultrasonic vocalizations, suggesting that benzodiazepine might regulate an efferent system of the vocalization at the level of periaqueductal grey on brain stem motor neuron as well as higher brain areas. Changes in frequency distribution caused by centrally acting agents might reflect mechanisms of action for each agent. Further studies will be necessary to clarify if various agents that affect the number of ultrasonic vocalizations alter the frequency distribution in different ways.
In conclusion, our results demonstrated that the emotional state of an isolated pup was reflected not only in the number but also in the frequency distribution of maternal separationinduced ultrasonic vocalizations. In particular, we revealed that the high-frequency component was sensitive to stress stimuli based on responses to lowering the ambient temperature, as well as activation of the CRF–CRF1 receptor and GABAergic systems.
4.
Experimental procedures
4.1.
Animals
Male and female 10-day-old Sprague–Dawley rat pups (Charles River Inc., Yokohama, Japan) were used. Newborn rats were culled to maintain less than 12 pups per dam on the day after birth (postnatal day 1). No sex differences in the number of vocalizations were observed in 10-day-old rat pups at various ambient temperatures in our preliminary study. The 10-day-old rat pups were used because the number of the ultrasonic
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vocalizations reached a peak at postnatal day 10 during 3 weeks of postnatal periods in our preliminary study. Pups were housed with the dam in their home cage in a controlled animal room (room temperature: 23.0 ± 2.0 °C, humidity: 55.0 ± 15.0%) on a 12 h light–dark cycle (lights on at 07:00). All animal procedures were approved by the Banyu Institutional Animal Care and Use Committee.
4.2. Measurement of maternal separation-induced ultrasonic vocalizations On the day of the study, the rat pups and their dam were moved to the experimental room from the animal housing room and left undisturbed for at least 1 h before initiation of the study. In order to minimize maternal and temporal effects, pups from one mother were randomly assigned to treatments. That is, for testing an effect of ambient temperatures, pups from two mothers were used and litters from each mother were randomly divided into three groups corresponding to three different ambient temperatures, another one was the same. Ambient temperature was set at 37 °C, 24 °C and then 15 °C to expose pups. We avoided reintroducing pups exposed to a lower temperature at first into the nest because pups exposed to lower ambient temperature might maintain more physical and psychological abnormalities than pups exposed to 37 °C when returned to the nest. In the case of pharmacological treatments, pups from three mothers were used for each drug. To minimize order effects and intra-mother's effects, both vehicle and one drug treated pups were at least assigned from each mother and the order of administration of vehicle and drugs were psudo-randomly conducted. After habituation, each pup received an intraperitoneal (i.p.) or subcutaneous (s.c.) injection of the vehicle and test drugs, and then was returned to the dam and littermates in the case of pharmacological manipulation. Thirty minutes after the administration, each pup was placed in a stainless-steel chamber (10.5 cm diameter × 16 cm height) on a Cool Plate® (NCP-2215; Nisshin Rika, Tokyo, Japan), which maintained the chamber at 37 °C or 24 °C or 15 °C in a soundproof room (AT-81; RION, Tokyo, Japan). Ultrasonic vocalizations were measured for 5 min. Individual calls made by each pup during this period were detected using an ultrasound-sensitive microphone (Inazu keisoku, Saitama, Japan) and amplified by a dedicated preamplifier. Analog signals were converted to digital signals using an A/D converter (CH-3150; Exacq Technologies, Indiana, USA) and stored on a personal computer (PRECISION 470®; Dell, Texas, USA). Analog signals were filtered (NF Corporation, Yokohama, Japan; settings: high-pass 30 kHz) before being digitized. Transferred digital data were automatically counted using recording software (Dasy Lab® 7.0; measX GmbH, Moenchengladbach, Germany). The threshold value was set at a signal amplitude of 0.1 V to exclude background noise. Each pup was immediately returned to its dam in the home cage after measurements. The digital data were transformed into the power spectrum using fast Fourier transformation and then converted into liner scale power spectrum in order to directly express the relationship between sound pressure and frequency (Dasy Lab® 7.0). In our preliminary study, ultrasonic vocalizations emitted by 10day-old rat pups were monitored up to 100 kHz. Power
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spectrum of full range showed that there was no clear peak fewer than 30 kHz and that large portions of power were found from 30 kHz to 50 kHz. Thus, analog signals were filtered by high-pass filter setting over 30 kHz and the power spectrum was analyzed ranging from 30 kHz to 50 kHz. In addition to these main portions at a range of 30 kHz to 50 kHz, power spectrum showed small portions at a ranging from 60 kHz to 100 kHz due to a harmonic overtone of the main portions. To assess frequency properties in the ultrasonic vocalization, we identified the predominant frequency peak between 30 kHz and 50 kHz and calculated the area under the curve (see detailed explanation in Fig. 1 legend).
4.3.
Drugs
A selective CRF1 receptor antagonist, NBI27914 was obtained from Tocris Bioscience (Bristol, UK) and dissolved in 100% dimethyl sulfoxide. NBI27914 was intraperitoneally injected in a volume of 1 ml/kg of body weight following previous report (Baram et al., 1997). Control group was received vehicle intraperitoneally at 1 ml/kg of body weight. A benzodiazepine receptor agonist, diazepam, was obtained from Wako Pure Chemicals (Osaka, Japan) and suspended in 0.5% methylcellulose solution. Diazepam was administered subcutaneously in a volume of 5 ml/kg of body weight. Control group received vehicle subcutaneously at 5 ml/kg of body weight.
4.4.
Data analysis
All data are expressed as mean ± standard error of the mean. Statistical analysis consisted of one-way analysis of variance followed by post-hoc multiple comparison tests and Williams test for ambient temperature- and dose-dependency. P < 0.05 was considered statistically significant.
Acknowledgments This research was supported by Dr. Norihiro Nagano (Pharmacology, Tsukuba Research Institute Banyu Pharmaceutical Co., Ltd.). He provided software for measuring the ultrasonic vocalization and the fast Fourier transform and calculating the power spectrum variables. We also thank him for his technical assistance.
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