Excessive Laughter-like Vocalizations, Microcephaly, and Translational Outcomes in the Ube3a Deletion Rat Model of Angelman Syndrome.

Angelman syndrome (AS) is a rare genetic neurodevelopmental disorder characterized by intellectual disabilities, motor and balance deficits, impaired communication, and a happy, excitable demeanor with frequent laughter. We sought to elucidate a preclinical outcome measure in male and female rats that addressed communication abnormalities of AS and other neurodevelopmental disorders in which communication is atypical and/or lack of speech is a core feature. We discovered, and herein report for the first time, excessive laughter-like 50 kHz ultrasonic emissions in the Ube3amat-/pat+ rat model of AS, which suggests an excitable, playful demeanor and elevated positive affect, similar to the demeanor of individuals with AS. Also in line with the AS phenotype, Ube3amat-/pat+ rats demonstrated aberrant social interactions with a novel partner, distinctive gait abnormalities, impaired cognition, an underlying LTP deficit, and profound reductions in brain volume. These unique, robust phenotypes provide advantages compared with currently available mouse models and will be highly valuable as outcome measures in the evaluation of therapies for AS.SIGNIFICANCE STATEMENT Angelman syndrome (AS) is a severe neurogenetic disorder for which there is no cure, despite decades of research using mouse models. This study used a recently developed rat model of AS to delineate disease-relevant outcome measures to facilitate therapeutic development. We found the rat to be a strong model of AS, offering several advantages over mouse models by exhibiting numerous AS-relevant phenotypes, including overabundant laughter-like vocalizations, reduced hippocampal LTP, and volumetric anomalies across the brain. These findings are unconfounded by detrimental motor abilities and background strain, issues plaguing mouse models. This rat model represents an important advancement in the field of AS, and the outcome metrics reported herein will be central to the therapeutic pipeline.

Contingent Social Interaction Does Not Prevent Habituation towards Playback of Pro-Social 50-kHz Calls: Behavioral Responses and Brain Activation Patterns.

Rats, which are highly social animals, are known to communicate using ultrasonic vocalizations (USV) in different frequency ranges. Calls around 50 kHz are related to positive affective states and promote social interactions. Our previous work has shown that the playback of natural 50-kHz USV leads to a strong social approach response toward the sound source, which is related to activation in the nucleus accumbens. In male Wistar rats, the behavioral response habituates, that is, becomes weaker or is even absent, when such playback is repeated several days later, an outcome found to be memory-dependent. Here, we asked whether such habituation is due to the lack of a contingent social consequence after playback in the initial test and whether activation of the nucleus accumbens, as measured by c-fos immunohistochemistry, can still be observed in a retest. To this end, groups of young male Wistar rats underwent an initial 50-kHz USV playback test, immediately after which they were either (1) kept temporarily alone, (2) exposed to a same-sex juvenile, or (3) to their own housing group. One week later, they underwent a retest with playback; this time not followed by social consequences but by brain removal for c-fos immunohistochemistry. Consistent with previous reports, behavioral changes evoked by the initial exposure to 50-kHz USV playback included a strong approach response. In the retest, no such response was found, irrespective of whether rats had experienced a contingent social consequence after the initial test or not. At the neural level, no substantial c-fos activation was found in the nucleus accumbens, but unexpected strong activation was detected in the anterior cingulate cortex, with some of it in GABAergic cells. The c-fos patterns did not differ between groups but cell numbers were individually correlated with behavior, i.e., rats that still approached in response to playback in the retest showed more activation. Together, these data do not provide substantial evidence that the lack of a contingent social consequence after 50-kHz USV playback accounts for approach habituation in the retest. Additionally, there is apparently no substantial activation of the nucleus accumbens in the retest, whereas the exploratory findings in the anterior cingulate cortex indicate that this brain area might be involved when individual rats still approach 50-kHz USV playback.

Response Calls Evoked by Playback of Natural 50-kHz Ultrasonic Vocalizations in Rats.

Rats are highly social animals known to communicate with ultrasonic vocalizations (USV) of different frequencies. Calls around 50 kHz are thought to represent a positive affective state, whereas calls around 22 kHz are believed to serve as alarm or distress calls. During playback of natural 50-kHz USV, rats show a reliable and strong social approach response toward the sound source. While this response has been studied in great detail in numerous publications, little is known about the emission of USV in response to natural 50-kHz USV playback. To close this gap, we capitalized on three data sets previously obtained and analyzed USV evoked by natural 50-kHz USV playback in male juvenile rats. We compared different rat stocks, namely Wistar (WI) and Sprague-Dawley (SD) and investigated the pharmacological treatment with the dopaminergic D2 receptor antagonist haloperidol. These response calls were found to vary broadly inter-individually in numbers, mean peak frequencies, durations and frequency modulations. Despite the large variability, the results showed no major differences between experimental conditions regarding call likelihood or call parameters, representing a robust phenomenon. However, most response calls had clearly lower frequencies and were longer than typical 50-kHz calls, i.e., around 30 kHz and lasting generally around 0.3 s. These calls resemble aversive 22-kHz USV of adult rats but were of higher frequencies and shorter durations. Moreover, blockade of dopamine D2 receptors did not substantially affect the emission of response calls suggesting that they are not dependent on the D2 receptor function. Taken together, this study provides a detailed analysis of response calls toward playback of 50-kHz USV in juvenile WI and SD rats. This includes calls representing 50-kHz USV, but mostly calls with lower frequencies that are not clearly categorizable within the so far known two main groups of USV in adult rats. We discuss the possible functions of these response calls addressing their communicative functions like contact or appeasing calls, and whether they may reflect a state of frustration. In future studies, response calls might also serve as a new read-out in rat models for neuropsychiatric disorders, where acoustic communication is impaired, such as autism spectrum disorder.

Limited generalizability, pharmacological modulation, and state-dependency of habituation towards pro-social 50-kHz calls in rats.

Communication constitutes a fundamental component of mammalian social behavior. Rats are highly social animals and emit 50-kHz ultrasonic vocalizations (USV), which function as social contact calls. Playback of 50-kHz USV leads to strong and immediate social approach responses in receiver rats, but this response is weak or even absent during repeated 50-kHz USV playback. Given the important role of 50-kHz USV in initiating social contact and coordinating social interactions, the occurrence of habituation is highly unexpected. It is not clear why a social signal characterized by significant incentive salience loses its power to change the behavior of the receiver so rapidly. Here, we show that the habituation phenomenon displayed by rats in response to repeated playback of 50-kHz USV (1) is characterized by limited generalizability because it is present in Wistar but not Sprague-Dawley rats, (2) can be overcome by amphetamine treatment, and (3) depends on the subject's internal state.

Antioxidant Treatment with N-acetyl Cysteine Prevents the Development of Cognitive and Social Behavioral Deficits that Result from Perinatal Ketamine Treatment.

Alterations of the normal redox state can be found in all stages of schizophrenia, suggesting a key role for oxidative stress in the etiology and maintenance of the disease. Pharmacological blockade of N-methyl-D-aspartic acid (NMDA) receptors can disrupt natural antioxidant defense systems and induce schizophrenia-like behaviors in animals and healthy human subjects. Perinatal administration of the NMDA receptor (NMDAR) antagonist ketamine produces persistent behavioral deficits in adult mice which mimic a range of positive, negative, and cognitive symptoms that characterize schizophrenia. Here we tested whether antioxidant treatment with the glutathione (GSH) precursor N-acetyl-cysteine (NAC) can prevent the development of these behavioral deficits. On postnatal days (PND) 7, 9 and 11, we treated mice with subanesthetic doses (30 mg/kg) of ketamine or saline. Two groups (either ketamine or saline treated) also received NAC throughout development. In adult animals (PND 70-120) we then assessed behavioral alterations in a battery of cognitive and psychomotor tasks. Ketamine-treated animals showed deficits in a task of cognitive flexibility, abnormal patterns of spontaneous alternation, deficits in novel-object recognition, as well as social interaction. Developmental ketamine treatment also induced behavioral stereotypy in response to an acute amphetamine challenge, and it impaired sensorimotor gating, measured as reduced prepulse inhibition (PPI) of the startle response. All of these behavioral abnormalities were either prevented or strongly ameliorated by NAC co-treatment. These results suggest that oxidative stress is a major factor for the development of the ketamine-induced behavioral dysfunctions, and that restoring oxidative balance during the prodromal stage of schizophrenia might be able to ameliorate the development of several major symptoms of the disease.

Microglomerular Synaptic Complexes in the Sky-Compass Network of the Honeybee Connect Parallel Pathways from the Anterior Optic Tubercle to the Central Complex.

While the ability of honeybees to navigate relying on sky-compass information has been investigated in a large number of behavioral studies, the underlying neuronal system has so far received less attention. The sky-compass pathway has recently been described from its input region, the dorsal rim area (DRA) of the compound eye, to the anterior optic tubercle (AOTU). The aim of this study is to reveal the connection from the AOTU to the central complex (CX). For this purpose, we investigated the anatomy of large microglomerular synaptic complexes in the medial and lateral bulbs (MBUs/LBUs) of the lateral complex (LX). The synaptic complexes are formed by tubercle-lateral accessory lobe neuron 1 (TuLAL1) neurons of the AOTU and GABAergic tangential neurons of the central body's (CB) lower division (TL neurons). Both TuLAL1 and TL neurons strongly resemble neurons forming these complexes in other insect species. We further investigated the ultrastructure of these synaptic complexes using transmission electron microscopy. We found that single large presynaptic terminals of TuLAL1 neurons enclose many small profiles (SPs) of TL neurons. The synaptic connections between these neurons are established by two types of synapses: divergent dyads and divergent tetrads. Our data support the assumption that these complexes are a highly conserved feature in the insect brain and play an important role in reliable signal transmission within the sky-compass pathway.

Transmedulla Neurons in the Sky Compass Network of the Honeybee (Apis mellifera) Are a Possible Site of Circadian Input.

Honeybees are known for their ability to use the sun's azimuth and the sky's polarization pattern for spatial orientation. Sky compass orientation in bees has been extensively studied at the behavioral level but our knowledge about the underlying neuronal systems and mechanisms is very limited. Electrophysiological studies in other insect species suggest that neurons of the sky compass system integrate information about the polarization pattern of the sky, its chromatic gradient, and the azimuth of the sun. In order to obtain a stable directional signal throughout the day, circadian changes between the sky polarization pattern and the solar azimuth must be compensated. Likewise, the system must be modulated in a context specific way to compensate for changes in intensity, polarization and chromatic properties of light caused by clouds, vegetation and landscape. The goal of this study was to identify neurons of the sky compass pathway in the honeybee brain and to find potential sites of circadian and neuromodulatory input into this pathway. To this end we first traced the sky compass pathway from the polarization-sensitive dorsal rim area of the compound eye via the medulla and the anterior optic tubercle to the lateral complex using dye injections. Neurons forming this pathway strongly resembled neurons of the sky compass pathway in other insect species. Next we combined tracer injections with immunocytochemistry against the circadian neuropeptide pigment dispersing factor and the neuromodulators serotonin, and γ-aminobutyric acid. We identified neurons, connecting the dorsal rim area of the medulla to the anterior optic tubercle, as a possible site of neuromodulation and interaction with the circadian system. These neurons have conspicuous spines in close proximity to pigment dispersing factor-, serotonin-, and GABA-immunoreactive neurons. Our data therefore show for the first time a potential interaction site between the sky compass pathway and the circadian clock.