Faculty Bibliography

CONTEXT: Best-estimate clinical diagnoses of specific autism spectrum disorders (autistic disorder, pervasive developmental disorder-not otherwise specified, and Asperger syndrome) have been used as the diagnostic gold standard, even when information from standardized instruments is available. OBJECTIVE: To determine whether the relationships between behavioral phenotypes and clinical diagnoses of different autism spectrum disorders vary across 12 university-based sites. DESIGN: Multisite observational study collecting clinical phenotype data (diagnostic, developmental, and demographic) for genetic research. Classification trees were used to identify characteristics that predicted diagnosis across and within sites. SETTING: Participants were recruited through 12 university-based autism service providers into a genetic study of autism. PARTICIPANTS: A total of 2102 probands (1814 male probands) between 4 and 18 years of age (mean [SD] age, 8.93 [3.5] years) who met autism spectrum criteria on the Autism Diagnostic Interview-Revised and the Autism Diagnostic Observation Schedule and who had a clinical diagnosis of an autism spectrum disorder. Main Outcome Measure Best-estimate clinical diagnoses predicted by standardized scores from diagnostic, cognitive, and behavioral measures. RESULTS: Although distributions of scores on standardized measures were similar across sites, significant site differences emerged in best-estimate clinical diagnoses of specific autism spectrum disorders. Relationships between clinical diagnoses and standardized scores, particularly verbal IQ, language level, and core diagnostic features, varied across sites in weighting of information and cutoffs. CONCLUSIONS: Clinical distinctions among categorical diagnostic subtypes of autism spectrum disorders were not reliable even across sites with well-documented fidelity using standardized diagnostic instruments. Results support the move from existing subgroupings of autism spectrum disorders to dimensional descriptions of core features of social affect and fixated, repetitive behaviors, together with characteristics such as language level and cognitive function

BACKGROUND:: Anesthetics are widely used to induce unconsciousness, pain relief, and immobility during surgery. It remains unclear whether the use of anesthetics has significant and long-lasting effects on synapse development and plasticity in the brain. To address this question, the authors examined the formation and elimination of dendritic spines, postsynaptic sites of excitatory synapses, in the developing mouse cortex during and after anesthetics exposure. METHODS:: Transgenic mice expressing yellow fluorescence protein in layer 5 pyramidal neurons were used in this study. Mice at 1 month of age underwent ketamine-xylazine and isoflurane anesthesia over a period of hours. The elimination and formation rates of dendritic spines and filopodia, the precursors of spines, were followed over hours to days in the primary somatosensory cortex using transcranial two-photon microscopy. Four to five animals were examined under each experimental condition. Student t test and Mann-Whitney U test were used to analyze the data. RESULTS:: Administration of either ketamine-xylazine or isoflurane rapidly altered dendritic filopodial dynamics but had no significant effects on spine dynamics. Ketamine-xylazine increased filopodial formation whereas isoflurane decreased filopodial elimination during 4 h of anesthesia. Both effects were transient and disappeared within a day after the animals woke up. CONCLUSION:: Studies suggest that exposure to anesthetics transiently affects the dynamics of dendritic filopodia but has no significant effect on dendritic spine development and plasticity in the cortex of 1-month-old mice

Glucocorticoids are a family of hormones that coordinate diverse physiological processes in responding to stress. Prolonged glucocorticoid exposure over weeks has been linked to dendritic atrophy and spine loss in fixed tissue studies of adult brains, but it is unclear how glucocorticoids may affect the dynamic processes of dendritic spine formation and elimination in vivo. Furthermore, relatively few studies have examined the effects of stress and glucocorticoids on spines during the postnatal and adolescent period, which is characterized by rapid synaptogenesis followed by protracted synaptic pruning. To determine whether and to what extent glucocorticoids regulate dendritic spine development and plasticity, we used transcranial two-photon microscopy to track the formation and elimination of dendritic spines in vivo after treatment with glucocorticoids in developing and adult mice. Corticosterone, the principal murine glucocorticoid, had potent dose-dependent effects on dendritic spine dynamics, increasing spine turnover within several hours in the developing barrel cortex. The adult barrel cortex exhibited diminished baseline spine turnover rates, but these rates were also enhanced by corticosterone. Similar changes occurred in multiple cortical areas, suggesting a generalized effect. However, reducing endogenous glucocorticoid activity by dexamethasone suppression or corticosteroid receptor antagonists caused a substantial reduction in spine turnover rates, and the former was reversed by corticosterone replacement. Notably, we found that chronic glucocorticoid excess led to an abnormal loss of stable spines that were established early in life. Together, these findings establish a critical role for glucocorticoids in the development and maintenance of dendritic spines in the living cortex

INTRODUCTION This protocol describes imaging of the living mouse brain through a thinned skull using two-photon microscopy. This transcranial two-photon laser-scanning microscope (TPLSM) imaging method allows high-resolution imaging of fluorescently labeled neurons, microglia, astrocytes, and blood vessels, as well as subcellular structures such as dendritic spines and axonal varicosities. The surgical procedure that is required to allow imaging thins the cranium so that it becomes optically transparent. Once learned, the surgery can be performed in approximately 30 min, and imaging can follow immediately. The procedure can be repeated multiple times, allowing brain cells and tissues to be studied in the same animals over short or long time intervals, depending on the design of the experiment. Two-photon imaging through a thinned and intact skull avoids side effects caused by skull removal and is a minimally invasive method for studying the living mouse brain under physiological and pathological conditions

Background: The focus of this presentation is to discuss the potential roles of microglia in regulating synaptic development and plasticity in the brain. Microglia are the resident immune cells of the central nervous system and display highly motile processes occupying a non-overlapping territory. Under physiological conditions, microglia may monitor the brain's microenvironment for damage signals and participate in the development and plasticity of neural circuits. Under pathological conditions, microglia undergo a series of morphological and functional changes, and may engage in containing tissue damage, phagocytosis and clearance of cellular debris, and/or the secretion of proinflammatory factors. Although microglia have been implicated in a multitude of physiological and pathological processes in the central nervous system, direct evidence of their roles in synaptic structure and functions remains elusive. Methods: Hampering efforts to delineate the role of microglia is the lack of tools to specifically perturb microglial function in vivo. To overcome this difficulty, we have recently generated mice with a targeted gene insertion allowing for the expression of tamoxifeninducible Cre recombinase in CX₃CR1 expressing microglial cells. By crossing CX₃CR1-CreER mice with mice harboring floxed alleles of the diphtheria toxin receptor (iDTR) under the control of the ubiquitous Rosa26 promoter, we have been able to specifically and efficiently ablate microglia in an inducible fashion. By ablating microglial cells and perturbing their functions in the living mice, we hope to elucidate the role of microglia in synapse development and learning-dependent synaptic plasticity. Results: Our preliminary results suggest that deletion of CX₃CR1 expressing microglial cells may cause a decrease in the turnover of postsynaptic dendritic spines in the living mouse cortex. Conclusions: Our findings indicate that the CX₃CR1-CreER mouse line provide a molecular handle for the in vivo manipulation of microglia including deletion and support an important role of microglia in synapse development and plasticity

Fragile X syndrome (FXS) is the most common inherited form of mental retardation and is caused by transcriptional inactivation of the X-linked fragile X mental retardation 1 (FMR1) gene. FXS is associated with increased density and abnormal morphology of dendritic spines, the postsynaptic sites of the majority of excitatory synapses. To better understand how lack of the FMR1 gene function affects spine development and plasticity, we examined spine formation and elimination of layer 5 pyramidal neurons in the whisker barrel cortex of Fmr1 KO mice with a transcranial two-photon imaging technique. We found that the rates of spine formation and elimination over days to weeks were significantly higher in both young and adult KO mice compared with littermate controls. The heightened spine turnover in KO mice was due to the existence of a larger pool of 'short-lived' new spines in KO mice than in controls. Furthermore, we found that the formation of new spines and the elimination of existing ones were less sensitive to modulation by sensory experience in KO mice. These results indicate that the loss of Fmr1 gene function leads to ongoing overproduction of transient spines in the primary somatosensory cortex. The insensitivity of spine formation and elimination to sensory alterations in Fmr1 KO mice suggest that the developing synaptic circuits may not be properly tuned by sensory stimuli in FXS

Microglial cells constitute the resident immune cell population of the mammalian central nervous system. One striking feature of these cells is their highly dynamic nature under both normal and pathological brain conditions. The highly branched processes of resting microglia display a constitutive mobility and undergo rapid directional movement towards sites of acute tissue disruption. Microglia can be converted by a large number of different stimuli to a chronically activated state by signaling through both purinergic and Toll-like receptor systems, among others. Recent work has uncovered some of the mechanisms underlying microglia dynamics and shed new light into the functional significance of this enigmatic member of the glial cell family

Imaging neurons, glia and vasculature in the living brain has become an important experimental tool for understanding how the brain works. Here we describe in detail a protocol for imaging cortical structures at high optical resolution through a thinned-skull cranial window in live mice using two-photon laser scanning microscopy (TPLSM). Surgery can be performed within 30-45 min and images can be acquired immediately thereafter. The procedure can be repeated multiple times allowing longitudinal imaging of the cortex over intervals ranging from days to years. Imaging through a thinned-skull cranial window avoids exposure of the meninges and the cortex, thus providing a minimally invasive approach for studying structural and functional changes of cells under normal and pathological conditions in the living brain

Changes in synaptic connections are considered essential for learning and memory formation. However, it is unknown how neural circuits undergo continuous synaptic changes during learning while maintaining lifelong memories. Here we show, by following postsynaptic dendritic spines over time in the mouse cortex, that learning and novel sensory experience lead to spine formation and elimination by a protracted process. The extent of spine remodelling correlates with behavioural improvement after learning, suggesting a crucial role of synaptic structural plasticity in memory formation. Importantly, a small fraction of new spines induced by novel experience, together with most spines formed early during development and surviving experience-dependent elimination, are preserved and provide a structural basis for memory retention throughout the entire life of an animal. These studies indicate that learning and daily sensory experience leave minute but permanent marks on cortical connections and suggest that lifelong memories are stored in largely stably connected synaptic networks