BTBR T+ tf/J (BTBR) is an inbred mouse strain that displays social abnormalities and repetitive behaviors analogous to the first and third diagnostic symptoms of autism. Here we investigate ultrasonic vocalizations in BTBR, to address the second diagnostic symptom of autism, communication deficits. As compared to the commonly used C57BL/6J (B6) strain, BTBR pups called more loudly and more frequently when separated from their mothers and siblings. Detailed analysis of ten categories of calls revealed an unusual pattern in BTBR as compared to B6. BTBR emitted high levels of harmonics, two-syllable, and composite calls, but minimal numbers of chevron-shaped syllables, upward, downward, and short calls. Because body weights were higher in BTBR than B6 pups, one possible explanation was that larger thoracic size was responsible for the louder calls and different distribution of syllable categories. To test this possibility, we recorded separation calls from FVB/NJ, a strain with body weights similar to BTBR, and 129X1/SvJ, a strain with body weights similar to B6. BTBR remained the outlier on number of calls, displaying low numbers of complex, upward, chevron, short, and frequency steps calls, along with high harmonics and composites. Further, developmental milestones and growth rates were accelerated in BTBR, indicating an unusual neurodevelopmental trajectory. Overall, our findings demonstrate strain-specific patterns of ultrasonic calls that may represent different lexicons, or innate variations in complex vocal repertoires, in genetically distinct strains of mice. Particularly intriguing is the unusual pattern of vocalizations and the more frequent, loud harmonics evident in the BTBR mouse model of autism that may resemble the atypical vocalizations seen in some autistic infants.

In neonatal mice ultrasonic vocalizations have been studied both as an early communicative behavior of the pup-mother dyad and as a sign of an aversive affective state. Adult mice of both sexes produce complex ultrasonic vocalization patterns in different experimental/social contexts. All these vocalizations are becoming an increasingly valuable assay for behavioral phenotyping throughout the mouse life-span and alterations of the ultrasound patterns have been reported in several mouse models of neurodevelopmental disorders. Here we also show that the modulation of vocalizations by maternal cues (maternal potentiation paradigm) -originally identified and investigated in ratscan be measured in C57Bl/6 mouse pups with appropriate modifications of the rat protocol and can likely be applied to mouse behavioral phenotyping. In addition we suggest that a detailed qualitative evaluation of neonatal calls together with analysis of adult mouse vocalization patterns in both sexes in social settings, may lead to a greater understanding of the communication value of vocalizations in mice. Importantly, both neonatal and adult USV altered patterns can be determined during the behavioural phenotyping of mouse models of human neurodevelopmental and neuropsychiatric disorders, starting from those in which deficits in communication are a primary symptom.

BTBR T+tf/J (BTBR) is an inbred mouse strain that displays social deficits and repetitive behaviors analogous to the first and third diagnostic symptoms of autism. We previously reported an unusual pattern of ultrasonic vocalizations in BTBR pups that may represent a behavioral homolog to the second diagnostic symptom of autism, impaired communication. This study investigated the social and vocal repertoire in adult BTBR mice, to evaluate the role of ultrasonic vocalizations in multiple social situations at the adult stage of development. Three different social contexts were considered: male-female, male-male (resident-intruder) and female-female interactions. Behavioral responses and ultrasonic vocalizations were recorded for BTBR and for the highly social control strain C57BL/6J (B6). No episodes of overt fighting or mating were observed during the short durations of the three different experimental encounters. BTBR displayed lower levels of vocalizations and social investigation in all three social contexts as compared with B6. In addition, the correlation analyses between social investigation and ultrasonic vocalization emission rate showed that in B6 mice, the two variables were positively correlated in all the three different social settings, whereas in BTBR mice, the positive correlation was significant only in the male-female interactions. These findings strongly support the value of simultaneously recording two aspects of the mouse social repertoire: social motivation and bioacoustic communication. Moreover, our findings in adults are consistent with previous results in pups, showing an unusual vocal repertoire in BTBR as compared with B6.

No abstract

Developmental exposure to the organophosphorous insecticide chlorpyrifos (CPF) induces long-term effects on brain and behavior in laboratory rodents. We evaluated in adult mice the behavioral effects of either fetal and/or neonatal CPF exposure at doses not inhibiting fetal and neonatal brain cholinesterase. CPF (3 or 6 mg/kg) was given by oral treatment to pregnant females on gestational days 15-18 and offspring were treated sc (1 or 3 mg/kg) on postnatal days (PNDs) 11-14. Serum and brain acetylcholinesterase (AChE) activity was evaluated at birth and 24 h from termination of postnatal treatments. On PND 70, male mice were assessed for spontaneous motor activity in an open-field test and in a socioagonistic encounter with an unfamiliar conspecific. Virgin females underwent a maternal induction test following presentation of foster pups. Both sexes were subjected to a plus-maze test to evaluate exploration and anxiety levels. Gestational and postnatal CPF exposure (higher doses) affected motor activity in the open field and enhanced synergically agonistic behavior. Postnatal CPF exposure increased maternal responsiveness toward pups in females. Mice of both sexes exposed to postnatal CPF showed reduced anxiety response in the plus-maze, an effect greater in females. Altogether, developmental exposure to CPF at doses that do not cause brain AChE inhibition induces long-term alterations in sex-specific behavior patterns of the mouse species. Late neonatal exposure on PNDs 11-14 was the most effective in causing behavioral changes. These findings support the hypothesis that developmental CPF may represent a risk factor for increased vulnerability to neurodevelopmental disorders in humans.

RhoGTPases are crucial molecules in neuronal plasticity and cognition, as confirmed by their role in non-syndromic mental retardation. Activation of brain RhoGTPases by the bacterial cytotoxic necrotizing factor 1 (CNF1) reshapes the actin cytoskeleton and enhances neurotransmission and synaptic plasticity in mouse brains. We evaluated the effects of a single CNF1 intracerebroventricular inoculation in a mouse model of Rett syndrome (RTT), a rare neurodevelopmental disorder and a genetic cause of mental retardation, for which no effective therapy is available. Fully symptomatic MeCP2-308 male mice were evaluated in a battery of tests specifically tailored to detect RTT-related impairments. At the end of behavioral testing, brain sections were immunohistochemically characterized. Magnetic resonance imaging and spectroscopy (MRS) were also applied to assess morphological and metabolic brain changes. The CNF1 administration markedly improved the behavioral phenotype of MeCP2-308 mice. CNF1 also dramatically reversed the evident signs of atrophy in astrocytes of mutant mice and restored wt-like levels of this cell population. A partial rescue of the overexpression of IL-6 cytokine was also observed in RTT brains. CNF1-induced brain metabolic changes detected by MRS analysis involved markers of glial integrity and bioenergetics, and point to improved mitochondria functionality in CNF1-treated mice. These results clearly indicate that modulation of brain RhoGTPases by CNF1 may constitute a totally innovative therapeutic approach for RTT and, possibly, for other disorders associated with mental retardation.

The neuropeptides oxytocin and vasopressin have been implicated in rodent social and affiliative behaviors, including social bonding, parental care, social recognition, social memory, vocalizations, territoriality, and aggression, as well as components of human social behaviors and the etiology of autism. Previous investigations of mice with various manipulations of the oxytocin and vasopressin systems reported unusual levels of ultrasonic vocalizations in social settings. We employed a vasopressin 1b receptor (Avpr1b) knockout mouse to evaluate the role of the vasopressin 1b receptor subtype in the emission of ultrasonic vocalizations in adult and infant mice. Avpr1b null mutant female mice emitted fewer ultrasonic vocalizations, and their vocalizations were generally at lower frequencies, during a resident-intruder test. Avpr1b null mutant pups emitted ultrasonic vocalizations similar to heterozygote and wildtype littermates when separated from the nest on postnatal days 3, 6, 9, and 12. However, maternal potentiation of ultrasonic vocalizations in Avpr1b null and heterozygote mutants was absent, when tested at postnatal day 9. These results indicate that Avpr1b null mutant mice are impaired in the modulation of ultrasonic vocalizations within different social contexts at infant and adult ages.

Rett syndrome (RTT) is a rare neurodevelopmental disorder affecting almost exclusively females, caused in the overwhelming majority of the cases by loss-of-function mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). High circulating levels of oxidative stress (OS) markers in patients suggest the involvement of OS in the RTT pathogenesis. To investigate the occurrence of oxidative brain damage in Mecp2 mutant mouse models, several OS markers were evaluated in whole brains of Mecp2-null (pre-symptomatic, symptomatic, and rescued) and Mecp2-308 mutated (pre-symptomatic and symptomatic) mice, and compared to those of wild type littermates. Selected OS markers included non-protein-bound iron, isoprostanes (F2-isoprostanes, F4-neuroprostanes, F2-dihomo-isoprostanes) and 4-hydroxy-2-nonenal protein adducts. Our findings indicate that oxidative brain damage 1) occurs in both Mecp2-null (both −/y and stop/y) and Mecp2-308 (both 308/y males and 308/+ females) mouse models of RTT; 2) precedes the onset of symptoms in both Mecp2-null and Mecp2-308 models; and 3) is rescued by Mecp2 brain specific gene reactivation. Our data provide direct evidence of the link between Mecp2 deficiency, oxidative stress and RTT pathology, as demonstrated by the rescue of the brain oxidative homeostasis following brain-specifically Mecp2-reactivated mice. The present study indicates that oxidative brain damage is a previously unrecognized hallmark feature of murine RTT, and suggests that Mecp2 is involved in the protection of the brain from oxidative stress.