50-kHz Ultrasonic Vocalizations Research Articles

Intracerebral injection of R-(-)-Apomorphine into the nucleus accumbens decreased carbachol-induced 22-kHz ultrasonic vocalizations in rats.

Rats can produce ultrasonic vocalizations (USVs) in a variety of different contexts that signal their emotional state to conspecifics. Under distress, rats can emit 22-kHz USVs, while during positive pro-social interactions rats can emit frequency-modulated (FM) 50-kHz USVs. It has been previously reported that rats with increasing emission of FM 50-kHz USVs in anticipation of rewarding electrical stimulation or positive pro-social interaction decrease the number of emitted 22-kHz USVs. The purpose of the present investigation was to determine, in a pharmacological-behavioural experiment, if the positive emotional arousal of the rat indexed by the number of emitted FM 50-kHz USVs can decrease the magnitude of a subsequent negative emotional state indexed by the emission of 22-kHz USVs. To induce a positive emotional state, an intracerebral injection of a known D1/D2 agonist R-(-)-apomorphine (3.0 μg/0.3 μl) into the medial nucleus accumbens shell was used, while a negative emotional state was induced by intracerebral injection of carbachol (1.0 μg/0.3 μl), a known broad-spectrum muscarinic agonist, into the anterior hypothalamic-medial preoptic area. Our results demonstrated that initiation of a positive emotional state was able to significantly decrease the magnitude of subsequently expressed negative emotional state measured by the number of emitted 22-kHz USVs. The results suggest the neurobiological substrates that initiate positive emotional state indirectly antagonize the brain regions that initiate negative emotional states.

Sex-dependent effects of Cacna1c haploinsufficiency on juvenile social play behavior and pro-social 50-kHz ultrasonic communication in rats.

As cross-disorder risk gene, CACNA1C is implicated in the etiology of all major neuropsychiatric disorders characterized by deficits in social behavior and communication and there is evidence for sex-dependent influences of single-nucleotide polymorphisms within CACNA1C on diagnosis, course, and recovery in humans. In this study, we aimed, therefore, at further exploring the role of Cacna1c in regulating behavioral phenotypes, focusing on sex-specific differences in social behavior and communication during the critical developmental period of adolescence in rats. Specifically, we compared rough-and-tumble play, concomitant emission of pro-social 50-kHz ultrasonic vocalizations, and social approach behavior in response to playback of 50-kHz ultrasonic vocalizations between constitutive heterozygous Cacna1c +/- females and wildtype Cacna1c +/+ littermate controls, and contrasted present female findings to data previously reported in males. Our results show for the first time that partial depletion of Cacna1c leads to sex-dependent alterations in social behavior and communication in rats. In females, Cacna1c haploinsufficiency led to hypermasculinization, with rough-and-tumble play behavior, in general, and pinning behavior, in particular, being even higher than in males without affecting concomitant 50-kHz ultrasonic vocalizations. In males, in contrast, rough-and-tumble play behavior was not altered, yet emission of 50-kHz ultrasonic vocalizations was diminished following partial Cacna1c depletion. The behavioral responses elicited by playback of 50-kHz ultrasonic vocalizations were reduced upon partial Cacna1c depletion in both sexes. It thus can be concluded that Cacna1c plays a prominent sex-dependent role in regulating juvenile rat social play behavior and pro-social 50-kHz ultrasonic communication with relevance to sex-specific effects seen in neuropsychiatric disorders.

Stimulus-seeking in rats is accompanied by increased c-Fos expression in hippocampal CA1 as well as short 22 kHz and flat 50 kHz calls

We determined CA1 hippocampal field to be involved in self-exposure, a type of novelty‑seeking behaviour that has also been associated with short 22 kHz and flat 50 kHz ultrasonic vocalizations (USV) in adult male Long-Evans rats. Rats were habituated for three days to a self-exposure cage with two nose-poke holes. On day four, the animals from the experimental group were allowed to turn the cage light off for 5 s with a nose‑poke (test/self‑exposure session), while rats from control-yoked group had changing light conditions coupled and identical to the experimental animals. The experimental rats performed more nose-pokes during self-exposure session than animals from the control group. This effect was accompanied by a higher density of c-Fos-positive nuclei in the hippocampal CA1. There were no significant group differences in c-Fos expression in other brain regions analysed. However, possible involvement of several other structures in self-exposure (i.e., CA3, the dentate gyrus, amygdala, prefrontal cortex, and nucleus accumbens) is also discussed, as their correlational activity, reflected by c-Fos immunoactivity, was observed in the experimental rats. During test sessions, there were more nose-pokes accompanied by short 22 kHz calls and 50 kHz calls performed by the rats of the experimental group than of the control group. The CA1 region has previously been associated with novelty; short 22 kHz USV and flat 50 kHz USV could be associated with self-exposure, also they appear to be emitted correlatively.

50-kHz ultrasonic vocalizations increase after sleep deprivation as mania-like elevated mood in rats: Effects of lithium

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19) has become a global health problem with pandemic character. Lung transplant recipients may be particularly at risk due to the high degree of immunosuppression and the lung being the organ primarily affected by COVID-19. We describe a 16-year-old male and a 64-year-old female recently lung transplanted patients with COVID-19 during inpatient rehabilitation. Both patients were receiving triple immunosuppressive therapy and had no signs of allograft dysfunction. Both patients had close contact with a person who developed COVID-19 and were tested positive for SARS-CoV-2. Subsequently, both patients underwent systematic screening and SARS-CoV-2 was ultimately detected. Although the 16-year-old boy was completely asymptomatic, the 64-year-old woman developed only mild COVID-19. Immunosuppressive therapy was unchanged and no experimental treatment was initiated. No signs of graft involvement or dysfunction were noticed. In conclusion, our report of patients with asymptomatic SARS-CoV-2 infection and mild COVID-19, respectively, may indicate that lung transplant recipients are not per se at risk for severe COVID-19. Further observations and controlled trials are urgently needed to study SARS-CoV-2 infection in lung transplant recipients.

22 kHz and 55 kHz ultrasonic vocalizations differentially influence neural and behavioral outcomes: Implications for modeling anxiety via auditory stimuli in the rat

The communicative role of ultrasonic vocalizations (USVs) in rats is well established, with distinct USVs indicative of different affective states. USVs in the 22 kHz range are typically emitted by adult rats when in anxiety- or fear-provoking situations (e.g. predator odor, social defeat), while 55 kHz range USVs are typically emitted in appetitive situations (e.g., play, anticipation of reward). Previous work indicates that USVs (real-time and playback) can effectively communicate these affective states and influence changes in behavior and neural activity of the receiver. Changes in cFos activation following 22 kHz USVs have been seen in cortical and limbic regions involved in anxiety, including the basolateral amygdala (BLA). However, it is unclear how USV playback influences cFos activity within the bed nucleus of the stria terminalis (BNST), a region also thought to be critical in processing anxiety-related information, and the nucleus accumbens, a region associated with reward. The present work sought to characterize distinct behavioral, physiological, and neural responses in rats presented with aversive (22 kHz) compared to appetitive (55 kHz) USVs or silence. Our findings show that rats exposed to 22 kHz USVs: 1) engage in anxiety-like behaviors in the elevated zero maze, and 2) show distinct patterns of cFos activation within the BLA and BNST that contrast those seen in 55 kHz playback and silence. Specifically, 22 kHz USVs increased cFos density in the anterodorsal nuclei, while 55 kHz playback increased cFos in the oval nucleus of the BNST, without significant changes within the nucleus accumbens. These results provide important groundwork for leveraging ethologically-relevant stimuli in the rat to improve our understanding of anxiety-related responses in both typical and pathological populations.

Effect of microinjections of dopamine into the nucleus accumbens shell on emission of 50 kHz USV: Comparison with effects of d-amphetamine

Systemic pharmacological manipulation of dopamine (DA) signaling has been central to many investigations of 50 kHz ultrasonic vocalizations (USVs) in the rat. In particular, the indirect DA releaser d-amphetamine (AMPH) has been used extensively in many such investigations. The possible unique character of the native transmitter relative to DA-stimulating drugs such as AMPH in inducing and modulating emission of 50 kHz USVs has not been investigated. Adult male Long Evans rats were tested with intracerebral application of DA into the nucleus accumbens shell at several doses (3.75 μg–120 μg) to determine its capacity to induce 50 kHz USV emission. Additionally, the call profile characteristics of intracerebral DA injections were compared with those of intracerebral application of AMPH. Results indicated that local increases in DA signaling within the nucleus accumbens shell are sufficient to increase 50 kHz call rate, reduce latency to call, and increase the degree of frequency modulation of emitted USVs. However, our results found that microinjections of DA were not as efficacious in either inducing 50 kHz USVs or increasing frequency modulation without antagonism of the dopamine reuptake transporter when compared with AMPH. In summary, these results support the notion that the native transmitter DA is driving the increase in frequency modulation seen after administration of DA stimulating drugs. These results also suggest that drugs affecting dopamine may be altering the 50 kHz call profile in a distinct manner from the native transmitter and thus caution should be used in interpreting their effects.

Differences in the dopaminergic reward system in rats that passively and actively behave in the Porsolt test

The aim of the study was to assess appetitive responses and central dopaminergic neurotransmission in passive and active rats divided according to their immobility time in the Porsolt swim test and exposed to restraint stress. Passive rats had more episodes of appetitive 50-kHz ultrasonic vocalization (USV) during rat encounter after social isolation and spent significantly more time in the amphetamine-associated context in conditioned place preference test, compared to active rats. Restraint stress decreased sucrose preference, but increased appetitive vocalization and reinforced the conditioned place preference only in passive animals that was associated with increased dopamine concentration in the amygdala. Restraint stress increased also the level of Cocaine- and Amphetamine Regulated Transcript (CART) peptide, a neuromodulator linked to dopamine neurotransmission, in the central nucleus of amygdala, while decreasing it the nucleus accumbens shell in passive rats. In the parvocellular region of paraventricular nucleus of the hypothalamus passive animals had a higher expression of CART compared to passive restraint rats and active control rats.The obtained results show that active and passive rats in the Porsolt test differ significantly in response to appetitive stimuli, which can be additionally changed under stress conditions. The underlying mechanisms are probably associated with differences in dopaminergic activity and CART signaling in reward system.

Non-pharmacological induction of rat 50 kHz ultrasonic vocalization: Social and non-social contexts differentially induce 50 kHz call subtypes

The emission of 50 kHz ultrasonic vocalizations (USVs) by rats is thought to represent a measurable expression of the individuals underlying emotional state. These calls are also posited as fulfilling important communicative functions among conspecifics. In addition to social situations, 50 kHz USVs are recorded in a variety of reward-related contexts including sugary foods and drink, consumable ethanol, and drugs of abuse. The current study sought to directly compare several of these behavioural contexts in their capability to induce and modulate 50 kHz USV emission in adult male rats. Using two social conditions (exposure of naïve rat to a naturally cycling female and reuniting with a same-sex cage partner) and two non-social conditions (access to consumables as Fruit Loops or 2% ethanol v/v), we analyzed USVs recorded in 6 stimulus-presentation sessions. Only the female-exposure condition was found to increase 50 kHz call rate significantly over baseline, and this induction sensitized across 4 standard recording sessions. The use of a same-sex cage-mate and the two consumable food rewards did not elicit higher than baseline 50 kHz calling. None of the behavioural contexts altered the acoustic parameters (peak frequency, duration, and bandwidth) of emitted 50 kHz calls. In counter-balanced recording sessions, calling across all groups was significantly reduced by pre-treatment with the dopamine antagonist haloperidol compared with vehicle. Non-social conditions appeared to induce a greater proportion of flat calls at the expense of non-trill FM calls, while the reverse was seen for social conditions. However, the type of food reward and the type of social context mattered for proportion of flat and trill calls respectively. When compared with a control, access to sugary food but not ethanol induced a greater proportion of flat calls, and the female but not the cage-mate stimulus induced a greater proportion of trill calls.

Investigation of social, affective, and locomotor behavior of adolescent Brattleboro rats reveals a link between vasopressin's actions on arousal and social behavior

Arginine vasopressin (AVP) has recently been implicated in juvenile and adolescent social development. How AVP influences social development, however, is not understood. Adolescent homozygous Brattleboro rats (Hom), which lack AVP due to a mutation in the Avp gene, exhibit fewer active social behaviors (e.g., social play) but more passive social behaviors (e.g., huddling) than their wild type and heterozygous (Het) littermates, raising the possibility that AVP impacts social development through an arousal mechanism. Here, we test whether the atypical social phenotype of adolescent Hom rats is associated with altered behavioral arousal, social approach, or affective behaviors and whether Brattleboro mothers impact these behavioral phenotypes. Male and female Het and Hom adolescents born to Het or Hom mothers were tested in social interaction, open field, novelty-seeking, social approach, and marble burying tests. As reported previously, Hom rats played less and emitted fewer 50 kHz ultrasonic vocalizations while huddling more than their Het littermates. No genotype differences were detected in novelty seeking or social approach, nor were consistent differences found between offspring from Het and Hom mothers. However, Hom rats were less active in the open field and buried fewer marbles than Het rats indicating a hypoaroused, low anxiety phenotype. Open field activity correlated with levels of social play indicating that the effects of the Brattleboro mutation on arousal and social behavior are linked. These data demonstrate that chronic AVP deficiency impacts behavioral arousal during adolescence and support the hypothesis that AVP influences adolescent social development, in part, through its regulation of arousal.

Practical rat tickling: Determining an efficient and effective dosage of heterospecific play

Laboratory rats may experience stress during handling which can reduce their welfare. Rat tickling, a handling technique that mimics aspects of rat rough-and-tumble play, has been found to induce positive affect based on production of 50-kHz ultrasonic vocalizations (USVs). However, current protocols for rat tickling are time-intensive, making implementation difficult. Our objective was to determine a time-efficient and effective dosage of rat tickling that could be practically implemented in the laboratory. We hypothesized that affect and handling can be improved by short, daily doses of tickling within a 5-day work week. Long-Evans rats (N = 72) of both sexes, housed in pairs were sampled. Each pair was randomly assigned a tickling duration (15, 30 or 60 s per rat) and frequency (1, 3, or 5 days). After the final day of tickling, rats were tested for ease of, and reaction to, handling via an intraperitoneal injection of saline following a tickling session for their assigned duration. On test day, we measured production of USVs, home cage behavior (60 min before/after testing), approach behavior (30 s before/after testing), and fecal corticosterone. Periods before and after testing measured anticipatory and reactionary responses, respectively. In cage behaviors included social play, activity, and location. Approach behaviors included indicators of fear or anxiety such as rearing, location, and contact with the hand. Data were analyzed using general linear models. We found that 3-day rat tickling was most efficient and effective as it produced a higher rate of 50-kHz USVs before and during tickling (p < 0.0001), and rats played more and were less inactive in their cage for the hour before tickling and injection (p < 0.003) compared to 1-day of rat tickling, but there was no difference between 3- and 5-days of tickling. Only one outcome (play behavior after tickling) showed more positive results after 5- vs 3-days of tickling (p = 0.002). Tickling duration did not impact any outcome measures (p > 0.05). Neither tickling duration nor frequency impacted approach behavior, injection duration, or fecal corticosterone (p > 0.05). In conclusion, a time-efficient and effective rat tickling dosage was identified to be 15 s for 3-days before any potentially aversive procedures are applied. This conclusion is based on increased 50-kHz USVs (a measure of positive affect) and positive anticipatory behavior, including play. Overall, our results suggest that minimal rat tickling can effectively habituate rats to handling and prepare them for research procedures within a work week.

Predator odour but not TMT induces 22-kHz ultrasonic vocalizations in rats that lead to defensive behaviours in conspecifics upon replay

Predator odours induce defensive behaviour in prey animals such as rats. The present study investigated (1) whether laboratory rats exposed to predator odours emit 22-kHz calls which may have an alarming function and (2) whether playback of such calls induces behavioural changes in conspecifics. For this, Sprague-Dawley rats were exposed to samples of fox and lion urine, as well as to the synthetic predator odour TMT. Despite that all odours induced defensive behaviour, only predator urine samples but not TMT were able to induce 22-kHz calls in a few rats. In a second experiment, naive rats were exposed to playback presentations of the 22-kHz calls recorded in the first experiment, as well as to phase-scrambled and frequency-shifted control stimuli. Low intensity playback presentations led to a reduction of locomotor activity during the presentation of the 22-kHz calls but not of the control stimuli. This effect was less specific under high intensity conditions. Taken together the present findings show that natural predator odours are able to induce emission of 22-kHz calls in rats and support the hypothesis that these calls have an alarming function.

Mania-like elevated mood in rats: Enhanced 50-kHz ultrasonic vocalizations after sleep deprivation

Mania is characterized by elevated drive and mood but animal models of mania have often neglected elevated mood. Ultrasonic vocalizations (USV) of 50-kHz emitted by rats are thought to index the subject's positive affective state. Fifty-kHz USV emission is increased by amphetamine, an effect blocked by lithium administration. Sleep deprivation (SD) is an environmental model of mania and the present study evaluated SD effects on behavioral activity and USV emission, together with the impact of lithium treatment. Adult rats were submitted to 24h or 72h SD, and locomotor activity and USV emission were assessed. To test their sensitivity to a standard antimanic drug, these behavioral parameters were also evaluated after acute administration of lithium carbonate (25, 50 or 100 mg/kg, i.p.). Striatal monoamine content was measured post-mortem. SD (24h and 72h) led to increased locomotor activity, rearing behavior and 50-kHz USV emission, together with a change in the call profile characterized by an increase in the percentage of frequency-modulated 50-kHz USV, which may indicate the mania-like consequences of SD. Importantly, all SD effects were reverted by lithium administration. SD also led to a decrease in dopamine content in the ventral striatum, while increasing dopamine turnover. In conclusion, SD increased 50-kHz USV emission, an effect prevented by acute lithium administration. This suggests 50-kHz USV as a new marker for mania-like elevated mood, which shows construct validity (associated with increased dopaminergic tone), face validity (reflecting increased positive affect) and predictive validity (high sensitivity to lithium treatment).

Revisiting the autoconditioning hypothesis for acquired reactivity to ultrasonic alarm calls.

Rats emit 22 kHz ultrasonic vocalizations (USVs) in association with pain, fear, or distress. Whereas the capacity to produce USVs is innate, reactivity to them appears to require experience. Specifically, 22 kHz USVs fail to elicit freezing behavior in naïve laboratory rats. However, these "alarm calls" do elicit freezing in rats that previously experienced foot shocks. These findings led to the hypothesis that acquired reactivity is based on "autoconditioning"-learning in which self-generated 22 kHz USVs serve as Pavlovian cues that become associated with foot shocks. The current study tested the autoconditioning hypothesis by devocalizing rats through a unilateral transection of the recurrent laryngeal nerve (Experimental group). Subsequently, animals in both the Experimental and sham-operated Control groups received five unsignaled foot shocks. One or two days later, both groups were tested for USV-elicited freezing in a novel context. Recurrent laryngeal nerve transection failed to prevent or even diminish USV-elicited freezing. In fact, both groups showed large and comparable increases in freezing to USV presentations. A subset of Control animals failed to vocalize during conditioning, while some Experimental animals did vocalize during conditioning. Animals were therefore re-grouped and reanalyzed based on whether they vocalized during conditioning. Again, both groups showed large and comparable increases in USV-elicited freezing. These results disconfirm the essential tenet or prediction of the autoconditioning hypothesis. Alternative mechanisms for acquired reactivity to 22 kHz USVs are therefore considered.

Effects of anxiogenic drugs on the emission of 22- and 50-kHz ultrasonic vocalizations in adult rats.

Adult rat 22-kHz vocalizations are often associated with alarm or distress, whereas a subset of 50-kHz calls is preferentially emitted in response to amphetamine and other rewarding stimuli. Whether any 50-kHz calls reflect anxiety is unknown. To determine the effects of anxiogenic drugs on 50-kHz call rate and call subtype profile, in comparison with D-amphetamine. Adult male rats received systemic amphetamine (1mg/kg) three times several days before testing. Ultrasonic vocalizations were then recorded after acute intraperitoneal injection of amphetamine or one of five anxiogenic drugs: yohimbine (2.5mg/kg), N-methyl-β-carboline-3-carboxamide (FG 7142, 5mg/kg), pentylenetetrazol (PTZ, 20mg/kg), m-chlorophenylpiperazine (mCPP, 1mg/kg), caffeine (25mg/kg), or vehicle. The duration of immobility was increased by FG 7142, PTZ, and mCPP; this measure was unchanged by yohimbine and reduced by the locomotor stimulant drugs amphetamine and caffeine. Conversely, the 50-kHz call rate was reduced by FG 7142, PTZ and mCPP, and increased by caffeine and amphetamine. Overall, the most common 50-kHz call subtypes were flat, trill, step-up, and complex. Consistent with previous reports, amphetamine increased the relative prevalence of trill calls while reducing the relative prevalence of flat calls. Yohimbine and caffeine reduced flat call prevalence, whereas mCPP reduced trill call prevalence. No other shifts in the call profile were observed, and no anxiogenic drug induced 22-kHz calls. Anxiogenic drugs, as a class, did not uniformly alter the 50-kHz call rate or subtype profile. Amphetamine-induced effects on 50-kHz call rate and profile do not reflect anxiety.

Maternal separation stress reduced prenatal-ethanol-induced increase in exploratory behaviour and extracellular signal-regulated kinase activity

In an attempt to better represent the aetiology of fetal alcohol spectrum disorder (FASD) and the associated psychological deficits, prenatal-ethanol exposure was followed by maternal separation in a rat model in order to account for the effects of early-life adversities in addition to in utero alcohol exposure. Extracellular signal-regulated kinase 1/2 (ERK1/2) and glycogen synthase kinase 3-β (GSK3β) are converging points for many signalling cascades and have been implicated in models of FASD and models of early-life stress. Therefore, these kinases may also contribute to the behavioural changes observed after the combination of both developmental insults. In this study, ethanol-dams voluntarily consumed a 0.066% saccharin-sweetened 10% ethanol (EtOH) solution for 10 days prior to pregnancy and throughout gestation while control-dams had ad libitumaccess to a 0.066% saccharin (sacc) solution. Whole litters were randomly assigned to undergo maternal separation (MS) for 3 h/day from P2 to P14 while the remaining litters were left undisturbed (nMS). This resulted in 4 experimental groups: control (sacc + nMS), MS (sacc + MS), EtOH (EtOH + nMS) and EtOH + MS. Throughout development, EtOH-rats weighed less than control rats. However, subsequent maternal separation stress caused EtOH + MS-rats to weigh more than EtOH-rats. In adulthood both MS- and EtOH-rats were hyperactive but the combination produced activity levels similar to that of control rats. All treated animals (MS-, EtOH- and EtOH + MS-rats) demonstrated a negative affective state shown by increased number and duration of 22 kHz ultrasonic vocalizations compared to control rats. Prenatal-ethanol exposure increased the P-GSK3β/GSK3β ratio in the prefrontal cortex (PFC) and maternal separation decreased the P-GSK3β/GSK3β ratio in the dorsal hippocampus (DH) of adult rats. However, maternal separation stress decreased the effect of prenatal-ethanol exposure on the P-ERK/ERK ratio in the PFC and DH and reduced prenatal-ethanol-induced hyperactivity. Therefore, indicating a significant interaction between prenatal-ethanol exposure and early-life stress on behaviour and the brain and may implicate P-ERK1/2 signalling in exploratory behaviour.

Individual behavioural predictors of amphetamine-induced emission of 50 kHz vocalization in rats

Measurement of ultrasonic vocalizations (USVs) produced by adult rats represents a highly useful index of emotional arousal. The associations found between 50 kHz USV production and a variety of behavioural and pharmacological protocols increasingly suggests they serve as a marker of positive motivational states. This study used a powerful within-subjects design to investigate the relationships among individual differences in approach to a sweet-food reward, predisposition to emit 50 kHz USVs spontaneously, and 50 kHz USVs emission following acute systemic administration of amphetamine. Both approach motivation and predisposition to call were found to not correlate with each other but did predict 50 kHz USV response to acute amphetamine. These two behavioural phenotypes appear to represent dissociable predictors of acute amphetamine-induced emission of 50 kHz USVs in a non-sensitization paradigm. In contrast to that, a measure of sucrose preference was not found to predict 50 kHz USV emission following amphetamine. Acute amphetamine was also found to increase average sound frequency of emitted USVs and selectively increase the proportion of Trill subtype 50 kHz USVs. Together, these data demonstrate that acute amphetamine-induced 50 kHz USVs in the adult rat represent more than just a univariate motivational state and may represent the product of dissociable subsystems of emotional behavior.

Pioglitazone abolishes autistic-like behaviors via the IL-6 pathway.

Autism is characterized by social deficits, communication abnormalities, and repetitive behaviors. The risk factors appear to include genetic and environmental conditions, such as prenatal infections and maternal dietary factors. Previous investigations by our group have demonstrated that prenatal exposure to lipopolysaccharide (LPS), which mimics infections by gram-negative bacteria, induces autistic-like behaviors. No effective treatment yet exists for autism. Therefore, we used our rat model to test a possible treatment for autism. We selected pioglitazone to block or ease the impairments induced by LPS because although this drug was designed as an anti-diabetic drug (it has an insulin effect), it also exerts anti-inflammatory effects. Juvenile offspring were treated daily with pioglitazone, and the main behaviors related to autism, namely, socialization (play behavior) and communication (50-kHz ultrasonic vocalizations), were studied. Biomarkers linked to autism and/or pioglitazone were also studied to attempt to understand the mechanisms involved, namely, IL-6, TNF-alpha, MCP-1, insulin, and leptin. Prenatal LPS exposure induced social deficits and communicational abnormalities in juvenile rat offspring as well as elevated plasma IL-6 levels. Daily postnatal pioglitazone treatment blocked the impairments found in terms of the time spent on social interaction, the number of vocalizations (i.e., autistic-like behaviors) and the elevated plasma IL-6 levels. Thus, pioglitazone appears to be a relevant candidate for the treatment of autism. The present findings may contribute to a better understanding and treatment of autism and associated diseases.

Effects of acute morphine withdrawal on ultrasonic vocalizations in adult rats: unchanged 50-kHz call rate and altered subtype profile.

Adult rat 22- and 50-kHz ultrasonic vocalizations (USVs) are commonly considered as indices of negative and positive affect, respectively. More specifically, we have proposed that positive affective states are revealed by a predominance of trill over flat 50-kHz call subtypes. However, the 50-kHz call subtypes emitted during aversive drug states remain largely uninvestigated. To determine whether acute morphine withdrawal affects 50-kHz call rates or alters the relative prevalence of trill and flat calls. In experiment 1, adult male rats were given saline or morphine (6mg/kg SC), then acutely challenged 4h later with saline or naloxone (1mg/kg SC), and recorded 10-30min post-injection. In experiments 2 and 3, rats received saline or morphine (6mg/kg), followed 4h later by acute saline or naloxone (0.1mg/kg) challenge; USVs were subsequently recorded during 30-min place conditioning sessions. Naloxone (0.1mg/kg) produced a strong conditioned place aversion only after acute morphine pretreatment, consistent with antagonist-precipitated morphine withdrawal. The morphine-naloxone combination decreased the relative prevalence of trills and promoted flat calls. Naloxone given alone (0.1 and 1mg/kg) inhibited trill calls but did not significantly alter the prevalence of flat calls, whereas morphine given alone (4h pre-session) was largely without effect. Fifty-kHz call rates were inhibited by naloxone given alone, but otherwise unaffected. Twenty-two-kHz calls were sparse. The 50-kHz call subtype shift seen during antagonist-precipitated morphine withdrawal was opposite in direction to that previously associated with rewards, and hence may reveal negative affect.

Diazepam blocks 50kHz ultrasonic vocalizations and stereotypies but not the increase in locomotor activity induced in rats by amphetamine.

We have recently shown that the benzodiazepine diazepam inhibits dopamine release in the NAc and blocks the increased release of dopamine induced by DL-amphetamine. Rewarding stimuli and many drugs of abuse can induce dopamine release in the nucleus accumbens as well as 50-kHz ultrasonic vocalizations (USVs) in rats. In the present study, we tested the hypothesis that diazepam can also block the increase in locomotor activity and USVs elicited by amphetamine. Fifty-kilohertz USVs, stereotypy, and locomotor behavior were scored in adult male Wistar rats treated with i.p. injections of saline, 3mg/kg DL-amphetamine, 2mg/kg diazepam, 0.2mg/kg haloperidol, or a combination of these drugs. In agreement with previous studies, amphetamine caused significant increases in the number of USV calls, stereotypies, and locomotor activity. The D2 dopamine receptor antagonist haloperidol blocked the effects of amphetamine on USVs, stereotypy, and locomotor activity. Diazepam blocked the effect of amphetamine on USV and stereotypy, but not on horizontal locomotion. These results suggest that diazepam blocks the rewarding effect of amphetamine. This finding is promising for basic research regarding treatments of substance use disorders and evaluation of the impact of benzodiazepines on motivation.

Emission of categorized 50-kHz ultrasonic vocalizations in rats repeatedly treated with amphetamine or apomorphine: Possible relevance to drug-induced modifications in the emotional state

The emission of 50-kHz ultrasonic vocalizations (USVs) is increasingly emerging as a potential behavioral marker of the subjective effects that psychoactive drugs elicit in rats. However, multiple categories of 50-kHz USVs have been identified, which are thought to possess different behavioral significance. Besides, limited information is available on how psychoactive drugs affect the emission of categorized 50-kHz USVs. To further elucidate this issue, we evaluated the numbers of multiple categories of 50-kHz USVs emitted by rats repeatedly treated with amphetamine (1 or 2 mg/kg, i.p.) or apomorphine (2 or 4 mg/kg, i.p.), two drugs that elicit similar and dissimilar subjective effects. Amphetamine- and apomorphine-treated rats emitted patterns of categorized 50-kHz USVs that varied according to the drug administered, drug dose, and number of drug administrations. Nevertheless, the numbers of several categories of 50-kHz USVs were positively correlated with the number of total calls emitted (i.e., the sum of categorized 50-kHz USVs). Moreover, a marked interindividual variability in the emission of categorized 50-kHz USVs was observed. Taken together, the present results may be relevant to further elucidating the interplay between calling of the 50-kHz USVs group and psychopharmacological profile of drugs.