Identification of a pheromone that increases anxiety in rats.

Chemical communication plays an important role in the social lives of various mammalian species. Some of these chemicals are called pheromones. Rats release a specific odor into the air when stressed. This stress-related odor increases the anxiety levels of other rats; therefore, it is possible that the anxiety-causing molecules are present in the stress-related odorants. Here, we have tried to identify the responsible molecules by using the acoustic startle reflex as a bioassay system to detect anxiogenic activity. After successive fractionation of the stress-related odor, we detected 4-methylpentanal and hexanal in the final fraction that still possessed anxiogenic properties. Using synthetic molecules, we found that minute amounts of the binary mixture, but not either molecule separately, increased anxiety in rats. Furthermore, we determined that the mixture increased a specific type of anxiety and evoked anxiety-related behavioral responses in an experimental model that was different from the acoustic startle reflex. Analyses of neural mechanisms proposed that the neural circuit related to anxiety was only activated when the two molecules were simultaneously perceived by two olfactory systems. We concluded that the mixture is a pheromone that increases anxiety in rats. To our knowledge, this is the first study identifying a rat pheromone. Our results could aid further research on rat pheromones, which would enhance our understanding of chemical communication in mammals.

The emission of stress-induced 22-kHz calls in female rats is independent of testosterone levels.

Although emission of ultrasonic calls in rats induced by stress, referred to as "22-kHz calls," is dependent on circulating testosterone levels in males, it is still unknown whether the same testosterone-based regulation is applicable to female rats. In this study, we investigated the sex difference in the emission of air-puff-induced 22-kHz calls in rats on the basis of the hypothesis that female rats would emit fewer 22-kHz calls, and assessed whether male-like circulating testosterone levels can also influence the emission of 22-kHz calls in females. The experimental results showed that female rats emit significantly fewer 22-kHz calls than male rats. However, male-like circulating testosterone levels have little effect on the emission of 22-kHz calls in females. Hence, it is suggested that there is a sex difference in stress-induced 22-kHz calls in rats and, in contrast to male rats, the emission of 22-kHz calls in female rats may be independent of circulating testosterone levels, as tested in response to air-puff stimulation.

Relationship between 22-kHz calls and testosterone in male rats.

Ultrasonic calls in rats induced by the presence of a predator, referred to as "22-kHz calls," are mainly emitted by socially dominant male rats. Testosterone levels are closely related to social dominance in male rats. In the present study, we investigated the relationship between the emission of stress-induced 22-kHz calls and circulating testosterone levels in male rats, using a combination of surgery (castration or sham operation) and chronic steroid administration (testosterone or cholesterol) to modify circulating testosterone levels. We also assessed the effects of androgen and/or estrogen receptor antagonists on the emission of 22-kHz calls in male rats. An air puff stimulus, known to reliably induce 22-kHz calls in rats, was used as a stressor. Castrated rats with cholesterol implants exhibited significantly fewer 22-kHz calls than rats that had received a sham operation and cholesterol implants, and there was no significant difference between castrated rats with testosterone implants and rats that had received a sham operation and cholesterol implants. Only male rats pretreated with a binary mixture of androgen and estrogen antagonists exhibited significantly fewer 22-kHz calls than controls. These results show that testosterone in male rats has a positive effect on the emission of stress-induced 22-kHz calls, and the calls may be regulated by the activation of both androgen and estrogen receptors.

The acoustic startle reflex as a tool for assessment of odor environment effects on affective states in laboratory mice.

Apart from self and conspecific odors, odors from other species also influence the affective states in laboratory mice (Mus musculus musculus) in their home cages and during experimental procedures, possibly inducing confusion and inconsistency in experimental data. Thus, it is important to detect the types of animal odors associated with housing, husbandry, and laboratory practice that can arouse different types of affective changes in mice. Here, we aimed to test the effectiveness of the acoustic startle reflex (ASR) in detecting changes in the affective states of laboratory mice due to animal-derived-odor as it has a non-zero baseline, and can be enhanced or attenuated by positive or negative affective shifts, respectively. We used ASR to examine the affective changes in mice that were induced by bedding odors and an alarm pheromone. The odor of bedding obtained from the mice' home cages significantly attenuated the ASR, suggesting positive affective shifts in the test mice, whereas that from bedding obtained from rat cages significantly enhanced the ASR, suggesting negative affective shifts. No significant changes in ASR were observed in mice presented with the odor of bedding obtained from cages of unfamiliar conspecifics. In contrast, there was significant ASR enhancement in mice exposed to volatile components of alarm pheromones trapped in water, suggesting negative affective shifts. Thus, our findings show that ASR may be a valuable tool in assessing the effects of odors on the affective states in laboratory mice.

The effect of playback of 22-kHz and 50-kHz ultrasonic vocalizations on rat behaviors assessed with a modified open-field test.

Juvenile and adult rats emit two affectively different types of ultrasonic vocalizations (USVs), namely aversive 22-kHz and appetitive 50-kHz USVs. Aversive 22-kHz USVs are considered to be alarm calls that communicate negative affective states to conspecific receivers. Although the alarming effects of playback of 22-kHz USVs were reported recently, behavioral data showing those effects are still not abundant. Appetitive 50-kHz USVs are considered to communicate positive affective states to conspecific receivers, to pace and coordinate social behavior. In line with this, playback of 50-kHz USVs has been found to initiate behavioral activation and induce approach behavior in receiver rats. However, most of these playback studies have used male 50-kHz USVs; thus, it seems to remain unclear whether female 50-kHz USVs exert a similar social attractant effect on male rats. To investigate these issues, we performed modified open-field tests, during which USVs were continuously presented for 15 min to male receivers. In these tests, if negative affective changes are evoked in subject rats, the time spent in the open arena decreases, while the time spent on defensive behaviors increases. In contrast, when positive affective changes are evoked, the opposite phenomenon is observed. Playback of male aversive 22-kHz USVs induced anxiety-related defensive responses in receivers. However, playback of female appetitive frequency-modulated (FM) 50-kHz USVs increased opposite, appetitive pattern of exploratory behavior with increased exploration. The results indicate that playback of male aversive 22-kHz and female appetitive 50-kHz USVs might induce behavioral responses probably associated with negative and positive affective states in male rats, respectively, suggesting the validity of rat USVs as an animal model of vocal communication of emotion.

The effect of vapor of propylene glycol on rats.

Propylene glycol (PG) is commonly used as a solvent for odorous chemicals employed in studies of the olfactory system because PG has been considered to be odorless for humans and other animals. However, if laboratory rats can detect the vapor of PG and if exposure to this influences behaviors, such effects might confound data obtained from experiments exposing conscious rats to odorants dissolved in PG. Therefore, we examined this issue using differences in the acoustic startle reflex (ASR) as an index. We also conducted a habituation/dishabituation test to assess the ability of rats to detect the vapor of PG. In addition, we observed Ca(2+) responses of vomeronasal neurons (VNs) in rats exposed to PG using the confocal Ca(2+)-imaging approach. Pure PG vapor significantly enhanced the ASR at a dose of 1 x 10(-4) M, which was much lower than the dose for efficiently detecting. In Ca(2+) imaging, VNs were activated by PG at a dose of 1 x 10(-4) M or lower. These results suggest that PG vapor acts as an aversive stimulus to rats at very low doses, even lower than those required for its detection, indicating that we should consider such effect of PG when it is employed as a solvent for odorants in studies using conscious rats. In addition, our study suggests that some non-pheromonal volatile odorants might affect animal behaviors via the vomeronasal system.

The volatility of an alarm pheromone in male rats.

The volatility of an alarm pheromone in male rats. PHYSIOL BEHAV 00(0) 000-000, 2008. We previously reported that an alarm pheromone released from the perianal region of male rats is perceived by the vomeronasal organ and evokes stress-induced hyperthermia and defensive and risk assessment behavior. In addition, we recently reported that the alarm pheromone enhances the acoustic startle reflex (ASR). However, in contrast to our knowledge about such biological aspects of the pheromone, information concerning the physical character of the alarm pheromone is extremely limited. In this study, we investigated the volatility of the alarm pheromone using enhancement of the ASR as an index of the pheromone effect. The alarm pheromone enhanced the ASR when it was presented at a distance of 10 mm but not at 200 mm. In addition, the pheromone effect was observed even after the pheromone was trapped in the adsorbent (Tenax) and then extracted using purified water. These results suggest that the alarm pheromone is both volatile and water soluble.

Lowering barometric pressure induces neuronal activation in the superior vestibular nucleus in mice.

Weather changes accompanied by decreases in barometric pressure are suggested to trigger meteoropathy, i.e., weather-related pain. We previously reported that neuropathic pain-related behavior in rats is aggravated by lowering barometric pressure, and that this effect is abolished by inner ear lesions. These results suggest that mechanisms that increase vestibular neuronal activity may parallel those that contribute to meteoropathy generation. However, it remains unknown whether changes in barometric pressure activate vestibular neuronal activity. To address this issue, we used expression of c-Fos protein as a marker for neural activation. Male and female mice were placed in a climatic chamber, and the barometric pressure was lowered by 40 hPa, from 1013 hPa, for 50 min (LP stimulation). The total number of c-Fos-positive cells in the vestibular nuclei was counted bilaterally after LP stimulation. We also video-recorded mouse behaviors and calculated the total activity score during the LP stimulation. LP stimulation resulted in significant c-Fos expression in the superior vestibular nucleus (SuVe) of male and female mice. There was no effect of LP stimulation on the total activity score. These data show that distinct neurons in the SuVe respond to LP stimulation. Similar mechanisms may contribute to the generation of meteoropathy in humans.

Enhancement of the acoustic startle reflex by an alarm pheromone in male rats.

Recently, we reported that an alarm pheromone released from the perianal region of male rats aggravated stress-induced hyperthermia and increased defensive and risk assessment behaviors in recipient male rats. Based on these results, we hypothesized that the primary effect of the alarm pheromone is to increase anxiety; however, there is still no clear evidence for this pheromone effect. Therefore, we examined this issue by assessing the effect of the alarm pheromone on the acoustic startle reflex (ASR), which is a useful index for studying negative emotions such as anxiety in rats. The alarm pheromone enhanced the ASR for 105-dB auditory stimuli, but not for those of 90 and 120 dB, when these three intensities of sound were used randomly. The same results were obtained when one of these three intensities was used repeatedly. In addition, pretreatment with diazepam (i.p.) at doses of 0.7 and 2.0 mg/kg suppressed the ASR of the pheromone recipients, whereas the lower dose (0.2 mg/kg) slightly attenuated the pheromone effect and the control injection (vehicle) had no effect. These results indicate that the alarm pheromone enhances the ASR by increasing anxiety in recipient rats, suggesting that the primary effect of the alarm pheromone is to increase the anxiety level.

The CCR4-NOT deadenylase complex controls Atg7-dependent cell death and heart function.

Shortening and removal of the polyadenylate [poly(A)] tail of mRNA, a process called deadenylation, is a key step in mRNA decay that is mediated through the CCR4-NOT (carbon catabolite repression 4-negative on TATA-less) complex. In our investigation of the regulation of mRNA deadenylation in the heart, we found that this complex was required to prevent cell death. Conditional deletion of the CCR4-NOT complex components Cnot1 or Cnot3 resulted in the formation of autophagic vacuoles and cardiomyocyte death, leading to lethal heart failure accompanied by long QT intervals. Cnot3 bound to and shortened the poly(A) tail of the mRNA encoding the key autophagy regulator Atg7. In Cnot3-depleted hearts, Atg7 expression was posttranscriptionally increased. Genetic ablation of Atg7, but not Atg5, increased survival and partially restored cardiac function of Cnot1 or Cnot3 knockout mice. We further showed that in Cnot3-depleted hearts, Atg7 interacted with p53 and modulated p53 activity to induce the expression of genes encoding cell death-promoting factors in cardiomyocytes, indicating that defects in deadenylation in the heart aberrantly activated Atg7 and p53 to promote cell death. Thus, mRNA deadenylation mediated by the CCR4-NOT complex is crucial to prevent Atg7-induced cell death and heart failure, suggesting a role for mRNA deadenylation in targeting autophagy genes to maintain normal cardiac homeostasis.

Changes in acoustic startle reflex in rats induced by playback of 22-kHz calls.

In aversive or dangerous situations, adult rats emit long characteristic ultrasonic calls, often termed "22-kHz calls," which have been suggested to play a role of alarm calls. Although the playback experiment is one of the most effective ways to investigate the alarming properties of 22-kHz calls, clear behavioral evidence showing the anxiogenic effects of these playback stimuli has not been directly obtained to date. In this study, we investigated whether playback of 22-kHz calls or synthesized sine tones could change the acoustic startle reflex (ASR), enhancement of which is widely considered to be a reliable index of anxiety-related negative affective states in rats. Playback of 22-kHz calls significantly enhanced the ASR in rats. Enhancement effects caused by playback of 22-kHz calls from young rats were relatively weak compared to those after calls from adult rats. Playback of synthesized 25-kHz sine tones enhanced ASR in subjects, but not synthesized 60-kHz tones. Further, shortening the individual call duration of synthesized 25-kHz sine tones also enhanced the ASR. Accordingly, it is suggested that 22-kHz calls induce anxiety by socially communicated alarming signals in rats. The results also demonstrated that call frequency, i.e., of 22kHz, appears important for ultrasonic alarm-signal communication in rats.

Air puff-induced 22-kHz calls in F344 rats.

Air puff-induced ultrasonic vocalizations in adult rats, termed "22-kHz calls," have been applied as a useful animal model to develop psychoneurological and psychopharmacological studies focusing on human aversive affective disorders. To date, all previous studies on air puff-induced 22-kHz calls have used outbred rats. Furthermore, newly developed gene targeting technologies, which are essential for further advancement of biomedical experiments using air puff-induced 22-kHz calls, have enabled the production of genetically modified rats using inbred rat strains. Therefore, we considered it necessary to assess air puff-induced 22-kHz calls in inbred rats. In this study, we assessed differences in air puff-induced 22-kHz calls between inbred F344 rats and outbred Wistar rats. Male F344 rats displayed similar total (summed) duration of air puff-induced 22 kHz vocalizations to that of male Wistar rats, however, Wistar rats emitted fewer calls of longer duration, while F344 rats emitted higher number of vocalizations of shorter duration. Additionally, female F344 rats emitted fewer air puff-induced 22-kHz calls than did males, thus confirming the existence of a sex difference that was previously reported for outbred Wistar rats. The results of this study could confirm the reliability of air puff stimulus for induction of a similar amount of emissions of 22-kHz calls in different rat strains, enabling the use of air puff-induced 22-kHz calls in inbred F344 rats and derived genetically modified animals in future studies concerning human aversive affective disorders.

Alarm pheromone does not modulate 22-kHz calls in male rats.

Rats are known to emit a series of ultrasonic vocalizations, termed 22-kHz calls, when exposed to distressing stimuli. Pharmacological studies have indicated that anxiety mediates 22-kHz calls in distressed rats. We previously found that exposure to the rat alarm pheromone increases anxiety in rats. Therefore, we hypothesized that the alarm pheromone would increase 22-kHz calls in pheromone-exposed rats. Accordingly, we tested whether exposure to the alarm pheromone induced 22-kHz calls, as well as whether the alarm pheromone increased 22-kHz calls in response to an aversive conditioned stimulus (CS). Rats were first fear-conditioned to an auditory and contextual CS. On the following day, the rats were either exposed to the alarm pheromone or a control odor that was released from the neck region of odor-donor rats. Then, the rats were re-exposed to the aversive CS. The alarm pheromone neither induced 22-kHz calls nor increased 22-kHz calls in response to the aversive CS. In contrast, the control odor unexpectedly reduced the total number and duration of 22-kHz calls elicited by the aversive CS, as well as the duration of freezing. These results suggest that the alarm pheromone does not affect 22-kHz calls in rats. However, we may have found evidence for an appeasing olfactory signal, released from the neck region of odor-donor rats.

The influence of social environmental condition on the production of stress-induced 22 kHz calls in adult male Wistar rats.

Adult rats emit 22 kHz ultrasonic vocalizations (USVs) in response to aversive stimuli, and these sounds are suggested to have communicative information among conspecifics. It is conceivable that social environment during development of rats has relevance to the emission of 22 kHz USVs. To examine the effects of social environment after weaning on production of stress-induced USVs, we compared the amount of emission of USVs among three groups of rats reared under different conditions after weaning. One group of rats was housed individually, and the other two groups were housed in pairs, in which social hierarchy of the pair was determined by social dominance-subordination relationships. The USVs were induced by acute mild somatic stimuli on the back and neck. Individually reared rats emitted much fewer USVs than pair-reared rats. In addition, socially subordinate rats emitted more USVs compared with socially dominant ones. These results suggest that not only social interaction but also the status in social hierarchy may play an important role in the process of the development of USVs induced by somatic stimuli.

Changes in autonomic control of heart associated with classical appetitive conditioning in rats.

The aim of this study was to examine the changes in autonomic control of the heart associated with classical appetitive conditioning in rats. We trained rats to learn that a movement into a test chamber was followed by delivery of reward (contextual conditioning) and performed power spectral analysis of heart rate variability from electrocardiograms recorded using the telemetry system. We investigated the sympathovagal balance of autonomic regulation of the heart in response to not only the conditioned stimulus (the movement into the test chamber), but also the unconditioned stimulus (reward), and compared the results of these two kinds of emotional states; it might be considered that "the reward-expecting state" is evoked by the conditioned stimulus and "the reward-receiving state" is evoked by the unconditioned stimulus in rats. The reward-expecting state resulted in a significant increase in both low frequency (LF) power and high frequency (HF) power with no change in heart rate (HR) and LF/HF ratio, indicating that both sympathetic and parasympathetic activity increased with no change in sympathovagal balance. The reward-receiving state resulted in a significant increase in HR and a significant decrease in LF power, HF power, and LF/HF ratio, indicating that both sympathetic and parasympathetic activity decreased with predominance in the parasympathetic activity. These results suggest that the method performed in our present study might be useful for distinguishing between two different emotional states evoked by classical appetitive conditioning in rats.

Temporary inactivation of the anterior part of the bed nucleus of the stria terminalis blocks alarm pheromone-induced defensive behavior in rats.

Rats emit an alarm pheromone in threatening situations. Exposure of rats to this alarm pheromone induces defensive behaviors, such as head out behavior, and increases c-Fos expression in brain areas involved in the mediation of defensive behaviors. One of these brain areas is the anterior bed nucleus of the stria terminalis (aBNST). The goal of the present study was to investigate if pharmacological inactivation of the aBNST by local microinjections of the GABAA receptor-agonist muscimol modulates alarm pheromone-induced defensive behaviors. We first established the behavioral paradigm of alarm pheromone-induced defensive behaviors in Sprague-Dawley rats in our laboratory. In a second experiment, we inactivated the aBNST, then exposed rats to one of four different odors (neck odor, female urine, alarm pheromone, fox urine) and tested the effects of the aBNST inactivation on the behavior in response to these odors. Our data show that temporary inactivation of the aBNST blocked head out behavior in response to the alarm pheromone. This indicates that the aBNST plays an important role in the mediation of the alarm pheromone-induced defensive behavior in rats.

Opioid mediated suppressive effect of milk-derived lactoferrin on distress induced by maternal separation in rat pups.

The present study assessed the effects of bovine milk-derived lactoferrin (bLf) on distress activities induced by maternal separation in 5- to 18-day-old rat pups. The rat pups were injected with BSA (100 mg/kg, i.p.; control) or bLf (100 mg/kg, i.p.) 30 min before the behavioral test. Distress activity was estimated by means of recording body movements or ultrasonic vocalizations (USVs). After 5 min of maternal separation, bLf significantly (P<0.01) suppressed body movements, particularly in the 10-day-old pups. This suppressive effect of bLf was reversed by pretreatment with naloxone, CTOP, and norBNI at doses of 0.1-1 mg/kg. Additionally, USVs were also suppressed by bLf, which was reversed by pretreatment with naloxone. Inhibition of nitric oxide synthase (NOS) with nitro-L-arginine methyl ester (L-NAME) dose dependently (3-10 mg/kg) suppressed separation-induced USV production in 10-day-old pups. Interestingly, the suppressive effect of bLf was completely reversed by pretreatment with a low dose (1 mg/kg) of L-NAME, which did not affect the USVs with single application. These findings demonstrate that milk-derived bLf suppresses distress induced by maternal separation via an opioid-mediated mechanism. Furthermore, bLf possibly activates NOS, and an elevated nitric oxide may cause some modification of the opioid system.

Effects of psychological stress on autonomic control of heart in rats.

The aim of this study was to examine the effects of psychological stress on autonomic control of the heart in rats. For this purpose, we evoked anxiety-like or fear-like states in rats by means of classical conditioning and examined changes in autonomic nervous activity using an implanted telemetry system and power spectral analysis of heart rate variability. Anxiety-like states resulted in a significant increase in heart rate (HR), low frequency (LF) power, and LF/HF ratio, with no change in high frequency (HF) power. Fear-like states resulted in a significant increase in HR and a significant decrease in HF power with no significant change in both LF power and LF/HF ratio, although LF/HF ratio increased slightly. These results suggest that autonomic balance becomes predominant in sympathetic nervous activity in both anxiety-like and fear-like states. These changes in rats correspond to changes which are relevant to cardiovascular diseases in humans under many kinds of psychological stress. Therefore, the experimental design of this study is a useful experimental model for investigating the effects of psychological stress on autonomic control of the heart in humans.

The critical point at which post-weaning individual housing conditions affect the emission of 22-kHz calls in male rats.

It is known that long-term post-weaning individual housing significantly reduces emissions of 22-kHz calls in male rats. In this study, we assessed post-weaning successive changes in 22-kHz calls emitted by male rats under two different types of post-weaning housing conditions (individually and socially). In addition, we evaluated the critical point at which a significant reduction in 22-kHz calls could be observed in male rats housed individually after weaning. Significantly fewer 22-kHz calls were emitted by individually housed rats compared to socially housed rats at 16 weeks of age, indicating that 13 weeks after weaning may be a critical point for the reduction of 22-kHz calls caused by post-weaning individual housing.

Effect of post-weaning individual housing on autonomic responses in male rats to sexually receptive female rats.

Post-weaning individual housing induces significant alterations in the reward system of adult male rats presented with sexually receptive female rats. In this study, we examined the effects of post-weaning individual housing on autonomic nervous activity in adult male rats during encounters with sexually receptive female rats to assess whether different affective states depending on post-weaning housing conditions are produced. Changes in heart rate and spectral parameters of heart rate variability indicated that in post-weaning individually housed male rats, both sympathetic and parasympathetic activity increased with no change in the sympathovagal balance, while in post-weaning socially housed male rats, both sympathetic and parasympathetic activity decreased with a predominance of parasympathetic activity. These two patterns of shifts in sympathovagal balances closely resembled changes in autonomic nervous activity with regard to classical appetitive conditioning in male rats. The autonomic changes in male rats housed individually after weaning corresponded to changes associated with the reward-expecting state evoked by the conditioned stimulus, and the autonomic changes observed in male rats housed socially after weaning corresponded to changes associated with the reward-receiving state evoked by the unconditioned stimulus. These results suggest that different affective states were induced in adult male rats during sexual encounters depending on male-male social interactions after weaning. The remarkable change caused by post-weaning individual housing may be ascribed to alteration of the reward system during sexual encounters induced by deficiency of intermale social communication after weaning.