Molecular and Cellular Mechanisms of Learning Disabilities: A

NE33CH11-Silva ARI 22 May 2010 18:58

Molecular and CellularMechanisms of LearningDisabilities: A Focus on NF1C. Shilyansky, Y.S. Lee, and A.J. SilvaDepartment of Neurobiology, Psychology, Psychiatry and Biobehavioral Sciences,Semel Institute, University of California, Los Angeles, California 90095;email: [email protected]

Annu. Rev. Neurosci. 2010. 33:221–43

First published online as a Review in Advance onMarch 26, 2010

The Annual Review of Neuroscience is online atneuro.annualreviews.org

This article’s doi:10.1146/annurev-neuro-060909-153215

Copyright c© 2010 by Annual Reviews.All rights reserved

0147-006X/10/0721-0221$20.00

Key Words

Ras, GABA, LTP, animal model, neurodevelopmental disorder,ADHD

Abstract

Neurofibromatosis Type I (NF1) is a single-gene disorder characterizedby a high incidence of complex cognitive symptoms, including learningdisabilities, attention deficit disorder, executive function deficits, andmotor coordination problems. Because the underlying genetic cause ofthis disorder is known, study of NF1 from a molecular, cellular, andsystems perspective has provided mechanistic insights into the etiologyof higher-order cognitive symptoms associated with the disease. In par-ticular, studies of animal models of NF1 indicated that disruption of Rasregulation of inhibitory networks is critical to the etiology of cognitivedeficits associated with NF1. Animal models of Nf1 identified mecha-nisms and pathways that are required for cognition, and represent animportant complement to the complex neuropsychological literatureon learning disabilities associated with this condition. Here, we reviewfindings from NF1 animal models and human populations affected byNF1, highlighting areas of potential translation and discussing the im-plications and limitations of generalizing findings from this single-genedisease to idiopathic learning disabilities.

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NF1:NeurofibromatosisType I

Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 222HUMAN GENETICS OF NF1 . . . . . . 223ROLE OF NEUROFIBROMIN

IN CELL SIGNALING . . . . . . . . . . . 223COGNITIVE PROFILE

OF NF1 PATIENTS . . . . . . . . . . . . . . 225GENETIC MODIFIERS

OF BEHAVIORAL EXPRESSIONOF NF1 . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

NF1 AND PHENOTYPESOF ANIMAL MODELS . . . . . . . . . . . 228

TASKS USED TO ASSESSBEHAVIOR IN ANIMALMODELS OF NF1 . . . . . . . . . . . . . . . . 229

PHENOTYPES OF NF1 ANIMALMODELS AND PARALLELS TOHUMAN COGNITIVESYMPTOMS. . . . . . . . . . . . . . . . . . . . . . 231

MULTIPLE PHENOTYPESLINKED TO THE SAMEMECHANISM . . . . . . . . . . . . . . . . . . . . 232

INCREASED GABA RELEASEFROM INTERNEURONSUNDERLIES Nf1 BEHAVIORALPHENOTYPES . . . . . . . . . . . . . . . . . . . 233

LEARNING DISABILITIES ASA DISTINCT PATHOLOGICALENTITY: LESSONS FROMNF1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236

ADULT TREATMENT OFNEURODEVELOPMENTALDISORDERS: THE CASE FORNF1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237

INTRODUCTION

Neurofibromatosis Type I (NF1) is one of anumber of genetically determined neurocuta-neous disorders that cause clusters of somaticand behavioral symptoms. Originally, these dis-orders were described according to their so-matic symptoms, for example cafe au lait spots,Lisch nodules, and neurofibromas in NF1.

Occurrence of these symptoms in a consistentpattern helped to identify a patient populationaffected by a single disease. Heading the humangenetics revolution, linkage studies mapped thedisease-causing gene in NF1 to chromosome17 (Barker et al. 1987, Seizinger et al. 1987). Asmall group of patients with NF1 were foundto carry translocation breakpoints on chromo-some 17, which greatly accelerated identifi-cation of the disease-causing gene (Fountainet al. 1989, Leach et al. 1989, Ledbetter et al.1989). Further gene mapping within this re-gion identified the Nf1 gene (Viskochil et al.1990, Wallace et al. 1990). Although early on,these studies focused on the somatic symptomsof NF1, additional work within the NF1 pa-tient population also revealed behavioral andlearning problems (Hyman et al. 2005, Northet al. 1997). These cognitive symptoms occuroften, and have a significant impact on quality oflife in individuals affected by NF1 (North et al.1997).

NF1 is a single gene–determined disordercharacterized by multiple complex cognitivesymptoms. Disease-causing mutations in theNf1 gene (Viskochil et al. 1990, Wallace et al.1990) result in loss of function of its proteinproduct, Neurofibromin. Individuals affectedby NF1 are heterozygous for the Nf1 genemutation, as homozygous mutations appear tobe lethal (Friedman 1999). Further study ofthese cognitive symptoms revealed that evenin this single-gene disease, a complex pictureof variable deficits and types of learning dis-abilities occurs. Here, we review human andanimal studies that together have provided in-sight into the nature of cognitive symptomsassociated with NF1 and the mechanisms un-derlying their expression and variability. SinceNF1 and other neurocutaneous disorders arelikely part of the broader cluster of learningdisability disorders, we also discuss the cur-rent understanding of the evolution of cognitivesymptoms in NF1 in the context of hypothe-ses regarding other learning disabilities withunknown etiology and poorly defined affectedpopulations.

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Human Nf1 NF1 (2818 aa)

9a

a

23a

neo

48a

Mouse Nf1 NF1 (2841 aa)

b

Nf1 null Null knockout

Nf1 flox Cell type –specificknockoutX Cre

Figure 1Schematic representation of human and mouse Nf1. (a) Human Nf1 on chromosome 17 encodes a 2818–amino acid protein calledneurofibromin. Small boxes represent exons. Filled boxes indicate exons 21 to 27a that encode the GAP-related domain (GRD).Alternatively spliced exons are marked by arrows. Disease-causing mutations are widely distributed throughout the entire Nf1 gene andmost of those result in loss of function of its protein product. (b) Top: the mouse Nf1 gene on chromosome 11 has a structure very similarto that of human Nf1 gene and encodes a protein with 98% identity to human NF1. Middle: Nf1 null mutant mice were generated byinserting a neomycin cassette in exon 31 ( Jacks et al. 1994). Light blue box indicates exon 31 including the neo gene. Bottom: conditionalmutants were engineered by inserting loxP sites flanking exons 31 and 32 (Zhu et al. 2001). loxP sites are indicated by triangles.Delivery of Cre recombinase by crossing with Cre-expressing transgenic line or viral vector enables cell type–specific deletion of Nf1.

HUMAN GENETICS OF NF1

The Nf1 gene, located on chromosome 17q, isone of the largest in the genome, encompassing60 exons (Figure 1) (Li et al. 1995, Marchuket al. 1991). It encodes multiple biochemicaldomains, including a Ras-GAP domain, whichrequires exon 23a for its activity. The Nf1 genehas four splice variants, two of which are ex-pressed in the CNS. The type I isoform ofthe Nf1 gene contains exon 23a, and as a re-sult encodes a version of Neurofibromin withefficient Ras-GAP activity. This variant is pre-dominantly expressed in neurons. The type IIisoform of the gene is a splice variant withoutexon 23a, and encodes a protein with ten timesless Ras-GAP activity than type I. This variant ishighly expressed in glia. Therefore, differentialsplicing of the Nf1 gene confers different func-tions to its protein product across cell types.

Additionally, Nf1 expression occurs acrossmultiple brain systems that participate in abroad variety of behaviors. Within the CNS,Nf1 transcription is seen in cortex, striatum,substantia nigra, brainstem, hippocampus, andcerebellum. In cortex and hippocampus, Nf1 isexpressed in pyramidal neurons, interneurons,

and glia. In cortex, Nf1 expression spans all cor-tical layers. In striatum, expression of Nf1 issparse, occurring in a pattern suggestive of in-terneuronal expression (Gutmann et al. 1995).It is also highly expressed in the Purkinje neu-rons of the cerebellum. This broad range ofbrain systems in which Nf1 is expressed is alsolikely to contribute to the range of cognitivesymptoms associated with its loss of function.

ROLE OF NEUROFIBROMININ CELL SIGNALING

The Nf1 protein product, Neurofibromin, hasmultiple biochemical roles (Figure 2). Thisprotein acts as a Ras-GAP (GTPase activat-ing protein) to negatively regulate Ras signaling(Weiss et al. 1999), and it can also serve as an ac-tivator of adenylate cyclase (Tong et al. 2002).Its role as a Ras-GAP has been most clearlyimplicated in regulating neuronal function inmammals.

The Ras protein is part of a large super-family of GTPases that mediate signaling frommembrane receptors to intracellular cascadesof kinases. Ras cycles between the inactive

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NMDARRTK

GEFs

SHP2

RasGDP

GPCR

Ras

Raf

MEK

MAPK

Neurotransmitterrelease

Transcriptionalregulation

cAMPGTP

?

NF1 AC

PKC

PKA

Figure 2Neurofibromin and cell signaling. Neurofibromin (NF1) is a GTPaseactivating protein (GAP) that functions as a negative regulator of Ras-MAPKsignaling cascade. Guanine-nucleotide exchange factors (GEFs), such as SOS,may counteract the GAP function of NF1. On the other hand, NF1 acts as anactivator of adenylate cyclase (AC). Note that the key pathways are grosslysimplified in this diagram. For example, the phosphatidylinositol 3-kinase(PI3K)-AKT-mTOR cascade is also modulated by NF1, but is omitted in thediagram. Arrows and barred lines indicate activation and suppression,respectively. MEK, mitogen-activated protein kinase or extracellularsignal-regulated kinase kinase; NMDAR, N-methyl-D-aspartate receptor;GPCR, G protein-coupled receptor; RTK, receptor tyrosine kinase; PKA,protein kinase A; PKC, protein kinase C; SHP2, Src homology 2-containingtyrosine phosphatase.

GDP-bound state and the active GTP-boundstate. The activity state of Ras is determined bya balance of activating proteins (GEFs, guaninenucleotide exchange factors, which allow boundGDP to be released so that GTP can bind) andinactivating proteins (GAPs, GTPase activat-ing proteins, which increase the endogenousGTP hydrolyzing activity of Ras) (Bernards &Settleman 2004). Receptor tyrosine kinases, in-cluding the growth factor receptors (e.g., Trk B,the receptor for BDNF), are common upstreamactivators of Ras signaling (Bernards & Set-tleman 2005). This family of transmembranereceptors is characterized by phosphorylationof intracellular tyrosines upon ligand binding.Tyrosine phosphorylation then leads either to

direct binding and inactivation of Ras-GAPssuch as Neurofibromin, or to indirect activationof Ras-GEFs (Patapoutian & Reichardt 2001,Weiss et al. 1999). Since this complex of recep-tors, Ras-GAPs, and Ras-GEFs resides at themembrane, Ras must also be tethered to the cellmembrane to be activated. At the membrane,Ras can interact with downstream effectorssuch as Raf kinase, which then activate signal-ing cascades such as the MEK/MAPK pathway(Boguski & McCormick 1993, Weiss et al.1999). These kinases then induce a number ofdownstream processes, including transcriptionof immediate early genes, which lead to short-and long-term changes in neuronal function.

In neurons, loss of Neurofibromin results inconstitutive increases in Ras intracellular sig-naling (Li et al. 2005). Further, Neurofibrominloss of function can allow Ras signaling tobecome decoupled from extracellular triggerssuch as growth factors. For example, Nf1−/−

sensory neurons no longer require the extracel-lular growth factor BDNF to survive and ma-ture. This is presumably due to constitutivelyincreased levels of activity of the Ras pathwaywhen it is no longer subject to negative reg-ulation by Neurofibromin (Vogel et al. 1995).It is currently unclear exactly which receptorsare upstream of Neurofibromin regulation ofRas signaling during behavior. However, Neu-rofibromin is known to be quickly regulated byPKC in response to growth factors that uti-lize the tyrosine kinase receptors (EGF), andby ubiquitin-dependent proteolysis in responseto both G-protein (LPA) and tyrosine kinasereceptor ligands (EGF, PDGF).

The Ca2+-sensitive PKC-alpha isoformquickly (within 1 min) phosphorylates the Nterminal region of Neurofibromin in neuronalcultures. This phosphorylation increases Neu-rofibromin association with actin and max-imizes its activity. Thus, this phosphoryla-tion could also make the Ras-GAP activity ofNeurofibromin activity dependent (Leondaritiset al. 2009). In this context, Neurofibromin maynormally act to downregulate the Ras responseto growth factors in an activity-dependent man-ner. The same growth factors can also quickly


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Working memory:a flexible, continuallyupdated system thatmaintains andmanipulatesinformation acrossshort delay periods

(by 5 min) but transiently induce ubiquitin-dependent proteolysis of Neurofibromin in anactivity-independent manner. Thus, followinggrowth factor release, Neurofibromin may playa key role in limiting and narrowing the time ofRas activation (Cichowski et al. 2003). As bothforms of regulation of Neurofibromin wereidentified in embryonic culture systems, it re-mains to be seen whether similar regulation ofNeurofibromin occurs in adult neuronal net-works, whether it occurs downstream of growthfactor receptors such as the BDNF receptors,and how either one or both mechanisms regu-late neuronal networks during behavior.

Finally, although the receptors that utilizeNeurofibromin to regulate Ras signaling inadult neurons have not been identified, one pos-sible upstream receptor outside the growth fac-tor receptors is the NMDA receptor. Neurofi-bromin is part of the NMDA receptor complexin the mouse forebrain (Husi et al. 2000). TheNMDA receptor does interact with intracellu-lar signaling cascades through its C terminus,in a manner that is independent of Ca2+ in-flux and critical to normal performance of de-layed alternation, a behavior related to workingmemory (Bannerman et al. 2008). Although thefunctional relevance of Neurofibromin’s local-ization to the NMDA receptor is unknown, itsuggests that this protein plays a role in NMDAreceptor dependent regulation of Ras signaling.

In addition to its role as a Ras-GAP,Neurofibromin acts as an activator of adenylatecyclase (Tong et al. 2002). Learning defects inNf1 null Drosophila melanogaster can be rescuedby expression of a constitutively active form ofPKA (Guo et al. 2000). These and other datasuggested that the associative learning impair-ments in Nf1 null flies are due to decreasedactivation of adenylate cyclase. Neurofibromincan increase adenylate cyclase activity in aRas-dependent manner. This form of signalingoccurs in response to growth factors such asEGF. Neurofibromin can also directly increaseadenylate cyclase activity in a Ras-independentmanner, through its C-terminal domain. Thisfunction of Nf1 is required for stimulationof adenylate cyclase by neurotransmitters

such as serotonin or histamine (Hannan et al.2006). Nf1 loss in Drosophila causes specificphenotypes, such as small body size, througha direct decrease of adenylate cyclase activity(Tong et al. 2002). Other behavioral functionsof Nf1, such as circadian rhythm modulation inDrosophila, are MAPK dependent and requirethe GAP domain (Williams et al. 2001). Stillunclear is whether these biochemical effectsof homozygous Nf1 deletion are relevant tothe behavioral and cognitive deficits associatedwith NF1, which are caused by heterozygousmutations. In both mouse and Drosophilamodels, heterozygous deletion of Nf1 does notgrossly affect regulation of the adenylate cy-clase pathway (Tong et al. 2002). Nevertheless,it is possible that Nf1 plays a role in maintainingthe relative balance between Ras- and cAMP-dependent signaling (Weeber & Sweatt 2002).

The Neurofibromin protein interacts with anumber of upstream regulators of Ras signal-ing, and has the potential to play multiple roleswithin neurons as part of various intracellularpathways. This may contribute to the range ofphenotypes that have been observed in Nf1+/−.

COGNITIVE PROFILEOF NF1 PATIENTS

NF1 is characterized by widespread symptomsaffecting multiple organ systems (Lynch &Gutmann 2002, Williams et al. 2009). Amongthese symptoms are prominent cognitive im-pairments, which pose one of the most signif-icant sources of lifetime morbidity for patients(North et al. 1997). NF1 specifically affects ex-ecutive and other higher-order cognitive func-tions. In contrast, NF1 does not cause globalcognitive impairments. In measures of globalcognitive function, performance of affected in-dividuals is comparable to unaffected siblings(Kayl & Moore 2000).

Executive function impairments are preva-lent and can be severe in NF1. When specificcognitive domains are probed in neuropsycho-logical batteries, 80%–90% of individuals af-fected by NF1 show impairments (Hyman et al.2005, Krab et al. 2008a). In particular, NF1


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affects planning, visuospatial function, read-ing/vocabulary, and motor coordination (Hof-man et al. 1994, Hyman et al. 2005). Additionalexecutive function deficits are prominently seenin working memory, cognitive flexibility, andinhibitory control (Rowbotham et al. 2009).There is also a high comorbidity between NF1and attention deficit disorder (Hofman et al.1994, Hyman et al. 2005, Mautner et al. 2002).In up to 40% of children with NF1 who areidentified as academic underachievers, under-achievement is likely due to deficits in atten-tion, planning, and organizational skills (Diltset al. 1996, Hyman et al. 2005, Kayl & Moore2000, Koth et al. 2000, North 2000). Comorbidattention deficit disorder affects not only aca-demic but also social development for childrenwith NF1 (Barton & North 2004). Childrenwith NF1 tend to have social problems and ap-pear socially awkward and withdrawn, in com-parison to their siblings and to children withother chronic, life-threatening illnesses (Kayl &Moore 2000). These social deficits could be re-lated to poor interpersonal skills resulting fromdecreased attention to social cues.

Developmental learning disabilities are alsohighly associated with NF1. Overall, individu-als with NF1 are fourfold more likely to requirespecial education (Krab et al. 2008a), and learn-ing disabilities are diagnosed in up to 65% ofindividuals affected by NF1(Rosser & Packer2003). Learning disabilities are characterizedby impairments restricted to specific domainsof mental function, leading to a discrepancybetween tests of intellectual capability and ac-tual achievement (Kelly 2004, Kronenberger &Dunn 2003). Both intellectual capability andachievement are typically assessed using stan-dardized, normalized tests. Learning disabilityis diagnosed by a discrepancy of one to twostandard deviations between achievement andintelligence test scores.

DSM-IV places learning disabilities intofive major categories: reading disorders, math-ematical disorders, disorders of written expres-sion, nonverbal learning disorders, and learn-ing disorders not otherwise specified (Kelly2004, Kronenberger & Dunn 2003, Palumbo &

Lynch 2006). In addition, learning disabilitiesmay be diagnosed according to specific symp-toms, such as in dyslexia (reading disorder) ordyscalculia (math learning disorder). Thus, thecriteria for diagnosis of learning disabilities arelargely based on different systems of represent-ing symptoms. However, a single learning dis-ability category is often insufficient to describeall the symptoms of a given individual with id-iopathic learning disability (Lagae 2008), or agroup of individuals with an inherited learningdisability such as NF1.

In fact, learning disabilities caused by NF1show some characteristics of both nonverbal-and verbal-type learning disability. Compo-nents of nonverbal learning disability seen inNF1 include consistently poor performancein tests of visuospatial functioning and spa-tial learning (Kelly 2004), notable impairmentsin the ability to perceive social cues, poororganizational skills, and increased impulsive-ness (Kayl & Moore 2000, North 2000). NF1patients also show aspects of verbal learn-ing disorder. Specifically, patients with NF1have deficits in expressive and receptive lan-guage, vocabulary, visual naming, and phono-logic awareness. In fact, reading and spellingare repeatedly found to be impaired moreseverely than predicted by IQ in NF1 pa-tients (North 2000, North et al. 1997). Con-sistent with these impairments in language-based learning, NF1 patients show pooreracademic achievement in reading and writ-ing compared to their unaffected siblings. Thepattern of learning disabilities seen in NF1does not fall cleanly into one of the DSM-IV defined categories, but rather shares fea-tures with multiple learning disability cate-gories. This shows that a single disorder, inthis case NF1, can cause learning disabili-ties of varying phenotypes and presentations.Therefore, the various classifications and sub-divisions of learning disabilities (for example,verbal versus nonverbal) may not actually rep-resent different disease entities but rather dif-ferent manifestations of a common diseasecluster caused by a common set of underly-ing factors. In NF1, the drastic variability of


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Genetic penetrance:the proportion ofindividuals carrying agenetic variant thatalso show pheno-typic/symptomaticmanifestationsassociated with thatvariant

Variable geneticexpression: indicatesthe range ofphenotypic/symptomatic severityassociated with thesame genetic variantacross phenotypes anddifferent individuals;frequently seen indominant geneticconditions

WT: wild type

symptom expression appears to be determinedlargely by independently inherited geneticmodifiers.

GENETIC MODIFIERSOF BEHAVIORAL EXPRESSIONOF NF1

Inheritance of NF1 shows an interesting pat-tern of complete genetic penetrance but vari-able expressivity (Ward & Gutmann 2005).Most people with an inactivating mutation inone allele of the Nf1 gene show some symp-toms of NF1. However, the clinical presenta-tion of NF1 ranges from minimal symptom loadto extremely high severity across many typesof symptoms. One important factor underlyingvariable expressivity of cognitive symptoms inthe NF1 population is the contribution of mod-ifying genes, some of which may significantlyalter behavior in the presence of mutant Nf1,but not in a wild-type (WT) background. Stud-ies in both mouse models and human patientpopulations suggest that inheritance of geneticmodifiers accounts for the majority of vari-ability in NF1 expression (Easton et al. 1993,Sabbagh et al. 2009).

In NF1 mouse models, the phenotypic effectof the Nf1 mutation depends on the backgroundstrain on which that mutation is expressed. Thishas been shown for both behavioral and somaticsymptoms of NF1. Different strains of mice (forexample, C57Bl/6J versus DBA/2J) carrying theNf1+/− mutation have been shown to have dif-ferent susceptibility to the formation of astrocy-tomas (Hawes et al. 2007). Similar studies haveshown that the background strain also affectsthe behavioral phenotype of the Nf1 mutation(Costa 2002). Since these strains are engineeredwith the same Nf1 gene mutation, phenotypicdifferences across strains are attributed to dif-ferential expression of modifier genes acrossmouse strains that were inbred from geneticallydifferent founders. Such modifiers may inter-act with the gene directly, altering its level ofexpression, or, more likely, may interact on amore functional level to exacerbate or compen-sate for the signaling changes caused by loss

of Nf1. The latter is likely to be most rele-vant as studies quantifying differences in lev-els of Nf1 expression across strains find thatalthough expression levels can vary dramati-cally, they do not always correlate with phe-notype expression (Hawes et al. 2007). On theother hand, introducing functional modifiers oflearning pathways can exacerbate the learningphenotype of Nf1+/− mice. This was demon-strated using a heterozygous null mutation of areceptor (NMDA; NR1+/−) critical for learn-ing and memory. Although the NR1+/− mu-tation alone does not have a spatial learningphenotype, it exacerbates the spatial learningphenotype of the Nf1+/− mutant mice (Silvaet al. 1997). Therefore, mouse studies indicatethat background genetic modifiers alter the ex-pression of Nf1+/−-related phenotypes, likelythrough functional interactions with the cellu-lar pathways altered by the Nf1 mutation.

In human patient studies, heritable geneticmodifiers have also been found to affect expres-sion of NF1, and current studies are in progressto identify specific loci encoding these modi-fiers. Analyses of large groups of extended fami-lies affected by NF1 suggest that the expressionof NF1 symptoms is heritable. Variation insymptom expression between family membersis consistent with a model of inheritance ofmodifier genes with little contribution fromspecific mutations to the Nf1 gene itself or fromvariations in the WT Nf1 allele (Easton et al.1993, Sabbagh et al. 2009). Among multipleclinical symptoms of NF1, including learningdisabilities and referral for remedial education,symptom expression shows high correlationamong first-degree family members (who share50% of their genes). Similarly, monozygotictwins affected by NF1 have a high correlationin symptom severity (Easton et al. 1993). Assecond- and third-degree family members areexamined (who share 25% and 12.5% of theirgenes, respectively), the correlation in symp-tom expression decreases sharply. The stronggenetic component in symptom expression inNF1 is consistent with modulation of diseasesymptoms by inherited genetic modifiers(Easton et al. 1993, Sabbagh et al. 2009). For


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example, proteins encoded by genetic modifiersmay interact with the Neurofibromin signalingpathway to exacerbate effects of Nf1 mutationsor confer protection by compensating for theloss of function.

These studies of NF1 highlight the reg-ulation of clinical presentation and symptomseverity by inheritable genetic modifiers, andsuggest that these modifiers may have a moredramatic impact in the presence of the Nf1 mu-tation than in a WT background. Similar find-ings have been made in other genetically de-termined learning disabilities, such as Noonansyndrome (NS). NS is another common auto-somal dominant genetic disorder with an in-cidence of 1 in ∼2500 live births. It is char-acterized by facial abnormalities, short stature,webbed neck, motor delay, and cardiac dis-ease (Noonan 1994, Tartaglia & Gelb 2005).Importantly, NS patients also show increasedrates of learning disabilities and mental re-tardation (Lee et al. 2005, Money & Kalus1979). Recently, Araki and colleagues gener-ated NS mouse models by engineering NS-associated PTPN11 mutants. They demon-strated that the gain-of-function mutants showphenotypes similar to that found in NS pa-tients (Araki et al. 2009, Araki et al. 2004).The penetrance of NS phenotypes is depen-dent on the specific Ptpn11 allele studied aswell as the genetic background of the mu-tant mice (Araki et al. 2009). Ptpn11 muta-tions that modify tyrosine phosphatase SHP2activity result in different heart defects. Half ofthe NS Ptpn11D61G/+ mutant mice are embry-onic lethal on a mixed 129S4/SvJae X C57BL/6genetic background (Araki et al. 2004). How-ever, the Ptpn11D61G/+ mutation backcrossed toC57BL/6 revealed high lethality. In contrast,when crossed into 129S6/SvEv genetic back-ground, this mutation showed normal viabil-ity, suggesting that there are modifier allele(s)that affect the viability of the NS Ptpn11D61G/+

mutation (Araki et al. 2009). The examples de-scribed above illustrate the dramatic influenceof modifier genes on the clinical presentationof a single-gene disorder.

NF1 AND PHENOTYPESOF ANIMAL MODELSMice with a heterozygous null mutation of theNf1 gene (Nf1+/−) have been the dominant ro-dent model used to study NF1. The Nf1+/−

mice show compelling genetic and behavioralparallels with human NF1, making them a use-ful system in which to study the mechanismsof behavioral phenotypes associated with NF1.Nevertheless, a word of caution is in order:There is a very large evolutionary gulf betweenmice and humans, and therefore the behav-ioral phenotypic parallels between animal mod-els and NF1 must not be overinterpreted. Muchneeds to be done to fully elucidate the apparentparallels between rodent and human behavior.

The sequence, transcriptional regulation,and downstream targets of Nf1 are conservedacross species, including mouse and human(Bernards et al. 1993, Hajra et al. 1994). Themajority of mutations (70%) of the Nf1 genein humans affected by NF1 lead to synthesisof a truncated, nonfunctional version of theencoded Neurofibromin protein (Shen et al.1996, Thomson et al. 2002). Accordingly, theNf1+/− mouse model was made by inserting aneo gene in exon 31 of the Nf1 gene, whichleads to an unstable, quickly degraded transcript(Figure 1) ( Jacks et al. 1994). Like patientswith NF1, the Nf1+/− mouse model is heterozy-gous for this loss-of-function mutation. Behav-ioral phenotypes of the Nf1+/− mouse show apattern of specific impairments in certain do-mains and preserved function in others. Thispattern of behavioral phenotypes is reminiscentof the pattern of behavioral and cognitive symp-toms seen in humans affected by NF1. Based onthe genetic, biochemical, and behavioral par-allels between the Nf1+/− mouse model andhuman NF1, it is thought that this mouse of-fers a useful model of the behavioral and cog-nitive symptoms associated with the disorder.The Nf1+/− mouse has therefore been utilizedto identify electrophysiological and molecularmechanisms that contribute critically to cog-nitive and behavioral changes associated withNF1.


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Long-termpotentiation (LTP):a form of synapticplasticity wherehigh-frequencystimulation leads toincreased strength ofsynaptic transmission.In hippocampus, LTPis thought to berequired for learningand memory

Conditional mutants of the Nf1 gene havealso been extensively studied to identify the ef-fects of Nf1 deletion within specific neuronaltypes. Conditional mutations can be createdin mice using the Cre-loxP system, a powerfultool widely used for restricting gene deletionsto specific time frames, cell types, or areas. Amouse line was engineered with loxP sites flank-ing exons 31–32 of the Nf1 gene (Nf1flox/flox)(Figure 1). The floxed Nf1 gene acts like aWT allele prior to expression of Cre recom-binase. Mice carrying one floxed Nf1 allele andone deleted Nf1 allele (Nf1flox/−) show the samephenotypes as Nf1+/− mice. For example, theyhave the same survival profile: Deletion of thefloxed Nf1 allele following delivery of Cre re-combinase caused embryonic lethality, similarto the Nf1−/− mutation (Zhu et al. 2001). Con-ditional Nf1 mutants have revealed cell-specificroles of Nf1 and interesting interactions be-tween neighboring cells with different Nf1 genedoses. Such interactions are critical to the de-velopment of some of the somatic events asso-ciated with NF1 such as neurofibromas (Zhuet al. 2002).

Finally, mouse models targeting specificbiochemical domains of the Nf1 gene have beenexamined to demonstrate the relative impor-tance of the various regulatory functions of theNf1 protein product, Neurofibromin. For ex-ample, exon 23a of the Nf1 gene is spliced outin peripheral tissue, but expressed in neuronalNf1. Presence of exon 23a increases the Rasregulatory activity of Neurofibromin, suggest-ing that this represents a uniquely importantaspect of Nf1 loss in the brain. Indeed, manyof the behavior deficits identified in Nf1+/−

mice were also found in Nf123a−/− mice, whichcarry a homozygous deletion restricted to exon23 of the Nf1 gene (Costa et al. 2001). Thisdeletion creates a version of Neurofibrominthat is functional but has diminished Ras reg-ulatory activity. Importantly, the Nf123a−/−

mice show learning disability–related behav-ioral deficits, but none of the other somatic con-sequences of Nf1 mutation such as increasedtumor predisposition (Costa et al. 2001). Thisnot only demonstrates that Neurofibromin has

a neuron-specific role as a Ras regulator, butalso shows that altered behavioral performancein these mutant mice is a direct function of theirNf1 mutation, rather than a secondary effect oftumor formation. Detailed studies have char-acterized the behavioral consequences of Nf1mutation.

TASKS USED TO ASSESSBEHAVIOR IN ANIMALMODELS OF NF1

The behavioral tasks used to study diseasemechanisms in Nf1+/− mice were chosen ac-cording to the following criteria: (a) face/construct validity to tasks performed less ac-curately by NF1 patients, (b) dependency onbrain areas that contribute critically to cog-nitive functions affected by NF1, and (c) be-haviors with well-established molecular andcellular underpinnings. To date, the behav-ioral paradigms used in the study of Nf1+/−

mice were predominantly tests sensitive to hip-pocampal and parietal/prefrontal cortical func-tion. This result reflects the prominence ofsymptoms thought to stem from dysfunction inthese brain areas in NF1 patients. The Morriswater maze was initially utilized in Nf1+/−

mice as a test of spatial learning and memorythat is highly sensitive to hippocampal func-tion (Morris et al. 1982). Additionally, per-formance in the Morris water maze requiresspecific synaptic and cellular mechanisms, suchas long-term potentiation (LTP) (Moser et al.1998; Silva et al. 1992a,b). As such, this be-havioral paradigm provided a bridge betweenfunctional/mechanistic changes in Nf1+/− miceand a mouse behavioral phenotype that couldbe related to the spatial learning deficits seen inNF1 patients. In the Morris water maze, miceare placed in a pool of opaque water in whichthere is an escape platform. The test subjectsmust use the spatial cues surrounding the poolto learn the location of the platform and nav-igate to it, a process that usually takes multi-ple trials over several days. Following training,memory of the spatial location of the platformis tested in probe trials where the platform is


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removed, and the experimenter measures theamount of time mice spend searching for themissing platform in the appropriate quadrantof the pool. In a control task (unperturbed bythe Nf1+/− mutation), the position of the es-cape platform is directly marked by an object,

ANIMAL MODELS OF HUMAN BEHAVIORS

A key question often raised by animal models of human diseaseis whether the model captures all of the essential features of thedisease. Despite the fact that some animal models only address asubset of the features of a disease, they can still be very useful forunderstanding the disease and developing treatments. To under-stand the complexity of the human condition a variety of differentmodels is needed, not necessarily one with all of the features of thedisorder. Another challenge is to extrapolate findings from animalmodels across species, despite differences between human androdent control over cognition, timing, lifespan, and organizationand development of brain structures such as prefrontal cortex.Currently, several approaches are being used in rodent modelsof disease. Behavioral batteries have been developed for animalsto test for disease-relevant clusters of phenotypes. Appropriateand high-throughput behavioral testing has been developed withdemonstrated face, construct, and some predictive validity withhuman disease symptoms (Chadman et al. 2009). Also, specific be-havioral tasks have been developed for animals to model cognitivefunctions commonly affected in human disease. For example, the5-choice serial reaction time task (5CSRTT) is one example ofa behavioral paradigm that is designed to test multiple attentionprocesses in a manner highly analogous to the probing of thoseprocesses in humans (Robbins 2002). Mechanisms contributingto normal performance of this task have been extensively charac-terized, including the neuromodulatory systems and brain areasthat mediate normal performance of the 5CSRTT in rodents(Robbins 2002). This paradigm has also been adapted to examineworking memory in rodents (Aarde & Jentsch 2006), in a manneranalogous to the Sternberg style working memory tests used inhumans (Cannon et al. 2005). Tasks are also being developed foruse in humans to parallel key features of commonly used rodenttasks (Demeter et al. 2008). The use of these tasks to obtain con-vergent evidence from multiple approaches offers exciting newprospects for understanding disease through the integration ofthe cognitive, clinical, and basic neurosciences (Chadman et al.2009, Fossella & Casey 2006).

and the mice just have to learn to go to it toescape from the water.

Extensive studies with the water maze andother hippocampal-dependent tasks helped todefine the behavioral deficits in Nf1 miceand unravel underlying mechanisms (Costaet al. 2001, 2002; Cui et al. 2008; Silva et al.1997). Subsequent behavioral studies have be-gun to address some of the prominent cog-nitive symptoms of NF1. Specific behavioralparadigms were utilized that have been de-veloped in rodents to model core cognitivefunctions commonly affected in human dis-ease. For example, the lateralized reaction timetask was used in Nf1+/− mice to test attentiondeficit/hyperactivity (Li et al. 2005), as atten-tion deficits are highly associated with NF1 inhumans. Although attention, like memory andother behaviors modeled in mice, has importantdifferences across species, there are also con-served elements that the behavioral tasks usedin rodent models utilize (see Sidebar: AnimalModels of Human Behaviors). In this case, thelateralized reaction time task was designed totest attention processes in a manner that cap-tures key features of tasks used to probe at-tention in humans (Robbins 2002). Further,performance of the rodent task requires the pre-frontal cortex, an area thought to contribute toattention in humans. Performance of rats in thelateralized reaction time task is also improvedby stimulants used to treat ADHD (atten-tion deficit and hyperactivity disorder) in hu-mans ( Jentsch et al. 2009), supporting commonmechanistic underpinnings. The lateralized re-action time task is implemented in an operantchamber and requires mice or rats to fixate ona central hole while attending to two potentialcues, one on either side of the center. In mul-tiple, serial, self-initiated trials, one of the cuesis lit for varying lengths of time, and the rodentmust maintain attention divided across bothsides of space in order to notice and respond tothe lit cues. Beyond attention, similar tasks havealso been adapted to examine working memoryin rodents (Aarde & Jentsch 2006), in a manneranalogous to the Sternberg style working mem-ory tests used in humans (Cannon et al. 2005).


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ADHD: attentiondeficit andhyperactivity disorder

These tasks provide very useful methods that al-low for parallel studies in rodents and humansof core symptoms of NF1, including attentiondeficit and the working memory impairmentsoften found in learning disabilities. As more ev-idence accumulates from the use of these tasksin rodents and humans, we will be able to bet-ter understand which behavioral mechanismshave been conserved across species and whichtasks are best able to probe conserved mecha-nisms in the context of disease. This offers excit-ing new prospects for understanding diseases,such as NF1, through an integration of cogni-tive, clinical, and basic neuroscience approaches(Chadman et al. 2009, Fossella & Casey 2006).

PHENOTYPES OF NF1 ANIMALMODELS AND PARALLELS TOHUMAN COGNITIVE SYMPTOMS

The phenotypes identified in Nf1 animal mod-els in the tasks described above demonstratethat mutation of the Nf1 gene in animal mod-els causes impairments with striking analogy to

that seen in the learning disabilities associatedwith NF1 in humans (Table 1). These phe-notypes of the Nf1+/− mice can be improvedwith extended training, a feature also observedin human learning disabilities.

Nf1+/− mice show spatial learning deficitsin the hidden version of the Morris water maze(Costa et al. 2001, 2002; Cui et al. 2008; Silvaet al. 1997). Probe trials given early on dur-ing water maze training revealed that Nf1+/−

mice require more training trials than controlsto learn the position of the hidden platform.Thus, when searching for the missing plat-form in probe trials, the mutant mice spent lesstime in the appropriate quadrant compared totheir WT littermates. Additional training led tocontinued improvement in spatial memory inNf1+/− mice, such that probe trials given afteradditional days of training no longer revealeddifferences between genotypes. This sensitiv-ity to overtraining is also a feature reportedin NF1-associated learning disabilities in pa-tients. Similarly, Nf1+/− mice also show deficitsin contextual conditioning, a test where mice

Table 1 Cognitive phenotypes of NF1 mouse models

Mouse Model Behavioral phenotypes Parallel human symptoms ReferencesNf1+/− Impaired in

Morris water maze (hidden platform)Contextual fear conditioningLateralized reaction time taskPre-pulse inhibition

Learning deficits in specific domain(visual-spatial)

Attention deficits

Silva et al. (1997)Cui et al. (2008)Li et al. (2005)

Normal inOpen field and visible platform water mazeCued fear conditioning

Nf123a−/− Impaired inMorris water maze (hidden platform)


Costa et al. (2001)

Contextual discrimination Motor deficitsRota-rod

Normal inOpen field and visible platform water mazeSocial transmission of food preference

Nf1flox/+; SynI-creNf1flox/+; Dlx5/6-cre

Impaired inMorris water maze (hidden platform)

Normal inVisible platform water maze


Cui et al. (2008)


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associated a novel chamber with a mild foot-shock (Cui et al. 2008). As with the water maze,contextual conditioning has a spatial learningcomponent and requires hippocampal function.

In contrast to the significantly impaired spa-tial learning seen in Nf1+/− mice, neither vi-sual learning nor simple associative learningis prominently disrupted in these mice (Costaet al. 2001, 2002; Cui et al. 2008; Silva et al.1997). Performance of the Nf1+/− mice in thevisual version of the Morris water maze is in-distinguishable from WT. Additionally, Nf1+/−

mice show no deficits in tone fear conditioning,where mice learn to associate a tone with a mildfootshock. Unlike the Morris water maze, tonefear conditioning is a single-trial, simpler asso-ciative learning paradigm that requires amyg-dala function. Therefore, Nf1 mutation has veryspecific effects on spatial learning while sparingsimple associative learning, possibly due to agreater effect on hippocampal function (Costaet al. 2002, Silva et al. 1997). This pattern ofspecific impairments is also seen in learning dis-abilities associated with NF1 in patients.

Finally, as NF1 subjects, Nf1+/− mice showattention deficits in the lateralized reaction timetask (Li et al. 2005). ADHD is highly associatedwith NF1, and thought to contribute to aca-demic and social changes in this patient pop-ulation. In the lateralized reaction time task,Nf1+/− mice perform as accurately as WT micewhen cues are lit for longer intervals (up to 1 s).However, as attention is taxed with shorterlight presentations (0.5 s), a failure to main-tain constant attention in the Nf1+/− mice isrevealed. The Nf1+/− mice make significantlymore omissions than do WT (Li et al. 2005).Humans with NF1 often show a type of ADHDcharacterized by increased omissions and lapsesof attention (Hyman et al. 2005, Mautner et al.2002).

These animal data demonstrate that muta-tion to the Nf1 gene is sufficient to directlycause a specific pattern of complex behav-ioral changes in mice. The behavioral pheno-types of the Nf1+/− mice in these tasks showanalogy to the learning disabilities and othercognitive symptoms that characterize NF1.

However, various differences clearly exist be-tween mouse and human. For example, theanatomical organization and connectivity ofprefrontal cortex and other regions likely tobe critical for the NF1 phenotype are differ-ent between the two species. Additionally, thecomplexity of the human NF1 phenotype canonly be partially captured in animal models.This and other factors make direct translationfrom mouse to human studies difficult and un-predictable. In the context of NF1, where alarge literature of both animal and human stud-ies exists, it will be interesting to follow futureexperiments evaluating which specific molecu-lar mechanisms, cellular effects, and behavioralparadigms carry translational value.

MULTIPLE PHENOTYPESLINKED TO THE SAMEMECHANISM

Cognitive effects of the Nf1+/− mutation inmouse have been seen in diverse processes,from hippocampal memory to quick, flexible,prefrontal/parietal cortex-dependent attentionprocesses. Although distinct underlying circuitsand brain mechanisms mediate these cognitivefunctions, in NF1 both memory and attentionare impaired by the same genetic change. InNf1+/− mice, both deficits in memory and in at-tention are caused by increased Ras signaling,resulting from the loss of regulation by Neu-rofibromin (Costa et al. 2002, Cui et al. 2008,Li et al. 2005). To demonstrate the Ras de-pendency of deficits in the Morris water maze,Nf1+/− mice were crossed to null Ras mutants(K-Ras+/− or N-Ras−/− mice). As a result of thedecreased levels of Ras activity in the Ras mu-tants, the Nf1/Ras double mutants performedat the same level as did WT mice (Costa et al.2002), even though each mutant individually(Nf1+/− and K-Ras+/−) showed deficits in thistask. Similarly, performance of Nf1+/− mice inthe Morris water maze was rescued with far-nesyl transferase inhibitors under conditionsthat did not enhance the performance of con-trols. These inhibitors pharmacologically de-crease levels of Ras signaling (Costa et al. 2002)


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by blocking the posttranslational modificationthat is necessary for Ras to associate with thecellular membrane and therefore be active. Thismechanism of action can also be targeted witha class of drugs, the statins, which also decreaseisoprenylation (Li et al. 2005). Statins are rela-tively safe and are widely used to control choles-terol levels. In both the Morris water maze andin the lateralized reaction time task, lovastatin,a drug in the statin class, normalized the perfor-mance of Nf1+/− mice to levels indistinguish-able from those of WT (Li et al. 2005). Impor-tantly, statins also normalized the signaling andthe neurophysiological deficits of the Nf1+/−

mice. Hence, regardless of method used, de-creases in Ras signaling rescue both the learn-ing impairments and the attention deficits ofthe Nf1+/− mice. These results also confirmthe wide range of cognitive dimensions affectedby alterations of Ras signaling. The observa-tion that multiple symptoms are caused by dis-ruption of a single biochemical process is notspecific to NF1, but is also seen following mu-tations to other Ras-regulatory genes. For ex-ample, SPRED1, another negative regulator ofRas-MEK/MAPK signaling, is responsible for aNF1-like syndrome (Legius syndrome) (Bremset al. 2007). Similar to Nf1, loss-of-functionmutations of SPRED1 also result in hyperac-tivity in MEK/MAPK signaling. The disor-der associated with Spred1 mutations involvessome of the same symptom dimensions (otherthan neurofibromas) seen in NF1(Pasmant et al.2009). This includes NF1-like cognitive deficitsin affected individuals (Brems et al. 2007,Pasmant et al. 2009, Spurlock et al. 2009).Spred1 knockout mice (Spred1−/−) have beengenerated (Brems et al. 2007, Denayeret al. 2008) and found to show deficits inhippocampus-dependent learning and memorytasks, which may model cognitive deficits inpatients with Spred1 mutation (Denayer et al.2008). Therefore, disruption of the Ras signal-ing pathway through SPRED1 can also lead toa complex, multifaceted disorder. These disor-ders demonstrate that multiple types of symp-toms can all be caused by alterations in a singlegene/biochemical component.

INCREASED GABA RELEASEFROM INTERNEURONSUNDERLIES Nf1 BEHAVIORALPHENOTYPES

Nf1 deletion and increased Ras signaling havecell type–specific physiological effects thatcontribute to behavioral symptoms. In par-ticular, Nf1 expression in interneurons seemsto be critical for behavior, whereas its role inpyramidal neurons appears to be nonessentialfor hippocampal learning in the strains of miceand behaviors tested (Cui et al. 2008). In theMorris water maze, heterozygous deletion ofNf1 from inhibitory interneurons is sufficientto cause behavioral impairments. Conversely,deletion of Nf1 from either pyramidal neuronsor glia does not alter behavior in this taskunder the conditions tested. Thus, regulationof Ras signaling by Nf1 is particularly criticalwithin interneurons, but perhaps less so inpyramidal neurons (Cui et al. 2008). Withinthe hippocampus of Nf1+/− mice, increased in-terneuronal Ras signaling causes an increase inactivity-dependent GABA release (Figure 3).This increased activity-dependent release ofGABA (Cui et al. 2008) leads to larger evokedinhibitory currents in CA1, shifting the balancebetween inhibitory and excitatory processeswithin hippocampal networks of the mutantmice. As a result, LTP is impaired in theNf1+/− mice, perhaps because the increasedinhibition prevents sufficient depolarization ofNMDARs during learning (Cui et al. 2008).In the hippocampus, there is strong evidencethat LTP is required for learning of spatialand contextual information (Lee & Silva 2009,Martin et al. 2000, Martin & Morris 2002,Richter-Levin et al. 1995). Additionally, thevery manipulations that reverse the learningdeficits of the Nf1+/− mice (decreases in Rassignaling with statins or farnesyl transferase in-hibitors), also reverse their LTP impairments.As additional training rescues the learningimpairments of the mutants, additional synap-tic stimulation (e.g., higher frequency) alsorescues their LTP deficits. Therefore, the LTPdeficits are thought to underlie the learning


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GEFs

RasGDP

Ras

MAPK

Synl

GABA

a

GTP

NF1GEFs

RasGDP

Ras

MAPK

Synl

GABA

b

GTP

NF1

Figure 3Proposed cellular mechanism underlying learning deficits of Nf1 mutant mice. (a) Learning triggersinterneuronal Ras signaling leading to increased GABA release. MAPK-dependent phosphorylation ofsynapsin I (SynI) plays a critical role in GABA release. Wild-type NF1 restricts the increase in GABA releasewithin an appropriate range that modulates learning. (b) In Nf1 mutants, reduced NF1 activity leads toabnormal hyperactivation of Ras signaling in inhibitory interneurons during learning, resulting inabnormally high GABA release. This increased activity-dependent GABA release shifts the balance betweenexcitatory and inhibitory processes in neuronetworks of the mutant mice and impairs synaptic plasticityneeded for learning and memory.

deficits seen in the Nf1+/− mice. Supportingthis assertion, both the Nf1+/− LTP andwater maze deficits can be improved usingpicrotoxin, a GABAA receptor antagonist (Cuiet al. 2008), at concentrations that do notaffect these phenomena in controls. Thesedata demonstrate that Neurofibromin is animportant regulator of inhibitory tone in hip-pocampal neuronal networks. This regulationof inhibition is functionally important as itsdisruption impairs behaviors, such as the ac-quisition of spatial and contextual information.Again, beyond manipulations of inhibition,Lovastatin, farnesyl transferase inhibitors, andthe N-ras and K-ras mutations, all reverse boththe LTP and the learning deficits of the Nf1+/−

mutant mice, strengthening the link betweenthe hippocampal LTP deficits of these miceand their hippocampal-dependent learningdeficits (Costa et al. 2002, Li et al. 2005).

In other brain areas such as the prefrontalcortex and striatum, inhibition also plays animportant role in regulating network phenom-ena that are critical for cognitive functions

affected in NF1, including working memoryand attention. In prefrontal cortex, workingmemory–related cellular activity involves abalance between a high degree of excitation(allowing inputs to induce persistent activity)and a coordinated set of inhibitory mechanisms(imposing specificity to the activity). Thisbalance between excitation and inhibitionallows prefrontal cortex to encode informationacross delays (Compte et al. 2000). Activityin prefrontal cortex is regulated by both feed-forward and feedback inhibitory microcircuits(Constantinidis et al. 2002, Hasenstaub et al.2005, Sanchez-Vives & McCormick 2000),which can affect the onset and duration of per-sistent activity (Compte et al. 2000, Fellous &Sejnowski 2003). In addition, inhibition acts totune persistent activity of prefrontal neurons tospecifically represent cue location, and to orga-nize temporal interactions between prefrontalneurons (Constantinidis & Goldman-Rakic2002, Constantinidis et al. 2002). Decreasinginhibition within prefrontal cortex leads to lossof spatial tuning, which results in inaccurate


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working memory performance. Decreasinginhibition also unmasks spatial tuning in previ-ously silent neurons. During a working memorytask, this spatial tuning results in inappropriate,pre-emptive behavioral responses (Rao et al.2000). Therefore, inhibitory interneurons inthe prefrontal cortex have multiple functionsduring behavior, an observation with interest-ing implications for attention and executivedeficits in both Nf1 mice and NF1 subjects.

Similarly, striatal inhibitory interneuronsmodulate the activity of local medium spinyneurons. Inhibitory currents induced at thesoma of medium spiny neurons by striatal in-terneurons can abolish or delay the onset ofspiking (Koos & Tepper 1999, Tepper et al.2008), making GABAergic interneurons a pri-mary modulator of activity flow in the striatum.Importantly, striatal inteneurons are driven byglutamatergic inputs, including those descend-ing from the cortex. Therefore, abnormallyhigh levels of inhibition seen in Nf1+/− micewould be expected to significantly alter activitywithin the striatum, the prefrontal cortex, andthe timing and flow of activity between striatumand cortex. Increased inhibition resulting fromNf1 mutation likely underlies not only learningand memory deficits but also other behavioralphenotypes of the Nf1+/− mice that are moreclosely related to dysfunction in frontal/corticalbrain areas (e.g., attention, executive function).

Increased inhibition can lead to behav-ioral deficits by altering either the ongoingfunction of neuronal networks in the adultbrain, or by affecting neuronal development.In the Nf1+/− mice, increased inhibition inthe adult brain contributes critically to behav-ioral deficits, since these deficits can be res-cued in adult Nf1+/− mice by decreasing inhibi-tion (e.g., using picrotoxin), or by normalizingRas signaling (e.g., with statins). This findingsuggests that these behavioral deficits occur asa result of a reversible increase in inhibition.However, it is important to also consider po-tential effects of increased inhibition on devel-opment of neuronal networks, since most Nf1gene mutations in patients and mice are presentfrom birth. GABAergic inhibition plays an

important role in the developmental patterningof neuronal networks, and so the Nf1 mutationmay cause developmental defects that correlatewith symptoms of NF1 such as learning disabil-ities. The distinction between developmentaland adult causes of NF1 is relevant for decid-ing which features of NF1 could be targeted fortreatment.

During development, experience-dependent plasticity mediates organization ofneuronal networks using mechanisms sharedwith adult experience-dependent plasticity.Therefore developmental plasticity is likely tobe disrupted in NF1 by the same increase ininhibition that disrupts LTP in adult Nf1+/−

mice. Further, GABAergic inhibitory networksplay a role in regulating the developmentalpatterning of cortical networks by modulatingthe length of developmental sensitive peri-ods. Sensitive periods are windows of timeduring development when experience has agreater effect on the organization of neuronalnetworks than in adulthood (Hensch 2004,Knudsen 2004). In primary sensory areas, thesensitive period is regulated by the maturationand activity of GABAergic interneurons.For example, tonic increases in GABAergicactivity correlate with the closure of the criticalperiod for ocular dominance in the visualcortex (Fagiolini & Hensch 2000, Hensch2005). In NF1, increased inhibition may causeinappropriate, early closure of sensitive periodsleading to altered patterning in cortical areas.Possibly related to increased inhibition, or toother effects of Nf1 deletion, cortical barrelsfail to form in mice where Nf1 is homozygouslydeleted from neurons and astrocytes of thesomtatosensory cortex (Lush et al. 2008). NF1may be associated with changes in primarysensory processing that are caused by increasedinhibition, but may not directly cause thehigher-order cognitive symptoms such aslearning disabilities. Consistent with thisincreased inhibition, learning disabilities areoften found in association, but not correlatedwith, alterations in primary sensory processing(Ramus 2003). Because the severity of learningdisabilities does not correlate well with the


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severity of alterations in sensory processing,such sensory processing deficits are not likelyto directly cause learning disabilities. In NF1,such associations may simply arise from acommon underlying mechanism. Alternatively,it is possible that the increase in inhibitioncharacterized in the Nf1+/− mutant mice issufficient to cause learning deficits, but notirreversible developmental changes. Furtherstudies are needed to better understand inter-actions between developmental effects of NF1,any associated sensory processing deficits, andbehavioral effects of constitutively altered Rassignaling/interneuronal regulation.

LEARNING DISABILITIES ASA DISTINCT PATHOLOGICALENTITY: LESSONS FROM NF1

A key question concerning learning disabilitiesis whether they represent a distinct diseasecluster or rather the low end of a spectrum ofperformance in the human population. This is aquestion that is common to discussions of manydisorders of cognitive function. For example,similar discussions in schizophrenia, autism,and Alzheimer’s have concluded that these con-ditions are not part of a behavioral and cognitivephenotypic continuum in the human popula-tion, but that instead represent separate patho-logical clinical entities. Accordingly, individualssuffering from each of these conditions accountfor a very small percentage of the population(1%–2%). With respect to learning disabilities,there is little agreement in the literature on theprevalence of learning disabilities, but estimateson incidence range from 1% to 17% (Altarac& Saroha 2007, Lagae 2008). It is argued thatan incidence of 10% is consistent with the ideathat learning disability is not a single clinicalentity, but reflects instead the wide distributionof cognitive traits in the population (normalspectrum hypothesis). Furthermore, the clas-sification criteria used for these estimates areoften broad, and these high frequencies oflearning disability are also argued to be consis-tent with the normal spectrum hypothesis.

The studies of learning disabilities associ-ated with single-gene disorders contradict thesearguments. These studies demonstrate that thecognitive deficits defined in these individualshave unique pathological causes and can beclassified into discrete clinical conditions withspecific diagnoses. Despite their broad clinicalmanifestations, the studies with NF1, FragileX, Tuberous Sclerosis, Noonan and Rett’s syn-drome, to name only a few, indicate that a widerange of cognitive symptoms can be caused bysingle-gene mutations. Thus, broad behavioraland cognitive profiles do not necessarily reflecteither the low end of the cognitive continuumof the general population, or the presence ofan equally broad and diffuse pathological etiol-ogy. Rather, in these diseases, it is clear that abroad range of cognitive phenotypes are causedby specific genetic mutations leading to distinctclinical entities.

The results from single-gene disorders alsodemonstrate that a wide range of severities canbe caused by the same mutation in a single gene.For example, learning disability and attentiondeficit symptoms occur in NF1 in a continuumof severity, and they are known to be causedby a single underlying genetic pathology. Infact, some individuals affected by NF1 showvery few cognitive manifestations, whereas oth-ers reveal profound, albeit often specific, cog-nitive deficits. In NF1, genetic factors modifythe severity and expression of symptoms, suchthat the variability in clinical presentation doesnot reflect the uniformity of the principal ge-netic cause (i.e., mutation in the NF1 gene).Among idiopathic learning disabilities, it is alsolikely that variability in expression between in-dividuals occurs as a result of other unknownmutations in the genetic background of affectedindividuals (i.e., modifier genes), as well as envi-ronmental factors. Therefore, variability in ex-pression of learning disabilities between indi-viduals cannot be taken to imply variability in,or lack of, a distinct underlying clinical pathol-ogy. These considerations also lend support tothe model of learning disabilities as a distinctsyndrome with underlying pathological mech-anisms that alter learning, rather than being a


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low end of the normal cognitive spectrum ofthe general population.

As in schizophrenia, autism, andAlzheimer’s, a small percentage of indi-viduals with learning disabilities have knowngenetic mutations (e.g., mutations in the Nf1gene), whereas the genetic etiology of mostindividuals is unknown. As in these otherdisorders, the severity and range of symptomsmanifested in any one affected individual canvary, perhaps reflecting the interplay betweengenetic and environmental factors. Again, asin these well-described disorders, there is acluster of phenotypes that represent the clinicaldiagnostic criteria, as well as other clinicallyimportant comorbidities that are not part ofthe disorder itself. For example, epilepsy andmental retardation are often associated withautism, but they are not an intrinsic part of thediagnosis of this condition, just as attentionproblems are often associated with learningdisabilities. Thus, learning disabilities sharethese and other features with other complexmental health problems. This complexity anddiversity are not the exception but the rulein current classifications of disorders of brainfunction.

Identifying learning disabilities as a dis-order, rather than a variation of “normal”learning, has more than purely academic impli-cations, as it bears on approaches to treatment.Learning disabilities have been identified as oneof the primary factors affecting quality of life inthe NF1 population, even given the high inci-dence of tumors and serious somatic symptomsthat are also associated with this condition.Additionally, without intervention, individualsaffected with NF1 more often than not havedifficulties in effectively compensating fortheir deficits in learning and attention. Fromthe perspective of affected individuals, familymembers, and professional care-givers, thecognitive and behavioral challenges associatedwith NF1 require professional interventionand are both unique and distinguishable fromthe normal cognitive spectrum in the generalpopulation. The difficulty in compensatingfor significant cognitive impairments also

differentiates the learning problems associatedwith these disorders from those seen in “nor-mal” populations. This need for specializedcare, as well as the considerable negative reper-cussions of underlying pathologies on the lifeof affected individuals, further emphasizes thenecessity to recognize learning disabilities as adistinct disorder that requires early detection,intervention, and treatment.

Importantly, early treatment in NF1 andother learning disabilities can be effective inimproving cognition, as measured by neuropsy-chological testing as well as academic and socialachievement. In the NF1 population, low dosesof methylphenidate, which may address theirattention problems, appear to improve perfor-mance in school as well as attention (Mautneret al. 2002). In idiopathic learning disabilities,current interventions include unified programs,involving phonology training, compensatorystrategy training, as well as involvement ofschool teachers and parents. Such cognitiveand behavioral therapy-based programs arealso effective in improving academic outcomes,especially when initiated early (Lagae 2008).The benefits of therapeutic intervention forlearning disabilities seem clearly to outweighthe risks of treatment. Therefore, it is critical torecognize learning disabilities as a disease entitythat requires early diagnosis and interventionto improve quality of life in affected individuals.

ADULT TREATMENT OFNEURODEVELOPMENTALDISORDERS: THE CASE FOR NF1

There is a reasonable and well-entrenchedassumption that neurodevelopmental disorderssuch as NF1 disrupt developmental processesthat cannot be fully reversed in adult indi-viduals, and that hope for treatment rests inearly (fetal stages) diagnosis and intervention(Ehninger et al. 2008). Indeed, there arecompelling data that many of the genes thataffect adult function also affect importantdevelopmental processes. For example, the Nf1gene affects trophic function during rodentdevelopment (Luikart et al. 2008, Vogel et al.


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1995). It is reasonable to propose that disrup-tion of these developmental processes couldlead to neurophysiological and neuroanatom-ical abnormalities that are irreversible in adultindividuals. Additionally, there is compellingevidence that the development of the brain hascritical periods, where key neurodevelopmentalevents must take place, outside of which thesesame processes no longer can be fully realized.For example, developmental studies of visionidentified critical periods with narrow temporalwindows, where high levels of sensory plasticityare important for the large-scale organizationof specific areas of the visual cortex (Hensch2005). The idea of developmental critical pe-riods has further strengthened the bias that itmay not be possible to reverse the pathologiesassociated with neurodevelopmental disordersin adult individuals.

Recent results with a number of animalmodels of neurodevelopmental disorders,including NF1, Fragile X, Tuberous Sclerosis,Rett syndrome, Lhermitte-Duclos disease/Cowden disease, Rubinstein-Taybi syndrome,Down syndrome, etc, demonstrate that it ispossible to dramatically improve, if not fullyreverse, the cognitive phenotypes associatedwith these neurodevelopmental disorders inadult animals (Ehninger et al. 2008). Whenthe underlying biochemical and physiologicalpathologies are reversed in adult individuals,the cognitive phenotypes could be eitherdramatically improved or even fully re-versed without necessarily intervening during

development (Ehninger et al. 2008)! Forexample, studies reviewed above showed thatbrief treatments in adult mutants with eitherLovastatin, a farnesyl transferase inhibitor, orpicrotoxin could reverse the physiological andbehavioral phenotypes of the Nf1+/− mutantmice. More importantly, recent clinical trialssuggested that statins may be effective at revers-ing at least some of the cognitive phenotypesof NF1 patients (Krab et al. 2008b). Similarly,findings in Fragile X also suggest that treatinga key physiological deficit in adult mice couldreverse clinically relevant phenotypes in mousemodels of this disease (Dolen et al. 2007). Ad-ditionally, results from pilot clinical trials arealso starting to suggest that similar treatmentsmay also be effective in Fragile X patients(Berry-Kravis et al. 2009, Hagerman et al.2009).

It is too early to gauge the efficacy of adulttreatments for neurodevelopmental disorders,since the interpretation of the few pilot clinicaltrials carried out so far is confounded by designflaws and limited numbers of subjects. Never-theless, studies in animal models are startingto uncover the molecular, cellular, and systemsmechanisms disrupted by neurodevelopmentaldisorders, as well as to use this information todevelop targeted treatments. Importantly, thesestudies have led to recent results raising theexciting possibility of dramatically improvingthe life of individuals afflicted with neurodevel-opmental disorders, even when treatments arestarted in adulthood.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

This work is supported by the CTF Young Investigators Award, ARCS Foundation, and NIHMCNB Training Grant (2T32MH019384-11A2) to C.S. This work was supported by grants fromthe NIH (R01 NS38480), Neurofibromatosis Inc., the Children’s Tumor Foundation, and UnitedStates Army (W81XWH-06-1-0174) to A.J.S.


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Annual Review ofNeuroscience

Volume 33, 2010Contents

Attention, Intention, and Priority in the Parietal LobeJames W. Bisley and Michael E. Goldberg � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

The Subplate and Early Cortical CircuitsPatrick O. Kanold and Heiko J. Luhmann � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �23

Fly Motion VisionAlexander Borst, Juergen Haag, and Dierk F. Reiff � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �49

Molecular Pathways of Frontotemporal Lobar DegenerationKristel Sleegers, Marc Cruts, and Christine Van Broeckhoven � � � � � � � � � � � � � � � � � � � � � � � � � � � � �71

Error Correction, Sensory Prediction, and Adaptationin Motor ControlReza Shadmehr, Maurice A. Smith, and John W. Krakauer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �89

How Does Neuroscience Affect Our Conception of Volition?Adina L. Roskies � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 109

Watching Synaptogenesis in the Adult BrainWolfgang Kelsch, Shuyin Sim, and Carlos Lois � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 131

Neurological ChannelopathiesDimitri M. Kullmann � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 151

Emotion, Cognition, and Mental State Representation in Amygdalaand Prefrontal CortexC. Daniel Salzman and Stefano Fusi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 173

Category Learning in the BrainCarol A. Seger and Earl K. Miller � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 203

Molecular and Cellular Mechanisms of Learning Disabilities:A Focus on NF1C. Shilyansky, Y.S. Lee, and A.J. Silva � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 221

Wallerian Degeneration, WldS, and NmnatMichael P. Coleman and Marc R. Freeman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 245

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Neural Mechanisms for Interacting with a World Fullof Action ChoicesPaul Cisek and John F. Kalaska � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 269

The Role of the Human Prefrontal Cortex in Social Cognitionand Moral JudgmentChad E. Forbes and Jordan Grafman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 299

Sodium Channels in Normal and Pathological PainSulayman D. Dib-Hajj, Theodore R. Cummins, Joel A. Black,and Stephen G. Waxman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 325

Mechanisms of Synapse and Dendrite Maintenance and TheirDisruption in Psychiatric and Neurodegenerative DisordersYu-Chih Lin and Anthony J. Koleske � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 349

Connecting Vascular and Nervous System Development: Angiogenesisand the Blood-Brain BarrierStephen J. Tam and Ryan J. Watts � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 379

Motor Neuron Diversity in Development and DiseaseKevin C. Kanning, Artem Kaplan, and Christopher E. Henderson � � � � � � � � � � � � � � � � � � � � � � 409

The Genomic, Biochemical, and Cellular Responses of the Retina inInherited Photoreceptor Degenerations and Prospects for theTreatment of These DisordersAlexa N. Bramall, Alan F. Wright, Samuel G. Jacobson, and Roderick R. McInnes � � � 441

Genetics and Cell Biology of Building Specific Synaptic ConnectivityKang Shen and Peter Scheiffele � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 473

Indexes

Cumulative Index of Contributing Authors, Volumes 24–33 � � � � � � � � � � � � � � � � � � � � � � � � � � � 509

Cumulative Index of Chapter Titles, Volumes 24–33 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 513

Errata

An online log of corrections to Annual Review of Neuroscience articles may be found athttp://neuro.annualreviews.org/

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Documents

Molecular and Cellular Mechanisms of Learning Disabilities: A