AbstractNEURONS respond to extracellular stimulation bymodulating the expression of certain immediate-earlygenes. Inducible transcription factors (ITFs), such as c-Fos and Zif268, are coded by this class of gene and areamong the first proteins to appear. The rapid accumu-lation of these products combined with histologicalmethods that offer detection at the cellular level arekey features that have led to their wide use in visual-izing activated neurons. However, neuroscientists havelong recognized two major drawbacks of ITFs that limittheir use in the CNS: cell-type expression specificityand stimulus–transcription coupling uncertainty. Inthis review, I discuss recent advances in the field thatbroaden our understanding of the molecularconstraints on ITF expression as well as in techniquesthat may help to extend their utility in functionalmapping.

IntroductionIt is 10 years since the discovery that sensory stim-ulation can initiate rapid and transient inductionof the c-fos gene in neurons. Since then, much effort has been directed at understanding the mol-ecular mechanisms that mediate the expression of c-fos as well as other so-called immediate earlygenes (IEGs). Indeed, we now know much aboutthe receptor systems and signal transduction path-ways that are involved in coupling synaptic stim-ulation to various IEG transcriptional responses. As a result of these efforts, the early notion that c-Fos, the protein product of the c-fos gene, may beused as an endogenous marker of neuronal activityhas now become widely accepted (for recentreviews1–3).

Although the products of both c-fos and anotherIEG, zif268, have become popular tools for map-ping functional activity in the brain, neuroscientists

have had to deal with two major drawbacks to theiruse: cell-type expression specificity and stimulus–coupling uncertainty. The first problem can pro-duce regional and cellular limitations in expressionthat constrain the use of IEGs as general markersof activity throughout the brain. The second prob-lem can affect the interpretation of IEG activitymaps once they are obtained. The purpose of thismini review is to examine recent developments inIEG mapping techniques and how continuingadvances in our understanding of the molecularmechanisms that govern IEG induction may helpto extend their utility in functional mapping.

Characteristics of inducibletranscription factorsThe various members of the IEG family are knownto encode proteins with diverse cellular functions.In the brain, IEGs that are linked to neural activityand that have mapping applications generallyencode proteins that serve as transcription factorsi.e. complexes that bind to the promoter regions of certain genes and either activate or repress their expression. These so-called inducible tran-scription factors (ITFs) are distinguished from otherproteins that normally reside in the nucleus andwhich also serve as regulators of ongoing tran-scriptional activity.

The ITFs c-Fos and Zif268 are both induced inneurons after extracellular stimulation by neuro-transmitters and trophic substances (Fig. 1). Thesequence of events that leads to ITF induction islargely coordinated by Ca2+ influx into the cell.4

This can occur either through the NMDAreceptor–Ca2+ ionophore complex after glutamate(G) binding or through voltage-sensitive calciumchannels (VSCCs) following membrane depolar-ization. Thereafter several different enzymesystems are marshalled by Ca2+. These includevarious protein kinases that activate the transcrip-tion factor CREB5 and phospholipases that initiatethe production of prostaglandins (PG) and leuko-trienes (LK).6,7 The precise manner by which PGsand LKs regulate IEG expression remains un-known, though each has been shown to havedifferent effects on c-fos and zif268 transcription.

NMDA receptor activation can also relay itseffects through a second pathway involving extra-cellular signal-regulated kinases (ERKs).8 Thispathway is generally linked to neurotrophins (NTs)which upon binding to Trk receptors ultimatelyactivate the transcription factors SRF and TCF thattogether regulate IEG transcription.9,10 There is also

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Neural activity mappingwith inducibletranscription factors

Avi ChaudhuriDepartment of Psychology, McGill University,1205 Doctor Penfield Avenue, Montreal,Quebec H3A 1B1, Canada

Key words: c-fos; CREB; cortex; immediate-earlygenes; NMDA; zif268

NeuroReport 8, 3 (1997)

evidence that Ca2+ entry through NMDA receptorsor VSCCs is sufficient to trigger SRF-mediated tran-scription of c-fos, possibly through the ERK signaltransduction cascade.11 CREB phosphorylation canalso be triggered by ERKs after NT stimulation.12

Thus, multiple signaling pathways appear to con-verge upon both SRF/TCF and CREB. The trans-activating effects of these complexes occur whenthey are bound to specific sequences (SRE andCaRE/CRE respectively) that are present in thepromoter regions of both the c-fos and zif268 genes.

After transcription is completed, the ITF mRNAsare translated into a protein product (c-Fos andZif268) in the cytosol. These products rapidlymigrate into the nucleus where they themselvesinfluence the expression of another set of genes, thelate-response genes. c-Fos must dimerize with amember of the Jun phosphoprotein family (c-Jun,JunB, or JunD) to produce a functional transcrip-tion factor, activating-protein 1 (AP-1).13 AP-1 mayeither stimulate or repress the candidate gene afterbinding to a specific promoter sequence (TRE). Byregulating the expression of a host of late-responsegenes, both AP-1 and Zif268 are able to have acommanding influence on short- and long-termcellular homeostasis. Regardless of the specificmolecular roles of ITFs, their accumulation in theneuron generally signifies a prior state of activityand thus forms the logic for obtaining functionalmaps based on ITF staining.

Neural activity and ITF expressionspecificityOne of the early concerns with ITF mapping wasthat in some cases c-Fos expression did not corre-late with 2-deoxyglucose (2-DG) accumulation,14,15

one of the standard procedures for imaging neuralactivity. We now know that only a subset ofneurons express a particular ITF and that neuronsin some brain structures may not express it atall.16,17 This is now recognized to be one of themajor drawbacks to ITF use since they cannot beconsidered to be a general marker of neural activitythat can be applied throughout the CNS.

A particularly striking example of the fact thatneuronal activation alone may not lead to ITFexpression was recently shown by Kimpo andDoupe.18 They examined c-Fos expression in thebirdsong system of zebra finches and found thatsinging induced c-Fos expression in two sensori-motor nuclei, HVc and RA. However, Kimpo andDoupe showed that it was the motor act of singingitself that induced this expression rather than audi-tory stimulation of the bird’s own song. This wasbased on the finding that normal and deafenedbirds that sang showed similarly robust c-Fosexpression in the two nuclei. Furthermore, expo-sure of normal birds to taped auditory stimuli, andin the absence of singing, failed to produce c-Fosexpression.

Although the Kimpo and Doupe study gives anelegant demonstration of how ITF maps canprovide a visual display of neural assemblies thatare involved in a particular behaviour, it also illus-trates the discordance that can exist between neuralactivity and c-Fos expression at several levels. First,they observed structural expression specificity inthat only the HVc and RA nuclei showed c-Fosinduction whereas other nuclei that are known tobe essential in birdsong learning and productiondid not. Second, cellular expression specificity wasapparent within a particular neural structure. HVccontains two separate but interconnected popula-

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FIG. 1. Schematic representation of molecular pathways involved in induction of two ITFs, c-Fos and Zif268, in neurons.

tion of neurons: those projecting to the downstreamRA nucleus and those projecting to a nucleus, areaX, in the anterior forebrain. Double labeling withretrograde tracers showed that the first set ofneurons expressed c-Fos after singing but thesecond set did not. And third, gene expressionspecificity was apparent in that certain auditoryareas that are related to the birdsong systemexpress Zif268 but not c-Fos.18,19 This finding maybe related to an important difference in thetemporal induction pattern of these two ITFs.

Sustained and transient patternsof ITF inductionWhile both c-fos and zif268 can be induced bysynaptic stimulation, the difference in their basalexpression levels has certain implications for theiruse in activity mapping. The relative instability ofc-fos mRNA and the presence of a negative feed-back loop whereby the protein down-regulates itsown transcription result in low constitutive c-Foslevels.20 As such, c-Fos staining is most informa-tive when novel stimuli are applied or when theanimal is stimulated after a period of sensory depri-vation. Only in these situations will those neuronsthat are specifically responsive to the stimulusundergo c-fos induction in a rapid and transientmanner. By the same argument, c-Fos staining after prolonged stimulation will yield negligibleresults. Because Zif268 does not appear to sharethe autoregulation features of c-Fos, it will displaypersistent expression and therefore high basallevels in many neural structures. This leads to thenotion that zif268 expression is linked to ongoingsynaptic activity whereas c-fos expression is reliant upon activity being triggered after a periodof neural quiescence or after exposure to a novelstimulus.It is now apparent that c-fos induction in stimu-lated neurons is also influenced by the temporaldynamics of CREB phosphorylation, as shownrecently by Liu and Graybiel.21 They studied thedeveloping rat striatum and found that the dura-tion of CREB phosphorylation was critical tosubsequent c-fos induction (i.e. transient phospho-rylation did not yield c-fos expression whereassustained phosphorylation favoured induction ofdownstream CRE-containing genes). The interplaybetween cell-type specific phosphatases and phos-phatase inhibitors turns out to be an importantdeterminant as to whether CREB phosphoryl-ation can be maintained after stimulation. Liu andGraybiel showed that D-1 class dopamine recep-tors activated a potent phosphatase inhibitor

(DARPP-32) that allows sustained CREB phos-phorylation, and subsequent c-Fos induction, tooccur in developing striosomes but not in the surrounding matrix neurons, which lack the in-hibitor. Similarly, Ca2+ influx through VSCCs activ-ates the phosphatase calcineurin and abbreviatesthe temporal span of CREB phosphorylation instriosomes but not in matrix neurons, which do not contain this phosphatase. Thus, the temporalcharacteristics of CREB phosphorylation in thedeveloping striatum, and its accompanying effectson c-fos expression, are governed by cellular gatingmechanisms that are spatially segregated in differ-ent cell types. The importance of the Liu andGraybiel study is that it provides insight into themolecular mechanisms that guide ITF expressionin the striatum, and perhaps elsewhere in the brain,and why certain types of neurons readily show c-fos induction in response to stimulation whereasothers do not.

Neuronal stimulation and geneexpression: the coupling uncertaintyGiven that IEG expression can be influenced by multiple receptor systems and coordinated bydifferent signal transduction pathways, a causallink between ITF gene expression and a particulartriggering event is often difficult to establish.Furthermore, functional mapping with in vivopreparations suffers from the usual uncertainty as to whether the pattern of ITF expression wasproduced by the specific behavioural task orsensory stimulus that was applied, by some non-specific feature of the experimental condition, orby an unrelated mental (or endocrin) event. Ofcourse suitable controls can be applied to verify thestimulus-response association, either at the stimu-lation stage or during evaluation of expressionmaps by comparison with other related neuralcompartments or activity markers (e.g. 2-DG,cytochrome oxidase, etc.). However, these controlscan only resolve the uncertainty at a regional level:they do not permit a positive association to bemade at the cellular level for any of the individualITF-stained neurons. However, one of the mostappealing features of this technique is the cellularresolution that ITF activity maps offer. This is afeature that often cannot be fully exploited due tothe stimulus-coupling uncertainty.

The recent development of an ITF dual activitymapping technique may allow for an internalcontrol to resolve such ambiguities at the cellularlevel.22 The principle behind dual-activity map-

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ping relies on the different spatial localization of ITF mRNA and protein (cytosol and nucleusrespectively) and the different temporal patterns of their expression. The mRNA levels are effec-tively regulated in either direction within 20 minof stimulus onset or offset whereas the proteinrequires a longer period that can be up to 90 min.The discrepant time course of these two productsmay be exploited to provide a visual display ofneurons that are stimulated under two differentconditions. By first applying a stimulus for aprolonged period (say 90 min or more) followed bya different stimulus for another 20 min, it is theo-retically possible to stain for the neurons that areselective to each stimulus by combined applicationof immunocytochemistry (ICC) and in situ hybrid-ization histochemistry (ISH). Neurons that weretriggered by the first stimulus (and unaffected by

the second) will be immunopositive only, neuronsstimulated by the second stimulus only will bemRNA-positive, and those with overlappingsensitivity will be double-labeled.

To test this idea, adult monkeys were givenselective light exposure in order to activate oculardominance columns in cortical area V1.22 The twosets of columns that represent each of the eyesremain spatially segregated in this area and there-fore provide an ideal system for examining theeffects of separate stimulus applications. A reverseocclusion procedure with appropriate exposuretimes showed differential zif268 mRNA and pro-tein accumulated in ocular dominance columns.The two sets of columns could be independentlystained by ISH and ICC procedures to producecomplementary, non-overlapping spatial profiles(Fig. 2). Simultaneous application of ICC and non-

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FIG. 2. Application of ITF dual activity mapping in primate visual system. Adult monkeys subjected to selective visual exposure showedelevated levels of zif268 mRNA and Zif268 protein in ocular dominance columns of area V1 (A). The two sets of stained columns werespatially coincident in a monocular deprivation condition whereas they were spatially complementary when reverse occlusion was applied. This is especially apparent in topographical view obtained after tangential sectioning (B). The delineated centres of mRNA andprotein columns (1 and 2 respectively) show a complementary relationship when superimposed (3). This is corroborated by optical densitymeasurements (4) taken from the rectangular windows in stained sections. Application of ICC and non-radioactive ISH can produce adisplay of activated neurons at the cellular level with either one-colour (1) or two-colour (2) processing (C). The border region of twoocular dominance columns from a reverse occlusion animal is shown here. Protein- and mRNA-positive neurons are indicated by blueand red arrowheads respectively whereas double-labeled cells are indicated with both. The first two are presumably monocular neuronsseparately driven by the first and second eyes whereas double-labeled cells were likely responding to stimulation through both eyes.Adapted from Chaudhuri et al.22

radioactive ISH procedures on the same tissuesection produced a visual display of activatedneurons at the cellular level. Neurons at the borderof two ocular dominance columns showed amixture of labeling: single labeling of protein(nuclear staining), of mRNA (cytoplasmic staining),and double-labeling. Thus, dual activity maps atthe cellular level allow identification of neuronsthat were presumably triggered by each stimulusas well as those neurons that were likely to havebeen stimulated by some measure of both. Thisfeature provides an internal control for codingspecificity of mRNA-positive neurons because it isunlikely that non-specific effects could arise onlyduring the terminal period of stimulation and notbefore.

SynthesisThe number and diversity of biological questionsto which c-fos and zif268 expression have alreadybeen applied is truly impressive. IEG expressionmaps can serve different purposes depending upon the brain structure being examined and thebehavioural or sensory condition being employed.Whether IEG expression is being used to map the neural correlates of learning and memory,developmental plasticity, or functional activity, theinformation contained in these profiles must beinterpreted within the molecular context by whichthese products are induced. Thus, continuingdevelopments in our understanding of the molec-ular mechanisms that guide ITF expression willlikely have considerable impact on future map-ping applications. For example, if we know whycertain types of neurons express various ITFproducts whereas others do not then this wouldassist in interpreting activity maps because themolecular ground rules that apply can be takeninto account in experimental design.

On the technical side, continuing refinements in non-radioactive ISH techniques should allowmultiple maps to be obtained from the same brainregion with greater ease than currently possible.The recent introduction of antibodies that specifi-cally recognize phosphorylated epitopes of tran-scription factors (e.g. c-Jun, CREB) may also findapplications in activity mapping. As discussedabove, the persistence of phosphorylation is veryshort because of the fine control that is exerted byphosphatases. If, however, the phosphorylationstate of a signaling enzyme or ITF can be veridi-cally captured, then activity mapping with theseprobes can be initiated after much shorter stimu-lation periods than currently possible. This wouldyield an additional time point that can be used inmultiple mapping strategies. Indeed, both technicaldevelopments in detection along with furtherunderstanding of molecular induction mechanismswill largely influence the next decade of activitymapping with ITFs.

References1. Hughes P and Dragunow M. Pharmacol Rev 47, 133–178 (1995).2. Herrera DG and Robertson HA. Prog Neurobiol 50, 83–107 (1996).3. Kaczmarek L and Chaudhuri A. Brain Res Rev 3, 237–256 (1997).4. Ghosh A, Ginty DD, Bading H and Greenberg ME. J Neurobiol 25, 294–303

(1994).5. Sheng M and Greenberg ME. Neuron 4, 477–485 (1990).6. Lerea LS, Carlson NG and McNamara JO. Mol Pharmacol 47, 1119–1125

(1995).7. Lerea LS and McNamara JO. Neuron 10, 31–41 (1993).8. Xia Z, Dudek H, Miranti CK and Greenberg ME. J Neurosci 16, 5425–5436

(1996).9. Treisman R. EMBO J 14, 4905–4913 (1995).

10. Whitmarsh AJ, Shore P, Sharrocks AD and Davis RJ. Science 269, 403–407(1995).

11. Ginty DD. Neuron 18, 183–186 (1997).12. Ginty DD, Bonni A and Greenberg ME. Cell 77, 713–725 (1994).13. Kaminska B, Kaczmarek L and Chaudhuri A. J Neurosci 16, 3968–3978

(1996).14. Sagar SM, Sharp FR and Curran T. Science 240, 1328–1331 (1988).15. Jorgenson M, Deckert J, Wright D and Gehlert D. Brain Res 484, 393–398

(1989).16. Labiner DM, Butler LS, Cao Z et al. J Neurosci 13, 744–751 (1993).17. Chaudhuri A, Matsubara JA and Cynader MS. Vis Neurosci 12, 35–50 (1995).18. Kimpo RR and Doupe AJ. Neuron 18, 315–325 (1997).19. Mello CV and Clayton DF. J Neurosci 14, 6652–6666 (1994).20. Sassone-Corsi P, Sisson JC and Verma IM. Nature 334, 314–319 (1988).21. Liu FC and Graybiel AM. Neuron 17, 1133–1144 (1996).22. Chaudhuri A, Nissanov J, Larocque S and Rioux L. Proc Natl Acad Sci USA

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