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Carbachol injections into the nucleus accumbens induce 50 kHz calls in rats
Neuroscience Letters 401 (2006) 10–15
Carbachol injections into the nucleus accumbens induce 50 kHz calls in rats Markus Fendt ∗ , Isabel Schwienbacher 1 , Hans-Ulrich Schnitzler Tierphysiologie, Zoologisches Institut, Fakult¨at f¨ur Biologie, Universit¨at T¨ubingen, Auf der Morgenstelle 28, D-72076 T¨ubingen, Germany Received 9 February 2006; received in revised form 22 February 2006; accepted 22 February 2006
Abstract In rats, different types of vocalization can be observed. High frequency vocalizations (so called 50 kHz calls) are believed to indicate an appetitive state of the emitting animal. This is supported by studies demonstrating that infusions of the dopamine agonist amphetamine into the nucleus accumbens (NAC), a key structure for appetitive behaviors, induce 50 kHz calls. Several studies during the last years demonstrated that not only infusions of dopamine agonists such amphetamine but also infusions of acetylcholine receptor agonists into the NAC stimulate the appetitive system. In present study, we tested whether infusions of the unspecific cholinergic agonist carbachol into the NAC are able to induce 50 kHz calls. Indeed, we observed a high number of 50 kHz calls after intra-NAC infusions of carbachol. The main frequency of the these calls was between 40 and 70 kHz, and the duration was mainly between 10 and 50 ms. We hypothesize that acetylcholine transmission within the NAC plays an important role in the induction of those ultrasonic calls indicating an appetitive state. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: 50 kHz calls; Acetylcholine; Appetitive system; Communication; Rats
Laboratory rats (Rattus norvegicus) emit different types of ultrasonic vocalization (USV) which are believed to play an important role in communication with conspecifics [5,26,37]. These USVs strongly vary in frequency and duration, but there two very often described variants which are thought to play an important role in the communication of the emotional state of the USV emitting animal. One type of these USVs has a high frequency (over 30 kHz), a duration shorter than 300 ms, and is usually termed as “50 kHz calls” [26]. The 50 kHz calls are mainly observable in appetitive situation: during sexual behavior and social contacts [2,10,16,32], as well as during anticipation of play [24], or food [11]. Furthermore, artificial rewarding stimuli such electrical brain stimulation or drugs that are experienced as rewarding as well as cues or contexts that predict such artificial reward induce 50 kHz calls [11,13,25,42]. The 50 kHz calls are clearly dissociated from another group of ultrasonic calls with much lower frequencies (between 18 and 25 kHz) and ∗ Corresponding author. Present address: Neuroscience Research, Novartis Institutes for BioMedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland. Tel.: +49 7071 2975347; fax: +49 7071 292618. E-mail address: [email protected] (M. Fendt). 1 Present address: Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Straße 65, D-88397 Biberach/Riß, Germany.
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.02.069
longer durations (more than 300 ms; [7]). These USV are usually termed “22 kHz calls” and are mainly emitted in and after the presence of aversive stimuli. For example, 22 kHz calls are observed during exposure to predators [3], to foot shocks [43], to startle stimuli [23], as well as after unfamiliar handling [9]. There are a small number of studies focusing on the neurobiology of USV in rats: whereas for 22 kHz calls the fear system including the amygdala and the mediobasal forebrain structures are crucial (e.g. [4,6,14]), the appetitive brain system appears to be important for 50 kHz calls. Systemic injections of amphetamine, a drug stimulating the appetitive system (e.g. [18]), induce 50 kHz calls [42]. The same effects are observed after intracranial injections of amphetamine into the Nucleus accumbens (NAC), one key structure of the appetitive system [12,41]. Furthermore, the anterior part of the hypothalamus seems to be involved in 50 kHz call production [15]. During the last years, a number of studies demonstrated that acetylcholine receptors within the NAC play an important role in the appetitive system: for example, appetitive learning is blocked by intra-NAC infusions of muscarinic receptor antagonists [35,36]. Furthermore, rats self-administer the unspecific acetylcholine receptor agonist carbachol directly into their NAC [19] and blockade of muscarinic receptors attenuates this carba-
M. Fendt et al. / Neuroscience Letters 401 (2006) 10–15
chol self-administration [19] indicating that carbachol infusions into the NAC stimulate the appetitive system. Based on these findings, we had the hypothesis that infusions of carbachol into the NAC are able to induce 50 kHz calls. The present study tested this hypothesis. Twelve male Sprague–Dawley rats (Charles River, Sulzfeld, Germany) weighing 220–260 g at the time of surgery were housed in groups of 5–6 rats/cage in a temperature controlled colony room under a 12:12 h light/dark cycle (lights on at 07:30). They were fed with 15 g standard rat chow/animal/day, and tap water was available ad libitum. Each rat was handled daily before and after surgery (handling started 5 days before surgery). The experiments were performed in accordance with international ethical guidelines for the care and use of animals for experiments and were approved by the local council of animal care (Regierungspr¨asidium T¨ubingen, ZP 4/02). Rats were anaesthetized with ketamine/xylazine (9:1, 100 mg/kg, i.p.) and placed into a stereotaxic frame. The skull was exposed and stainless steel guide cannulae (0.7 mm diameter) were implanted bilaterally aiming at the NAC (toothbar + 5 mm above the interaural plane) with the following distances to Bregma: rostrocaudal + 3.4 mm, mediolateral ± 1.5 mm, dorsoventral −7.2 mm (coordinates from [34]). The guide cannulae were fixed to the skull with acrylic cement (Kulzer, Wehrheim, Germany) and three anchoring screws. After surgery, the cannulae were fitted with stylets in order to maintain patency. The rats were allowed to recover completely from surgery (4–5 days). The unspecific acetylcholine receptor agonist carbachol (carbamylcholine chloride 99%, Acros, Geel, Belgium) was dissolved in saline and was administered bilaterally (4 ng/0.5 l per side) into the NAC with a velocity of 0.5 l/min. The injection cannulae were left in place for additional 2 min to allow diffusion of the solution away from the cannulae. The dose of 4 ng/0.5 l carbachol was chosen on the basis of a previous study showing that this dose leads to significant changes in the emotional state but only slightly affect motor behavior of the rats [38]. Latter study also showed that doses in the range of 1–4 ng carbachol had very similar behavioral effects. Higher doses which can be found in the literature (e.g. [19,27]) have very intensive effects on spontaneous motor behavior in our laboratory. Therefore, we decided to infuse only one weak dose of carbachol into the NAC. Immediately after injections into the NAC, the animals were put in a small open field (30 cm × 60 cm × 30 cm). During the next 30 min, USVs were recorded. All animals were tested twice and got saline or carbachol in a pseudorandomized order. The second test was carried out two days later and was identical to the first test. To record USVs, a custom made condensor microphone (University of T¨ubingen) was fixed at the top of each compartment. The microphones were sensitive between 10 and 200 kHz, with a flat frequency response (±3 dB) between 30 and 120 kHz and ±6 dB between 15 and 160 kHz. From 30 to 10 kHz and 120 to 200 kHz the sensitivity dropped by 0.2 dB/kHz. The signals were digitized (sample rate 192 kHz, 16 bit) and stored for subsequent analysis as .wav-files on a laptop computer. Sound analysis was performed with the colour sonograph software Selena (custom-
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made, T¨ubingen University). Signals were processed through a fast Fourier-transformation (512, Hann window) and displayed as colour sonograms. Frequency resolution and time resolution were determined by the sampling rate and the FFT points. In our analysis frequency resolution was 375 Hz and time resolution was 2.7 ms. For each signal, duration and best frequency (in the following just named “frequency”) were measured. Frequency was measured on the point of the highest sound intensity and duration was defined as the length between the beginning and the end of the USVs. USVs with a frequency of ≥30 kHz and duration of ≤300 ms were classified as so called 50 kHz USVs (cf. [26]). To analysis the USVs, the frequencies were sorted in different frequency ranges (30–40 kHz, >40–50 kHz, >50–60 kHz, etc.) and the durations in different duration ranges (<10 ms, 10–50 ms, >50–100 ms, etc.) and the percentage of emitted USVs of each frequency and duration range were calculated for each individual animal. Finally, the average over all animals was determined. After the tests, the rats were sacrificed with an overdose of pentobarbital (Nembutal). Their brains were removed and immersion-fixed in 8% paraformaldehyde and 20% sucrose. Frontal sections (50 m) were cut on a freezing microtome and Nissl stained with thionine. The injection sites were localized under a light microscope and were drawn onto plates taken from a rat brain atlas [33]. Since the number of USVs was not normally distributed (Kolmogorov-Smirnov test with Lillifors adaptation: p < 0.0001 for saline, p = 0.07 for carbachol infusions), a chi-square test was used for the statistical analysis of the effect of intra-accumbal carbachol on USVs. A value of p < 0.05 was considered to represent a significant effect. Fig. 1 shows the cannula placements for the rats that were included in the analysis of the effects of carbachol injections into the NAC (n = 11). One rat had misplaced cannulas (located in dorsal striatum) and was excluded from further analysis. The different symbols of the injection sites symbolize the amount of carbachol effect on USV (see below).
Fig. 1. Serial drawing of a frontal section of the NAC (+3.4 mm rostral to Bregma) depicting the injection sites of carbachol into the NAC. Abbreviations: AcbC—nucleus accumbens core, AcbSh—nucleus accumbens shell.
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M. Fendt et al. / Neuroscience Letters 401 (2006) 10–15
Fig. 2. Effects of acute carbachol injections into the NAC on the emission of 50 kHz calls. (A) Medians of the mean amount of 50 kHz USVs after injections with saline or carbachol. Carbachol infusions into the NAC increased the emission of 50 kHz USVs. *** p < 0.001, chi-square test. (B) Distribution of the mean frequency of the USVs. (C) Distribution of the mean duration of the USVs.
All recorded USVs had durations shorter than 300 ms and frequencies higher than 30 kHz and thus met the criterion for 50 kHz calls (cf. [26]). No other USVs were found. The emission of 50 kHz calls was strongly biased to carbachol infusions into the NAC; there were almost no calls after saline treatment (see Fig. 2A; chi-square test: comparison saline versus carbachol: χ2 = 4455; p < 0.001). Those injection sites which elicit a high number of 50 kHz calls (>500 calls/20 min) were exclusively located in the core region of the NAC.
Most of these 50 kHz USVs were emitted in the frequency range between 40 and 70 kHz with durations between 10 and 50 ms (see Fig. 2B and C). The basic structure of the USVs was rather similar in all individuals (Fig. 3). The signals often consisted of three elements with very variable durations. The first and the third element were frequency-modulated and covered frequency ranges above 55 kHz. The modulation direction was upward in the first and downward in the third element. The second element had a rather constant frequency always in the frequency range between 40 and 50 kHz. The rats also emitted combinations of only two of these three elements. Here, all possible combinations, i.e. the first and the second element, the first and third element, and the second and third element, have been observed. Furthermore, each of the three elements was also produced alone. Quite rarely, we found single elements which did not fit into the describe pattern, e.g. the single element in rat 4 in Fig. 3. In the present study, we investigated whether infusions of the cholinergic agonist carbachol into the NAC induce 50 kHz calls. Our results clearly show that rats emit a very high amount of 50 kHz calls after carbachol infusions into the NAC. It is likely that carbachol in the low dose we used here is acting mainly via muscarinic receptors since the behavioral effects of much higher doses of carbachol can totally be blocked by coadministration of the muscarinic antagonist scopolamine [19]. Thus, the present study supports previous studies demonstrating that cholinergic stimulation of the appetitive system induce 50 kHz calls [12,41,42]. It was postulated that 50 kHz calls provide a sensitive marker for a stimulated appetitive system [25]: rats emit 50 kHz calls during play and anticipation of play [24], sexual and mating behavior [2,32] and other appetitive forms of social contact [10] as well as during anticipation of food [11]. Fifty-kilohertz calls are not only observed during natural rewarding stimuli but also in the presence of artificial rewarding stimuli like pharmacological treatments and electrical stimulation of the medial forebrain bundle [11,12,25,41,42]. Furthermore, USV calls are also emitted during conditioned rewarded states, e.g. in contexts previously paired with rewarding drugs [25]. Some of the studies quoted above point to different brain structure which appears to be important for the induction of 50 kHz calls. The facts that systemic infusions of amphetamine is very potent in inducing 50 kHz calls [42] and that 50 kHz are emitted in appetitive states (e.g. [25]) suggest that the appetitive dopaminergic system is involved in the production of 50 kHz calls. This is supported by two studies measuring a high amount of 50 kHz calls after direct infusions of amphetamine into the NAC [12,41]. The present study clearly shows that also a stimulation of cholinergic receptors within the NAC is able to induce 50 kHz calls. In contrast to the amphetamine studies quoted above, those injection sites of carbachol in the present study, which were most effective in inducing 50 kHz calls, were exclusively located in the core region of the NAC (cf. Fig. 1). However, since infused drugs are able to diffuse about 1 mm in the brain [31], the individual numbers of induced 50 kHz calls were quite variable, and we did not infuse carbachol into adjacent areas (as an anatomical
M. Fendt et al. / Neuroscience Letters 401 (2006) 10–15
Fig. 3. Sonographs showing typical examples of USVs of five different individual rats recorded after carbachol injections into the NAC.
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control) such a localization of the carbachol effects is rather questionable. Beside the NAC, the anterior part of the hypothalamus/preoptic area is a further brain site, which was discussed to be important for 50 kHz call production [15]. This area is functionally connected with the NAC [1], i.e. pharmacological manipulation of this area affects the activity of the appetitive system (e.g. [8]). Taken together, the present study support the hypotheses that (1) the appetitive system, especially the NAC, is important for the generation of 50 kHz calls (cf. [26]) and that [37] infusions of acetylcholine agonists into the NAC activate the appetitive system (cf. [19]). It should be mentioned here that the exact function of acetylcholine within the NAC is still unclear. For example, acetylcholine transmission plays an important role in the processing of both, appetitive [28,35] and aversive stimuli ([30], but see also [29]). Against that, dopamine transmission within the NAC is thought to be determinant for the appetitive behavioral arousal (summarized in [20]). Since carbachol infusions into the NAC enhance dopamine transmission [17], we suggest that the carbachol effect on USV observed in the present experiment is mediated by an enhancement of dopamine transmission within the NAC. The 50 kHz calls we observed are consistent with previously characterized 50 kHz calls (cf. [10]), regarding to the frequency and duration range. Usually, these USVs are reported as calls in a range of 30–70 kHz. Most of the USVs we observed were in that frequency range, even though we also found USVs between 70 and 96 kHz. We cannot exclude that there were additional calls above the frequency of 96 kHz, since the frequency range of our system was limited to 96 kHz. The structure of the carbachol-induced 50 kHz calls measured in the present study varied strongly within and between individuals. Interestingly, the most USVs had a similar basic structure with three elements, seen in all the individuals. Such USVs with three elements and a similar frequency range were also described in previous study from our laboratory [21,22]. In latter studies, these calls were observed during sexual behavior but also during agonistic behavior (cf. [39,40]). In summary, the present study demonstrated that carbachol infusions into the NAC core region induce 50 kHz calls in male rats. This support the idea that the appetitive system, especially the NAC, is important for 50 kHz calls and that infusion of acetylcholine receptor agonists into the NAC stimulates the appetitive system. References [1] G.V. Allen, D.F. Cechetto, Functional and anatomical organization of cardiovascular pressor and depressor sites in the lateral hypothalamic area. II. Ascending projections, J. Comp. Neurol. 330 (1993) 421– 438. [2] R.J. Barfield, D.A. Thomas, The role of ultrasonic vocalizations in the regulation of reproduction in rats, Ann. N.Y. Acad. Sci. 474 (1986) 33–43. [3] R.J. Blanchard, D.C. Blanchard, R. Agullana, S.M. Weiss, Twentytwo kHz alarm cries to presentation of a predator, by laboratory rats living in a visible burrow system, Physiol. Behav. 50 (1991) 967– 972.
[4] S.M. Brudzynski, Ultrasonic vocalization induced by intracerebral carbachol in rats: localization and a dose-response study, Behav. Brain Res. 63 (1994) 133–143. [5] S.M. Brudzynski, Principles of rat communication: quantitative parameters of ultrasonic calls in rats, Behav. Genet. 35 (2005) 85–92. [6] S.M. Brudzynski, F. Barnabi, Contribution of the ascending cholinergic pathways in the production of ultrasonic vocalization in the rat, Behav. Brain Res. 80 (1996) 145–152. [7] S.M. Brudzynski, F. Bihari, D. Ociepa, X.W. Fu, Analysis of 22 kHz ultrasonic vocalization in laboratory rats: long and short calls, Physiol. Behav. 54 (1993) 215–221. [8] S.M. Brudzynski, G.J. Mogenson, Inhibition of amphetamine-induced locomotor activity by injection of carbachol into the anterior hypothalamic/preoptic area: pharmacological and electrophysiological studies in the rat, Brain Res. 376 (1986) 47–56. [9] S.M. Brudzynski, D. Ociepa, Ultrasonic vocalization of laboratory rats in respond to handling and touch, Physiol. Behav. 52 (1992) 655–660. [10] S.M. Brudzynski, A. Pniak, Social contacts and production of 50-kHz short ultrasonic calls in adult rats, J. Comp. Psychol. 116 (2002) 73– 82. [11] J. Burgdorf, B. Knutson, J. Panksepp, Anticipation of rewarding electrical brain stimulation evokes ultrasonic vocalization in rats, Behav. Neurosci. 114 (2000) 320–327. [12] J. Burgdorf, B. Knutson, J. Panksepp, S. Ikemoto, Nucleus accumbens amphetamine microinjections unconditionally elicit 50-kHz ultrasonic vocalizations in rats, Behav. Neurosci. 115 (2001) 940–944. [13] J. Burgdorf, B. Knutson, J. Panksepp, T.S. Shippenberg, Evaluation of rat ultrasonic vocalizations as predictors of the conditioned aversive effects of drugs, Psychopharmacology (Berl) 155 (2001) 35–42. [14] J.S. Choi, T.H. Brown, Central amygdala lesions block ultrasonic vocalization and freezing as conditional but not unconditional responses, J. Neurosci. 23 (2003) 8713–8721. [15] X.W. Fu, S.M. Brudzynski, High-frequency ultrasonic vocalization induced by intracerebral glutamate in rats, Pharmacol. Biochem. Behav. 49 (1994) 835–841. [16] L.A. Geyer, R.J. Barfield, Influence of gonadal hormones and sexual behavior on ultrasonic vocalization in rats: I. Treatment of females, J. Comp. Physiol. Psychol. 92 (1978) 438–446. [17] A.M. Gray, J.H. Connick, Clozapine-induced dopamine levels in the rat striatum and nucleus accumbens are not affected by muscarinic antagonism, Eur. J. Pharmacol. 362 (1998) 127–136. [18] C.J. Harmer, G.D. Phillips, Enhanced conditioned inhibition following repeated pretreatment with d-amphetamine, Psychopharmacology 142 (1999) 120–131. [19] S. Ikemoto, B.S. Glazier, J.M. Murphy, W.J. McBride, Rats selfadminister carbachol directly into the nucleus accumbens, Physiol. Behav. 63 (1998) 811–814. [20] S. Ikemoto, J. Panksepp, The role of nucleus accumbens dopamine in motivated behavior: a unifying interpretation with special reference to reward-seeking, Brain Res. Brain Res. Rev. 31 (1999) 6–41. [21] M.T. Kaltwasser, Vergleichende Untersuchungen zum akustischen Kommunikationsverhalten und zur Orientierung bei Rattus rattus und Rattus norvegicus [Comparitive studies on acoustic communication and navigation in Rattus rattus and Rattus norvegicus], Ph.D. thesis, University of T¨ubingen, 1985. [22] M.T. Kaltwasser, Acoustic signaling in the black rat (Rattus rattus), J. Comp. Psychol. 104 (1990) 227–232. [23] M.T. Kaltwasser, Acoustic startle induced ultrasonic vocalization in the rat: a novel animal model of anxiety? Behav. Brain Res. 43 (1991) 133–137. [24] B. Knutson, J. Burgdorf, J. Panksepp, Anticipation of play elicits highfrequency ultrasonic vocalizations in young rats, J. Comp. Psychol. 112 (1998) 65–73. [25] B. Knutson, J. Burgdorf, J. Panksepp, High-frequency ultrasonic vocalizations index conditioned pharmacological reward in rats, Physiol. Behav. 66 (1999) 639–643. [26] B. Knutson, J. Burgdorf, J. Panksepp, Ultrasonic vocalizations as indices of affective states in rats, Psychol. Bull. 128 (2002) 961–977.
M. Fendt et al. / Neuroscience Letters 401 (2006) 10–15 [27] N. Koshikawa, Y. Yoshida, M. Kitamura, T. Saigusa, M. Kobayashi, A.R. Cools, Stimulation of acetylcholine or dopamine receptors in the nucleus accumbens differentially alters dopamine release in the striatum of freely moving rats, Eur. J. Pharmacol. 303 (1996) 13–19. [28] G.P. Mark, A.E. Kinney, M.C. Grubb, A.S. Keys, Involvement of acetylcholine in the nucleus accumbens in cocaine reinforcement, Ann. N.Y. Acad. Sci. 877 (1999) 792–795. [29] G.P. Mark, P.V. Rada, T.J. Shors, Inescapable stress enhances extracellular acetylcholine in the rat hippocampus and prefrontal cortex but not the nucleus accumbens or amygdala, Neuroscience 74 (1996) 767–774. [30] G.P. Mark, J.B. Weinberg, P.V. Rada, B.G. Hoebel, Extracellular acetylcholine is increased in the nucleus accumbens following the presentation of an aversively conditioned taste stimulus, Brain Res. 688 (1995) 184–188. [31] J.H. Martin, Autoradiographic estimation of the extent of reversible inactivation produced by microinjection of lidocaine and muscimol in the rat, Neurosci. Lett. 127 (1991) 160–164. [32] T.K. McIntosh, R.J. Barfield, L.A. Geyer, Ultrasonic vocalisations facilitate sexual behaviour of female rats, Nature 272 (1978) 163–164. [33] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, 1997. [34] L.J. Pellegrino, A.S. Pellegrino, A.J. Cushman, A Stereotaxic Atlas of the Rat Brain, Plenum Press, New York, 1979. [35] W.E. Pratt, A.E. Kelley, Nucleus accumbens acetylcholine regulates appetitive learning and motivation for food via activation of muscarinic receptors, Behav. Neurosci. 118 (2004) 730–739.
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[36] L. Ramirez-Lugo, S. Zavala-Vega, F. Bermudez-Rattoni, NMDA and muscarinic receptors of the nucleus accumbens have differential effects on taste memory formation, Learn. Mem. 13 (2006) 45–51. [37] C. Sanchez, Stress-induced vocalisation in adult animals. A valid model of anxiety? Eur. J. Pharmacol. 463 (2003) 133–143. [38] I. Schwienbacher, M. Fendt, H.-U. Schnitzler, R.F. Westbrook, R. Richardson, Carbachol injections into the nucleus accumbens disrupts acquisition and expression of fear-potentiated startle and freezing in rats, Neuroscience, in press. [39] L.K. Takahashi, D.A. Thomas, R.J. Barfield, Analysis of ultrasonic vocalizations emitted by residents during aggressive encounters among rats (Rattus norvegicus), J. Comp. Psychol. 97 (1983) 207– 212. [40] D.A. Thomas, L.K. Takahashi, R.J. Barfield, Analysis of ultrasonic vocalizations emitted by intruders during aggressive encounters among rats (Rattus norvegicus), J. Comp. Psychol. 97 (1983) 201– 206. [41] B. Thompson, K.C. Leonard, S.M. Brudzynski, Amphetamine-induced 50kHz calls from rat nucleus accumbens: a quantitative mapping study and acoustic analysis, Behav. Brain Res. 168 (2006) 64–73. [42] A.J. Wintink, S.M. Brudzynski, The related roles of dopamine and glutamate in the initiation of 50-kHz ultrasonic calls in adult rats, Pharmacol. Biochem. Behav. 70 (2001) 317–323. [43] M. Wohr, A. Borta, R.K. Schwarting, Overt behavior and ultrasonic vocalization in a fear conditioning paradigm: a dose-response study in the rat, Neurobiol. Learn. Mem. 84 (2005) 228–240.
Carbachol injections into the nucleus accumbens induce 50 kHz calls in rats Markus Fendt ∗ , Isabel Schwienbacher 1 , Hans-Ulrich Schnitzler Tierphysiologie, Zoologisches Institut, Fakult¨at f¨ur Biologie, Universit¨at T¨ubingen, Auf der Morgenstelle 28, D-72076 T¨ubingen, Germany Received 9 February 2006; received in revised form 22 February 2006; accepted 22 February 2006
Abstract In rats, different types of vocalization can be observed. High frequency vocalizations (so called 50 kHz calls) are believed to indicate an appetitive state of the emitting animal. This is supported by studies demonstrating that infusions of the dopamine agonist amphetamine into the nucleus accumbens (NAC), a key structure for appetitive behaviors, induce 50 kHz calls. Several studies during the last years demonstrated that not only infusions of dopamine agonists such amphetamine but also infusions of acetylcholine receptor agonists into the NAC stimulate the appetitive system. In present study, we tested whether infusions of the unspecific cholinergic agonist carbachol into the NAC are able to induce 50 kHz calls. Indeed, we observed a high number of 50 kHz calls after intra-NAC infusions of carbachol. The main frequency of the these calls was between 40 and 70 kHz, and the duration was mainly between 10 and 50 ms. We hypothesize that acetylcholine transmission within the NAC plays an important role in the induction of those ultrasonic calls indicating an appetitive state. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: 50 kHz calls; Acetylcholine; Appetitive system; Communication; Rats
Laboratory rats (Rattus norvegicus) emit different types of ultrasonic vocalization (USV) which are believed to play an important role in communication with conspecifics [5,26,37]. These USVs strongly vary in frequency and duration, but there two very often described variants which are thought to play an important role in the communication of the emotional state of the USV emitting animal. One type of these USVs has a high frequency (over 30 kHz), a duration shorter than 300 ms, and is usually termed as “50 kHz calls” [26]. The 50 kHz calls are mainly observable in appetitive situation: during sexual behavior and social contacts [2,10,16,32], as well as during anticipation of play [24], or food [11]. Furthermore, artificial rewarding stimuli such electrical brain stimulation or drugs that are experienced as rewarding as well as cues or contexts that predict such artificial reward induce 50 kHz calls [11,13,25,42]. The 50 kHz calls are clearly dissociated from another group of ultrasonic calls with much lower frequencies (between 18 and 25 kHz) and ∗ Corresponding author. Present address: Neuroscience Research, Novartis Institutes for BioMedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland. Tel.: +49 7071 2975347; fax: +49 7071 292618. E-mail address: [email protected] (M. Fendt). 1 Present address: Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Straße 65, D-88397 Biberach/Riß, Germany.
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.02.069
longer durations (more than 300 ms; [7]). These USV are usually termed “22 kHz calls” and are mainly emitted in and after the presence of aversive stimuli. For example, 22 kHz calls are observed during exposure to predators [3], to foot shocks [43], to startle stimuli [23], as well as after unfamiliar handling [9]. There are a small number of studies focusing on the neurobiology of USV in rats: whereas for 22 kHz calls the fear system including the amygdala and the mediobasal forebrain structures are crucial (e.g. [4,6,14]), the appetitive brain system appears to be important for 50 kHz calls. Systemic injections of amphetamine, a drug stimulating the appetitive system (e.g. [18]), induce 50 kHz calls [42]. The same effects are observed after intracranial injections of amphetamine into the Nucleus accumbens (NAC), one key structure of the appetitive system [12,41]. Furthermore, the anterior part of the hypothalamus seems to be involved in 50 kHz call production [15]. During the last years, a number of studies demonstrated that acetylcholine receptors within the NAC play an important role in the appetitive system: for example, appetitive learning is blocked by intra-NAC infusions of muscarinic receptor antagonists [35,36]. Furthermore, rats self-administer the unspecific acetylcholine receptor agonist carbachol directly into their NAC [19] and blockade of muscarinic receptors attenuates this carba-
M. Fendt et al. / Neuroscience Letters 401 (2006) 10–15
chol self-administration [19] indicating that carbachol infusions into the NAC stimulate the appetitive system. Based on these findings, we had the hypothesis that infusions of carbachol into the NAC are able to induce 50 kHz calls. The present study tested this hypothesis. Twelve male Sprague–Dawley rats (Charles River, Sulzfeld, Germany) weighing 220–260 g at the time of surgery were housed in groups of 5–6 rats/cage in a temperature controlled colony room under a 12:12 h light/dark cycle (lights on at 07:30). They were fed with 15 g standard rat chow/animal/day, and tap water was available ad libitum. Each rat was handled daily before and after surgery (handling started 5 days before surgery). The experiments were performed in accordance with international ethical guidelines for the care and use of animals for experiments and were approved by the local council of animal care (Regierungspr¨asidium T¨ubingen, ZP 4/02). Rats were anaesthetized with ketamine/xylazine (9:1, 100 mg/kg, i.p.) and placed into a stereotaxic frame. The skull was exposed and stainless steel guide cannulae (0.7 mm diameter) were implanted bilaterally aiming at the NAC (toothbar + 5 mm above the interaural plane) with the following distances to Bregma: rostrocaudal + 3.4 mm, mediolateral ± 1.5 mm, dorsoventral −7.2 mm (coordinates from [34]). The guide cannulae were fixed to the skull with acrylic cement (Kulzer, Wehrheim, Germany) and three anchoring screws. After surgery, the cannulae were fitted with stylets in order to maintain patency. The rats were allowed to recover completely from surgery (4–5 days). The unspecific acetylcholine receptor agonist carbachol (carbamylcholine chloride 99%, Acros, Geel, Belgium) was dissolved in saline and was administered bilaterally (4 ng/0.5 l per side) into the NAC with a velocity of 0.5 l/min. The injection cannulae were left in place for additional 2 min to allow diffusion of the solution away from the cannulae. The dose of 4 ng/0.5 l carbachol was chosen on the basis of a previous study showing that this dose leads to significant changes in the emotional state but only slightly affect motor behavior of the rats [38]. Latter study also showed that doses in the range of 1–4 ng carbachol had very similar behavioral effects. Higher doses which can be found in the literature (e.g. [19,27]) have very intensive effects on spontaneous motor behavior in our laboratory. Therefore, we decided to infuse only one weak dose of carbachol into the NAC. Immediately after injections into the NAC, the animals were put in a small open field (30 cm × 60 cm × 30 cm). During the next 30 min, USVs were recorded. All animals were tested twice and got saline or carbachol in a pseudorandomized order. The second test was carried out two days later and was identical to the first test. To record USVs, a custom made condensor microphone (University of T¨ubingen) was fixed at the top of each compartment. The microphones were sensitive between 10 and 200 kHz, with a flat frequency response (±3 dB) between 30 and 120 kHz and ±6 dB between 15 and 160 kHz. From 30 to 10 kHz and 120 to 200 kHz the sensitivity dropped by 0.2 dB/kHz. The signals were digitized (sample rate 192 kHz, 16 bit) and stored for subsequent analysis as .wav-files on a laptop computer. Sound analysis was performed with the colour sonograph software Selena (custom-
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made, T¨ubingen University). Signals were processed through a fast Fourier-transformation (512, Hann window) and displayed as colour sonograms. Frequency resolution and time resolution were determined by the sampling rate and the FFT points. In our analysis frequency resolution was 375 Hz and time resolution was 2.7 ms. For each signal, duration and best frequency (in the following just named “frequency”) were measured. Frequency was measured on the point of the highest sound intensity and duration was defined as the length between the beginning and the end of the USVs. USVs with a frequency of ≥30 kHz and duration of ≤300 ms were classified as so called 50 kHz USVs (cf. [26]). To analysis the USVs, the frequencies were sorted in different frequency ranges (30–40 kHz, >40–50 kHz, >50–60 kHz, etc.) and the durations in different duration ranges (<10 ms, 10–50 ms, >50–100 ms, etc.) and the percentage of emitted USVs of each frequency and duration range were calculated for each individual animal. Finally, the average over all animals was determined. After the tests, the rats were sacrificed with an overdose of pentobarbital (Nembutal). Their brains were removed and immersion-fixed in 8% paraformaldehyde and 20% sucrose. Frontal sections (50 m) were cut on a freezing microtome and Nissl stained with thionine. The injection sites were localized under a light microscope and were drawn onto plates taken from a rat brain atlas [33]. Since the number of USVs was not normally distributed (Kolmogorov-Smirnov test with Lillifors adaptation: p < 0.0001 for saline, p = 0.07 for carbachol infusions), a chi-square test was used for the statistical analysis of the effect of intra-accumbal carbachol on USVs. A value of p < 0.05 was considered to represent a significant effect. Fig. 1 shows the cannula placements for the rats that were included in the analysis of the effects of carbachol injections into the NAC (n = 11). One rat had misplaced cannulas (located in dorsal striatum) and was excluded from further analysis. The different symbols of the injection sites symbolize the amount of carbachol effect on USV (see below).
Fig. 1. Serial drawing of a frontal section of the NAC (+3.4 mm rostral to Bregma) depicting the injection sites of carbachol into the NAC. Abbreviations: AcbC—nucleus accumbens core, AcbSh—nucleus accumbens shell.
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M. Fendt et al. / Neuroscience Letters 401 (2006) 10–15
Fig. 2. Effects of acute carbachol injections into the NAC on the emission of 50 kHz calls. (A) Medians of the mean amount of 50 kHz USVs after injections with saline or carbachol. Carbachol infusions into the NAC increased the emission of 50 kHz USVs. *** p < 0.001, chi-square test. (B) Distribution of the mean frequency of the USVs. (C) Distribution of the mean duration of the USVs.
All recorded USVs had durations shorter than 300 ms and frequencies higher than 30 kHz and thus met the criterion for 50 kHz calls (cf. [26]). No other USVs were found. The emission of 50 kHz calls was strongly biased to carbachol infusions into the NAC; there were almost no calls after saline treatment (see Fig. 2A; chi-square test: comparison saline versus carbachol: χ2 = 4455; p < 0.001). Those injection sites which elicit a high number of 50 kHz calls (>500 calls/20 min) were exclusively located in the core region of the NAC.
Most of these 50 kHz USVs were emitted in the frequency range between 40 and 70 kHz with durations between 10 and 50 ms (see Fig. 2B and C). The basic structure of the USVs was rather similar in all individuals (Fig. 3). The signals often consisted of three elements with very variable durations. The first and the third element were frequency-modulated and covered frequency ranges above 55 kHz. The modulation direction was upward in the first and downward in the third element. The second element had a rather constant frequency always in the frequency range between 40 and 50 kHz. The rats also emitted combinations of only two of these three elements. Here, all possible combinations, i.e. the first and the second element, the first and third element, and the second and third element, have been observed. Furthermore, each of the three elements was also produced alone. Quite rarely, we found single elements which did not fit into the describe pattern, e.g. the single element in rat 4 in Fig. 3. In the present study, we investigated whether infusions of the cholinergic agonist carbachol into the NAC induce 50 kHz calls. Our results clearly show that rats emit a very high amount of 50 kHz calls after carbachol infusions into the NAC. It is likely that carbachol in the low dose we used here is acting mainly via muscarinic receptors since the behavioral effects of much higher doses of carbachol can totally be blocked by coadministration of the muscarinic antagonist scopolamine [19]. Thus, the present study supports previous studies demonstrating that cholinergic stimulation of the appetitive system induce 50 kHz calls [12,41,42]. It was postulated that 50 kHz calls provide a sensitive marker for a stimulated appetitive system [25]: rats emit 50 kHz calls during play and anticipation of play [24], sexual and mating behavior [2,32] and other appetitive forms of social contact [10] as well as during anticipation of food [11]. Fifty-kilohertz calls are not only observed during natural rewarding stimuli but also in the presence of artificial rewarding stimuli like pharmacological treatments and electrical stimulation of the medial forebrain bundle [11,12,25,41,42]. Furthermore, USV calls are also emitted during conditioned rewarded states, e.g. in contexts previously paired with rewarding drugs [25]. Some of the studies quoted above point to different brain structure which appears to be important for the induction of 50 kHz calls. The facts that systemic infusions of amphetamine is very potent in inducing 50 kHz calls [42] and that 50 kHz are emitted in appetitive states (e.g. [25]) suggest that the appetitive dopaminergic system is involved in the production of 50 kHz calls. This is supported by two studies measuring a high amount of 50 kHz calls after direct infusions of amphetamine into the NAC [12,41]. The present study clearly shows that also a stimulation of cholinergic receptors within the NAC is able to induce 50 kHz calls. In contrast to the amphetamine studies quoted above, those injection sites of carbachol in the present study, which were most effective in inducing 50 kHz calls, were exclusively located in the core region of the NAC (cf. Fig. 1). However, since infused drugs are able to diffuse about 1 mm in the brain [31], the individual numbers of induced 50 kHz calls were quite variable, and we did not infuse carbachol into adjacent areas (as an anatomical
M. Fendt et al. / Neuroscience Letters 401 (2006) 10–15
Fig. 3. Sonographs showing typical examples of USVs of five different individual rats recorded after carbachol injections into the NAC.
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control) such a localization of the carbachol effects is rather questionable. Beside the NAC, the anterior part of the hypothalamus/preoptic area is a further brain site, which was discussed to be important for 50 kHz call production [15]. This area is functionally connected with the NAC [1], i.e. pharmacological manipulation of this area affects the activity of the appetitive system (e.g. [8]). Taken together, the present study support the hypotheses that (1) the appetitive system, especially the NAC, is important for the generation of 50 kHz calls (cf. [26]) and that [37] infusions of acetylcholine agonists into the NAC activate the appetitive system (cf. [19]). It should be mentioned here that the exact function of acetylcholine within the NAC is still unclear. For example, acetylcholine transmission plays an important role in the processing of both, appetitive [28,35] and aversive stimuli ([30], but see also [29]). Against that, dopamine transmission within the NAC is thought to be determinant for the appetitive behavioral arousal (summarized in [20]). Since carbachol infusions into the NAC enhance dopamine transmission [17], we suggest that the carbachol effect on USV observed in the present experiment is mediated by an enhancement of dopamine transmission within the NAC. The 50 kHz calls we observed are consistent with previously characterized 50 kHz calls (cf. [10]), regarding to the frequency and duration range. Usually, these USVs are reported as calls in a range of 30–70 kHz. Most of the USVs we observed were in that frequency range, even though we also found USVs between 70 and 96 kHz. We cannot exclude that there were additional calls above the frequency of 96 kHz, since the frequency range of our system was limited to 96 kHz. The structure of the carbachol-induced 50 kHz calls measured in the present study varied strongly within and between individuals. Interestingly, the most USVs had a similar basic structure with three elements, seen in all the individuals. Such USVs with three elements and a similar frequency range were also described in previous study from our laboratory [21,22]. In latter studies, these calls were observed during sexual behavior but also during agonistic behavior (cf. [39,40]). In summary, the present study demonstrated that carbachol infusions into the NAC core region induce 50 kHz calls in male rats. This support the idea that the appetitive system, especially the NAC, is important for 50 kHz calls and that infusion of acetylcholine receptor agonists into the NAC stimulates the appetitive system. References [1] G.V. Allen, D.F. Cechetto, Functional and anatomical organization of cardiovascular pressor and depressor sites in the lateral hypothalamic area. II. Ascending projections, J. Comp. Neurol. 330 (1993) 421– 438. [2] R.J. Barfield, D.A. Thomas, The role of ultrasonic vocalizations in the regulation of reproduction in rats, Ann. N.Y. Acad. Sci. 474 (1986) 33–43. [3] R.J. Blanchard, D.C. Blanchard, R. Agullana, S.M. Weiss, Twentytwo kHz alarm cries to presentation of a predator, by laboratory rats living in a visible burrow system, Physiol. Behav. 50 (1991) 967– 972.
[4] S.M. Brudzynski, Ultrasonic vocalization induced by intracerebral carbachol in rats: localization and a dose-response study, Behav. Brain Res. 63 (1994) 133–143. [5] S.M. Brudzynski, Principles of rat communication: quantitative parameters of ultrasonic calls in rats, Behav. Genet. 35 (2005) 85–92. [6] S.M. Brudzynski, F. Barnabi, Contribution of the ascending cholinergic pathways in the production of ultrasonic vocalization in the rat, Behav. Brain Res. 80 (1996) 145–152. [7] S.M. Brudzynski, F. Bihari, D. Ociepa, X.W. Fu, Analysis of 22 kHz ultrasonic vocalization in laboratory rats: long and short calls, Physiol. Behav. 54 (1993) 215–221. [8] S.M. Brudzynski, G.J. Mogenson, Inhibition of amphetamine-induced locomotor activity by injection of carbachol into the anterior hypothalamic/preoptic area: pharmacological and electrophysiological studies in the rat, Brain Res. 376 (1986) 47–56. [9] S.M. Brudzynski, D. Ociepa, Ultrasonic vocalization of laboratory rats in respond to handling and touch, Physiol. Behav. 52 (1992) 655–660. [10] S.M. Brudzynski, A. Pniak, Social contacts and production of 50-kHz short ultrasonic calls in adult rats, J. Comp. Psychol. 116 (2002) 73– 82. [11] J. Burgdorf, B. Knutson, J. Panksepp, Anticipation of rewarding electrical brain stimulation evokes ultrasonic vocalization in rats, Behav. Neurosci. 114 (2000) 320–327. [12] J. Burgdorf, B. Knutson, J. Panksepp, S. Ikemoto, Nucleus accumbens amphetamine microinjections unconditionally elicit 50-kHz ultrasonic vocalizations in rats, Behav. Neurosci. 115 (2001) 940–944. [13] J. Burgdorf, B. Knutson, J. Panksepp, T.S. Shippenberg, Evaluation of rat ultrasonic vocalizations as predictors of the conditioned aversive effects of drugs, Psychopharmacology (Berl) 155 (2001) 35–42. [14] J.S. Choi, T.H. Brown, Central amygdala lesions block ultrasonic vocalization and freezing as conditional but not unconditional responses, J. Neurosci. 23 (2003) 8713–8721. [15] X.W. Fu, S.M. Brudzynski, High-frequency ultrasonic vocalization induced by intracerebral glutamate in rats, Pharmacol. Biochem. Behav. 49 (1994) 835–841. [16] L.A. Geyer, R.J. Barfield, Influence of gonadal hormones and sexual behavior on ultrasonic vocalization in rats: I. Treatment of females, J. Comp. Physiol. Psychol. 92 (1978) 438–446. [17] A.M. Gray, J.H. Connick, Clozapine-induced dopamine levels in the rat striatum and nucleus accumbens are not affected by muscarinic antagonism, Eur. J. Pharmacol. 362 (1998) 127–136. [18] C.J. Harmer, G.D. Phillips, Enhanced conditioned inhibition following repeated pretreatment with d-amphetamine, Psychopharmacology 142 (1999) 120–131. [19] S. Ikemoto, B.S. Glazier, J.M. Murphy, W.J. McBride, Rats selfadminister carbachol directly into the nucleus accumbens, Physiol. Behav. 63 (1998) 811–814. [20] S. Ikemoto, J. Panksepp, The role of nucleus accumbens dopamine in motivated behavior: a unifying interpretation with special reference to reward-seeking, Brain Res. Brain Res. Rev. 31 (1999) 6–41. [21] M.T. Kaltwasser, Vergleichende Untersuchungen zum akustischen Kommunikationsverhalten und zur Orientierung bei Rattus rattus und Rattus norvegicus [Comparitive studies on acoustic communication and navigation in Rattus rattus and Rattus norvegicus], Ph.D. thesis, University of T¨ubingen, 1985. [22] M.T. Kaltwasser, Acoustic signaling in the black rat (Rattus rattus), J. Comp. Psychol. 104 (1990) 227–232. [23] M.T. Kaltwasser, Acoustic startle induced ultrasonic vocalization in the rat: a novel animal model of anxiety? Behav. Brain Res. 43 (1991) 133–137. [24] B. Knutson, J. Burgdorf, J. Panksepp, Anticipation of play elicits highfrequency ultrasonic vocalizations in young rats, J. Comp. Psychol. 112 (1998) 65–73. [25] B. Knutson, J. Burgdorf, J. Panksepp, High-frequency ultrasonic vocalizations index conditioned pharmacological reward in rats, Physiol. Behav. 66 (1999) 639–643. [26] B. Knutson, J. Burgdorf, J. Panksepp, Ultrasonic vocalizations as indices of affective states in rats, Psychol. Bull. 128 (2002) 961–977.
M. Fendt et al. / Neuroscience Letters 401 (2006) 10–15 [27] N. Koshikawa, Y. Yoshida, M. Kitamura, T. Saigusa, M. Kobayashi, A.R. Cools, Stimulation of acetylcholine or dopamine receptors in the nucleus accumbens differentially alters dopamine release in the striatum of freely moving rats, Eur. J. Pharmacol. 303 (1996) 13–19. [28] G.P. Mark, A.E. Kinney, M.C. Grubb, A.S. Keys, Involvement of acetylcholine in the nucleus accumbens in cocaine reinforcement, Ann. N.Y. Acad. Sci. 877 (1999) 792–795. [29] G.P. Mark, P.V. Rada, T.J. Shors, Inescapable stress enhances extracellular acetylcholine in the rat hippocampus and prefrontal cortex but not the nucleus accumbens or amygdala, Neuroscience 74 (1996) 767–774. [30] G.P. Mark, J.B. Weinberg, P.V. Rada, B.G. Hoebel, Extracellular acetylcholine is increased in the nucleus accumbens following the presentation of an aversively conditioned taste stimulus, Brain Res. 688 (1995) 184–188. [31] J.H. Martin, Autoradiographic estimation of the extent of reversible inactivation produced by microinjection of lidocaine and muscimol in the rat, Neurosci. Lett. 127 (1991) 160–164. [32] T.K. McIntosh, R.J. Barfield, L.A. Geyer, Ultrasonic vocalisations facilitate sexual behaviour of female rats, Nature 272 (1978) 163–164. [33] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, 1997. [34] L.J. Pellegrino, A.S. Pellegrino, A.J. Cushman, A Stereotaxic Atlas of the Rat Brain, Plenum Press, New York, 1979. [35] W.E. Pratt, A.E. Kelley, Nucleus accumbens acetylcholine regulates appetitive learning and motivation for food via activation of muscarinic receptors, Behav. Neurosci. 118 (2004) 730–739.
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[36] L. Ramirez-Lugo, S. Zavala-Vega, F. Bermudez-Rattoni, NMDA and muscarinic receptors of the nucleus accumbens have differential effects on taste memory formation, Learn. Mem. 13 (2006) 45–51. [37] C. Sanchez, Stress-induced vocalisation in adult animals. A valid model of anxiety? Eur. J. Pharmacol. 463 (2003) 133–143. [38] I. Schwienbacher, M. Fendt, H.-U. Schnitzler, R.F. Westbrook, R. Richardson, Carbachol injections into the nucleus accumbens disrupts acquisition and expression of fear-potentiated startle and freezing in rats, Neuroscience, in press. [39] L.K. Takahashi, D.A. Thomas, R.J. Barfield, Analysis of ultrasonic vocalizations emitted by residents during aggressive encounters among rats (Rattus norvegicus), J. Comp. Psychol. 97 (1983) 207– 212. [40] D.A. Thomas, L.K. Takahashi, R.J. Barfield, Analysis of ultrasonic vocalizations emitted by intruders during aggressive encounters among rats (Rattus norvegicus), J. Comp. Psychol. 97 (1983) 201– 206. [41] B. Thompson, K.C. Leonard, S.M. Brudzynski, Amphetamine-induced 50kHz calls from rat nucleus accumbens: a quantitative mapping study and acoustic analysis, Behav. Brain Res. 168 (2006) 64–73. [42] A.J. Wintink, S.M. Brudzynski, The related roles of dopamine and glutamate in the initiation of 50-kHz ultrasonic calls in adult rats, Pharmacol. Biochem. Behav. 70 (2001) 317–323. [43] M. Wohr, A. Borta, R.K. Schwarting, Overt behavior and ultrasonic vocalization in a fear conditioning paradigm: a dose-response study in the rat, Neurobiol. Learn. Mem. 84 (2005) 228–240.