Genes, brain, and behavior: development gone awry in...

Ann. N.Y. Acad. Sci. ISSN 0077-8923

ANNALS OF THE NEW YORK ACADEMY OF SCIENCESIssue: Autism: Integrating Genes, Brain, and Behavior

Genes, brain, and behavior: development gone awryin autism?

A report on the 23rd Annual International Symposium of the Centerfor the Study of Gene Structure and Function

Michael J. Lewis1,a and Jason B. Dictenberg2,a

Departmements of 1Psychology and 2Biological Sciences, Hunter College of the City University of New York, New York,New York

Addresses for correspondence: Michael J. Lewis, Department of Psychology, Hunter College of the City University of NewYork, 695 Park Avenue, New York, NY 10065. [email protected]; Jason B. Dictenberg, Department ofBiological Sciences, Hunter College of the City University of New York, 695 Park Avenue, New York, NY [email protected]

Autism and its highly variable symptomology were the themes of the 23rd Annual International Symposium of theCenter for the Study of Gene Structure and Function at Hunter College in New York City, held 15 January 2010.The meeting explored the extensive research on autism from several perspectives—integrating research on genetics,neuroscience, and behavior—from researchers presenting new and innovative approaches to understanding theautism spectrum. Early diagnosis, intervention, and genetics were major themes because they are seen as essentialareas in which progress is needed before the rise in numbers of cases of autism throughout the world, which somedescribe as approaching an epidemic, can be stemmed. Several genetic, neurobiological, and behavioral markers ofautism have been identified that may ultimately provide the basis for early identification, and that presently definethe key areas requiring intensive intervention.

Keywords: autism spectrum disorder (ASD); neuron; synapse; epilepsy; endophenotype; fragile X syndrome (FXS);

microcephaly with seizures (MCSZ); mental retardation; connectome; brain circuit; axon; dendrite; Early Start Denver

Model (ESDM); mirror neurons; Asperger’s syndrome; specific language impairment (SLI); joint attention

Autism—Integrating Genes, Brain and Behavior,was a day-long symposium held at Hunter Collegeof the City University of New York. It was the 23rdAnnual International Symposium of Hunter’s Cen-ter for the Study of Gene Structure and Function andwas cosponsored by the Clinical and TranslationalScience Center at Weill Cornell Medical College. Thesymposium provided an exciting exploration of thebasic research on the molecular genetics and neu-robiological mechanisms of autism, as well as thesocial and cognitive research on autism. The speak-ers, in their respective fields, shared an appreciation

aJason B. Dictenberg wrote the first part of this reportdealing with the morning session, and Michael J. Lewiscovered the second part or afternoon session.

for the complexity of autism and the challenges ofunderstanding it. Each speaker offered a unique ap-proach to these challenges using a wide range of re-search tools and skills. The research and analysis cov-ered ranged from molecular biology and real-timeneuronal function to therapeutic early interventionand parental training. The symposium ended with apresentation on the epidemiology of autism; in thecontext of the data on prevalence, the questions ofwhether incidence of autism is increasing, and if so,why, were at the forefront of the conference.

The conference, organized by faculty from HunterCollege, Weill Cornell Medical College, MemorialSloan-Kettering Cancer Center, and the Hospitalfor Special Surgery, originated from discussions be-tween faculty from the Psychology and Biologi-cal Sciences Departments at Hunter College. The

doi: 10.1111/j.1749-6632.2010.05723.xAnn. N.Y. Acad. Sci. 1205 S1 (2010) E21–E36 c© 2010 New York Academy of Sciences. E21

Autism meeting report Lewis & Dictenberg

presentations were divided into two sections: onefocused on cellular and molecular research, whilethe other focused on behavioral and therapeuticresearch. However, the two sections were linkedby the recognition that progress in our under-standing and ability to alleviate the often devas-tating impact to the family of the autism spec-trum disorders will require parallel progress in bothareas.

Introduction

Autism spectrum disorders (ASD) comprise a widerange of brain dysfunctions that result in alteredsocial behavior, self-awareness, and language-basedinteractions. The genetic, biochemical, and cell bi-ological bases of these brain-altered states are onlybeginning to be understood. Recent focus in theseareas of research has been on connecting genes im-plicated in autism to their function at the synapse,the structure that ensures the transfer of signals fromone brain cell (neuron) to the next. Recent data sug-gest that genes implicated in autism affect the cir-cuitry of the brain, that is, the wiring that definesthe particular geography of synaptic connectionsbetween neurons. Hence, genes whose products reg-ulate the structure and function of the synapse arelikely to be implicated in neuronal wiring and ASDpathogenesis. The idea that many genes may be im-plicated independently in autism is supported by re-cent evidence from cases of inherited autism, whichoccur secondary to multiple, distinct mutations inneuronal genes required for signaling and synapseregulation. The heterogeneous nature of these ge-netic abnormalities is consistent with the spectrumof phenotypes and severity of altered behaviors thatare observed clinically in autism.

In the early stages of brain development, neu-rons of the central nervous system (CNS) associateto form billions of connections or synapses. Whilethis process is dynamic and just a fraction of theseconnections persist into adulthood, the retention ofimportant synapses for brain function involves thefine-tuned expression of genes at precise momentsin development. Any cellular or physiological er-rors that disrupt the proper regulation of synapsesmay cause brain dysfunction. Current research ef-forts seek to determine if synaptic dysfunction, asseen in autism and related disorders such as fragileX syndrome, contributes to the mis-wiring of braincircuitry. Efforts to link cognitive dysfunction to

circuity changes in the brain have focused on manylevels, including the structure of dendrites that me-diates the presynaptic/postsynaptic connection inthe majority of excitatory synapses in the brain, thedendritic spine. Pioneering work of Purpura andMarin-Padilla during the 1970s showed changes inthe morphology of synapses in the brains of chil-dren with mental retardation.1,2 These changes werecharacterized by fewer numbers of synapses (i.e.,dendritic spines) and greater spine length. A de-crease in the number of spines, and thus the numberof synapses, has been found in other disease statesinvolving cognition, such as Down syndrome andAlzheimer’s disease.3 A change in the morphologyof spines seems to be a unifying theme in diseasesof cognition, in that the normal pathway of den-dritic spine maturation appears disrupted. This re-sults in longer, thinner spines that resemble imma-ture filopodia, or spine precursors. The challengenow is to link these changes in spine architectureand biochemistry to defects in brain organization,function, and ultimately, behavior.

Autism research has expanded to encompassmany of the major behavioral and neurobiologi-cal research areas. The recognition that the disorderis not just a problem of social or cognitive devel-opment has led to the mobilization of research ingenetics, biochemistry, and neurophysiology withbehavior, cognition, and development to produce apanoply of research wisdom and skills against whatwe now know is a spectrum of disorders. The ap-plication of research findings from developmentalpsychology in the areas of perception, cognition, andsocial skills have provided a foundation upon whichautism research has progressed. Likewise the recog-nition by the autism field of learning and memoryprocesses, and the application of strategies that haveemanated from them, have provided insight intothe etiology and treatment of affected children. Theblend of applied behavioral analysis with cognitiveneuroscience approaches has been successfully ap-plied to autism with considerable success. Currentautism research employs techniques from discretetrial learning, imitation, language comprehension,and perception, among others. The combinations ofthese with electrophysiological and imaging meth-ods have linked behavioral and cognitive process af-fected by autism to discrete brain mechanism. Thereis recognition that the stages of early developmen-tal processes, with the emergence of behavioral and

E22 Ann. N.Y. Acad. Sci. 1205 S1 (2010) E21–E36 c© 2010 New York Academy of Sciences.

Lewis & Dictenberg Autism meeting report

Figure 1. Dan Geschwind, keynote speaker.

physiological changes, have provided the timing forsuch investigations.

In the morning session, five investigators fromaround the country highlighted their approachesto autism using a range of genetics and diversebrain imaging approaches. The first cohort of re-searchers at the symposium discussed recent ad-vances that point to activity-dependent genes thatfunction at the synapse, linking the pre- and postsy-naptic compartments. Often, these genes are alsomaster regulators of other genes through con-trol of gene expression at the transcriptional andtranslational level. The products of these activity-dependent genes may be defective in patients withautism since they are highly heritable, and, inter-estingly, some correlate with language processingfunction.

In the afternoon session, six other investigatorsdiscussed their research. They reported on brainimaging approaches combined with cognitive test-ing, behavioral intervention strategies, and parent-based support to better diagnose autism earlier andto understand and ameliorate the loss of typical lan-guage and joint attention in autism. The symposiumconcluded with epidemiological research showingpossible causes for the increased incidence of autismas characterized today.

The genetics of autism

Dan Geschwind, of the UCLA School of Medicine,opened the conference with a discussion of the

“autisms” as a spectrum of developmental behav-ioral abnormalities that results from a spectrumof genetic disruptions (Fig. 1). While many geneshave been implicated in the etiology of ASD, anddespite the disease being highly heritable (up to90% penetrant), the mode of inheritance is not wellunderstood. As many as 25% of patients will de-velop epilepsy at some point in their life, and manyalso have intellectual disabilities. It appears to affectmales more commonly, at a ratio of 4:1 (males tofemales) and at an overall rate of 1:150–200 births,being more common than any other childhood dis-ease. While many genes have been implicated, it isimportant to note that no single gene appears toaccount for more than 5% of autism cases. There-fore Geschwind’s lab focuses on endophenotypes, orbiomarkers of behavioral phenotypes that are stablyassociated with a genetic component.

Geschwind and colleagues discovered that a sin-gle gene mutation co-segregated with a languagedelay in children with autism, and that this mu-tation encoded polymorphisms in the contactin-associated protein-like 2 (CNTNAP2) gene.4

CNTNAP2, a member of the neurexin gene fam-ily encoding a trans-synaptic protein involved insynapse formation and maintenance, is expressedin brain regions that are more evolved in primates.Patients with intellectual disabilities, as well as anAmish family with children having focal epilepsy,also carry mutations in this gene; in addition, CNT-NAP2 mutations are also highly correlated with

Ann. N.Y. Acad. Sci. 1205 S1 (2010) E21–E36 c© 2010 New York Academy of Sciences. E23


specific language impairment (SLI), another highlyheritable condition that carries no other noticeabledevelopmental abnormalities. Intriguingly, suchdata connect the CNTNAP2 gene implicated in ahighly specific language learning and memory func-tion with autism; more generally, this connectionexemplifies the potential of a gene to impact onecomponent on the spectrum of endophenotypescharacteristic of a given disorder (such as autism).Given that language impairment is such a preva-lent feature of autism, much work has focused ongenes that can account for specific language dis-orders; other genes, such as those encoding thetranscriptional control protein FOXP2, are knownto cause a monogenic language disorder, althoughmutations within this gene are rare. This repre-sents another case where gene network interactionshighlight a developmentally regulated program tocontrol complex human behaviors. Therefore itis no surprise that the gene encoding CNTNAP2functions “downstream” of FOXP2 regulation inneurons.5

As a member of the neurexin gene family, CNT-NAP2 is one of the best candidates known to linkautism with synapses and brain circuits. Neurexinsare presynaptic binding partners for postsynapticproteins termed neuroligins, which have mutatedforms in isolated families with multiple autistic fam-ily members.6,7 These proteins are important forsynaptic formation and maintenance, and are im-plicated in synapse function and proper brain cir-cuit formation. CNTNAP2 appears to be expressedin highly evolved frontal regions of the humanbrain and may have played a key role in the evo-lution of language-based regions in humans (e.g.,the anterior perisylvian cortex and the basal gan-glia) since this protein is expressed in these areasin non-human primates. Collaborative research be-tween Geschwind’s lab and those of Mirella Daprettoand Susan Bookheimer at UCLA has investigated theproposed role for CNTNAP2 in autism pathogen-esis. This research has shown that risk-allele carri-ers of CNTNAP2 do not display a typical responseto externally directed task attention. These patientsdisplay a stronger local and weaker long-range con-nectivity in the frontal-striatal circuits than normalsubjects using fMRI techniques, lending credence tothe hypothesis of disconnections on a circuit level asa basis for part of the behavioral pathology in autismpatients.8

Following the genetic heterogeneity of brain dis-eases of closely related social groups has been ofgreat value in tracing the origin of autism and otherrelated developmental disorders of intellectual dis-ability, including seizure and mental retardation.Of particular significance are studies of rare reces-sive mutations that are carried within families formany generations, such as within consanguineousmarriages. One such study has been pioneered byChristopher Walsh of Harvard Medical School, whodiscovered a disorder termed microcephaly withseizures (MCSZ). Children with this disease typi-cally exhibit a reduced head size, developmental de-lay, and mild seizure disorder. By recognizing a sim-ilar phenotype in the offspring of consanguineousmarriages, such as in certain Middle Eastern pop-ulations, they were able to discover the recessivemutation on chromosome 19 that is responsible forthis disorder.9 While the mutations in the separatepopulations were distinct, they both affected thePNKP gene. How the PNKP gene, which encodesfor a DNA repair protein, plays a role in the patho-genesis of MCSZ is not yet known. What is clear isthat population-specific mutations in a given geneor genes within the same pathway can give rise to aspectrum of phenotypes depending on the effect ofthe mutations on protein function.

Using a similar approach to discover the genes in-volved in autism pathogenesis, Walsh and colleaguesformed the Homozygosity Mapping Collaborativefor Autism, where clinicians and geneticists in theUnited States and the Middle East study consan-guineous families with autism. They found largecopy number variation in a section of chromosome22, an area previously associated with intellectualdisability when mutated.10 Five regions were deletedon both parental sets of chromosomes, impactingsix genes, although none were completely disabledin function. These genes include several of unknownfunction, ionic gating membrane channel proteins,axonal growth factors, and a transcription factor.Significantly, four of the six genes are expressed inthe hippocampus, a region of the brain central tolearning and memory function, and an area that isalready implicated in dysfunction in autism stud-ies. In addition, some of these genes were previ-ously shown to be activated in response to neuronalactivity. This activity-dependent response may bea key mechanism to facilitate the proper regula-tion of synapse firing during experience-dependent



changes in synaptic strength (the cellular correlateof learning and memory). A dysregulation of anyof these genes could have direct implications for al-tered synapse formation and function during braindevelopment.5,11

Further probing the nature of the deletions andthe effect on these genes led Walsh and colleaguesto use microarray technology, focusing on a singlecopy number variant that deleted a region near theRNF8 gene, a transcriptional co-regulator.10 Tran-scription factors including RNF8 are proteins thatcan rapidly switch genes on or off in response tocellular signaling and growth cues. The deletion af-fected the transcription site of the RNF8 gene in aregion that contained a CREB binding site. CREB isanother major transcription factor that is mobilizedin response to activity to turn on genes that supportlong-term changes in synaptic strength. Removal ofthis binding site for CREB will likely have significanteffects on RNF8 gene expression and subsequentlyon the genes that require this cofactor for their ex-pression. Future studies will determine which genesthese are and in which direction they are dysregu-lated in response to the RNF8 mutation.

Taken together, these genetic studies reveal theheterogeneity of causes of autism that parallel thespectrum of observed phenotypes of human be-haviors among carriers of these mutations. Whilethe nature of these mutations is only beginning tobe revealed, family pedigrees of recessive mutationshave implicated genes central to neuronal function,such as those functioning at synapses. A paradigmexample of this is the CNTNAP2 gene encoding atrans-synaptic protein mediating synapse formationand function, and its proposed partner neuroligin,which binds to CNTNAP2 family members to bridgepre- and postsynaptic compartments. Loss of func-tion of these genes has direct implications for loss ofproper circuit formation, and may lead to synapticdysconnection during development.12 In addition,it appears that seizure disorders such as epilepsy arecommon to several distinct brain pathologies thatinvolve developmental delay, and therefore this ex-cessive activity may contribute to defects in learningand behavior that manifest in ASD.

Gene expression, synapses, and circuitformation

Coming from the other end of the molecular spec-trum, Jason Dictenberg of Hunter College is pio-

neering work on the monogenic causes of autismat the molecular level of the synapse. Fragile Xsyndrome (FXS) is the leading single-gene causeof autism, presently estimated at ∼3–5%. Strik-ingly ∼60% of children with FXS have autistic be-havior.13 The fragile X protein FMRP, an mRNA-binding protein, is a master regulator of mRNAtransport to dendrites. It is also involved in thetranslation of synaptic genes in response to neuronalactivity.14 FXS is caused by a trinucleotide(CGG)-repeat expansion in the 5’ untranslated region ofthe FMRP gene that leads to transcriptional silenc-ing secondary to hypermethylation. The mutationis highly heritable and appears to be exaggeratedupon generational inheritance. Dictenberg and col-leagues use a mouse model of FXS to study synapticdefects in genes implicated in circuit formation andfunction.

A hallmark of synaptic changes in FXS is the pre-ponderance of long, thin spines that are reminiscentof immature stages during development. Dictenbergand colleagues used a novel RNA-tagging method todemonstrate the role of FMRP in mRNA transportto dendrites in response to neuronal activity. Theyfound that loss of FMRP led to decreased mRNAtargeting to dendrites and altered spine morphol-ogy.15 One example of a specific mRNA affectedis the CaMKII-alpha mRNA, which is transportedto dendrites in response to glutamatergic signal-ing to facilitate learning and memory. However, ex-periments carried out in the mouse model of FXSdemonstrated that the processivity (run length) ofindividual CaMKII-alpha mRNA particles was di-minished following metabotropic glutamate recep-tor (mGluR) signaling. Previous work has shownthat, in the absence of FMRP, hippocampal CA1neurons undergo excessive long-term depression ofsynaptic responses upon mGluR signaling in themouse model, a process that normally requires localprotein synthesis within dendrites.16 Therefore lossof mRNA transport and subsequent dysregulationof local protein synthesis may contribute to defectsin synaptic plasticity, learning, and memory func-tion observed in FXS. Dictenberg hypothesizes that aloss of stimulus-induced mRNA transport may leadto precocious mRNA translation before the mRNAsreach the dendrite, with proteins therefore made atthe wrong place and time. This mechanism may becommon to many of the heterogeneous causes ofautism.



Figure 2. Cultured hippocampal neuron (21 days in vitro) from mouse brain showing how super-resolution microscopy canhighlight individual synapses. Here neurons are stained using immunofluorescence with antibodies to synapsin (red), microtubule-associated protein 2 (MAP2, blue), and the actin cytoskeleton (green). Note the colocalization of actin-rich dendritic spines (green)with the presynaptic axonal boutons (red) along the length of the dendrites (blue) where hundreds of synapses can be visualized.Courtesy Jason Dictenberg, Hunter College.

Recent data from the Dictenberg lab exploresthe role of trans-synaptic cell adhesion moleculesand their potential dysregulation in FXS. Neu-roligins (NLs) are postsynaptic proteins that areimplicated in autism pathogenesis in cases of famil-ial inheritance. The proteins are binding partners forthe CNTNAP2 family of presynaptic neurexins.6,7,17

Importantly, Dictenberg made the connection thatone of the known mRNA targets of FMRP, PSD-95,is a scaffold protein that can regulate the levels ofNLs at synapses.18 Therefore, dysregulation of PSD-95 expression, as seen in neurons from the mousemodel of autism, implicates NL dysfunction. The labuses super-resolution microscopy methods to quan-tify changes in proteins at individual synapses uponneuronal activation (Fig. 2). The data show thatmGluR stimulation causes a rapid increase (within15 minutes) in dendritic PSD-95 protein levels inwild-type hippocampal neurons, but that this up-regulation is absent in neurons derived from the FXSmouse model of autism. NL1 protein also appears

altered in its expression in FXS neurons, and this al-tered the ratio of excitatory to inhibitory synapses.Live cell imaging of PSD-95-GFP and NL1-GFP fu-sion proteins expressed in hippocampal neuronsshows dynamic movements within filopodia andspines. Analysis of these protein movements in neu-rons from FXS mice will lead to a greater under-standing of the defects that may give rise to alteredplasticity and synapse structure. Future work fromthe Dictenberg lab aims to determine how an al-teration of the balance of excitatory and inhibitoryinputs in diverse brain regions contributes to theobserved physiological and behavioral changes inFXS and autism, and how excessive activity such asin epilepsy may contribute to synaptic defects andaltered circuit architecture.

Looking into the visual system for clues oncircuit formation and function, Hollis Cline andher group at Scripps Research Institute turned tothe tadpole eye and the innervation pattern ofthe optic tectum. They used fluorescent proteins



expressed in these animals and, owing to the relativetransparency of these animals, were able to visualizethe axonal projections and their changes in connec-tivity over time. The projections were modified byvisual experience as predicted by early experimentsby LeVay, Hubel, and Wiesel.19 These pioneeringscientists showed that sensory experience shapedthe developing visual cortex during a critical periodin brain development and that sensory deprivationled to loss of connectivity in that represented re-gion. Cline used this approach to look into den-dritic arborization in response to synaptic activity, aprocess that is less understood than axonal innerva-tion. Her group showed a dramatic rearrangementof dendritic arbors in less than an hour and, overlonger periods, demonstrated an extensive elabo-ration with almost every process undergoing somechange. Through careful analysis this work showedan iterative process whereby branches were elabo-rated and then subsequently pruned to allow stabi-lization of only a select few processes until the ma-ture circuit formed.20 This was consistent with thesynaptotrophic hypothesis, whereby synaptic con-nections control the exploratory behavior of devel-oping neurons, the development of neuronal arbors,and the establishment of neuronal circuits. Visualstimulation was shown to enhance this arboriza-tion, and electrophysiological experiments showedthe strengthening of existing synapses and the addi-tion of new synapses as well, further confirming thevalidity of the hypothesis.20

Cline’s lab observed not only these changes inneuronal excitability and arborization upon sen-sory stimulation, but also modification of gene ex-pression. In searching for an activity-dependentmechanistic cause to explain these broad-rangingeffects of visual stimulation on the brain, her grouplooked to mRNA-binding proteins like FMRP thatcontrol mRNA stability, transport, and translation.They focused on CPEB, which binds to cytoplasmicpolyadenylated mRNA tails to control their subse-quent translation upon neuronal activity. Using adominant-negative form of the CPEB protein thatinterferes with normal CPEB function, they lookedto the effects on arborization in the tadpole visualcircuit. The mutant protein diminished the overallgrowth rate of the dendrites and the branch length,which dampened the circuit formation.21

Together, the studies of Cline and Dictenberghighlight the role of gene expression in controlling

synapse formation, structure and function, and howsynaptic activity can alter gene expression to mod-ify synaptic inputs and the topography of the overallcircuit. Cline’s elegant work highlights the plastic-ity of the circuit with regard to sensory input anddendritic morphology, and shows how this can af-fect the brain on a macroscopic level over longerperiods of time.

The connectome

Bringing the theory of genomics to the study ofsynaptic connections, Jeffrey Lichtman of HarvardUniversity discussed the architecture of the wiringof human brains and suggested that most diseasesof the nervous system could be a result of aberranteffects on the circuitry of neurons. The work of hislab aims to build on the observations of the earlyneuroscientists Golgi and Ramon y Cajal, who dis-covered the interconnectivity of the neurons in thebrain and speculated on the directionality of infor-mation flow from one neuron to the next withina circuit. The wiring of individual neurons withinthe human brain is astoundingly complex. There-fore Lichtman’s lab uses advances in imaging tech-nologies to better assess connectivity. Similar to theway that geneticists have mapped out the genome,his group seeks to map the human “connectome”to have a working diagram of an apparently nor-mally wired brain. He acknowledged that ”normallywired” may be inaccurate, since genes are expresseduniquely within individuals despite sharing over99% genetic similarity. These individual expressionpatterns may affect neuronal connections so thateach person has refined connections that are uniqulybased on experience-dependent learning and plas-ticity. Despite these limitations, Lichtman’s grouphas advanced the connectome project by applyingcomputer-assisted image acquisition and analysis tothe structural mapping of sets of neuronal circuitsand the nervous system as a whole. Once the con-nectome is mapped, withstanding some accepteddiversity in the wiring, this knowledge could be ap-plied to animal models of neurological diseases, likeautism, to determine the defects in wiring architec-ture that may give rise to altered learning, memory,and behavior.

Lichtman’s group has shown differences in the de-velopmental connectivity of mouse brain using theneuromuscular junction (NMJ) as a model system.The NMJ is where the motor nerves that originate in



the spinal cord innervate muscle peripherally. Earlyafter birth there exist multiple neurons (axons) thatcontact the same muscle fiber junction.22 After a fewweeks in mice (and months in humans), a pruningof these connections takes place such that eventu-ally only a single axon innervates the junction. Theother axons retract and find other targets, resultingin a smaller motor unit in the fiber that is stablethroughout the lifetime of the animal.23 This pro-cess of multiple innervation followed by pruningduring development occurs in the central nervoussystem as well, and the mechanisms for this processappear to be similar.

To better follow the individual connections ofaxons with the muscle fibers during development,the Lichtman lab used spectrally distinct fluorescentprotein markers to visualize individual fibers. Usingtime-lapse imaging methods, they observed in vivothe initial innervation and synaptic competition,subsequent synaptic formation, and finally synapseretraction during development of the mouse. Ex-panding on this approach, they created a new lineof transgenic mouse that expresses three differentspectral varieties of fluorescent proteins within thebrain. By driving the expression of each color (blue,green, and red) randomly within each cell, they wereable to create a “brainbow” mouse where each cell

has a distinct color variation from the next.24 Giventhe complexity and density of packing of individualfibers within the central nervous system, this tech-nique serves to disambiguate each neuronal processeven within the context of hundreds or thousandsof neighboring processes (Fig. 3). Using these mice,Lichtman discovered that an axonal connection atthe NMJ that is dominant, or one that occupies mostof the junctional space, may not always win out overthe lesser-connected axons. The neuron of the “win-ning” axon, however, was always observed to be suc-cessful at adjacent synapses where another branch ofits axon innervated a distinct NMJ.25 These resultssuggest that the “successful” neuron is able to ex-press synaptic molecules that enable it to strengthenits synapses more efficiently than other competingneurons in response to activity at the NMJ.

Applying new technological advances in tissuesectioning along with the fluorescent labeling tech-nique, Lichtman’s group was able to demonstratethe highly individualized nature of axonal innerva-tion by mapping the connectome of the interscutu-laris muscle of the mouse ear. Using a serial sectiontechnique that preserves a continuous connectionbetween subsequent slices, they generated a stripof tissue that spans the whole muscle, leaving itsneuronal processes intact. Upon examination of the

Figure 3. Connectomics with no tracing necessary! Brainbow mouse sections of the peripheral nervous system neuromuscularjunction (NMJ) highlight the use of randomized fluorescent protein expression within each neuron. Individual axons can bedistinguished from each other as they enter the muscle (left) and at the synapse (right) using this technique as they innervate theNMJ during development. Slide courtesy of Jeff Lichtman.



connectivity, they found that, strikingly, between ge-netically identical animals, the patterns were distinct(i.e., like the distinct fingerprints of identical twins).This individuality included arbor shapes, branchingpatterns, and the number of branches.26 Also no-ticed was a suboptimal pattern of connectivity, withbranches often passing a connection before loopingback to make a synapse, which Lichtman says is ev-idence that the connectome is to some degree built“on the fly” without pre-programming. He believesthat the patterning of synapse elimination “unfettersthe mammalian nervous system from the tyranny ofthe genes.” In the end, each animal ends up with avery different nervous system, largely due to envi-ronmental influences and experiences, through ba-sic learning and memory mechanisms. Ultimately,connectomics research may allow investigators tocompare brains of autistic patients to normal in-dividuals to determine which circuits are disruptedand how this correlates with behavioral differences.

New directions in early detection andintervention in autism

The second keynote address was presented by Geral-dine Dawson (Fig. 4), chief science officer of Autism

Speaks, an austism research and advocacy organiza-tion. She provided an overview of the research onearly diagnosis of ASD and clearly articulated theconsensus in the field that there is a need to accel-erate the identification and treatment of ASD. Shepointed out that while there have been deficienciesof the health care system in recognizing the need forearly diagnosis,27 there are now several early child-hood screening options, including some that appearto be useful in identifying signs of autism as earlyas six months. In agreement with the speakers ofthe morning session, Dawson indicated the impor-tance of the genetic components of ASD and the factthat siblings of autistic children are at much higherrisk for the disorder. This provides an opportunityfor prospective research on these infants that mayassist with the detection of signs of autism evenearlier.

Dawson and others have shown that up to sixmonths of age, children who will go on to developautism typically seem fairly normal. Deficits in at-tention and selective engagement with human facesand voices begin to appear at this age and increasein severity to one year when the social and commu-nication deficits of autism can clearly be identified

Figure 4. Geraldine Dawson, keynote speaker.



in most ASD children. It is important to note, how-ever, that between a quarter and a third of childrenwho develop ASD do not show deficits until one ortwo years of age.

Dawson described innovative research that showspromise for recognizing the early signs of autism.First, she discussed research indicating that high-risk infants have diminished ability to distinguishlightness and darkness in the visual field and thatthis deficit may play a role in the inability to re-spond to human faces.28 Next, Dawson presentedground-breaking research identifying deficits inevent-related potentials (ERPs) to human faces in aspecific cortical region, the fusiform gyrus, of high-risk infants. The deficit is characterized as dimin-ished amplitude in this electrophysiological patternthat is not lateralized to the right hemisphere as itis in low-risk infants.29 She states that the challengeis to determine whether these and other promisingfindings are predictive of the disease or whether theyare endophenotypes indicating a predisposition tothe disease.

Early intervention has been shown to have animpact on learning and language development ofASD. Dawson asserts that along with the successfulstrategies from applied behavior analysis and dis-crete trials training, the use of methods from devel-opmental psychology that take advantage of the “in-tuitive” learning of children as they actively exploretheir environment might produce the most effectiveintervention strategy.30 This thinking emphasizescomprehensive approaches to intervention and isat the heart of Dawson’s collaboration with SallyRogers in the Early Start Denver Model (ESDM),a comprehensive intervention program for youngchildren with autism. The ESDM Program is a col-laborative effort of experts—physicians, psycholo-gists, behavioral therapists, speech pathologists, andoccupational therapists—who work to intervene onas many ASD deficits as possible. Social develop-ment with language, motor, and cognitive skills areintensively applied to autistic children. She states,“We’re building a baby from the ground up in allsenses of the word.” Their promising findings31 us-ing this approach have encouraged Dawson, Rogers,and their team to expand the program to other sites.They believe that programs that bring together ther-apists, experts, and parents in early intervention of-fer the greatest promise of improvement for thosewith ASD.

Social motivation, attention, and learningin the autistic brain

Early in development, infants preferentially fo-cus their attention on human faces and voices—particularly the face and voice of their mother—while infants with ASD do not show this pref-erence. Dawson discussed how the ERP responseto faces is diminished in autistic children. Thefusiform “face area” and the amygdala, a keybrain region involved with emotion, exhibit re-duced amplitude ERP responses in children withASD.

Mirella Dapretto, at the University of California,Los Angeles, and colleagues have investigated thesefindings from the perspective that autism may in-volve the alteration of brain function in areas thatmediate “theory of mind,” or the interpretation ofothers’ intentions, actions, and emotional states.Her research has shown that although deficits inthe brain regions associated with theory of mindare observed when unfamiliar faces are presented toautistic children,32 the response is normative whenfamiliar faces are presented. The deficit appears tobe associated with attentional mechanisms: whenautistic children are instructed to pay attention dur-ing testing, they exhibit normal levels of activity inthese brain areas.33

With this evidence that brain function is essen-tially intact in these areas, Dapretto shifted herfocus to explore the lack of preference for facialand vocal stimuli in ASD. “According to the socialmotivation hypothesis, this lack of an attentionalpreference for these stimuli may reflect that they’renot ‘rewarding’,” she said. These stimuli typicallyproduce reinforcement in unaffected children butnot in children with ASD. Her research showedthat social and nonsocial reinforcement that acti-vates the brain reward regions, such as the ven-tral striatum, fails to do so in children with ASD.The difference was particularly striking with socialrewards.

These deficits suggest that brain centers thatmediate emotion, especially emotion generated insocial situations, may be impaired. Dapretto andcolleagues have performed experiments in whichASD children were instructed to mimic facial ex-pressions that show different emotions. Althoughthe children performed well on the task behav-iorally, functional magnetic resonance imaging



Figure 5. Mirror neuron system (MNS) activity in ASD children during imitation. The effect of imitation of emotional expressionson a MNS. Event-related fMRI images of typically developing children (TD) and autism spectrum disorder (ASD) children and thedifferential comparisons of these children (TD vs. ASD). Higher activity is found in the right pars opercularis of the inferior frontalgyrus in TD children but not in the ASD children. The between-group comparison shows the significant effects.8 Differences in theMNS are believed to underlie the deficits in social and emotional development of ASD children. Lower image: P < 0.05, correctedat cluster level.

(fMRI) shows that the children exhibited deficits inimportant brain regions in comparison to unaf-fected children (Fig. 5). In particular, they showedlower levels of activity in brain areas with “mirrorneurons.”8 These are regions of the brain that arethought to be necessary for following and interpret-ing movements and intentions of others, and maybe key to understanding social and emotional situa-tions. Dapretto’s research is consistent with others’in suggesting that this system may be impaired inautistic children. Dapretto indicated that there maybe a link between the deficits in mirror neuron activ-ity and attentional bias seen with autism; recent datafrom her lab indicates that there is a lack of coordina-tion between the reward system and mirror neuroncenters with ASD. According to Dapretto, childrenwho have highest activity in the ventral striatum(when they were getting the smiling faces as positivefeedback) were also the children who showed greateractivity in those mirroring regions that are thoughtto be important for interpreting another’s facial ex-pression and feelings. Dapretto concluded by statingthat the social and emotional impairments observedwith ASD may be due to deficits in the mechanismsthat mediate long-range integration between func-tioning brain regions. This could be exacerbated byimpaired attentional bias. Her current research isfocused on the role these differences may play in thedeficits observed with the development of linguisticabilities.

Language in ASD: from behavioralphenotypes to neurobiology and genetics

Language deficits are a common feature of ASD. Ex-ploring the range of these deficits is the focus ofwork by Helen Tager-Flusberg (Boston University)and her colleagues. They have examined the dif-ferences of those who have intact language skills(autism language normal; ALN), who may stillhave diminished abilities to understand social cuesand the embedded meaning of speech (often la-beled Asperger’s syndrome) compared with thosewith more severe language deficits (autism languageimpaired; ALI). ALI children were found to ex-hibit deficits that are surprisingly similar to thosediagnosed with SLI on language tests.34,35 Tager-Flusberg and colleagues showed that ALN did notshow these deficits. With these findings as a clue, sheand her colleagues explored the brain regions thatmediate language to determine if the deficits pro-duced identifiable anomalies. She found that ALIchildren show impairments in the left hemispherelateralization of speech. Typically, the developmentof language is associated with increased left hemi-sphere dominance of Broca’s area in the frontal cor-tex (the speech production area) and Wernicke’sarea in the temporal cortex (the speech recogni-tion area). Autistic children exhibit little left hemi-sphere dominance or even reversed dominance ofBroca’s area concomittant with an exaggerated left



dominace in Wernicke’s area.36 Tager-Flusberg andcolleagues found that this profile was associatedwith ALI children, and not ALN children. She andher colleagues investigated other language regions,exploring the arcuate fasiculus, the major pathwayconnecting Broca’s and Wernicke’s areas.37 Usingdiffusion tensor imaging of the arcuate fasiculus,they found no apparent differences between ADSchildren and unaffected controls. However, whenASD children were divided on the basis of linguisticability, it was apparent that ALI showed diminishedconnectivity between the two speech areas.

Siblings of autistic children are at greater risk fordeveloping autism, and they exhibit reduced lan-guage performance and similar alterations of brainlanguage centers as their affected siblings.38 This wasexemplified by Tager-Flusberg and colleagues’ workin which they found that siblings showed lower lan-guage and reading performance and similar anoma-lies of Broca’s and Wernicke’s areas as ASD childen.Since not all of these siblings will go on to haveASD, it suggests that the language impairments andaltered brain systems that mediate language repre-sent an endophenotype for ASD.

Tager-Flusberg also reported preliminary data onidentification of basic speech sounds at ages sixmonths and nine months as they relate to autism.With normal development, there is a change inrecognition of these sounds associated with greaterbrain lateralization at the later stage of development.Infants at risk for ASD exhibited no change in recog-nition and a lack of asymmetry in brain develop-ment at age nine months. These data suggest thatthere may be a seven- to nine-month period duringwhich developmental abnormalities can be detectedand followed as indicators of risk for the develop-ment of ASD. Identification of genetic markers thatare associated with the language deficits during thisperiod may lead to markers useful in identifyingrisk for ASD. A finding such as that reported byGeschwind earlier in the symposium indicating amutation in the CNTNAP2 gene that is passed onmaternally and is associated with language develop-ment is very encouraging and is being pursued byTager-Flusberg and colleagues.

Integrating neuropsychology,development, behavior, and treatment

Sally Rogers, at the University of California, Davis,described how we have come to understand the es-

sential features of autism. We now understand thatASD is manifested differently in early childhood incomparison to school age. She described how thevarious cognitive impairments of affected two tofive year olds can be grouped in “clusters” of social,emotional, action-oriented, linguistic, perceptual,and learning deficits.

In the 1990s, autism research emphasized a modelin which the symptoms of autism were seen asa function of the impact of biological and envi-ronmental factors on cognitive processes. There-fore, treatment strategies tended to selectively tar-get the problem areas with the hope that therewould be improvements in symptoms associatedwith them. Along these lines, strategies that teachimitation to ASD children have been shown to im-prove symbolic play, joint attention, and languageskills.

More recently, an alternative model has gainedfavor: one that ties the symptoms of ASD to dys-functions in the structure and connectivity of spe-cific brain regions. Rogers reflected on Geschwind’searlier presentation as an example of this approach.There is a growing body of information that ASD isassociated with a diminished capacity for the forma-tion of long-range circuits that are essential for theintegration of complex brain functions. She believesthat these circuits are essential for the developmentof complex skills that are deficient in ASD. In ad-dition, she described a different potential problemassociated with the development of these critical cir-cuits. As discussed in Lichtman’s presentation in themorning session, there may be deficits in the for-mation of brain circuits associated with a lack of“pruning” of local brain circuits that result in over-connectedness within these systems, thereby leadingto disorganization at the local level. Rogers believesthat interventions that include teaching imitationbehavior provide the stimulation for the develop-ment of efficient circuits. There is evidence thatwhen engaging in these tasks, ASD children (eventhe high functioning individuals) show diminishedand more localized brain activity than their unaf-fected peers. She believes that early learning of theseand other skills is the key to producing the essen-tial neuronal circuits. Early interventions can bolsterthe development of neuronal circuits that are prob-ably less robust in those with ASD. Although timeconsuming, the repeated exercises of discrete trialtraining and other behavioral methods of applied



behavioral analysis are consistent with this brain-oriented approach.

A series of other approaches, particularly thosethat include naturalistic teaching, show greatpromise. Allowing children to determine the choiceand focus of activities in the context of daily pro-grams with adults leads to the development of im-portant skills and is similar to what parents do withtheir children. In this context, Rogers explored herresearch with Dawson on the ESDM.31 Using a videopresentation of an ESDM training session, she illus-trated the range of situations and reinforcementsemployed by the program. Social interactions, in-cluding eye contact, motor activity, and affirmingemotional feedback, provide effective reinforcementthat is evident. The focus is primarily on the vari-ous neuropsychological deficits of the early stagesof ASD. The hope is that active play in the contextof the developmental training program will lead tothe acquisition of skills that then permits cognitiveand social advancement, which may in turn allowthem to catch up to their peers. She presented addi-tional data from a University of Washington studydescribed by Dawson showing that the percentage ofchildren that met diagnostic criteria for intellectualdisability in the sample decreased from 70% to 30%after two years of the program. She concludes thatthese findings are evidence of the potency of earlylearning and experiences in the acquisition of cogni-tive, social, and motor skills. Early intervention withESDM and similar programs that emphasize an in-tensive “integrative” approach will prove invaluablein the development of the essential neural networksin young ASD children and lead to better treatment.

A parent-mediated intervention

The establishment of joint attentional focus betweeninfant and adult is essential for the development ofearly language acquisition. Michael Siller at HunterCollege views early language learning as a collabo-rative process in which a child and adult both attendto the same object or task in parallel. The basic pro-cesses associated with this state are quite powerfuland the failure of their development may be a ma-jor factor in the language deficits in autism. Siller’sresearch suggests that the deficits in joint attention,which are common in ASD children, make it exceed-ingly difficult for children to effectively pair wordswith their meanings. Typically, children between 15and 19 months are able to recognize the focus ofanother person’s attention, and as a result label the

object. Before this age, language as well as otherlearning depends on the parental establishment ofjoint attention. “What you hear a lot is parents’ label-ing objects to which the child is already attending, orcommenting on actions or intentions or goals thatthe child is pursuing,” he said.

Siller, in collaboration with Marian Sigman atUCLA, has shown that a child’s capacity for jointattention correlates directly with their subsequentacquisition of language skills.39 Siller and Sigmandescribed how joint attention deficits are observedin children with ASD, probably disrupting languagedevelopment. Longitudinal studies by Siller andSigman also suggested that autistic children ben-efit from a process sometimes used by motherswith their autistic children, a process called mater-nal “synchronization.” With synchronization, themother continually uses engaging language to talkabout and describe the objects and actions of achild’s attention, thereby mitigating their languagedeficits.

Recently Siller and colleagues have investigatedthe processes that are the basis of the benefits ofsynchronization and have been developing an in-tervention program to train parents of ASD chil-dren in the effective use of responsive languagewith their children. Data from his recently com-pleted randomized controlled trial indicate that anintervention designed to effectively train individu-als to use these techniques could be implementedand prove effective in stimulating language devel-opment in autistic children. The program involves12 in-home training sessions in which both parent–child and interventionist–child interactions are em-ployed. Conventional teaching, live modeling, andcoaching methods are used in the program. Eachsession is recorded on video and subsequently re-viewed, enabling parents and interventionists to re-view progress and make adjustments to improve thesuccess of the intervention.

Siller reported on a pilot study to aid in the de-velopment of the study guide for the interventioninvolving a small sample. His team partnered withthe California Regional Centers to recruit a largersample of families from the Los Angeles metropoli-tan area for a randomized controlled trial. Chil-dren in the trial had a clinical diagnosis of ASD, se-vere language impairment, and were six years of ageor younger. The group using the training showedimprovements in the use of maternal synchro-nization over their performance at the beginning



of the study, thus indicating that the interventionprocedures were effective in training parents in theuse of responsive language. Further examination ofthe data is required to determine if there were long-term gains in language resulting from this enhancedsynchronization. Siller and colleagues are currentlyanalyzing the first year follow-up data. He is alsocollaborating with Connie Kasari at UCLA to ex-plore the possibility of a similar intervention withyounger children (18 to 30 months old).

Epidemiology and the changing paradigmof ASD

The concluding presentation given by MarshalynYeargin-Allsopp provided a review of ongoing ef-forts of the CDC to determine the prevalence ofASD in the United States and provide informationon the factors that contribute to changes in thesestatistics (Fig. 6). She began by listing some of thefactors that have confounded previous efforts to ac-curately measure the incidence of autism. Most ofthese confounds are related to the quality of the mea-surement criteria, including quantification of onsetbased on diagnosis at a relatively late age (averaging4 to 6 years old), failure to confirm diagnoses overthe course of a study, and the use of inaccurate oroutdated diagnostic standards.

This latter factor is a crucial consideration incurrent attempts to measure prevalence. Yeargin-Allsopp provided a brief update on the change incriteria. This change is especially important consid-ering that autism was first described in 1956 by LeoKanner and that in 1980 this disorder was reclassi-

fied as a developmental disability rather than a men-tal illness. Other milestones are the inclusion of ASDin the World Health Organization’s ICD-10 in the1990s and in the American Psychiatric Association’sDSM-IV, both of which considerably broadened thedescription of ASDs and included high-functioningindividuals such as those with Asperger’s syndrome.She indicated that an increase in prevalence has oc-curred in parallel with the use of the broader, morenuanced diagnostic criteria. This has made it diffi-cult to understand the changes in prevalence overtime, which have been dramatic: increasing from 4–5 in 10,000 individuals prior to 1990s standards to 6cases per 1,000 individuals more recently. Yeargin-Allsopp stated that prevalence trends are limited bythe changes in the characterization of the illness,frequently leading to contradictory interpretations.

Yeargin-Allsopp’s team at the CDC uses a mul-tiple source record review in their efforts to accu-rately determine trends. Using this strategy to mon-itor ASD along with other developmental disorders,they found that the prevalence among eight year-olds in the five counties of metropolitan Atlantashowed a prevalence of 6.5 per 1000 in the year2000.40 These data were surprising because its peakprevalence exceeded all disorders being studied, in-cluding diseases such as cerebral palsy, hearing loss,or visual impairment.

In light of such statistics, the U.S. Congressexpanded the funding of this program and theAutism and Developmental Disabilities Monitor-ing (ADDM) Network to now encompass multiplesites across the nation. Surveillance at all locations

Figure 6. Summary and historical perspective on autism prevalence before 2009, as assessed by the CDC (Yeargin-Allsopp).



incorporates records from multiple health and edu-cation sources, with all cases subject to review andconfirmation by clinicians using DSM-IV criteria.“Our results have really become the standard forsetting ASD prevalence estimates for the UnitedStates,” said Yeargin-Allsopp.

Surveillance data from 14 different sites for theyear 2002 monitored nearly 10% of American chil-dren for ASD.41 The average prevalence was 6.6 per1,000—a measurement that is the basis for the 1:150statistic that was cited throughout the symposium.The conclusion, based on this rate of prevalence,is that approximately 560,000 Americans under theage of 21 are affected by the disorder. In December2010, the CDC reported on data collected from 11sites in 200642 that indicated an even-higher preva-lence of 1 in 110 children, a 57% increase since 2002.As with previous surveys, the disparity between boysand girls remains approximately the same—boys aremore than four times more likely to be diagnosedwith ASD than girls. These data are consistent withthose of Europe and Asia. The current criteria for de-velopment of ASD include onset by the age of three;however, the average age of diagnosis of the disor-der is considerably later at four and one-half yearsof age. In agreement with many of the other pre-sentations, Yeargin-Allsopp stated, “We obviouslystill have a lot of work to do in terms of identifyingchildren with these behaviors early.”

Conclusion

Having Marshalyn Yeargin-Allsopp of the CDCconclude the symposium put ASD in the contextof the national debate. The CDC data and othersources around the world suggest an epidemic ofASD among various demographic groups across theUnited States. In the past few decades, the num-ber of children with ASD has gone from around 4cases per 10,000 children to as high as 1 in 150 insome areas. Although the precise reasons for thisprecipitous rise are unclear, previous efforts to ac-curately measure autism were flawed, and the def-inition of those included on the spectrum has re-cently broadened considerably. She concluded thatfully exploring and addressing this problem will re-quire a concerted effort among scientists, clinicians,and governmental and non-governmental agencies.New detection methods now show potential to iden-tify high-risk infants as early as 6 months of age. Thedetection of changes in language recognition withdevelopment in typical children was notably absent

in autistic children, and a novel paradigm of mater-nal “synchronization” with an ASD child’s attentionwas shown to be effective in stimulating languagedevelopment. A new understanding of the geneticabnormalities that may contribute to the cause ofautism was highlighted, with both large mutations(such as with copy number variations that deletethe RNF8 gene) and single gene mutations (such aswith the fragile X gene and the contactin-associatedprotein, CNTNAP2) playing a significant role in theetiology of ASD. In addition, the function of thesegenes in the formation of circuits, which are beingmapped in the human brain currently, is an excitingarea of research that is just beginning to open up newpossibilities for treatment. The fact that epilepsy is ahighly comorbid factor with ASD suggests that ex-cessive excitatory activity during brain developmentmay contribute to the defects in learning, behavior,and brain wiring observed in ASD; this must be ad-dressed in future studies using anticonvulsant drugsto determine which types of ASD may respond fa-vorably. The presenters and attendees of the sym-posium benefited from the knowledge exchange atthe intersection of cell biology, neuroscience, devel-opmental psychology, and public health on the un-derstanding and treatment of autism. Sharing theresults in these disparate fields should fuel new andexciting research that may one day alleviate the painand suffering experienced by patients and familiesaffected by autism.

Acknowledgments

This publication was made possible by Grant Num-ber RR003037 from the National Center for Re-search Resources (NCRR), a component of the Na-tional Institutes of Health (NIH).

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