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Contribution of CaMKIV to injury and fear- induced ultrasonic vocalizations in adult mice
© Ko et al; licensee BioMed Central Ltd. 2005
Received: 15 December 2004
Accepted: 22 March 2005
Published: 22 March 2005
Calcium-calmodulin dependent protein kinase IV (CaMKIV) is a protein kinase that activates the transcription factor CREB. Our previous work demonstrated that mice lacking CaMKIV had a defect in fear memory while behavioral responses to noxious stimuli were unchanged. Here, we measured ultrasonic vocalizations (USVs) before and after fear conditioning and in response to a noxious injection of capsaicin to measure behavioral responses to emotional stimuli. Consistent with previous findings, behavioral nociceptive responses to capsaicin were undistinguishable between wild-type and CaMKIV-/- mice. Wild-type animals showed a selective increase in 50 kHz USVs in response to capsaicin while such an increase was absent in CaMKIV-/- mice. The foot shock given during fear conditioning caused an increase in 30 kHz USVs in both wild-type and CaMKIV-/- mice. When returned to the context one hour later, USVs from the wild-type were significantly decreased. Additionally, the onset of a tone, which had previously been paired with the foot shock, caused a significant decrease in USVs during auditory conditioning. CaMKIV-/- mice showed significantly less reduction in USVs when placed in the same context three days after receiving the shock, consistent with the decrease in freezing reported previously. Our results provide a new approach for investigating the molecular mechanism for emotional vocalization in mice and suggest that CaMKIV dependent signaling pathways play an important role in the emotional response to pain and fear.
The transcription factor CREB (cAMP response-element binding protein) has been implicated in several aspects of higher brain function, such as spatial memory, fear conditioning, conditioned taste aversion and social transmission of food preference [1–7]. Recently, Barrot et al. (2002) reported that changes in CREB expression in the nucleus accumbens shell altered behavioral responses to emotional stimuli. Both the rewarding stimulus of morphine and the aversive stimulus of stress activated CRE-mediated transcription in the nucleus accumbens shell . This study suggests an important role for CREB activation in the behavioral responses to emotional stimuli.
CaMKIV, along with other kinases, activates CREB by phosphorylating it at Ser 133. Phosphorylated CREB recruits the transcriptional co-activator CBP (CREB binding protein) which leads to the activation of CRE (cAMP response element) containing promoters and ultimately to gene expression [9–11]. CaMKIV is a calcium dependent protein kinase that is detected predominantly in the nuclei of neurons [12, 13]. The nuclear location of CaMKIV, its broad distribution in forebrain areas related to pain, attention and emotion, [10, 14–18] and its activation of CREB make it an attractive candidate for the processing of higher brain functions. Our recent study of CaMKIV knockout mice (CaMKIV-/-) showed that fear memory was impaired [10, 19], while behavioral responses to noxious stimuli were indistinguishable from that of wild-type mice. CREB activation by fear conditioning was absent in the amygdala of CaMKIV-/- mice . This suggests that CREB activation in response to emotional events may be impaired in CaMKIV-/- mice as well. Therefore, we hypothesize that CaMKIV-/- mice may exhibit a defect in behavioral responses to emotional stimuli.
Currently, responses to physiologically important stimuli, such as pain and fear, are based on the assessment of motor activities, such as licking and freezing. Here we propose to look at an additional measure, ultrasonic vocalizations (USVs), in order to uncover emotional responses less related to the motor response. USVs are an important form of communication in rodents [20–22]. USVs from rats have been recorded in response to painful or startling stimuli , drugs of abuse [24–26], stressful situations , and as a measure of fear memory . USVs are considerably less studied in adult mice. Sonographic analysis of USVs emitted during male-female pairings show that the majority of USVs were at 70 kHz with some calls at 40 kHz . USVs have not been studied in adult mice in response to the noxious stimulus of capsaicin or as a measure of fear memory.
In this study, we measured USVs emitted from isolated, adult male mice in parallel with conventional behavioral parameters to evaluate responses to emotionally relevant stimuli. We report here that USVs emitted from adult male mice may be used as an index of internal emotional states and that CaMKIV has a significant contribution to the USVs produced in response to capsaicin and fear conditioning.
CaMKIV-/- mice were derived as described  and bred for several generations (F12-F16) on C57Bl/6 background. Control wild-type mice were adult male (8–12 weeks old) C57Bl/6 mice from Jackson Laboratories. All efforts were made to minimize the number of animals used in this study and to minimize their suffering. At the conclusion of experiments, animals were sacrificed by an overdose of inhaled anesthesia (halothane). The animals were housed on a 12 h: 12 h light: dark cycle with food and water available ad libitum. All mouse protocols are in accordance with NIH guidelines and approved by the Animal Care and Use Committee of University of Toronto. No visual difference between wild-type and CaMKIV-/- mice is noticeable, and experiments were performed blind to the genotype.
Open field activity monitor
To record horizontal locomotor activity we used the Activity Monitor system from Med Associates (43.2 × 43.2 × 30.5 cm; MED-associates, St. Albans, VT). Briefly, this system uses paired sets of photo beams to detect movement in the open field and movement is recorded as beam breaks. The open field is placed inside an isolation chamber with dim illumination and a fan. Each subject was placed in the center of the open field and activity was measured for 30, 60 or 120 minutes.
Ultrasonic vocalizations (USVs) were detected using four mini-3 ultrasonic detectors (Ultrasound Advice, London UK), each tuned to a different frequency level ranging from 20 kHz to 130 kHz. The detectors were suspended in one row of four along one side of the open field 19 cm from the floor. The total duration and number of USVs were measured using UltraVox data acquisition software (Noldus Information Technology, Leesburg, Va.). Each transition from vocalization to silence is time stamped, written as a new record and exported into a database for analysis. Before each experiment, the ultrasonic detectors were calibrated so that no USVs were recorded when no animal was present in the chamber. Four frequencies were recorded for each experiment. We set our recording frequencies to represent the range of sound possible from mice. The ultrasonic detectors detect signals within 4 kHz of the set dial frequency. We therefore represent a range from 26 to 124 kHz with our frequency settings at 30, 50, 90, and 120 kHz. The individual frequencies were selected both to reflect a wide distribution of calls and to mirror work established in rats which report USVs emitted selectively at 22 and 50 kHz [25, 30]. We choose to focus on USVs at 30 kHz since preliminary studies showed only a small amount of USVs detected at 20 kHz. USV data are presented as a rate (ms/sec).
All mice were habituated to the test chamber for 3 minutes before conditioning. During conditioning, mice were presented with a 30 s tone, the conditioned stimulus (85 dB, 2800 Hz) and 28 s later received the foot shock (0.75 mA) for 2 seconds, unconditioned stimulus. Animals were returned to the same chamber 1 hour, 1, 3, and 7 days later and USVs were recorded. One hour after being placed in the contextual environment, animals were placed into a novel chamber for a total of 6 min and the tone was played for the last 3 minutes. USVs were recorded in the novel environment before and during the tone 1 hour, 1, 3, and 7 days after training.
All animals were gently restrained by the experimenter before intraplantar injection. Capsaicin (1 μg/10 μl) was dissolved in 7.5 % DMSO and injected into the right hind paw at a volume of 10 μl. Formalin (5%, 10 μl) was dissolved in saline and injected into the right hind paw at a volume of 10 μl. Saline was injected as a control.
Data analysis and statistics
Open field activity was analyzed using the Activity monitor program. USVs were analyzed using the UltraVox program. Statistical comparisons were made using the t- test, paired t-test or One way or two way ANOVA (Student-Newmann-Keuls test was used for post hoc comparison). All data is represented by the mean +/- S.E.M. In all cases, p < 0.05 is considered statistically significant.
USVs from adult male mice in a novel, open field
During 30 minutes in the open field, mice emitted significantly more USVs at 30 kHz (n = 17) compared to 20 (n = 8) or 40 kHz (n = 9) (p < 0.05 for both, Figure 1B). In general there were very few USVs at frequencies higher than 50 kHz. For example, the highest rate of USV production was at 30 kHz (mean 11.52 ± 2.04 ms/sec, n = 17), which was significantly higher than USVs at 50 and 90 kHz (mean 0.53 ± 0.19 ms/sec; 0.14 ± 0.09 ms/sec, respectively, n = 9 and 7, p < 0.001) (Figure 1B–C). Only one 120 kHz USVs was detected (mean 0.03 ± 0.03 ms/sec, n = 7). There was also a significant difference in the duration of a single USV between frequencies. USVs at 30 kHz (83 ± 9 ms) were significantly longer than USVs at 50 kHz (56 ± 2 ms), 90 kHz (49 ± 4 ms), and 120 kHz (60 ± 9 ms) (Figure 1A).
USVs induced by a noxious stimulus
USVs after classic fear conditioning
In general, USVs were higher in the absence of the tone than when the tone was played. USVs were significantly decreased at the onset of the tone one hour (t = 2.24, p < 0.05), one day (t = 2.75, p < 0.01), but not three and seven days after conditioning (Figure 5D,E). Some animals emitted more USVs in the last three minutes than in the first (4 of 17 mice 1 hour after training). This added high variability when calculating the percentage of reduction at the tone, therefore, these few animals were excluded from Figure 4F. The tone decreased USVs by 55.4 ± 6.3 % one hour after conditioning; seven days later USVs were only decreased by 28.1 ± 2.9% (Figure 5F).
CaMKIV-/- mice in a novel open field
USVs of CaMKIV-/- mice induced by a noxious stimulus
USVs from CaMKIV-/- mice after fear conditioning
In the present study, we describe USVs from isolated adult male mice under different conditions. We show that the majority of USVs emitted in the open field occur within the 30 kHz range (26–34 kHz) and that the duration of a single call varies between frequencies. A noxious hind paw injection of capsaicin decreased locomotion and had no effect on 30 kHz USVs while significantly increasing 50 kHz USVs. While the foot shock significantly increased USVs at all frequencies, 30 kHz USVs made up the vast majority of all vocalization before and after fear conditioning. Additionally, 30 kHz USVs were significantly decreased in the context after conditioning and at the onset of the tone in a novel environment. CaMKIV-/- mice did not differ from wild-type in vocalization in the open field or in 30 kHz USVs in response to capsaicin. However, the increase in 50 kHz USVs found in the wild-type in response to capsaicin was absent in the knockout, while licking and locomotor responses were unchanged. No difference was found in USV production during training or auditory conditioning; however, USVs were significantly less reduced in CaMKIV-/- mice in the context where they had received the shock-tone pairing three days prior. Taken together, these results suggest a role for CaMKIV in the vocal behavioral responses to capsaicin and fear conditioning, and imply that the USV response can distinguish physiological responses to stimuli.
We have previously reported that CaMKIV-/- mice have unaltered responses to acute noxious stimuli while having a defect in fear memory . However, these results were based solely off of traditional measures of pain and fear-related behaviors (i.e. licking and freezing). Here, we employ an additional measure (USVs) to characterize the role of CaMKIV in the behavioral responses to pain and fear. A great deal of evidence supports the use of USVs as an emotional indicator in rats; they have been used as a measure of anxiety, fear memory, and as a tool to predict the impact of drug withdrawal [25, 27, 28]. Mice also use USVs as a form of communication, thus, it is reasonable to assume that mice would emit USVs in response to physiologically relevant events. Advances in genetic technology have increased the production of transgenic mice and USV analysis may allow for a more thorough neurobehavioral characterization. Also, current methods of behavioral observation require manual scoring and direct observation, which include human error and bias. We propose that USV analysis of mice will compliment existing methods of behavioral characterization.
In order to use USVs in mice as a useful measure of behavior, we first observed USV production at four frequency ranges in the open field. We chose to observe USVs along with open field activity since it allows us to directly correlate exploratory and vocal behavior, and provides a good context in which to observe responses in an isolated environment. Patterns of movement in the open field are widely regarded to reflect anxiety and provide a quantifiable and unbiased measure when considering the effects of various treatments on behavior. In general, there is an inverse relationship between activity and anxiety in the open field. Therefore, simultaneous recording of open field locomotor activity and vocalization will allow us to compare changes in USVs with changes in anxiety-related activity levels.
Both saline and capsaicin injections decreased locomotor activity in the open field when compared to animals that did not receive an injection (Fig 2C,D). Capsaicin has also been shown to decrease exploratory activity in rats . However, only capsaicin-injected animals showed an increase in 50 kHz USVs, while other frequencies were unchanged as compared to controls and saline-injected mice (Fig. 2A,B). This suggests that the stress from handling and the hind paw injection decreases locomotor activity in mice whether they receive capsaicin or saline. The fact that only capsaicin-injected animals show an increase in 50 kHz USVs suggests that this is a response to the noxious character of the capsaicin and not to the injection itself. CaMKIV-/- mice, which have previously been shown not to have a defect in motor responses to noxious stimuli , were similar to wild-type mice in the reduction of locomotor activity as well as in the behavioral licking response to capsaicin (Fig. 6A,B). Again, the only difference between the groups occurred at 50 kHz USVs. In this case, CaMKIV-/- mice failed to show the 50 kHz increase in response to capsaicin, while USVs were unchanged at other frequencies (Figure 7C,D). These results imply that CaMKIV may play a role in emotional, and not motor, responses to noxious stimuli. Also, 50 kHz USVs may index a negative state in response to capsaicin that is independent of locomotor and licking responses. The increase in 50 kHz USVs was also seen after formalin injection (Figure 3), strengthening our hypothesis that 50 kHz USVs may reflect the animal's reaction to the noxious character of the injection.
Several studies report that 50 kHz USVs may represent a positive affective state in rats [25, 35, 39]. While we cannot speculate as to why there would be such a species difference in the modulation of 50 kHz USVs, we suggest that mice and rats use distinct frequencies to communicate negative and positive affective states. For example, 50 kHz USVs are emitted from male rats when mounting the female , in contrast, male mice emit 70 kHz USVs when mounting . A previous study reports that USVs are notaffected by carrageenan induced inflammation, or chronic pain caused by Complete Freund's adjuvant (CFA), when measured in rats . Further studies are needed todetermine if these models of chronic pain can elicit a change in 50 kHz USVs in mice.
Fear memory of a noxious foot shock was measured by recording behavioral freezing and was shown to be decreased in CaMKIV-/- mice . In these experiments, we used the same procedures, but instead measured USVs. USVs have been used along with freezing, heart rate, locomotion, and defecation as a measure of fear in rats after conditioning . CaMKIV-/- mice did not differ in their response to the foot shock or in vocalization to the tone as compared to wild-type. However, the knockout showed significantly less reduction in 30 kHz USVs in the context three days after fear conditioning. This suggests that changes in USVs after fear conditioning were more sensitive to the context than to the auditory tone, and that USVs are quite different from behavioral freezing responses in that no difference has been reported between contextual and auditory fear memory in CaMKIV-/- mice.
We propose that 30 kHz USVs can be used as a measure of fear memory in mice. Freezing responses in CaMKIV-/- mice were decreased by one day after fear conditioning , but USVs were not significantly decreased until three days after conditioning in this study. USVs after fear conditioning in rats are considered to be a slowly acquired measure of fear memory since three training sessions were required to condition them to the context . It is possible that USVs, as a measure of fear memory in mice, may be a slower measure than the freezing response. The decrease in USVs emitted in the context after conditioning was still present seven days later, further studies are needed to determine how long this affect lasts.
In summary, our results demonstrate that capsaicin, formalin and fear conditioning induced changes in vocalization at different frequencies. In the case of an acute insult like capsaicin, enhancement of vocalization is mostly at 50 kHz. For fear conditioning, only vocalizations at 30 kHz were dramatically altered. We also report a role for CaMKIV in mediating responses to emotional stimuli. CaMKIV-/- mice did not emit capsaicin-induced 50 kHz vocalizations while having the same behavioral response measured by licking. Lastly, CaMKIV-/- mice showed an increase in vocalization over the wild-type three days after fear conditioning. This correlates with our previous results in CaMKIV-/- mice that show a decrease in fear memory, and the fact that CaMKIV is widely distributed in cognition-related forebrain areas .
CaMKIV is an important member of the signal transduction pathway leading to CREB activation and gene expression and has been reported to participate in activation of the ERK pathway . We believe that CaMKIV plays a role in mediating emotional responses to painful and fearful stimuli and that USV analysis will provide a model by which we can quantitatively measure an animal's internal affective state. The current manuscript focuses on characterizing USVs as a response to a noxious chemical injection and as a measure of fear memory. The evaluation of anxiolytic and analgesic drugs is important and should be considered in future studies. Our present results suggest that USV analysis may allow us to elucidate the molecular mechanisms underlying emotional responses.
We would like to thank Keiko Ishioka, Fanny Shum, Hoi Ki Ding, and everyone in the labs of M.Z. and T.C for their help and advice. Supported by grants from the EJLB-CIHR Michael Smith Chair in Neurosciences and Mental Health in Canada, Canadian Research Chair to M.Z, and the NIH NINDS 42733 and 38680.
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