Shank2 Mutant Mice Display Hyperactivity Insensitive to Methylphenidate and Reduced Flexibility in Social Motivation, but Normal Social Recognition

Mouse models of autism can be used to study evolutionarily conserved mechanisms underlying behavioral abnormalities in social communication and repetitive behaviors. SHANK genes code for synaptic scaffolding proteins at excitatory synapses and mutations in all SHANK genes have been associated with autism. Here, we present three behavioral aspects of the mutant mice deleted for exon 16 in Shank2. First, we treated Shank2 mutant mice with methylphenidate to rescue the hyperactivity. Our failure to do so suggests that the hyperactivity displayed by Shank2 mutant mice is not related to the one displayed by the typical mouse models of hyperactivity, and might be more closely related to manic-like behaviors. Second, by testing the effect of group housing and social isolation on social interest, we highlighted that Shank2 mutant mice lack the typical flexibility to modulate social interest, in comparison with wild-type littermates. Finally, we established a new protocol to test for social recognition in a social context. We used this protocol to show that Shank2 mutant mice were able to discriminate familiar and unknown conspecifics in free interactions. Altogether, these studies shed some light on specific aspects of the behavioral defects displayed by the Shank2 mouse model. Such information could be used to orient therapeutic strategies and to design more specific tests to characterize the complex behavior of mouse models of autism

Toll-Like Receptor 3 (TLR3) Plays a Major Role in the Formation of Rabies Virus Negri Bodies

Human neurons express the innate immune response receptor, Toll-like receptor 3 (TLR3). TLR3 levels are increased in pathological conditions such as brain virus infection. Here, we further investigated the production, cellular localisation, and function of neuronal TLR3 during neuronotropic rabies virus (RABV) infection in human neuronal cells. Following RABV infection, TLR3 is not only present in endosomes, as observed in the absence of infection, but also in detergent-resistant perinuclear inclusion bodies. As well as TLR3, these inclusion bodies contain the viral genome and viral proteins (N and P, but not G). The size and composition of inclusion bodies and the absence of a surrounding membrane, as shown by electron microscopy, suggest they correspond to the previously described Negri Bodies (NBs). NBs are not formed in the absence of TLR3, and TLR3−/− mice—in which brain tissue was less severely infected—had a better survival rate than WT mice. These observations demonstrate that TLR3 is a major molecule involved in the spatial arrangement of RABV–induced NBs and viral replication. This study shows how viruses can exploit cellular proteins and compartmentalisation for their own benefit

The Autism ProSAP1/Shank2 mouse model displays quantitative and structural abnormalities in ultrasonic vocalisations.

International audienceMouse ultrasonic vocalisations have been often used as a paradigm to extrapolate vocal communication defects observed in patients with autism spectrum disorders (ASD). The role of these vocalisations as well as their development, structure and informational content, however, remain largely unknown. In the present study, we characterised in depth the emission of pup and adult ultrasonic vocalisations of wild-type mice and their ProSAP1/Shank2(-/-) littermates lacking a synaptic scaffold protein mutated in ASD. We hypothesised that the vocal behaviour of ProSAP1/Shank2(-/-) mice not only differs from the vocal behaviour of their wild-type littermates in a quantitative way, but also presents more qualitative abnormalities in temporal organisation and acoustic structure. We first quantified the rate of emission of ultrasonic vocalisations, and analysed the organisation of vocalisations sequences using Markov models. We subsequently measured duration and peak frequency characteristics of each ultrasonic vocalisation, to characterise their acoustic structure. In wild-type mice, we found a high level of organisation in sequences of ultrasonic vocalisations, suggesting a communicative function in this complex system. Very limited significant sex-related variations were detected in their usage and acoustic structure, even in adult mice. In adult ProSAP1/Shank2(-/-) mice, we found abnormalities in the call usage and the structure of ultrasonic vocalisations. Both ProSAP1/Shank2(-/-) male and female mice uttered less vocalisations with a different call distribution and at lower peak frequency in comparison with wild-type littermates. This study provides a comprehensive framework to characterise abnormalities of ultrasonic vocalisations in mice and confirms that ProSAP1/Shank2(-/-) mice represent a relevant model to study communication defects

Social communication in mice - Are there optimal cage conditions?

Video and audio recordings of social interactions in adult male C57BL/6J mice under different conditions (habituation time to the test cage and cage shape and size

Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging

Antibodies normally do not cross the blood-brain barrier (BBB) and cannot bind an intracellular cerebral antigen. We demonstrate here for the first time that a new class of antibodies can cross the BBB without treatment. Camelids produce native homodimeric heavy-chain antibodies, the paratope being composed of a single-variable domain called VHH. Here, we used recombinant VHH directed against human glial fibrillary acidic protein (GFAP), a specific marker of astrocytes. Only basic VHHs (e.g., pI=9.4) were able to cross the BBB in vitro (7.8 vs. 0% for VHH with pI=7.7). By intracarotid and intravenous injections into live mice, we showed that these basic VHHs are able to cross the BBB in vivo, diffuse into the brain tissue, penetrate into astrocytes, and specifically label GFAP. To analyze their ability to be used as a specific transporter, we then expressed a recombinant fusion protein VHH-green fluorescent protein (GFP). These "fluobodies" specifically labeled GFAP on murine brain sections, and a basic variant (pI=9.3) of the fusion protein VHH-GFP was able to cross the BBB and to label astrocytes in vivo. The potential of VHHs as diagnostic or therapeutic agents in the central nervous system now deserves attention. Li, T., Bourgeois, J.-P., Celli, S., Glacial, F., Le Sourd, A.-M., Mecheri, S., Weksler, B., Romero, I., Couraud, P.-O., Rougeon, F., and Lafaye, P. Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging

Sevoflurane Anesthesia Alters Exploratory and Anxiety-like Behavior in Mice Lacking the β2 Nicotinic Acetylcholine Receptor Subunit

International audienceBackgroundPreexisting cognitive impairment and advanced age are factors that increase the risk of developing postoperative cognitive dysfunction. Because anesthetic agents interfere with cholinergic transmission and as impairment of cholinergic function is associated with cognitive decline, the authors studied how the volatile anesthetic sevoflurane affects exploratory and anxiety-like behavior in young and aged animals with a genetically modified cholinergic system.MethodsYoung and aged wild-type and mutant mice lacking the beta2 subunit of the nicotinic cholinergic receptor (beta2KO) were anesthetized for 2 h with 2.6% sevoflurane in oxygen and compared with nonanesthetized controls. Locomotor activity and organization of movement in the open field model were assessed before and 24 h after anesthesia. Locomotor activity and anxiety-like behavior in the elevated plus maze were assessed 24 h after anesthesia. High- and low-affinity nicotinic receptor and cholinergic uptake site densities were evaluated in the hippocampus, amygdala, and forebrain regions using receptor autoradiography.ResultsSevoflurane anesthesia significantly reduced locomotor activity, altered temporospatial organization of trajectories, and increased anxiety-like behavior in young beta2KO mice, whereas no such changes were observed in young wild-type mice. Aged wild-type and beta2KO mice displayed reactions that were similar, but not identical, to the reactions of young mice to sevoflurane anesthesia. However, behavioral changes were not associated with differences in nicotinic receptor or cholinergic uptake site densities.ConclusionIn conclusion, sevoflurane anesthesia altered exploratory and anxiety-like behavior in mice lacking the beta2 nicotinic acetylcholine receptor subunit

Recording Mouse Ultrasonic Vocalizations to Evaluate Social Communication.

International audienceMice emit ultrasonic vocalizations in different contexts throughout development and in adulthood. These vocal signals are now currently used as proxies for modeling the genetic bases of vocal communication deficits. Characterizing the vocal behavior of mouse models carrying mutations in genes associated with neuropsychiatric disorders such as autism spectrum disorders will help to understand the mechanisms leading to social communication deficits. We provide here protocols to reliably elicit ultrasonic vocalizations in pups and in adult mice. This standardization will help reduce inter-study variability due to the experimental settings. Pup isolation calls are recorded throughout development from individual pups isolated from dam and littermates. In adulthood, vocalizations are recorded during same-sex interactions (without a sexual component) by exposing socially motivated males or females to an unknown same-sex conspecific. We also provide a protocol to record vocalizations from adult males exposed to an estrus female. In this context, there is a sexual component in the interaction. These protocols are established to elicit a large amount of ultrasonic vocalizations in laboratory mice. However, we point out the important inter-individual variability in the vocal behavior of mice, which should be taken into account by recording a minimal number of individuals (at least 12 in each condition). These recordings of ultrasonic vocalizations are used to evaluate the call rate, the vocal repertoire and the acoustic structure of the calls. Data are combined with the analysis of synchronous video recordings to provide a more complete view on social communication in mice. These protocols are used to characterize the vocal communication deficits in mice lacking ProSAP1/Shank2, a gene associated with autism spectrum disorders. More ultrasonic vocalizations recordings can also be found on the mouseTube database, developed to favor the exchange of such data

Ultrastructural localization of the α4-subunit of the neuronal acetylcholine nicotinic receptor in the rat substantia nigra

The distribution of the alpha4-subunit of the neuronal nicotinic acetylcholine receptor (nAChR) in the rat brain was examined at light and electron microscopy levels using immunohistochemical staining. In the present study we demonstrate the specificity, in both tissue homogenates and brain sections, of a polyclonal antibody raised against the rat nAChR alpha4-subunit. The characterization of this antibody involved: (1) Western blot analysis of rat brain homogenates and membrane extracts from cells previously transfected with diverse combinations of neuronal nAChR subunits, and (2) immunohistochemistry using transfected cells and rat brain tissue. At the light microscope level, the alpha4-subunit-like-immunoreactivity (LI) was widely distributed in the rat brain and matched the distribution of the alpha4-subunit transcripts observed previously by in situ hybridization. Strong immunohistochemical labeling was detected in the mesencephalic dopaminergic nuclei. The nAChRs in this region are thought to be responsible for the modulation of dopaminergic transmission. The neurotransmitter identity of alpha4-immunolabeled neurons in the substantia nigra pars compacta (SNpc) and the ventral tegmental area was thus assessed by investigating the possible colocalization of the nAChR alpha4-subunit with tyrosine hydroxylase using confocal microscopy. The double labeling experiments unambiguously indicated that the alpha4-subunit-LI is present in dopaminergic neurons. At the electron microscope level, the neurons in the SNpc exhibited alpha4-subunit-LI in association with a minority of postsynaptic densities, suggesting that the alpha4-subunit may be a component of functional nAChRs mediating synaptic transmission between midbrain cholinergic neurons and mesencephalic dopaminergic neurons.This research was supported by grants from The College de France, the Association Française contre la Myopathie, the Council for Tobacco Research, Reynolds Pharmaceutics, the European Union (Biomed BMH1-CT94–1060 and Biotech 960236), the French Embassy in Spain, the Ministerio de Educacion y Cultura (PB94–0219-CO2-01), the Comunidad de Madrid (AE00268/95), and the National Alliance for Research on Schizophrenia and Depression.Peer reviewe