Molecular Analysis of Developmental Plasticity in Neocortex

Elly Nedivi Department of Brain and Cognitive Sciences, Center for Learning and Memory, Massachusetts Institute of Technology, 45 Carleton St., E25-435, Cambridge, Massachusetts 02139

Received 19 March 1999; accepted 25 May 1999

ABSTRACT: Gene expression studies indicate that ticity. Knockout mice with deletions of such genes have during activity-dependent developmental plasticity, N- allowed analyzing their function in the context of differ- 2؉ methyl-D-aspartate receptor activation causes a Ca - ent systems and in different paradigms. Studies of mu- dependent increase in expression of transcription factors tant mice reveal that activity-dependent plasticity is not and their downstream targets. The products of these necessarily a unified phenomenon. The relative impor- plasticity genes then operate collectively to bring about tance of a gene can vary with the context of its expres- the structural and functional changes that underlie oc- sion during different forms of plasticity. Forward ge- ular dominance plasticity in visual cortex. Identifying netic screens provide additional new candidates for and characterizing plasticity genes provides a tool for testing, some with well-defined cellular functions that molecular dissection of the mechanisms involved. Mem- provide insight into possible plasticity mechanisms. bers of second-messenger pathways identified in adult © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 135–147, 1999 plasticity paradigms and elements of the transmission Keywords: gene expression; development; cerebral cor- machinery are the first candidate plasticity genes tested tex; activity-dependent plasticity; gene knockout for their role in activity-dependent developmental plas-

Since the groundbreaking work of Hubel and Weisel ing new insight as to the components of cellular in the 1960s and 1970s on how the environment plasticity mechanisms and the level to which these influences development of the visual cortex (Wiesel, components are essential. 1982), studies of ocular dominance column (ODC) formation have come to play a leading role in the field of developmental plasticity. Concepts emerging from DEVELOPMENTAL PLASTICITY these studies have been extended to other parts of the visual system and to other sensory and motor systems. True to precedent, the application of molecular tools Developmental plasticity is typified by a critical pe- toward elucidating mechanisms of developmental riod when manipulating input will cause dramatic plasticity has also started predominantly in visual changes in cortical connectivity (Hubel and Wiesel, cortex. This review discusses gene expression studies 1970; Hubel et al., 1977; Jeanmonod et al., 1981). In and molecular genetic analysis of developmental plas- the visual system, neural activity generated by both ticity in the neocortex, and how this complementary eyes during the critical period drives the final pattern- approach to electrophysiology and anatomy is provid- ing of neuronal connections (reviewed in Constantine- Paton et al., 1990; Goodman and Shatz, 1993; Katz and Shatz, 1996; Shatz, 1990). Formation of eye- Contract grant sponsor: NIH; contract grant number: EY11894 Contract grant sponsor: Ellison Medical Foundation specific layers in the lateral geniculate nucleus (LGN) Contract grant sponsor: Alfred P. Sloan of the thalamus and ODCs in the cortex both occur © 1999 John Wiley & Sons, Inc. CCC 0022-3034/99/010135-13 through an activity-driven process of axon terminal 135 136 Nedivi remodeling (LeVay et al., 1978, 1980; Shatz, 1990; of activity-dependent plasticity in the developing vi- Shatz and Sretavan, 1986; Shatz and Stryker, 1978). sual cortex (Bear et al., 1987). Inappropriate connections are gradually withdrawn, while appropriate ones develop extensive terminal arbors (Antonini and Stryker, 1993; Guillery, 1972; DOWNSTREAM OF THE N-METHYL-D- Sretavan and Shatz, 1986). In this context, an appro- ASPARTATE (NMDA) RECEPTOR priate connection is defined as a connection where pre- and postsynaptic activity are correlated—in other In visual cortex, as in the adult hippocampus, the words, a Hebbian synapse (Fregnac et al., 1988; Shulz ability of postsynaptic cells to detect coincident ac- and Fregnac, 1992; Stryker and Strickland, 1984). tivity during LTP and LTD apparently resides in the In principle, the Hebbian synapse hypothesis pro- NMDA receptor (Artola and Singer, 1987; Kirkwood vides a theoretical basis for linking developmental and Bear, 1994a,b). The NMDA receptor is essential plasticity that occurs during ODC formation in visual not only for LTP in hippocampal CA1 synapses and cortex with adult plasticity that occurs in the hip- for spatial memory (Morris et al., 1986; Tsien et al., pocampus and neocortex (Constantine-Paton et al., 1996), but also for ocular dominance plasticity that 1990; Goodman and Shatz, 1993; Shatz, 1990). In the occurs in cortical neurons in response to visual ma- developing visual system there are correlated patterns nipulations during the critical period (Bear et al., of activity (Maffei and Galli-Resta, 1990; Meister et 1990; Kleinschmidt et al., 1987). In hippocampal neu- rons, activation of the NMDA receptor causes a rise in al., 1991; Schwartz et al., 1998; Yuste et al., 1992) 2ϩ and there are cortical synapses that are capable of intracellular Ca , thereby triggering several second- detecting such activity and responding with functional messenger systems, among them cyclic adenosine changes (Kirkwood et al., 1993; Miller et al., 1989; monophosphate (cAMP) and a variety of kinases such as protein kinase A (PKA) and Ca2ϩ/calmodulin- Shulz and Fregnac, 1992). These Hebbian character- dependent protein kinase II (CaMKII) (Bliss and Col- istics of visual cortical synapses during the critical lingridge, 1993). Second messengers can have both period resemble those of synapses in the adult hip- short- and long-term effects within the postsynaptic pocampus that are capable of undergoing long-term cell. One of the long-term effects is activation of potentiation (LTP) (Brown et al., 1990; Madison et immediate early genes (IEGs). Tetanic stimulation al., 1991; Wigstrom and Gustafsson, 1985). Since its that produces LTP in the hippocampus has been discovery in the hippocampus, LTP, the sustained shown to cause an NMDA receptor-dependent in- increase in synaptic transmission resulting from high- crease in mRNA for the IEG zif268 (Cole et al., 1989; frequency stimulation of excitatory pathways, has Wisden et al., 1990). During the late phase of LTP been the primary experimental model for studies of and for hippocampal-dependent long-term memory, the synaptic basis of learning and memory in verte- cAMP induces new gene transcription through an- brates (Bliss and Collingridge, 1993). The same type other IEG: the cAMP-responsive element binding of stimulation protocols that elicit LTP in the CA1 protein (CREB) (Bourtchuladze et al., 1994; Huang et region of a hippocampal slice can elicit LTP in layer al., 1994). Many IEGs such as zif268 and CREB III, when applied to the optic radiation or to layer IV encode transcription factors that subsequently activate of a visual cortical slice, (Artola and Singer, 1987; additional downstream genes containing specific reg- Kirkwood and Bear, 1994; Kirkwood et al., 1993). ulatory elements in their promoters. For example, Another form of synaptic plasticity found in the hip- CREB can induce transcription of any gene that con- pocampus, long-term depression (LTD), can be elic- tains a cAMP-responsive element (CRE). It has been ited electrophysiologically in a visual cortical slice suggested that activation of IEGs that are transcription (Kirkwood and Bear, 1994) or in vivo in the develop- factors provides a link between extracellular signals ing visual cortex by monocular deprivation (Ritten- and synthesis of the mRNA and protein required for house et al., 1999). Susceptibility to both LTP and long-term changes in synaptic connections (Goelet et LTD in visual cortical slices correlates with the crit- al., 1986; Sheng and Greenberg, 1990). ical period for development of ocular dominance In cats and primates, the physiological plasticity (Dudek and Friedlander, 1996; Kirkwood et al., manifested by cortical cells after monocular depriva- 1995). These and additional studies showing that vi- tions during the critical period for ODC formation is sual experience can enhance or diminish LTP and accompanied by clear activity-driven rearrangements LTD in visual cortical slices (Kirkwood et al., 1996) of geniculocortical afferents (Antonini and Stryker, have lent support to the theory that the properties of 1993, 1996; Shatz and Stryker, 1978). The obvious synaptic LTP and LTD can account for many aspects structural changes required for remodeling of axon Molecular Analysis of Plasticity 137

Table 1 CPGs Implicated in Cortical Plasticity Adult Expression Developmental Expression Seizure Visually During CP for Activity- Gene Induced Induced OD Plasticity Dependent Protein zif268 ϩϩ ϩ ϩTranscription factor c-fos ϩ ND ϩϩTranscription factor jun B ϩ ND ϩϩTranscription factor BDNF ϩϩ ϩ ϩNeurotrophin GAP-43 ϩϩ ϩ ϩAxon-specific growth-associated protein CaMKII Ϫϩ ϩ ϩCa2ϩ calmodulin–dependent kinase GAD ϩϩ ϩ ϩGABA synthetic enzyme COX-2 ϩϩ ϩ ϩRate-limiting prostaglandin synthetic enzyme Egr3 ϩϩ ϩ ϪTranscription factor Arc ϩϩ ϩ ϩDendrite-specific cytoskeletal-associ- ated protein Narp ϩϩ ϩ ϩCa2ϩ-dependent lectin that promotes neuronal migration and dendrite growth cpg2 ϩϩ ϩ ND Structural protein cpg22 (homer) ϩϩ ϩ ϩEVH domain protein that binds to mGLuR c-terminus rheb ϩϩ ϩ ϪSmall G protein cpg15 ϩϩ ϩ ϩMembrane-bound extracellular sig- naling molecule that promotes dendritic growth cpg29 ϩϩ ϩ ND ? MHC ϩ ND ϩϩMajor histocompatability protein arbors suggest that the underlying cellular processes ulated in the developing visual cortex. For example, involve new protein synthesis and therefore regulation Ca2ϩ-dependent regulation of brain-derived neurotro- at the level of gene expression. Indeed, visual manip- phic factor (BDNF) transcription in cortical neurons is ulations during the critical period for ODC formation mediated by CREB through a CRE in the BDNF gene have been shown to regulate expression of transcrip- promoter (Tao et al., 1998). Thus, BDNF, whose tion factor IEGs (Table 1). Brief visual experience mRNA levels are regulated by visual activity in the after dark rearing induces expression of the IEGs developing and adult visual cortex (Castren et al., zif268,c-fos, and junB specifically in visual cortex 1992; Schoups et al., 1995), is an IEG-activated ef- (Rosen et al., 1992). Developmental and adult laminar fector molecule that can profoundly effect the mor- distributions of the Zif268 and Fos proteins are re- phology of developing cortical neurons (McAllister et sponsive to visual manipulations that are correlated al., 1995), and has been implicated in formation of with plasticity rather than neural activity (Kaplan et ODCs (Cabelli et al., 1995). Other potential activity- al., 1996). Most conclusively, transgenic mice carry- regulated IEG targets are effector genes whose prod- ing a CRE-lacZ reporter were used to demonstrate ucts such as BDNF are regulated in the developing that monocular deprivation leads to activation of visual system by sensory input (Table 1). One exam- CRE-mediated transcription (presumably through ple is the NMDA receptor, whose level in layer 4 of CREB binding) in visual cortex during the critical visual cortex is regulated by activity during the criti- period (Pham et al., 1999). cal period (Catalano et al., 1997). Others are GAP-43, Expression and regulation of IEG transcription fac- CaMKII, and glutamic acid decarboxylase (GAD), tors during developmental plasticity in visual cortex whose mRNA levels are all regulated by visual input implies that through their activity a cohort of down- during early postnatal development (Mower and stream effector genes are being mobilized to execute Rosen, 1993; Neve and Bear, 1989). Additional genes long-term modification of neuronal properties. There identified as induced by seizure activity in the hip- are indications that IEG downstream targets are reg- pocampus, COX-2, Arc, Narp, homer (identified in 138 Nedivi parallel as cpg22), and cpg15, were subsequently deprivations during a critical period shift the re- shown to be regulated by visual activity in the devel- sponses of neurons in the binocular zone toward the oping striate cortex (Table 1; discussed in more detail open eye (Fagiolini et al., 1994; Gordon and Stryker, later in this review) (Brakeman et al., 1997; Corriveau 1996). The beautiful quantitative studies by Gordon et al., in preparation; Lyford et al., 1995; Tsui et al., and Stryker defining a critical period for developmen- 1996; Yamagata et al., 1993, 1994). The plenitude of tal plasticity in mouse visual cortex provide a basis for IEGs and effector genes regulated by visual input in comparison when mutant mice are used to investigate the developing neocortex makes it easy to imagine the role of individual molecules in activity-dependent that the similarity between developmental plasticity development (Gordon and Stryker, 1996). and adult forms of plasticity such as LTP does not Adult mice with an ␣-CaMKII knockout were used stop at the NMDA receptor. During development, to confirm a role for this gene in certain forms of similarly to the adult, synaptic modifications brought learning and memory (Bach et al., 1995; Silva et al., about by correlated activity may result from activation 1992), in the ability of adult synapses to undergo LTP of gene expression via this receptor. in both hippocampus and neocortex (Bach et al., 1995; Hinds et al., 1998; Kirkwood et al., 1997; Silva et al., 1992), and in experience-dependent plasticity of MOLECULAR GENETIC ANALYSIS OF barrel cortex (Glazewski et al., 1996). The ␣-CaMKII DEVELOPMENTAL PLASTICITY knockouts develop normal visual cortical responses, including receptive field properties, maximum re- The cAMP second-messenger system and compo- sponse strength, orientation selectivity, and ocular nents thereof—CREB, PKA, and CaMKII—have dominance (Gordon et al., 1996). Recording field been repeatedly shown in a variety of organisms to be potentials in layer II/III evoked by stimulation of layer involved in long-term synaptic plasticity, during IV showed that in slices from visual cortex of young learning and memory as well as LTP (Frank and ␣-CaMKII knockout mice there is a reduced proba- Greenberg, 1994; Martin and Kandel, 1996; Stevens, bility of obtaining LTP and a reduction in the mag- 1994). Recently transgenic and gene-transfer technol- nitude of potentiation (Kirkwood et al., 1997). The ogies in mice have allowed genetic manipulation of LTP deficits are less drastic than in older mutants, genes suspected to be involved in plasticity. Knockout since potentiation could be obtained in most slices. In mice lacking genes for CREB, PKA, ␣-CaMKII, monocular deprivation experiments, the ␣-CaMKII PKC-␥, metabotropic glutamate receptor 1 (mGLuR1), knockouts exhibit dramatically reduced but variable BDNF, and double mutants in endothelial and neuro- plasticity (Gordon et al., 1996). The shift in eye nal nitric-oxide synthase (eNOS and nNOS, respec- preference toward the open eye is significantly less in tively) have been generated and tested in adult forms approximately half the ␣-CaMKII knockouts, the of plasticity such as learning and memory and/or LTP same fraction of animals severely impaired in a spatial (Abeliovich et al., 1993a,b; Aiba et al., 1994; learning task (Gordon et al., 1996). The finding that Bourtchuladze et al., 1994; Brandon et al., 1995; ␣-CaMKII knockouts display severe deficits in ocular Huang et al., 1995; Patterson et al., 1996; Silva et al., dominance plasticity argues that ␣-CaMKII does play 1992a,b; Son et al., 1996). Some of these genes are a role in activity-dependent aspects of visual cortex responsive to activity in the visual system (Neve and development. The variability of the in vivo deficits Bear, 1989; Tighilet et al., 1998) and/or have been and the mildness of the in vitro ones argue that this otherwise implicated in developmental plasticity (Ca- role is not pivotal to the developmental plasticity belli et al., 1995; Cramer et al., 1996; Pham et al., occurring during establishment of eye preference; as 1999; Reid et al., 1996). It is therefore reasonable that in most cases, the system is capable of adequate candidate plasticity gene knockout mice were tested performance in its absence. for a role not only in adult plasticity, but also during The ␣-CaMKII knockouts are thus far the only development of ocular dominance plasticity in visual plasticity gene knockouts that have been tested for cortex. Although in the rodent visual cortex there is involvement in both ocular dominance plasticity and no anatomical segregation of neurons according to developmental plasticity of somatosensory cortex. In function such as the orientation or ocular dominance experience-dependent plasticity of the somatosensory columns seen in cats and primates, neurons in their barrel cortex, when sensory input is restricted to a visual cortex do have well-defined functional proper- single whisker on the rodent snout, the cortical rep- ties and a certain degree of eye preference (Fagiolini resentation of this spared vibrissa is increased (Fox, et al., 1994; Gordon and Stryker, 1996; Maffei et al., 1992, 1994). Similarly to ODC plasticity, vibrissa 1992; Parnavelas et al., 1981). Moreover, monocular deprivation plasticity in layer IV is restricted to the Molecular Analysis of Plasticity 139 critical period, while layers 2/3 show a prolonged prived eye can be restored by reverse suture of eyelids propensity to shift receptive field properties in re- during the critical period (Hensch et al., 1998). In sponse to peripheral manipulations (Daw et al., 1992; contrast, in visual cortical slices from the mutant Fox, 1994; Glazewski and Fox, 1996). In contrast to mice, profound deficiencies in synaptic plasticity are adult mice lacking the ␣-CaMKII gene, where there is manifested by the total absence of theta burst–induced a significant decrease in experience-dependent plas- LTP, LTD, or paired pulse facilitation (Hensch et al., ticity of the barrel cortex, plasticity in the developing 1998). barrel cortex seems wholly intact (Glazewski et al., The most severe disruption of ocular dominance 1996). Since development of eye preference is dis- plasticity in vivo is manifested in GAD65 knockout rupted in approximately half the animals while mice (Hensch et al., 1998). This is perhaps not sur- vibrissa deprivation plasticity during development prising in light of the serious perturbations to ODC seems unaffected, ␣-CaMKII may not be required for formation induced in kitten visual cortex by the activity-dependent development of somatosensory GABA-A agonist muscimol (Hata and Stryker, 1994; cortex, while it is necessary for some aspects, albeit Reiter and Stryker, 1988). In mice lacking the 65-kD noncrucial ones, of activity-dependent development isoform of GAD, cortical morphology and adult of visual cortex. GABA concentrations are normal presumably owing Another study testing for the role of a plasticity to the presence of the constitutively expressed molecule, nitric oxide (NO), in development of both GAD67 isoform (Kash et al., 1997). GAD65 is nor- visual and somatosensory cortex only partially in- mally localized to synaptic terminals (Fukuda et al., volved the use of knockout mice (Finney and Shatz, 1998) and serves as a reservoir recruited when addi- 1998). NO has been a prime candidate for the role of tional GABA synthesis is required (Martin et al., retrograde messenger communicating between the 1991). In the GAD65 knockouts, extracellular GABA post and the presynaptic cell during synaptic plasticity concentrations are similar to those in wild types but (Bliss and Collingridge, 1993). NO effects hippocam- release in response to a brief depolarization with high pal LTP in vivo and in vitro (O’Dell et al., 1991; potassium is significantly compromised, supporting Schuman and Madison, 1991; Son et al., 1996). In the the notion that the GAD65 isoform function may be developing visual system, blockade of NOS activity specialized for a rapid response to intense neuronal inhibits segregation of retinogeniculate axons into activity (Hensch et al., 1998). Despite a tendency for ON-OFF sublaminae (Cramer et al., 1996), and NOS prolonged discharge after visual stimulation, cortical expression in the visual subplate and cortex of ferrets cells in the binocular zone of visual cortex in these correlates well with ODC formation (Finney and mutants appear to develop normally by all other visual Shatz, 1998). Nevertheless, NOS blockade in ferrets parameters tested: spontaneous activity, habituation, fails to prevent formation of ODCs, and pharmaco- retinotopic organization, orientation and direction se- logical NOS blockade in NOS knockout mice fails to lectivity, and receptive field size. The distribution of prevent formation of cortical barrels or barrel field ocular dominance within the binocular zone is iden- plasticity induced by whisker ablation (Finney and tical in the GAD65 knockout and wild-type mice Shatz, 1998). In this case, pharmacological and (Hensch et al., 1998). However, the GAD65 knockout knockout analysis concur that NO is unlikely to be mice do not show the shift in eye preference that essential for developmental plasticity of thalamocor- normally occurs in response to monocular depriva- tical connections in either visual or somatosensory tion: they continue to show a preferred response to systems, despite playing a role in hippocampal LTP. contralateral (deprived) eye input (Hensch et al., Adult mice deficient for the RI␤ regulatory subunit 1998). LTP and LTD in layers 2/3 of visual cortical of another candidate-plasticity gene, PKA, display slices from the mutant animals are indistinguishable defective hippocampal LTD (Brandon et al., 1995) from those in wild-type controls (Hensch et al., 1998). and mossy fiber LTP (Huang et al., 1995), but appear In the ␣-CaMKII knockouts, deficits are significant normal in spatial and contextual learning and long- in all cases of adult plasticity irrespective of whether term memory (Huang et al., 1995). Primary visual the assay is behavioral or electrophysiological. Learn- cortex in these mice develops with apparently normal ing and memory are defective, as well as LTP and receptive field size, retinotopy, and ocular dominance LTD in both hippocampus and neocortex. Develop- (Hensch et al., 1998). In monocular deprivation ex- mental plasticity in these mutants, assayed in vivo and periments, the shift in eye preference toward the open in vitro, is not impaired to the same degree. The eye is significant and comparable to wild-type con- severity of the deficits in different types of plasticity trols. In the mutants as in the wild-type mice, loss of in these knockouts splits along developmental lines, responsiveness to stimulation of the originally de- demonstrating that a role for a specific molecule in 140 Nedivi adult plasticity does not necessarily have predictive clones for sequences specific to the tissue from which value for its participation in activity-dependent as- one wants to isolate differentially expressed genes. pects of development. The differential sensitivity of The main difficulty in designing a differential screen the developing visual cortex as opposed to somato- is selecting the two tissue sources to be compared. sensory cortex in response to loss of CAMKII sug- The greater the number of differences between two gests that mechanisms of plasticity may not be totally tissue sources, the more difficult it is to select from overlapping in these two cortical regions. In the RI␤- the cloned genes those that are relevant to the specific PKA knockouts the dividing line is not between adult difference of interest. The more complex a tissue and developmental plasticity, but between in vivo source is, containing multiple cell types or multiple versus in vitro models of plasticity. Learning and functional regions, the more differences will likely be memory in the adult and ocular dominance plasticity detected. For these reasons, it is best to compare cell during development seem unaffected by the RI␤-PKA populations that are as similar and homogenous as knockout, while LTP and LTD in hippocampus and possible. In the case of plasticity during development, visual cortex are both impaired. Analysis of these such a comparison is inherently confounded by the particular knockouts provides evidence contrary to the developmental changes unrelated to synaptic plastic- growing consensus that the in vitro models of LTP ity that are still occurring in the cortex between time and LTD faithfully represent in vivo synaptic mech- points that may be compared. In addition, a complex anisms of plasticity that occur during ocular domi- tissue such as neocortex is not homogenous as to the nance development or, for that matter, during learning timing of activity-dependent plasticity. Experience- and memory in the adult. The predictive value of in dependent plasticity in rat barrel cortex precedes that vitro LTP and LTD paradigms is also undermined in of ocular dominance plasticity in visual cortex by the GAD65 knockouts, where conversely to the RI␤- approximately 2 weeks (Fox, 1992; Maffei et al., PKA knockouts, LTP and LTD are intact despite 1992). Even if one were to confine the screen to a severe deficits in developmental ocular dominance functional region of neocortex such as striate cortex, plasticity. all aspects of activity-dependent plasticity would not The analysis of knockout mice demonstrating that necessarily be temporally synchronized or spatially the same gene may be involved to a different degree discrete. It is not clear that the critical period for in paradigms of adult versus developmental plasticity, ocular dominance plasticity overlaps critical periods in behavioral versus electrophysiological paradigms, for activity-dependent refinement of other visual during development of visual versus somatosensory properties. Experience-independent activity may systems, or perhaps even in hippocampus versus neo- guide development of orientation columns prior to the cortex, provides insight not only as to the mechanisms critical period for ODC formation (Chapman et al., involved in each individual case, but also as to the 1996; Crair et al., 1998). Upper-layer organization extent of overlap between them. Such informative may precede and guide thalamocortical organization genetic experiments are possible only once genes of (Chapman and Stryker, 1993; Crair et al., 1998), and potential interest are identified. Elements of the basic in cat, persists past the critical period for ODC for- transmission machinery or components of second- mation (Daw et al., 1992; Gilbert and Wiesel, 1992; messenger pathways previously implicated in plastic- Singer et al., 1981). Development and susceptibility ity have been the first obvious candidates. However, of cells within a given layer to the effects of monoc- discovery of unknown or unsuspected participants in ular deprivation are not uniform. A substantial frac- neuronal plasticity can only be achieved through un- tion of layer 4 cells do not shift their eye preference in prejudiced forward genetic screens. response to monocular deprivation, and weakening of deprived eye responses are far from homogenous across the superficial layers (Crair et al., 1997; Shatz SCREENS FOR CANDIDATE and Stryker, 1978). In general, cells that are able to PLASTICITY-RELATED GENES (CPG)S rearrange connections in response to correlated activ- ity at any given time or place may be a minority Screening for genes that are specifically expressed in within the general population of cortical cells. The cells that are undergoing activity-dependent plasticity dilution problem is more severe in rodents, where the is an individual example of a more general strategy binocular region is small and activity-dependent seg- for differential screening for genes that are expressed regation of geniculocortical afferents has not been in one population of cells versus another. Differential demonstrated. On the other hand, working in cats or screening is often combined with subtractive hybrid- primates would greatly restrict the amount of tissue ization methods that are used to enrich the screened available for cloning, imposing the use of amplifica- Molecular Analysis of Plasticity 141 tion-based technologies that introduce bias problems 1990; Sloviter, 1992; Tauck and Nadler, 1985) that and a higher percent of false positives. In addition, in are accompanied by activation of gene expression vivo testing of feline or primate candidate plasticity (Bugra et al., 1994; Gall et al., 1991; Morgan and genes would require isolation of their murine ho- Curran, 1991). The second practical simplification is mologs. To make matters more complicated, if one that using relatively low investment stimulation pro- were looking at developmental plasticity as that oc- tocols allows harvesting of the large amount of ma- curring at the geniculocortical synapse, the mRNA terial necessary for subtractive technologies that are containing cell bodies of the presynaptic partners at not polymerase chain reaction (PCR) based. Once this synapse would be located in the LGN. Using the candidate genes are cloned, they can then be further cortex as a tissue source for a differential screen screened by in situ hybridization in a variety of spe- would limit the screen toward isolation of exclusively cific plasticity paradigms by which it would be ex- postsynaptic components. tremely difficult to clone. The expression of a specific The above considerations illustrate some of the gene in correlation with the critical period and in difficulties in temporal and regional selection of tissue response to visual manipulations can be tested at sources for a differential screen in a complex system various times in multiple locations in parallel without where multiple variables are at play. Perhaps because resorting to preconceptions about temporal and spatial of these difficulties, as yet there have been no screens regulation patterns. devised for directly identifying candidate genes that A total of 362 different CPGs were isolated in a may be specifically involved in neocortical develop- uniquely sensitive subtractive and differential cloning mental plasticity. The main premise of all searches for procedure that selected genes induced by the gluta- CPGs thus far has been that because activity is the mate analog kainate in the rat hippocampal dentate driving force for plasticity, regulation by activity is a gyrus (Hevroni et al., 1998; Nedivi et al., 1993). prerequisite for any gene involved in plasticity. Partial sequence analysis showed that 70 of the cloned Screens for identifying and isolating genes that are CPGs encode known proteins, while 292 are novel. induced by activity have successfully generated a Despite the large number of genes that are induced by large pool of candidates. These candidates can now seizure activity (approximately 5% of the genes ex- undergo secondary screens for expression during spe- pressed in dentate gyrus neurons) (Nedivi et al., cific developmental or adult plasticity paradigms be- 1993), induction is not random. The CPGs encoding fore ultimately being selected for testing in knockout known proteins can be classified into distinct func- or transgenic animals. tional categories: for example, IEGs, proteins partic- All but one of the CPGs (class I MHC; see below) ipating in second-messenger pathways, growth fac- have been generated by differential screens for activ- tors, and structural proteins. Housekeeping genes ity-regulated genes that are induced by seizure activ- related to ubiquitous metabolic functions are not in- ity in the adult rat hippocampus (Nedivi et al., 1993; duced (Hevroni et al., 1998; Nedivi et al., 1993). Qian et al., 1993; Yamagata et al., 1993). The ratio- Additional candidate plasticity genes have been nale of this approach is to simplify the cloning pro- isolated from the hippocampus in related approaches cedure in two important ways. The first is by speci- using metrazol (Qian et al., 1993) or electrically in- fying a well-defined differential selection criterion duced seizures (Yamagata et al., 1993) as the inducing between two tissues that are as similar and homoge- stimulus. Since these screens were intended for iden- nous as possible. Using adult tissue circumvents the tification of novel CPGs that were IEGs, the inducing problem of interference from developmental pro- stimulus was administered in the presence of the cesses unrelated to activity. The hippocampus is a protein synthesis inhibitor cycloheximide. Some of simpler tissue source than neocortex as far as cell the genes identified in these two screens encode tran- types and circuitry, but it shares structural similarities scription factors (Qian et al., 1993; Yamagata et al., and common forms of synaptic plasticity with the 1994), but many encode IEGs that can directly affect neocortex (Kirkwood et al., 1993). It is reasonable to synaptic structure or function (Brakeman et al., 1997; assume that the hippocampus, a region of the brain Link et al., 1995; Lyford et al., 1995; Qian et al., involved in learning and memory (Squire and Zola- 1993; Tsui et al., 1996; Yamagata et al., 1994a,b). In Morgan, 1991), also shares with neocortex molecular all the above screens, the predominant category of mechanisms that underlie the ability to undergo known CPGs is that of membrane-, vesicle-, and change in response to activity. Seizures provide a synapse-related proteins. The constructive nature of strong, temporally synchronized activation of dentate these molecules suggests that seizure-induced gene gyrus neurons that result in prominent physiological expression is not necessarily related to excitotoxicity and morphological changes (Ben-Ari and Represa, or cell death. Moreover, induction of molecules that 142 Nedivi could be used for synaptic restructuring provides mo- al., 1997; Nedivi et al., 1996). zif268, COX-2, Egr3, lecular genetic support for the idea that morphological Arc, Narp, cpg2, cpg22 (homer), rheb, cpg15, and rearrangement may also play a role in activity-depen- cpg29 were thus shown to be regulated by activity dent plasticity in the adult (Bailey and Kandel, 1993; levels that are within the realm of everyday neocor- Merzenich, 1998). Structural reorganization is obvi- tical function (Table 1). ously less dramatic than that seen during development A similar although not totally overlapping set of of ODCs, but there is anatomical evidence for mossy CPGs could be induced when screened for induction fiber growth in the hippocampus after seizure (Ben- in the hippocampal plasticity paradigm of LTP or by Ari and Represa, 1990; Sloviter, 1992; Tauck and high-frequency stimulation that elicits LTP (Brake- Nadler, 1985) and for extensive sprouting of horizon- man et al., 1997; Hevroni et al., 1998; Link et al., tal connections during functional rearrangement of 1995; Lyford et al., 1995; Nedivi et al., 1993; Qian et primary sensory maps in the adult cortex (Darian- al., 1993; Tsui et al., 1996; Yamagata et al., 1994). Smith and Gilbert, 1994; Florence et al., 1998). A third screen of the CPG pool, and perhaps most The one rather heroic attempt to directly identify relevant to the topic of this review, was for develop- genes regulated by activity in the developing visual mental expression in neocortex, specifically in corre- system resulted in cloning of the previously charac- lation with the critical period for ocular dominance terized class I major histocompatibility antigen (class plasticity. The same CPGs that were tested for light I MHC) (Corriveau et al., 1998). In this screen, dif- induction in the adult neocortex were also analyzed by ferential display was used to compare gene expression in situ hybridization for expression in the postnatally in kitten LGN during the period of eye-specific layer developing cortex. Approximately 70% of the CPGs formation, in the presence or absence of action poten- tested were found to be transiently expressed in the tial blockade with tetrodotoxin (TTX) (Corriveau et developing cortex between postnatal days 1 and 21, at al., 1998). Identification of class I MHC in this screen levels significantly higher than in normal adult rat is another example of how forward genetic screens cortex (Nedivi et al., 1996). Up-regulation of COX-2, can bring to attention in addition to new molecules Arc, Narp, cpg22 (homer), cpg2, and cpg15 during also known ones not previously associated with ner- cortical development correlates with eye opening and vous system function or plasticity. With class I MHC, the onset of the critical period for OD plasticity in rat as with the other CPGs, it remains to be shown that (Brakeman et al., 1997; Lyford et al., 1995; Nedivi et regulation by activity in correlation with activity- al., 1996; Tsui et al., 1996; Yamagata et al., 1993), dependent plasticity is due to participation in these and MHC up-regulation is correlated with the critical events. period for OD plasticity in kittens (Corriveau et al., 1998) (Table 1). Of these, COX-2, Arc, Narp, cpg22 SECONDARY SCREENS OF (homer), and cpg15 are regulated by visual activity at THE CPG POOL this time (Brakeman et al., 1997; Corriveau et al., in press; Lyford et al., 1995; Tsui et al., 1996; Yamagata A substantial fraction of candidate genes have been et al., 1993) (Table 1). tested subsequent to their isolation by criteria de- These secondary screens using in situ hybridiza- signed to more closely correlate them with plasticity tion as an assay show that the CPG pool generated by as a prelude to later in vivo testing in transgenic or a seizure paradigm in the adult contains a subgroup of knockout mice. genes that is also regulated by visual activity during Since none of the CPGs were isolated from neo- development, in correlation with critical period plas- cortex, and except MHC all had been isolated using a ticity. As it turns out, class I MHC mRNA, identified seizure protocol, it was important to show that these in a developmental activity-based screen, can also be genes could be induced by a normal physiological induced in the adult hippocampus and neocortex fol- stimulus in the neocortex. The first physiological test lowing kainate-induced seizure (Corriveau et al., in all cases was whether these genes’ expression could 1998). The existence of an overlapping set of genes be regulated specifically in visual cortex by manipu- that are induced by seizure activity in the adult and lating visual activity. In some cases, expression in also by normal action potential activity during devel- visual cortex was monitored after activity blockade by opment has several implications. Theoretically, the intraocular injection of TTX (Brakeman et al., 1997; implication is that overlapping gene sets reflect an Lyford et al., 1995; Tsui et al., 1996; Worley et al., overlap in activity-dependent mechanisms in the adult 1990; Yamagata et al., 1993, 1994), in others by and during development. The practical implication is exposure to light after dark adaptation (Brakeman et that the CPG pool, with appropriate subscreens, may Molecular Analysis of Plasticity 143 provide useful candidates for testing in developmental tiated aspects of plasticity: for example, negative reg- plasticity paradigms. ulation of mGluR signaling by homer, or regulation of Candidate plasticity-related genes passing at least coordinated growth of pre- and postsynaptic elements two of the three above screens were partially or fully by cpg15. An integrated picture of combined CPG characterized. COX-2 encodes an enzyme that cata- functions can hint at the mechanisms they represent. lyzes the first and rate-limiting step in prostaglandin For example, the preponderance of CPGs associated synthesis. Since levels of prostanoids are determined with structural remodeling both during development mainly by their rate of synthesis, COX-2 regulation by and in the adult implicates process outgrowth as a neuronal activity could dynamically regulate the level plasticity mechanism. In addition, as more candidate of these signaling proteins (Yamagata et al., 1993). plasticity genes are analyzed, we can use them to Arc encodes a cytoskeletal-associated protein local- address in a comprehensive way the extent of overlap ized to neuronal dendrites (Link et al., 1995; Lyford et between different manifestations of activity-depen- al., 1995). Narp encodes a secreted calcium-depen- dent plasticity. Using gene expression studies and dent lectin that promotes neuronal migration and den- mouse knockout mutants, it should eventually be pos- dritic outgrowth (Tsui et al., 1996). The activity- sible to form a picture of combinatorial gene sets that regulated form of Homer encodes an EVH domain are regulated as groups during activity-dependent protein that competes with constitutively regulated plasticity. Although some of the same genes may be Homer for binding to the C-terminus of mGLuR1 and involved in different cases, their combination in a mGLuR5 (Brakeman et al., 1997; Xiao et al., 1998), discrete set would be unique to a specific system and thus interfering with their ability to activate the ino- stimulus and would define the underlying molecular sitol triphosphate receptor (Tu et al., 1998). The EVH mechanism. domain in Homer suggests that it may also interact with the actin-based cytoskeleton. cpg15 encodes a The author thanks Dr. Hollis Cline for critical reading of small secreted protein that is bound to the extracellu- the manuscript. Research in the author’s laboratory is sup- lar membrane surface by a glycosylphosphatidylinosi- ported by NIH Grant EY11894, a Young Scholar Award tol (GPI) linkage (Naeve et al., 1997) and promotes from the Ellison Medical Foundation, and an Alfred P. growth of dendritic arbors in neighboring neurons Sloan Fellowship. through an intercellular signaling mechanism that re- quires its GPI link (Nedivi et al., 1998). CPG15 may therefore represent a new class of activity-regulated REFERENCES membrane-bound ligands that can permit exquisite temporal and spatial control of neuronal structure Abeliovich A, Chen C, Goda Y, Silva AJ, Stevens CF, (Nedivi et al., 1998). Immunohistochemistry in the Tonegawa S. 1993a. 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