Neuroanatomy and behavior in mice with a haploinsufficiency of AT-rich interactive domain 1B (ARID1B) throughout development.

BACKGROUND: One of the causal mechanisms underlying neurodevelopmental disorders (NDDs) is chromatin modification and the genes that regulate chromatin. AT-rich interactive domain 1B (ARID1B), a chromatin modifier, has been linked to autism spectrum disorder and to affect rare and inherited genetic variation in a broad set of NDDs. METHODS: A novel preclinical mouse model of Arid1b deficiency was created and validated to characterize and define neuroanatomical, behavioral and transcriptional phenotypes. Neuroanatomy was assessed ex vivo in adult animals and in vivo longitudinally from birth to adulthood. Behavioral testing was also performed throughout development and tested all aspects of motor, learning, sociability, repetitive behaviors, seizure susceptibility, and general milestones delays. RESULTS: We validated decreased Arid1b mRNA and protein in Arid1b+/- mice, with signatures of increased axonal and synaptic gene expression, decreased transcriptional regulator and RNA processing expression in adult Arid1b+/- cerebellum. During neonatal development, Arid1b+/- mice exhibited robust impairments in ultrasonic vocalizations (USVs) and metrics of developmental growth. In addition, a striking sex effect was observed neuroanatomically throughout development. Behaviorally, as adults, Arid1b+/- mice showed low motor skills in open field exploration and normal three-chambered approach. Arid1b+/- mice had learning and memory deficits in novel object recognition but not in visual discrimination and reversal touchscreen tasks. Social interactions in the male-female social dyad with USVs revealed social deficits on some but not all parameters. No repetitive behaviors were observed. Brains of adult Arid1b+/- mice had a smaller cerebellum and a larger hippocampus and corpus callosum. The corpus callosum increase seen here contrasts previous reports which highlight losses in corpus callosum volume in mice and humans. LIMITATIONS: The behavior and neuroimaging analyses were done on separate cohorts of mice, which did not allow a direct correlation between the imaging and behavioral findings, and the transcriptomic analysis was exploratory, with no validation of altered expression beyond Arid1b. CONCLUSIONS: This study represents a full validation and investigation of a novel model of Arid1b+/- haploinsufficiency throughout development and highlights the importance of examining both sexes throughout development in NDDs.

Abnormal electrophysiological phenotypes and sleep deficits in a mouse model of Angelman Syndrome.

BACKGROUND: Angelman Syndrome (AS) is a rare genetic disorder characterized by impaired communication, motor and balance deficits, intellectual disabilities, recurring seizures and abnormal sleep patterns. The genetic cause of AS is neuronal-specific loss of expression of UBE3A (ubiquitin-protein ligase E6-AP), an imprinted gene. Seizure and sleep disorders are highly prevalent (> 80%) in the AS population. The present experiments were designed to identify translational, neurophysiological outcome measures in a model of AS. METHODS: We used the exon-2 deletion mouse (Ube3a-del) on a C57BL/6J background to assess seizure, sleep and electrophysiological phenotypes. Seizure susceptibility has been reported in Ube3a-del mice with a variety of seizure induction methods. Here, we provoked seizures by a single high-dose injection of 80 mg/kg pentylenetetrazole. Novel experiments included the utilization of wireless telemetry devices to acquire global electroencephalogram (EEG) and neurophysiological data on electrographic seizures, power spectra, light-dark cycles, sleep stages and sleep spindles in Ube3a-del and WT mice. RESULTS: Ube3a-del mice exhibited reduced seizure threshold compared to WT. EEG illustrated that Ube3a-del mice had increased epileptiform spiking activity and delta power, which corroborates findings from other laboratories and recapitulates clinical reports in AS. This is the first report to use a cortical surface-based recording by a wireless telemetry device over tethered/fixed head-mount depth recordings. Less time in both paradoxical and slow-wave sleep, longer latencies to paradoxical sleep stages and total less sleep time in Ube3a-del mice were observed compared to WT. For the first time, we detected fewer sleep spindles in the AS mouse model. LIMITATIONS: This study was limited to the exon 2 deletion mouse model, and future work will investigate the rat model of AS, containing a complete Ube3a deletion and pair EEG with behavior. CONCLUSIONS: Our data enhance rigor and translatability as our study provides important corroboration of previous reports on epileptiform and elevated delta power. For the first time we report neurophysiological phenotypes collected via translational methodology. Furthermore, this is the first report of reduced sleep spindles, a critical marker of memory consolidation during sleep, in an AS model. Our results are useful outcomes for therapeutic testing.

Translational outcomes in a full gene deletion of ubiquitin protein ligase E3A rat model of Angelman syndrome.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by developmental delay, impaired communication, motor deficits and ataxia, intellectual disabilities, microcephaly, and seizures. The genetic cause of AS is the loss of expression of UBE3A (ubiquitin protein ligase E6-AP) in the brain, typically due to a deletion of the maternal 15q11-q13 region. Previous studies have been performed using a mouse model with a deletion of a single exon of Ube3a. Since three splice variants of Ube3a exist, this has led to a lack of consistent reports and the theory that perhaps not all mouse studies were assessing the effects of an absence of all functional UBE3A. Herein, we report the generation and functional characterization of a novel model of Angelman syndrome by deleting the entire Ube3a gene in the rat. We validated that this resulted in the first comprehensive gene deletion rodent model. Ultrasonic vocalizations from newborn Ube3am-/p+ were reduced in the maternal inherited deletion group with no observable change in the Ube3am+/p- paternal transmission cohort. We also discovered Ube3am-/p+ exhibited delayed reflex development, motor deficits in rearing and fine motor skills, aberrant social communication, and impaired touchscreen learning and memory in young adults. These behavioral deficits were large in effect size and easily apparent in the larger rodent species. Low social communication was detected using a playback task that is unique to rats. Structural imaging illustrated decreased brain volume in Ube3am-/p+ and a variety of intriguing neuroanatomical phenotypes while Ube3am+/p- did not exhibit altered neuroanatomy. Our report identifies, for the first time, unique AS relevant functional phenotypes and anatomical markers as preclinical outcomes to test various strategies for gene and molecular therapies in AS.

GABAB Receptor Agonist R-Baclofen Reverses Social Deficits and Reduces Repetitive Behavior in Two Mouse Models of Autism.

Autism spectrum disorder (ASD) is diagnosed by two core behavioral criteria, unusual reciprocal social interactions and communication, and stereotyped, repetitive behaviors with restricted interests. Excitatory/inhibitory imbalance is a prominent hypothesis for the etiology of autism. The selective GABAB receptor agonist R-baclofen previously reversed social deficits and reduced repetitive behaviors in a mouse model of Fragile X syndrome, and Arbaclofen improved some clinical symptoms in some Fragile X and ASD patients. To evaluate R-baclofen in a broader range of mouse models of ASD, we tested both the R-baclofen enantiomer and the less potent S-baclofen enantiomer in two inbred strains of mice that display low sociability and/or high repetitive or stereotyped behaviors. R-baclofen treatment reversed social approach deficits in BTBR T+ Itpr3tf/J (BTBR), reduced repetitive self-grooming and high marble burying scores in BTBR, and reduced stereotyped jumping in C58/J (C58), at nonsedating doses. S-baclofen produced minimal effects at the same doses. These findings encourage investigations of R-baclofen in other preclinical model systems. Additional clinical studies may be warranted to further evaluate the hypothesis that the GABAB receptor represents a promising pharmacological target for treating appropriately stratified subsets of individuals with ASD.

Cognitive abilities on transitive inference using a novel touchscreen technology for mice.

Cognitive abilities are impaired in neurodevelopmental disorders, including autism spectrum disorder (ASD) and schizophrenia. Preclinical models with strong endophenotypes relevant to cognitive dysfunctions offer a valuable resource for therapeutic development. However, improved assays to test higher order cognition are needed. We employed touchscreen technology to design a complex transitive inference (TI) assay that requires cognitive flexibility and relational learning. C57BL/6J (B6) mice with good cognitive skills and BTBR T+tf/J (BTBR), a model of ASD with cognitive deficits, were evaluated in simple and complex touchscreen assays. Both B6 and BTBR acquired visual discrimination and reversal. BTBR displayed deficits on components of TI, when 4 stimuli pairs were interspersed, which required flexible integrated knowledge. BTBR displayed impairment on the A > E inference, analogous to the A > E deficit in ASD. B6 and BTBR mice both reached criterion on the B > D comparison, unlike the B > D impairment in schizophrenia. These results demonstrate that mice are capable of complex discriminations and higher order tasks using methods and equipment paralleling those used in humans. Our discovery that a mouse model of ASD displays a TI deficit similar to humans with ASD supports the use of the touchscreen technology for complex cognitive tasks in mouse models of neurodevelopmental disorders.

Long-term exposure to intranasal oxytocin in a mouse autism model.

Oxytocin (OT) is a neuropeptide involved in mammalian social behavior. It is currently in clinical trials for the treatment of autism spectrum disorder (ASD). Previous studies in healthy rodents (prairie voles and C57BL/6J mice) have shown that there may be detrimental effects of long-term intranasal administration, raising the questions about safety and efficacy. To investigate the effects of OT on the aspects of ASD phenotype, we conducted the first study of chronic intranasal OT in a well-validated mouse model of autism, the BTBR T+ Itpr3tf/J inbred strain (BTBR), which displays low sociability and high repetitive behaviors. BTBR and C57BL/6J (B6) mice (N=94) were administered 0.8 IU/kg of OT intranasally, daily for 30 days, starting on day 21. We ran a well-characterized set of behavioral tasks relevant to diagnostic and associated symptoms of autism, including juvenile reciprocal social interactions, three-chambered social approach, open-field exploratory activity, repetitive self-grooming and fear-conditioned learning and memory, some during and some post treatment. Intranasal OT did not improve autism-relevant behaviors in BTBR, except for female sniffing in the three-chambered social interaction test. Male saline-treated BTBR mice showed increased interest in a novel mouse, both in chamber time and sniffing time, whereas OT-treated male BTBR mice showed a preference for the novel mouse in sniffing time only. No deleterious effects of OT were detected in either B6 or BTBR mice, except possibly for the lack of a preference for the novel mouse's chamber in OT-treated male BTBR mice. These results highlight the complexity inherent in understanding the effects of OT on behavior. Future investigations of chronic intranasal OT should include a wider dose range and early developmental time points in both healthy rodents and ASD models to affirm the efficacy and safety of OT.

Chronic desipramine treatment rescues depression-related, social and cognitive deficits in Engrailed-2 knockout mice.

Engrailed-2 (En2) is a homeobox transcription factor that regulates neurodevelopmental processes including neuronal connectivity and elaboration of monoaminergic neurons in the ventral hindbrain. We previously reported abnormalities in brain noradrenergic concentrations in En2 null mutant mice that were accompanied by increased immobility in the forced swim test, relevant to depression. An EN2 genetic polymorphism has been associated with autism spectrum disorders, and mice with a deletion in En2 display social abnormalities and cognitive deficits that may be relevant to multiple neuropsychiatric conditions. This study evaluated the ability of chronic treatment with desipramine (DMI), a selective norepinephrine (NE) reuptake inhibitor and classical antidepressant, to reverse behavioral abnormalities in En2−/− mice. Desipramine treatment significantly reduced immobility in the tail suspension and forced swim tests, restored sociability in the three-chambered social approach task and reversed impairments in contextual fear conditioning in En2−/− mice. Our findings indicate that modulation of brain noradrenergic systems rescues the depression-related phenotype in En2−/− mice and suggest new roles for NE in the pathophysiology of the social and cognitive deficits seen in neuropsychiatric disorders such as autism or schizophrenia.

AMPAKINE enhancement of social interaction in the BTBR mouse model of autism.

Autism is a neurodevelopmental disorder in which the first diagnostic symptom is unusual reciprocal social interactions. Approximately half of the children diagnosed with an autism spectrum disorder also have intellectual impairments. General cognitive abilities may be fundamental to many aspects of social cognition. Cognitive enhancers could conceivably be of significant benefit to children and adults with autism. AMPAKINE compounds are a novel class of pharmacological agents that act as positive modulators of AMPA receptors to enhance excitatory glutamatergic neurotransmission. This class of compounds was reported to improve learning and memory in several rodent and non-human primate tasks, and to normalize respiratory abnormalities in a mouse model of Rett syndrome. Here we evaluate the actions of AMPA compounds in adult male and female BTBR mice, a well characterized mouse model of autism. Acute treatment with CX1837 and CX1739 reversed the deficit in sociability in BTBR mice on the most sensitive parameter, time spent sniffing a novel mouse as compared to time spent sniffing a novel object. The less sensitive parameter, time in the chamber containing the novel mouse versus time in the chamber containing the novel object, was not rescued by CX1837 or CX1739 treatment. Preliminary data with CX546, in which ß-cyclodextrin was the vehicle, revealed behavioral effects of the acute intraperitoneal and oral administration of vehicle alone. To circumvent the artifacts introduced by the vehicle administration, we employed a novel treatment regimen using pellets of peanut butter for drug delivery. Absence of vehicle treatment effects when CX1837 and CX1739 were given in the peanut butter pellets, to multiple cohorts of BTBR and B6 control mice, confirmed that the pharmacologically-induced improvements in sociability in BTBR were not confounded by the administration procedures. The highest dose of CX1837 improved the cognitive deficit in novel object recognition in BTBR. No drug effects were detected on the high levels of repetitive self-grooming in BTBR. In open field tests, CX1837 and CX1739 did not induce hyperactivity or sedation in either strain. It is interesting to speculate that the ability of CX1837 and CX1739 to restore aspects of sociability in BTBR mice could utilize synaptic mechanisms regulating social cognition, suggesting a potential pharmacological target for interventions to treat symptoms of autism. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

Absence of deficits in social behaviors and ultrasonic vocalizations in later generations of mice lacking neuroligin4.

Mutations in NLGN4X have been identified in individuals with autism spectrum disorders and other neurodevelopmental disorders. A previous study reported that adult male mice lacking neuroligin4 (Nlgn4) displayed social approach deficits in the three-chambered test, altered aggressive behaviors and reduced ultrasonic vocalizations. To replicate and extend these findings, independent comprehensive analyses of autism-relevant behavioral phenotypes were conducted in later generations of the same line of Nlgn4 mutant mice at the National Institute of Mental Health in Bethesda, MD, USA and at the Institut Pasteur in Paris, France. Adult social approach was normal in all three genotypes of Nlgn4 mice tested at both sites. Reciprocal social interactions in juveniles were similarly normal across genotypes. No genotype differences were detected in ultrasonic vocalizations in pups separated from the nest or in adults during reciprocal social interactions. Anxiety-like behaviors, self-grooming, rotarod and open field exploration did not differ across genotypes, and measures of developmental milestones and general health were normal. Our findings indicate an absence of autism-relevant behavioral phenotypes in subsequent generations of Nlgn4 mice tested at two locations. Testing environment and methods differed from the original study in some aspects, although the presence of normal sociability was seen in all genotypes when methods taken from Jamain et al. (2008) were used. The divergent results obtained from this study indicate that phenotypes may not be replicable across breeding generations, and highlight the significant roles of environmental, generational and/or procedural factors on behavioral phenotypes.

Low stress reactivity and neuroendocrine factors in the BTBR T+tf/J mouse model of autism.

Autism is a neurodevelopmental disorder characterized by abnormal reciprocal social interactions, communication deficits, and repetitive behaviors with restricted interests. BTBR T+tf/J (BTBR) is an inbred mouse strain that displays robust behavioral phenotypes with analogies to all three of the diagnostic symptoms of autism, including low social interactions, reduced vocalizations in social settings, and high levels of repetitive self-grooming. Autism-relevant phenotypes in BTBR offer translational tools to discover neurochemical mechanisms underlying unusual mouse behaviors relevant to symptoms of autism. Because repetitive self-grooming in mice may be a displacement behavior elevated by stressors, we investigated neuroendocrine markers of stress and behavioral reactivity to stressors in BTBR mice, as compared to C57BL/6J (B6), a standard inbred strain with high sociability. Radioimmunoassays replicated previous findings that circulating corticosterone is higher in BTBR than in B6. Higher basal glucocorticoid receptor mRNA and higher oxytocin peptide levels were detected in the brains of BTBR as compared to B6. No significant differences were detected in corticotrophin releasing factor (CRF) peptide or CRF mRNA. In response to behavioral stressors, BTBR and B6 were generally similar on behavioral tasks including stress-induced hyperthermia, elevated plus-maze, light ↔ dark exploration, tail flick, acoustic startle and prepulse inhibition. BTBR displayed less reactivity than B6 to a noxious thermal stimulus in the hot plate, and less immobility than B6 in both the forced swim and tail suspension depression-related tasks. BTBR, therefore, exhibited lower depression-like scores than B6 on two standard tests sensitive to antidepressants, did not differ from B6 on two well-validated anxiety-like behaviors, and did not exhibit unusual stress reactivity to sensory stimuli. Our findings support the interpretation that autism-relevant social deficits, vocalizations, and repetitive behaviors are not the result of abnormal stress reactivity in the BTBR mouse model of autism.

Hepatic angiomyolipoma: two case reports of caudate-based lesions and review of the literature.

Two case reports of hepatic angiomyolipoma, both originating in the caudate lobe, are reported with a review of the literature. The liver is the second most common site of angiomyolipoma, an uncommon benign tumor of mixed mesenchymal origin. It is commonly diagnosed following abdominal pain or as an asymptomatic mass discovered on abdominal ultrasound or computed tomography scan. Of 74 cases reported, the lesions ranged from 0.3 to 36 cm in diameter and are noted between the first and eighth decade, with predominant female predilection. The right lobe is the most common site, with lesions arising in the caudate lobe comprising only five cases. The natural history of the hepatic lesion is unknown. Malignant invasion or metastatic disease has not been documented. Hepatic and renal angiomyolipoma can occur concurrently (13 of 60 cases), although the majority are not biopsy proven. Multicentric hepatic disease occurs. The correlation between tuberous sclerosis and hepatic angiomyolipoma is not confirmed histologically and occurs rarely. These lesions have a characteristic radiographic appearance due to high fat content. Histologically, angiomyolipoma are characterized by an admixture of adipose tissue, blood vessels, and smooth muscle cells. These lesions cannot reliably be differentiated from a malignant lesion based on clinical history, radiologic examination, and/or pathologic interpretation. If clinical suspicion for malignancy is low, then careful observation with serial radiologic follow-up is performed. The treatment for a symptomatic or suspicious lesion is resection, if feasible. Liver transplantation may be considered for large or centrally located lesions not amenable to resection.

Needle electromyographic evaluation of the diaphragm.

Needle electromyography of the diaphragm is a useful tool in diagnosis and prognosis of patients with diaphragmatic dysfunction. Spontaneous activity, polyphasic motor units and decreased recruitment can be found in phrenic nerve and spinal cord injury. We describe a safe technique for studying the diaphragm using needle electrodes.

Quantitative cervical flexor strength in healthy subjects and in subjects with mechanical neck pain.

Although weakness of anterior cervical muscles is postulated to contribute to persistent neck pain in patients with mechanical neck pain, quantitation of weakness has never been reported. We compared anterior cervical muscle strength in 30 subjects with mechanical neck pain and in 30 asymptomatic control subjects. Testing was performed with the subject supine, chin retracted, and neck flexed. Assessment was made using a hand-held dynamometer with head held at the midline and with rotation left and right within a pain-free range. Analysis with Wilcoxon scores showed that patients with neck pain had significantly less (p less than .05) strength (N.Kg-1) in all three positions than controls (1.16 +/- 0.49 vs 1.71 +/- 0.42, sagittally; 1.01 +/- 0.52 vs 1.47 +/- 0.41, rotation left; .99 +/- 0.46 vs 1.43 +/- 0.43 rotation right; neck pain vs control, respectively.) This weakness and its role in persistent neck pain should be recognized. The efficiency and effect of cervical muscle strengthening in treatment of chronic neck pain should be further defined.

Needle electromyographic evaluation of patients with myofascial or fibromyalgic pain.

Several past studies have evaluated the electromyographic activity of myofascial trigger points with conflicting results. This study was performed to determine whether spontaneous activity or motor unit activity was present in patients with focal myofascial pain or fibromyalgia. Using routine needle electromyographic techniques, we sampled reproducibly tender focal areas (tender points), similar tender areas with pain referral (trigger points), associated muscle bands and adjacent uninvolved musculature. Twenty-five subjects (14 females, 11 males, mean +/- SD age 43 +/- 14 years) were studied; twenty-one subjects with focal myofascial pain and four with fibromyalgia. Spontaneous fibrillatory or positive sharp wave potentials were not found in any muscle in the 25 subjects. There was no evidence of focal motor unit activity in the tender points, trigger points or associated muscle bands in either group. Motor unit recruitment was similar in all areas sampled. We conclude that no electrodiagnostic evidence of ongoing denervation or focal muscle spasm is found in association with focal myofascial pain or fibromyalgia.