The effects of selective breeding for differential rates of 50-kHz ultrasonic vocalizations on emotional behavior in rats

Fifty-kHz ultrasonic vocalizations have previously been shown to be positively correlated with reward and appetitive social behavior in rats, and to reflect a positive affective state. In this study, rats selectively bred for high and low rates of 50-kHz vocalizations as juveniles were tested as adults in a battery of behavioral tests for social/emotional behaviors. We found that animals selectively bred for high rates of 50-kHz vocalizations exhibited more crosses into the center area of the open field apparatus, were more likely to show a preference for a dilute sucrose solution (.8%) compared to tap water, and were less aggressive than randomly bred animals. Conversely, animals bred for low rates of 50-kHz calls produced more fecal boli during both open field testing and "tickling" stimulation, and made less contact with conspecifics in a social interaction test compared to randomly bred animals. We also observed that low line rats have elevated brain levels of cholecystokinin (CCK) in the cortex, which is consistent with literature showing that CCK content in the cortex is positively correlated with rates of aversive 22-kHz USVs. Conversely, high line animals had elevated levels of met-enkephalin in several brain regions, which is consistent with the role of endogenous-opioids in the generation 50-kHz USVs and positive affect. These results suggest that animals bred for high rates of 50-kHz may show a stress resilient phenotype, whereas low line rats may show a stress prone phenotype. As such these animals could provide novel insights into the neurobiology of emotion.     

Neurogenesis in the dentate gyrus of the rat hippocampus enhanced by tickling stimulation with positive emotion

Hippocampal neurogenesis is influenced by many factors. In this study, we examined the effect of tactile stimulation (tickling), which induced positive emotion, on neurogenesis in the dentate gyrus (DG) of the hippocampus. Four week-old rats were tickled for 5 min/day on 5 consecutive days and received 5-bromo-2′-deoxyuridine (BrdU) administration for 4 days from the second tickling day. Then they were allowed to survive for 18 h or 3 weeks after the end of BrdU treatment. Neurogenesis in the DG was compared between the tickled and untickled rats by using immunohistochemistry with anti-BrdU antibody. The result showed that the number of BrdU- and NeuN (neural cell marker)-double positive neurons on 18 h as well as 3 weeks of the survival periods was significantly increased in the tickled group as compared with the untickled group. The expression of mRNA of brain-derived neurotrophic factor (BDNF) in the hippocampus of the tickled rats was not altered when compared with the control rats. In conclusion, tickling stimulation which induces positive emotion may affect the generation and survival of new neurons of the DG through the BDNF-independent pathway.     

Playback of 22-kHz and 50-kHz ultrasonic vocalizations induces differential c-fos expression in rat brain

Rodent ultrasonic vocalizations, which serve as sensitive measures in a number of relevant individual and social behaviours, have become increasingly interesting for biopsychological studies on emotion and motivation. Of these, high frequency (50-kHz) ultrasonic vocalizations can index a positive emotional state, and induce approach, whereas low frequency (22-kHz) ultrasonic vocalizations can induce avoidance and may index anxiety, since they are emitted during various unconditioned and conditioned aversive situations. While cholinergic and dopaminergic systems have been implicated, specific neural substrates that sub-serve these vocalization-dependent states remain to be elucidated. Using c-fos immunocytochemistry, we revealed neural activity in brain areas of naïve male Wistar rats in response to playback of 22-kHz and flat and frequency-modulated 50-kHz ultrasonic vocalizations. Presentation of background noise or no acoustic stimulus at all constituted the controls. Playback of 50-kHz ultrasonic vocalizations led to approach behaviour. Acoustically stimulated animals demonstrated differential activation in auditory areas, with a frequency-dependent activation in the auditory cortex. Specific forebrain, thalamic, hypothalamic and brainstem areas were also activated differentially. While 50-kHz playback induced sparse fos-like immunoreactivity in frontal association cortex, nucleus accumbens, thalamic parafascicular and paraventricular nuclei, 22-kHz playback elicited c-fos expression in the perirhinal cortex, amygdalar nuclei and the periaqueductal gray. This study unveils neural substrates that are activated during ultrasonic playback perception, which could sub-serve the affective states elicited by these vocalizations.     

The effects of buspirone and diazepam on aversive context- and social isolation-induced ultrasonic vocalisation

Rats emit two types of high-frequency vocalisations (aversive and appetitive calls) in different behavioural situations. The aims of this paper were to examine an animal model of appetitive behaviour (as an element of social interaction) and to study the effects of selected psychotropic drugs on appetitively evoked ultrasonic vocalisation (USVs) and aversive context-evoked USVs. Specifically, we analysed the impact of the encounter of pairs of adult rats after long-term isolation on ultrasound vocalisation. It was found that isolation of the adult rats significantly enhanced the appetitive ultrasound vocalisations (50-kHz) during encounters between pairs of rats. In the pharmacological part of the study, we found that diazepam (1.0 mg kg^− 1) significantly increased isolation-induced appetitive USVs (50 kHz) and decreased aversive context-evoked USVs (22-kHz). Buspiron (3.0 mg kg^− 1) decreased the aversive context-evoked USVs and had no effect on isolation-induced appetitive USVs. These data indicate that long-term, isolation-induced 50-kHz USVs in adult rats represent a new behavioural parameter under control of the central GABAergic system, which can be used to study the effects of anxiolytic drugs.     

On the relationships between ultrasonic calling and anxiety-related behavior in rats

In the present review, the phenomenon of ultrasonic vocalization in rats will be outlined, including the three classes of vocalizations, namely 40-kHz calls of pups, and 22- and 50-kHz calls of juvenile and adult rats, their general relevance to behavioral neuroscience, and their special relevance to research on anxiety, fear, and defense mechanisms. Here, the emphasis will be placed on 40- and 22-kHz calls, since they are typical for various situations with aversive properties. Among other topics, we will discuss whether such behavioral signals can index a certain affective state, and how these signals can be used in social neuroscience, especially with respect to communication. Furthermore, we will address the phenomenon of inter-individual variability in ultrasonic calling and what we currently know about the mechanisms, which may determine such variability. Finally, we will address the current knowledge on the neural and pharmacological mechanisms underlying 22-kHz ultrasonic vocalization, which show a substantial overlap with mechanisms known from other research on fear and anxiety, such as those involving the periaqueductal gray or the amygdala.     

Tickling induces reward in adolescent rats

In adolescent rats, 50-kHz vocalizations are most evident during tickling and rough-and-tumble play. The following experiments evaluated whether 50-kHz vocalizations reflect positive social affect by determining (1) if tickling is a rewarding event, (2) if social or isolate housing conditions differentially influence the response (since housing condition has been found to effect the reward magnitude of social encounters), and (3) if drugs that work on mu-opiate receptors, which has been hypothesized to control positive social affect, modulate tickling. Tickling was positively reinforcing as demonstrated by elevated operant behavior, conditioned place preference, and approach measures. A significant negative correlation between vocalization rate and approach latency measures was found. Social housing reduced tickle-induced vocalizations and approach speeds compared to isolate housing. Naloxone (1 mg/kg) increased vocalization in the socially housed rats and decreased it in isolated Subjects (Ss). These findings suggest that tickling can be used to induce positive social affect in rodents, and that it is modulated by endogenous opioids.     

Ultrasonic vocalizations in male rats following acquisition of copulation-illness associations

Brief 50-kHz ultrasonic vocalizations are emitted by male rats during copulatory encounters with sexually receptive females. These vocalizations also occurred during pairings with estrous females in male rats whose copulatory behaviors had been associatively inhibited by LiCl-induced aversive contingencies. Longer 22-kHz calls, typically emitted during the postejaculatory refractory period, were not observed in these males. These results demonstrate a dissociation of behaviors that were (mounts, intromissions, and ejaculations) and were not (50-kHz vocalizations) associatively inhibited by LiCl.     

Breeding for 50-kHz Positive Affective Vocalization in Rats

Adolescent and adult rats exhibit at least two distinct ultrasonic vocalizations that reflect distinct emotional states. Rats exhibit 22-kHz calls during social defeat, drug withdrawal, as well as in anticipation of aversive events. In contrast, 50-kHz calls are exhibited in high rates during play behavior, mating, as well as in anticipation of rewarding events. The neurochemistry of 22-kHz and 50-kHz calls closely matches that of negative and positive emotional systems in humans, respectively. The aim of this study was to replicate and further evaluate selective breeding for 50-kHz vocalization, in preparation for the analysis of the genetic underpinnings of the 50-kHz ultrasonic vocalization (USV). Isolate housed adolescent rats (23–26 days old) received experimenter administered tactile stimulation (dubbed tickling), which mimicked the rat rough-and-tumble play behavior. This stimulation has previously been shown to elicit high levels of 50-kHz USVs and to be highly rewarding in isolate-housed animals. Each tickling session consisted of 4 cycles of 15 seconds stimulation followed by 15 seconds no stimulation for a total of 2 min, and was repeated once per day across 4 successive days. Rats were then selected for either High or Low levels of sonographically verified 50-kHz USVs in response to the stimulation, and a randomly selected line served as a control (Random group). Animals emitted both 22-kHz and 50-kHz types of calls. After 5 generations, animals in the High Line exhibited significantly more 50-kHz and fewer 22-kHz USVs than animals in the Low Line. Animals selected for low levels of 50-kHz calls showed marginally more 22-kHz USVs then randomly selected animals but did not differ in the rate of 50-kHz calls. These results extend our previous findings that laboratory rats could be bred for differential rates of sonographically verified 50-kHz USVs.     

Tickling in juvenile but not adult female rats conditions sexual partner preference

Female rats display a conditioned partner preference for males that bear odors paired with different types of rewarding unconditioned stimuli (UCS). Here we examined whether tickling constitutes a rewarding UCS that supports the development of partner preferences. In Experiment 1, we tested the possibility that odors associated with a tickling UCS in prepubescent rats would induce a conditioned partner preference in adulthood. Two groups were formed with 31-day-old, single-housed females, tickled for 6 min daily for 10 days, by a hand that wore a scented glove (almond or lemon). At 47 days of age, females were ovariectomized (OVX), hormone-primed (EB+P), and tested for sexual partner preference with two scented stud males (one almond and one lemon). In each group, females displayed a sexual preference toward males bearing the odor paired with tickling, as observed with longer visits, more solicitations, hops & darts, and receiving more intromissions and ejaculations from the preferred male. In Experiment 2, we used 3-month old, OVX, hormone-primed rats conditioned every 4 days for 10 trials. In contrast to juvenile females, adult females failed to prefer males that bore the odor paired with tickling but instead preferred the novel male. These results suggest that tickling has opposite age-dependent effects in the conditioning of partner preference. Tickling in juvenile females appears to act as a rewarding UCS, whereas in adult females it may act as an aversive UCS. Further research is needed to understand brain mechanisms that might account for such differences.     

Rats selectively bred for low levels of 50 kHz ultrasonic vocalizations exhibit alterations in early social motivation

In rats, the rates of 50 kHz ultrasonic vocalizations (USVs) can be used as a selective breeding phenotype and variations in this phenotype can be an indicator of affective states. The 50 kHz USV is elicited by rewarding stimuli (e.g., food, sexual behavior) and therefore can express a positive affective state. Conversely, the 22 kHz USV is elicited by aversive stimuli (e.g., presence of a predator, social defeat) indicating a negative affective state. In the present study, we tested the effect of selectively breeding for 50 kHz USVs on a variety of maternal social/emotional behaviors in young rat pups (PND 10-12). These measures consisted of an assessment of isolation calls and conditioned odor preference paradigm. Results indicate that animals selected for low levels of 50 kHz USVs show the greatest alterations in social behaviors compared to the control animals. The low line animals had an increase in isolation calls tested during place preference conditioning and a decrease in 50 kHz ultrasonic calls in all conditions. These same low line animals failed to show a typical preference for a maternally-associated odor during the place preference test. The different social behaviors of the high line animals did not consistently vary from those of the control group. These results have important implications for the study of genetic and epigenetic mechanisms underlying emotional states, and possibly contribute to the research underlying the emotional changes in developmental disorders such as autistic spectrum disorder by providing a novel animal model that displays communication deficits that are interdependent with significant social behavioral impairments.     

Maternal care, isolation-induced infant ultrasonic calling, and their relations to adult anxiety-related behavior in the rat

In the rat, variations in maternal care affect the development of stable individual differences in anxiety-related behavior. Here, it was asked whether such experience-dependent differences can be detected already during early life. As a measure for anxiety in pups, isolation-induced ultrasonic vocalizations were used, and their dependency on different maternal behaviors, namely licking, retrieval behavior, and responsiveness to playback of pup calls, was tested. Consistent with reported differences of adult rats with high or low levels of maternal care experienced, the rarely licked offspring appeared to be more anxious, since they emitted more calls when separated from their mother and litter. Based on these findings, it was examined whether infant calling can be used as a predictor of adult anxiety-related behavior. Results show that infant call emission was negatively correlated with immobility and calling during fear conditioning. These relationships seem to be mediated at least partly by maternal care. In total, measuring ultrasonic vocalizations can provide information about an affective trait of infant and adult rats, which gives the opportunity to study the development of emotionality from early life onward.     

Psychophysiology and Psychopathology: A Motivational Approach

The current zeitgeist strongly emphasizes genetic and biochemical approaches to psychopathology over psychological and psychobiological approaches. It is argued that a psychobiological model, involving a theory of motivation derived from the animal learning literature, offers an attractive theoretical bridge between neurochemical influences and the phenotypic features of psychiatric disorders. This model involves separate but interactive appetitive and aversive motivational systems that control behavioral activation (appetitive) and inhibition (aversive). Ways in which these motivational constructs can be relevant to psychopathology are discussed for anxiety, psychopathy, childhood disorders, depression, mania, drug abuse, and schizophrenia. Because of this general application, motivational constructs offer an attractive theoretical framework for understanding psychopathology. Application of the motivational theory to psychophysiology suggested that heart rate may be significantly influenced by appetitive motivation. A series of studies have shown that heart rate during performance of a continuous motor task does respond to appetitive motivation in the form of performance-contingent monetary incentives, but does not respond to aversive stimulation in the form of failure feedback. Nonspecific skin conductance fluctuations have not responded to appetitive motivation in this paradigm, but this failure could possibly be due to ceiling effects. Nonspecific skin conductance fluctuations do respond to aversive stimulation in other contexts. These findings suggest that under the right circumstances appetitive motivation can be assessed via heart rate and aversive motivation via skin conductance.     

营养不良可增加幼鼠齿状回颗粒下层的细胞增殖和神经生发(英文)

为了观察营养不良对幼鼠海马齿状回 (DG)和脑室下层 (SVZ)的细胞增殖和神经发生的影响 ,采用 5 -溴 -2 -脱氧尿苷(Brd U)标记结合免疫组织化学方法对脑切片分别进行 Brd U、Tu J1(β tubulin,β微管蛋白 )及 GFAP(胶质纤维酸性蛋白 )反应或双重反应。结果表明 ,营养不良幼鼠齿状回的细胞增殖和神经生发明显高于营养良好的幼鼠而脑室下层的细胞增殖数量在两者却无明显差异。在齿状回 ,新生的细胞中大约有 5 0 %为新生的神经元 ,10～ 2 0 %为神经胶质细胞。本文结果提示 ,幼鼠海马齿状回的细胞增殖和神经生发可能因营养不良而增加 ,这些新生的细胞可能对日后某些海马依赖性行为产生一定的影响     

Changes in the categorization of appetitive and aversive events during postnatal development of the rat

Environmental stimuli are not clearly categorized into appetitive and aversive classes during the first postnatal week. Three- and 6-day-old rats are highly activated by nominally appetitive (milk infusions) and aversive (foot shock) stimuli. Both evoked the same generalized behavioral responses (rolling, curling). By 12 days of age, these stimuli were responded to differently; mouthing and probing occurred in response to milk while flinching and escape responses were observed to shock. The affective properties of mild shock were found to be hedonically opposite for 6- and 12-day-old pups. Six-day-old showed an acquired preference for an odor paired with shock which increased with intensity (.1–.5 mA) similar to that observed with milk infusions. An acquired odor aversion was displayed by 12 days old using these shock parameters. These results suggest that young rat pups may not differentiate many appetitive and aversive events behaviorally or affectively until the second postnatal week.     

Ultrasonic vocalizations and aggressive behavior in male rats

The aim of the present study was to know whether 22-28-kHz vocalizations have any communicatory role in the regulation of aggressive behavior in male rats of the Wistar strain. In pairs of intact rats 22-28-kHz vocalizations showed a positive correlation with the extent of aggressive behavior. The pattern of aggressive behavior during ultrasonic vocalizations was different from that just before and just after the vocalizations. However, surgically deafened rats were less active in aggressive behavior and more active in ambulatory activity in the open field than the controls. Muted rats were not different from the controls in both aggressive behavior and ambulatory activity. The present result that the deprivation of ultrasonic signals failed to increase aggressive behavior does not support the classical hypothesis that ultrasonic vocalizations inhibit the initiation of aggressive behavior. It is concluded that ultrasounds emitted during aggressive encounters may have little communicative value in male rats.     

"Laughing" rats and the evolutionary antecedents of human joy?

Paul MacLean's concept of epistemics-the neuroscientific study of subjective experience-requires animal brain research that can be related to predictions concerning the internal experiences of humans. Especially robust relationships come from studies of the emotional/affective processes that arise from subcortical brain systems shared by all mammals. Recent affective neuroscience research has yielded the discovery of play- and tickle-induced ultrasonic vocalization patterns ( approximately 50-kHz chirps) in rats may have more than a passing resemblance to primitive human laughter. In this paper, we summarize a dozen reasons for the working hypothesis that such rat vocalizations reflect a type of positive affect that may have evolutionary relations to the joyfulness of human childhood laughter commonly accompanying social play. The neurobiological nature of human laughter is discussed, and the relevance of such ludic processes for understanding clinical disorders such as attention deficit hyperactivity disorders (ADHD), addictive urges and mood imbalances are discussed.     

Nucleus accumbens amphetamine microinjections unconditionally elicit 50-kHz ultrasonic vocalizations in rats

The authors have hypothesized that, in adult rats, 50-kHz ultrasonic vocalizations (USVs) index a state characterized by high arousal and expectations of reward. This study was conducted to investigate whether dopamine agonism of the nucleus accumbens (NAcc) could evoke such an appetitive state, by examining the effects of NAcc amphetamine (AMPH) microinjections on USVs. Intra-NAcc AMPH injections (0.3, 1.0, 3.0, 10.0 microg unilaterally) produced robust, dose-dependent increases in 50-kHz USVs, which could not be accounted for by concomitant increases in locomotor activity (LA). However, AMPH injections into dorsal control caudate putamen sites produced a modest, dose-dependent increase in LA without significant increases in 50-kHz USVs. These findings indicate that NAcc AMPH microinjections selectively evoke 50-kHz USVs in rats, supporting the notion that dopamine elevations in the NAcc may unconditionally elicit a state of reward anticipation.     

Electrophysiological and ultrasonic correlates of reproductive behavior in the male rat

The purpose of the present study was to examine the electrophysiological correlates of copulatory behavior in the rat and correlate brain electroencephalographic (EEG) activity with the ultrasonic vocalizations that have been shown to play a major role in the integration of mating behavior. Ultrasonic vocalizations of male rats with chronically implanted hippocampal and cortical electrodes were monitored during mating with a female rat. Hippocampal theta rhythms were significantly correlated with high activity, mounting, intromissions, and preejaculatory excitatory behavior and were also significantly associated with 50-kHz short ultrasonic vocalizations. Postmount or postintromission behaviors (grooming, exploration) were closely correlated with an absence of ultrasonic vocalizations and the onset of irregular low-amplitude hippocampal EEG recordings. Long 22-kHz vocalizations occurred in all tests during the postejaculatory refractory period. Shorter 22-kHz vocalizations occurred during mating and were associated with unsuccessful intromissions or mounting attempts. Postejaculatory long 22-kHz vocalizations were significantly associated with irregular high-amplitude hippocampal EEG tracings, and preejaculatory short 22-kHz vocalizations were also accompanied by sleep-like irregular high-amplitude hippocampal EEG tracings with cortical spindling. The correlation of ultrasonic vocalizations with electroencephalographic and behavioral manifestations of central arousal and inhibitory processes suggests that these vocalizations are indicators of the sexual arousal of the mating pair.     

High-frequency ultrasonic vocalizations index conditioned pharmacological reward in rats.

We have proposed that short (<0.5 s), high-frequency (approximately 50 kHz) ultrasonic vocalizations ("50-kHz USVs") index a positive affective state in adult rats, because they occur prior to rewarding social interactions (i.e., rough-and-tumble play, sex). To evaluate this hypothesis in the case of nonsocial stimuli, we examined whether rats would make increased 50-kHz USVs in places associated with the administration of rewarding pharmacological compounds [i.e., amphetamine (AMPH) and morphine (MORPH)]. In Experiment 1, rats made a greater percentage of 50-kHz USVs on the AMPH-paired side of a two-compartment chamber than on the vehicle-paired side, even after statistical correction for place preference. In Experiment 2, rats made a higher percentage of 50-kHz USVs on the MORPH-paired side than on the vehicle-paired side, despite nonsignificant place preference. These findings support the hypothesis that 50-kHz USVs mark a positive affective state in rats and introduce a novel and rapid marker of pharmacological reward.     

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.