comparative distribution of myristoylated alanine-rich c kinase substrate (marcks) and f1/gap-43...

Comparative Distribution ofMyristoylated Alanine-Rich C KinaseSubstrate (MARCKS) and F1/GAP-43Gene Expression in the Adult Rat Brain

ROBERT K. MCNAMARA1 AND ROBERT H. LENOX1,2,3*1Department of Psychiatry, University of Florida College of Medicine,

Gainesville, Florida 32610-02562Department of Pharmacology, University of Florida College of Medicine,

Gainesville, Florida 32610-02563Department of Neuroscience, University of Florida College of Medicine,

Gainesville, Florida 32610-0256

ABSTRACTMyristoylated alanine-rich C-kinase substrate (MARCKS) and F1/GAP-43 (B-50/

neuromodulin) are both major specific substrates for protein kinase C (PKC) and appear toplay an important role in the regulation of neuroplastic events during development and in theadult brain. Since PKC isozymes are differentially expressed in brain and the expression ofF1/GAP-43 and MARCKS mRNAs are differentially regulated by PKC through posttransla-tional mechanisms, the present study examined the relative distribution of both mRNAs inthe adult rat brain by using in situ hybridization histochemistry. MARCKS hybridization wasmost pronounced in the olfactory bulb, piriform cortex (layer II), medial habenular nucleus,subregions of the amygdala, specific hypothalamic nuclei, hippocampal granule cells, neocor-tex, and cerebellar cortex, intermediate in the superior colliculus, hippocampal CA1, andcertain brainstem nuclei including the locus coeruleus, and low-absent in regions of thecaudate-putamen, geniculate nuclei, thalamic nuclei, lateral habenular nucleus, and hippo-campal CA3 pyramidal and hilar neurons. Consistent with previous reports, prominentF1/GAP-43 hybridization was observed in neocortex, medial geniculate, piriform cortex (layerII), substantia nigra pars compacta, hippocampal CA3 pyramidal cells, thalamic andhypothalamic nuclei, lateral habenular nucleus, locus coeruleus, raphe nuclei, and cerebellargranule cells, intermediate in regions of the thalamus, hypothalamus, and amygdala, andlow-absent in regions of the olfactory bulb, caudate-putamen, medial habenular nucleus,hippocampal granule cells, and superior colliculus. Overall, F1/GAP-43 was highly expressedin a greater number of regions compared to MARCKS and, in a number of regions, includingthe hippocampus, habenular complex, ventral tegmentum, geniculate, and certain brain stemnuclei, a striking inverse pattern of expression was observed. These results indicate thatMARCKS gene expression, like that of F1/GAP-43, remains elevated in select regions of theadult rat brain which are associated with a high degree of retained plasticity. The potentialrole of PKC in the regulation ofMARCKS and F1/GAP-43 gene expression in brain is assessed.J. Comp. Neurol. 379:48–71, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: in situ hybridization; neuroanatomy; PKC; plasticity

Protein kinase C (PKC) represents a family of 12 iso-zymes which are predominantly calcium- and/or phospho-lipid-dependent protein kinases that differ in their tissuedistribution and subcellular localization. These isozymeshave been implicated in a broad spectrum of cellularresponses including neurotransmitter release and regula-tion of ion channels and gene expression (reviewed in

Contract grant sponsor: NIMH; Contract grant number: MH-50105;Contract grant sponsor: The Stanley Foundation; Contract grant sponsor:MRC/CIBA-GEIGY.*Correspondence to: Robert H. Lenox, University of Florida, Department

of Psychiatry, P.O. Box 100256, Gainesville, FL 32610-0256.E-mail: [email protected] 19 June 1996; Revised 18 October 1996; Accepted 1 November

1996

THE JOURNAL OF COMPARATIVE NEUROLOGY 379:48–71 (1997)

r 1997 WILEY-LISS, INC.

Nishizuka, 1995; Tanaka and Nishizuka, 1994). Activationof PKC by endogenous (diacylglycerol) or exogenous (phor-bol ester) factors results in the translocation of PKC fromthe cytosol to the plasmamembrane where it becomes fullyactivated and phosphorylates membrane-associated phos-phoprotein substrates. Two prominent PKC substrates arethe growth-associated protein GAP-43 (a.k.a. F1, P57,pp46, B-50, neuromodulin, 43–57 kDa, pI 4.5) and themyristoylated alanine-rich C-kinase substrate (MARCKS,80–87 kDa, pI ,4.0). Both proteins are predominantlymembrane-associated, MARCKS via myristoylation at theN-terminus region and phosphorylation site domain(Aderem et al., 1988; Swierczynski and Blackshear, 1995)and F1/GAP-43 via palmitoylation at the N-terminusregion (Skene andVirag, 1989). Both proteins bind calcium-calmodulin, MARCKS in a calcium-dependent manner(Graff et al., 1989) and F1/GAP-43 in a calcium-indepen-dent manner (Alexander et al., 1988). MARCKS binds aswell as cross-links filamentous actin (F-actin; Hartwig etal., 1992), whereas F1/GAP-43 binds but does not cross-link F-actin (Hens et al., 1993). Within the cell, F1/GAP-43protein expression is most prevalent in axons and axonterminals (Gispen et al., 1985; Goslin et al., 1988), thoughit has also been localized to dendritic processes and spines(Difiglia et al., 1990;Verhaagenetal., 1989), and theMARCKSprotein is expressed in axons and axon terminals, smalldendrites and dendritic spines (Ouimet et al., 1990). Al-though F1/GAP-43 is expressed exclusively in brain (Neveet al., 1987; Rosenthal et al., 1987), MARCKS is also ex-pressed in other tissues (Albert et al., 1986; Blackshear etal., 1986; Stumpo et al., 1989). In brain, both proteins areexpressed predominantly in neurons and to a lesser degreein glial cells (Ouimet et al., 1990; Vitkovic et al., 1988).Posttranslational modification of MARCKS and F1/

GAP-43 proteins via PKC phosphorylation appears to playan important role in modulating their function. PKCphosphorylation of MARCKS inhibits its capacity to cross-link F-actin without affecting binding of F-actin (Hartwiget al., 1992), inhibits calcium-calmodulin binding (Graff etal., 1989), and results in its reversible translocation fromthe membrane to the cytosol (Thelen et al., 1991; Wang etal., 1989). PKC phosphorylation of F1/GAP-43 similarlyinhibits its capacity to bind calcium-calmodulin (Alex-ander et al., 1987) but does not affect F-actin binding(Hens et al., 1993). Phosphorylation of both proteins byPKC is correlated with neurotransmitter release (Dekkeret al., 1989; Nichols et al., 1987; reviewed in Robinson,1992). Phosphorylation by PKC of both F1/GAP-43 and an80-kDa (pI 4.0), likely MARCKS based on its apparentmolecular weight, isoelectric point, and phosphorylationby PKC, is correlated with the persistence of long-termpotentiation in vivo (Nelson et al., 1989), an electrophysi-ological model of memory associated in part with enhancedneurotransmitter release (Dolphin et al., 1982; Weisskopfand Nicoll, 1995). Endogenous phosphorylation ofMARCKS, but not F1/GAP-43, is increased in recognitionmemory regions of the chick brain following imprinting(Sheu et al., 1993) and endogenous phosphorylation ofF1/GAP-43 and an 81-kDa (pI 4.0) protein is greater intemporal lobe regions implicated in visual informationstorage than in primary sensory cortex in monkey brain(Nelson et al., 1987). In addition to phosphorylating F1/GAP-43 and MARCKS proteins, PKC also plays an impor-tant role in the regulation of F1/GAP-43 and MARCKSgene expression in cultured cell systems without affecting

its transcription rate; PKC activation down-regulatesMARCKS mRNA and protein (Brooks et al., 1992; Linderet al., 1992; Watson et al., 1994) but increases F1/GAP-43mRNA (Perrone-Bizzozero et al., 1991, 1993). Regulationof mRNA stability by PKC may entail phosphorylation-induced alterations in the binding of proteins to the38-untranslated region (Amara et al., 1994; Kohn et al.,1996; Sachs, 1993).Both MARCKS and F1/GAP-43 proteins appear to be

vital for neuronal plasticity during brain development.MARCKS and F1/GAP-43 proteins are localized in axonalgrowth cones and filopodic extensions (Katz et al., 1985;Meiri et al., 1986; Nelson et al., 1989; Rosen et al., 1990),are developmentally regulated (Dani et al., 1991; De laMonte et al., 1989; Mahalik et al., 1992; Neve et al., 1987;Patel and Kligman, 1987), and are necessary for normalbrain development and perinatal survival (Strittmatter etal., 1995; Stumpo et al., 1995). However, despite a precipi-tous decline in expression during the perinatal period(Dani et al., 1991; De la Monte et al., 1989; Mahalik et al.,1992; Patel and Kligman, 1987), both proteins remainelevated in specific regions of the adult brain (Benowitz etal., 1988; Ouimet et al., 1990; Neve et al., 1987). While theregional distribution of F1/GAP-43 mRNA and protein inbrain has been extensively documented (Bendotti et al.,1991; Benowitz et al., 1988; Dani et al., 1991; De la Monteet al., 1989; Kruger et al., 1992, 1993; Mahalik et al., 1992;Meberg and Routtenberg, 1991; Neve et al., 1987, 1988;Oestreicher and Gispen, 1986; Verhaagen et al., 1989,1990; Yao et al., 1993), the distribution of MARCKSmRNAin adult rat brain has not yet been determined. In thepresent study, we report on the distribution of MARCKSgene expression assessed by in situ hybridization histo-chemistry in the adult rat brain. Based on the differentialregulation of MARCKS and F1/GAP-43 mRNA by PKCdescribed above, the distribution of F1/GAP-43 gene expres-sion was also examined and compared with MARCKSexpression across specific brain regions to determine rela-tive distribution patterns. An abstract of this work hasappeared elsewhere (McNamara et al., 1995).

MATERIALS AND METHODS

Tissue preparation

Five adult male Sprague-Dawley rats (Harlan Farms)200–250 g were anesthetized with sodium pentobarbitol(70 mg/kg) and perfused transcardially with phosphate-buffered saline (PBS) and then with 4% paraformaldehyde(PFA, pH 7.3) in 0.1 M sodium phosphate buffer. Brainswere removed and postfixed for 1 hour by immersion into4% PFA in 0.1 M sodium phosphate buffer. Brains werethen immersed in 20% sterile sucrose in 0.1 M sodiumphosphate buffer until saturated (,16 hours) and thenquick-frozen in 280°C N-methyl butane. Brains weresectioned coronally at 12 µm and thaw-mounted ontopoly-L-lysine-treated slides which were stored at 280°C.

Probe construction

MARCKS sense and antisense riboprobes were synthe-sized from a 1.15-kb Xho II subclone of the murineMARCKS cDNAinserted into a pcDNAI/Neo vector flankedby T7 and SP6 promotors (Seykora et al., 1991). The1.15-kb fragment spans bases 354–1505 of the murineMARCKS cDNA which encompasses 32 bases of the 58-UTR, the entire 927 bases of the protein coding region, and

MARCKS mRNA EXPRESSION IN RAT BRAIN 49

191 bases of the 38-UTR. F1/GAP-43 sense and antisenseriboprobes corresponding to bases 779–1295 of the ratF1/GAP-43 cDNA inserted into a pSEA vector flanked byT7 and SP6 promotors were used (Rosenthal et al., 1987).Linearized DNA (1 µg) was labeled with 35S-UTP (Amer-sham) by in vitro transcription. A 1-µl aliquot of labeledmRNA was suspended in 3 ml scintillation fluid andactivity in c.p.m. determined by a scintillation counter.

In situ hybridization

Prior to hybridization, mounted sections were immersedin 4°C 4% PFA (10 minutes), 0.1 M DEPC (rinse), 0.1 Mtriethanolamine (TEA, pH 8, rinse), 0.1 M TEA 1 aceticanhydride (0.25%; 10 minutes), 23 SSC (sodium chloride/sodium citrate; rinse), ethanol series (50, 70, 95, 100%, 3minutes each), and air-dried. Sense and antisense ribo-probes were added to slides (6 3 105 c.p.m./slide) in a 60-µlvolume of hybridization buffer (50% formamide, 300 mMNaCl, 20 mM Tris (pH 8.0), 5 mM EDTA, 13 Denhardt’s,10% dextran sulfate (50% W/V), 10 mM dithiolthreitol).Slides were then coverslipped, placed in a sealable con-tainer moistened with box buffer (50% formamide and 43SSC) and incubated for 16 hours at 60°C. Coverslips wereremoved and slides washed in 23 SSC (2 3 10 minutes),pancreatic RNaseAsolution (20 µg/ml in 500 mMNaCl, 10mM Tris, 30 minutes at 37°C), 23 SSC (2 3 10 minutes),0.13 SSC (65°C for 2 hours), 0.53 SSC (2 3 10 minutes),and ethanol (50%, 70%, 95%, 100% each containing 0.3 Mammonium acetate 2 min each). All wash solutions con-tained 10 mM 2-mercaptoethanol and 1 mM EDTA, exceptfor the RNase A, 0.53 SSC washes, and ethanol solutions.Air-dried slides were apposed to X-ray film (Hyperfilm-bmax,Amersham) for 3 days. For both probes, the distribu-tion pattern observed from the X-ray film after 3-dayexposure did not differ from the pattern observed fromslides exposed to emulsion (NTB-2, Kodak) for 7 days.Distribution and expression levels of both probes wererated qualitatively from autoradiographs enlarged ontoKodak paper (F3 Glossy) as well as from emulsion-dippedslides. Signal intensity was rated according to the follow-ing scale: 0, grain density at background levels; 1, lowdensity;11, moderate density;111, high density;1111,highest density. Quantitation was conducted by an evalua-tor who was knowledgeable of the probe being quantitatedas well as by two evaluators who were blind to the probecondition. A criterion of .95% agreement within one ‘1’was established between individual evaluators. The inten-sity of each probe within a specific region was ratedrelative to regions expressing either background levels orregions exhibiting maximal expression levels. Approximate section coordinates and neuroanatomical nomencla-ture were derived from Paxinos and Watson (1988). Rela-tive expression levels in the different brain regions exam-ined are summarized Table 1.

Probe specificity

In accordance with previously described procedures(Tecott et al., 1987), a series of control experiments wasconducted to verify the specificity of the MARCKS anti-sense riboprobe, as follows: (1) incubation with a senseoriented riboprobe, (2) incubation of the labeled antisenseriboprobe in the presence of excess (10- to 20-fold, calcu-lated from A260) unlabeled (cold) antisense riboprobe, (3)pretreatment of tissue with pancreatic RNase A (20 µg/ml,30 minutes at 37°C) followed by washing in 0.1MNaOH in

diethyl pyrocarbonate (DEPC) treated water to inactivatethe enzyme, to determine binding to non-RNA tissueelements, and (4) hybridization with a second partiallyoverlapping probe (pMG80K2.4; Stumpo et al., 1989).Relative to the signal level observed following hybridiza-tion with the antisense riboprobe in Figure 1A, the ab-sence of hybridization signal after hybridization with thesense riboprobe under identical treatment conditions isapparent (Fig. 1B). Moreover, antisense riboprobe hybrid-ization was completely blocked in the presence of excessunlabeled MARCKS antisense probe (Fig. 1C) but not inthe presence of excess (,10-fold) of an unlabeledMARCKS-related protein (MRP) antisense probe (F52/MacMARCKS,data not shown), a protein which exhibits moderate se-quence homology with MARCKS (Aderem, 1992; Black-shear et al., 1992; Blackshear, 1993). MARCKS antisenseriboprobe hybridization was also completely prevented byprior treatment of tissue with RNase (Fig. 1D). Hybridiza-tion with a second partially overlapping MARCKS DNAprobe exhibited the same hybridization distribution asthat observed following hybridization with the MARCKSantisense riboprobe (data not shown). The specificity of theMARCKS probe was also assessed by Northern blot analy-sis which demonstrated that no antisense riboprobe hybrid-ization occurred with total RNA extracted from rat liverbut hybridization did occur with total RNA extracted fromrat brain and spleen (data not shown), consistent with aprevious report on the tissue distribution of MARCKSmRNA (Stumpo et al., 1989). Collectively, these resultsconfirm the specificity of the MARCKS antisense ribo-probe. The specificity of the F1/GAP-43 antisense ribo-probe has been demonstrated previously (Meberg andRouttenberg, 1991) but we again verified the absence ofsignal following hybridization with the sense F1/GAP-43probe (data not shown).

RESULTS

Telencephalon

Olfactory bulb. In the olfactory bulb,MARCKShybrid-ization was pronounced in the internal granular layer,whereas F1/GAP-43 hybridization was at background lev-els in this region (Fig. 2, See also Table 1). Interestingly,prominent MARCKS hybridization was also present in theglomerular layer, composed primarily of mitral, periglo-merular, and tufted cell dendrites, and olfactory nervelayer, composed primarily of axons arriving from olfactoryepithelium, suggesting both axonal and dendritic localiza-tion of MARCKS mRNA (see Discussion). F1/GAP-43hybridization was also present in the glomerular layer, butto a lesser degree, and may also represent expression indendrites, and absent from the olfactory nerve layer.F1/GAP-43 hybridization was pronounced in the anteriorolfactory nucleus, whereas MARCKS hybridization wasmoderate-low. In the accessory olfactory bulb, prominentMARCKS hybridization was detected as was F1/GAP-43hybridization, though not apparent in Figure 2D. Themitral cell layer exhibited high levels of both MARCKSand F1/GAP-43 hybridization, whereas the internal plexi-form layer exhibited low levels of both mRNAs. BothMARCKS and F1/GAP-43 hybridization were moderate inthe olfactory tubercle (Fig. 3).Cerebral cortex. In each of the cerebral cortical re-

gions examined, MARCKS, but not F1/GAP-43, hybridiza-tion was observed in the most superficial aspect of layer I

50 R.K. MCNAMARA AND R.H. LENOX

(Figs. 2–9 and Fig. 10C vs. F) which may representexpression in glial cells located in this region (see Discus-sion). Pronounced labeling for both MARCKS and F1/GAP-43 was present in piriform cortex along the entireanterior-posterior extension and was largely restricted tolayer II (Figs. 3–5). In frontal cortex, MARCKS hybridiza-tion was absent from the majority of layer I, except themost superficial aspect, low-moderate in layers II–III, andlow in layers IV, V, and VI; F1/GAP-43 hybridization wasabsent from layer I but high in the remaining 5 layers (Fig.2). In angular insular cortex, MARCKS hybridization wasabsent from layer I, except the most superficial aspect, lowin layer II, and absent from the remaining layers; F1/GAP-43 hybridization was absent from layer I and moder-ate to high in the remaining layers (Fig. 3). In cingulatecortex, MARCKS hybridization was absent from layer I,except the most superficial aspect, and low in the remain-ing layers; F1/GAP-43 hybridization was absent from layerI, moderate in layers II–IV, and high in layers V and VI(Fig. 3). In parietal and temporal cortices, both MARCKSand F1/GAP-43 hybridization was but moderate-high inall layers except layer I (Fig. 5). In entorhinal cortex, bothMARCKS and F1/GAP-43 hybridization was present athigh levels, principally in layers II and III (Fig. 6). In the

TABLE 1. Summary of MARCKS and F1/GAP-43 mRNAExpressionin the Adult Rat Brain

Brain region MARCKS2 F1/GAP-432

AmygdalaACo Anterior cortical amyg n. 11111 01BL Basolateral amyg n. 11 1111

BM Basomedial amyg n. 111 11Ce Central amyg. n. 11 11Me Medial amyg. n. 11 111MePV Medial posteroventral amyg.

n. 111 11MePD Medial posterodorsal

amyg. n. 111 11PMCo Postermedial cortical

amyg. n. 11 1Basal ganglia/Basal

forebrainAcb Accumbens n. 1 11B Basal nucleus Meynert 0 0CPu Caudate-putamen 1 1DB Diagonal band nucleus 1 1111GP Globus pallidus 0 0VP Ventral pallidum 1 11

Cerebral cortexAI Agranular insular 1 1111Cg Cingulate 1 1111Ent Entorhinal 111 111Fr Frontal 11 1111IL infralimbic 1 1111Par Parietal 111 111Pir Piriform 1111 1111Te Temporal 111 111

ClaustrumCL 1 1111

Endopiriform nucleusDEn Dorsal endopiriform n. 11 111VEn Ventral endopiriform n. 111 111

Geniculate nucleusMGd Medial geniculate n. dorsal 0 1111MGv Medial geniculate n. ventral 1 1111

Habenula nucleusLHb Lateral 1 111MHb Medial 1111 0

Hippocampal formationA. Septal aspectCA1 11 111CA2 1 1CA3 1 1111gc Granule cells 1111 0h Hilar cells 1 11

B. Temporal aspectCA1 111 1111CA2 111 111CA3 11 1111gc Granule cells 1111 0h Hilar cells 1 111

HypothalamusDMH Dorsomedial hy n. 111 111MP Medioposterior mammil-

lary n. 0 0pvh Paraventricular hy n. 1111 111VMH Ventromedial hy n. 1111 1111

Olfactory systemAO Anterior olfactory n. 11 1111AOB Accessory olfactory bulb 1111 1111E Ependyma & subependyma l. 0 0EPl External plexiform layer 0 1Gl Glomerular layer 1111 1IGr Int granular layer 1111 1IPl Int plexiform layer 1 1Mi Mitral cell layer 111 1111ON Olf nerve layer 1111 0OV Olfactory ventricle 1111 0tu Olfactory tubercle 111 111

SeptumLS Lateral septal n. 0 111MS Medial septal n. 0 11SHi Septohippocampal n. 11 1111

Substantia nigraPN Paranigral n. 1 111SNC Substantia nigra, compacta 1 1111SNR Substantia nigra, reticular 0 0

Superior colliculusOp Optic nerve layer 11 1SuG Superficial gray layer 11 1Zo Zonal layer 11 1

TectumAPT Anterior pretectal n. 1 11

TegmentumLDT Laterodorsal tegmental n. 1 11VTA Ventral tegmental area 0 1111

TABLE 1. (continued)

Brain region MARCKS2 F1/GAP-432

ThalamusAD Anterodorsal thal. n. 0 111AV Anteroventral thal. n. 0 11IMD Intermediodorsal thal. n. 1 11LD Laterodorsal thal. n. 1 11MD Mediodorsal thal. n. 0 111PVP Paraventricular thal. 1 1111Re Reuniens thal. n. 1 111VM Ventromedial thal. n. 1 111

Brainstem nuclei7 Facial motor n. 1 0amb Ambiguus n. 0 1111CG Central gray 1 111CLi Caudal linear raphe n. 1 1111DR Dorsal raphe n. 1 1111GiA Gigantocell reticular n. 1 111IP Interpeduncular n. 1 111IO Inferior olive 11 1111LC Locus coeruleus 111 1111LDT Laterodorsal tegmental n. 1 1111LRt Lateral reticular n. 11 0LSO Lateral superior olive 0 111MVe Medial vestibular n. 1 111PCRt Parvocellular reticular n. 0 11PrH Prepositus hypoglossal n. 1 111ROb Raphe obscurus n. 0 1RMg Raphe magnus n. 1 1111RPa Raphe pallidus n. 11 1111Ru Red n. 0 1Sol Solitary tract n. 11 1111

CerebellumA. CortexGr Granule cell layer 111 1111Pkj Purkinje cell layer 111 0Ml Molecular layer 0 0

B. Deep cerebellarnuclei

Int Interposed 1 1Lat Lateral 11 11Med Medial 1 1

Spinal cordMd Medullary reticular field 1 11Ramb Retroambiguus n. 1 111SpVe Spinal vestibular n. 0 1Sp5C Spinal trigeminal n., caudal 111 1111Sp5O Spinal trigeminal n., oral 1 11

10, grain density at background levels; 1, low density; 11, moderate density; 111,high density; 1111, very high density.2MARCKS,myristoylated alanine-rich C kinase substrate; F1/GAP-43, B-50/neuromodu-lin.


Fig. 1. Controls demonstrating the specificity of the myristoylatedalanine-rich C-kinase substrate (MARCKS) riboprobe. Distribution ofMARCKS antisense riboprobe hybridization is presented in A forcomparison withB–D. Specificity of the MARCKS antisense riboprobewas confirmed according to the following criteria: (1) incubation withsense oriented riboprobe did not result in detectable signal, verifyingthat antisense probe binding was the result of its base sequenceorientation and not other physical properties (B); (2) incubation of the

labeled antisense riboprobe in the presence of excess unlabeled proberesulted in a significant reduction of signal, indicating a fixed numberof target sites are available for probe binding and the blockade ofspecific hybridization with target mRNA (C); and (3) pretreatment oftissue with RNase eliminates antisense hybridization signal indicat-ing that the probe is hybridizing to RNA rather than to unknowncellular components (D).

Figure 1 (Continued.)


claustrumand endopiriform nucleus, MARCKS hybridiza-tion was low-moderate, whereas F1/GAP-43 hybridizationwas pronounced (Fig. 4).Basal ganglia and basal forebrain. In the caudate-

putamen, MARCKS hybridization was present at lowlevels rostrally (Fig. 3A) but at moderate levels caudally(Figs. 4C, 5A). However, F1/GAP-43 hybridization wasmoderate rostrally (Fig. 4B) but low-absent caudally (Figs.4D, 5B). MARCKS and F1/GAP-43 hybridization werelargely absent from globus pallidus (Fig. 4C,D), the basalnucleus of Meynert and ventral pallidum (Fig. 4A,B).MARCKS hybridization was also absent from the nucleusof the diagonal band, whereas F1/GAP-43 hybridizationwas highly expressed in this region (Fig. 4A,B).Septum and hippocampus. In the septum, MARCKS

expression was low-absent in the lateral and medial septalnuclei; F1/GAP-43 hybridization was moderate in thesenuclei (Figs. 3C,D, 4A,B). In the septohippocampal nucleus,MARCKS hybridization was moderate, whereas F1/GAP-43 hybridization was high (Fig. 3C,D). Along theseptotemporal hippocampal axis, MARCKS hybridizationwas very high in the granule cell layer unlike F1/GAP-43which was not expressed in this cell layer (Figs. 4C,D–6C,D). In area CA1, MARCKS hybridization was low-moderate and F1/GAP-43 hybridization was moderate-

high. In area CA2, both mRNAs were expressed at lowlevels. In areaCA3a–c of the septal hippocampus,MARCKShybridization was low, whereas in the temporal hippocam-pus MARCKS expression in CA3 was moderate (Fig. 5C),possibly due to the increased density of cells in the CA3layer of the temporal hippocampus (Cavazos et al., 1994).F1/GAP-43 hybridization was pronounced in area CA3a–cin the septal hippocampus as well as in the temporalhippocampus (Figs. 4C,D–6C,D). MARCKS hybridizationwas largely absent from the hilus region, whereas F1/GAP-43 hybridization was punctate in correspondancewith the large neuronal cell bodies in this region.Amygdala. MARCKS hybridization was very high in

the anterior cortical amygdaloid nucleus, whereas F1/GAP-43 hybridization was absent from this region (Fig.4C,D). In the posteromedial cortical nuclei, low-moderatelevels of both MARCKS and F1/GAP-43 hybridizationwere observed. In themedial posterodorsal and posteroven-tral amygdaloid nuclei, MARCKS hybridization was high,whereas F1/GAP-43 hybridization was moderate (Fig.5A,B). In themedial amygdaloid nucleus,MARCKShybrid-ization was low, whereas F1/GAP-43 hybridization washigh. In the basomedial amygdaloid nucleus, bothMARCKS and F1/GAP-43 hybridization was moderate,whereas in the basolateral amygdaloid nucleus MARCKS

Fig. 2. Autoradiographs illustrating MARCKS (A,C) and B-50/neuromodulin (F1/GAP-43) (B,D) hybridization distribution at thelevel of Bregma 6.7 mm (A,B) and Bregma 4.7 mm (C,D). aci, anteriorcommissure, intrabulbar; AO, anterior olfactory nucleus; AOB, acces-

sory olfactory bulb; E, ependyma layer; EPl, external plexiform layer;Fr, frontal cortex; Gl, glomerular layer; IGr, internal granule layer;IPl, internal plexiform layer; Mi, mitral cell layer; ON, olfactory nervelayer; OV, olfactory ventricle. Scale bars 5 1 mm.


hybridization was low and F1/GAP-43 hybridization washigh (Fig. 5A,B). In the central amygdaloid nucleus, bothMARCKS and F1/GAP-43 hybridization was low-moderate(Fig. 4C,D).

Diencephalon

In the epithalamus, MARCKS hybridization was veryhigh in the medial habenular nucleus and low-absent inthe lateral habenular nucleus (Figs. 5A,B; 10A). Con-versely, F1/GAP-43 signal was high in the lateral habenu-lar nucleus and absent from the medial habenular nucleus(Figs. 5A,B; 10B). In the thalamus, MARCKS hybridiza-tion was low-absent in the anterodorsal, anteroventral,and mediodorsal thalamic nuclei, whereas F1/GAP-43hybridization was high in these nuclei (Fig. 4C,D). In theintermediodorsal, paraventricular, reuniens, and ventro-medial thalamic nuclei, MARCKS hybridization was pres-ent only at low levels, whereas F1/GAP-43 hybridizationwas high in these nuclei (Figs. 4C,D; 5A,B). However, inthe laterodorsal thalamic nucleus, low-moderateMARCKSand F1/GAP-43 hybridization was observed. In the medialgeniculate, MARCKS hybridization was low-absent inboth the dorsal and ventral regions, whereas F1/GAP-43hybridization was very high in both regions (Fig. 6A,B). Inthe hypothalamus, both MARCKS and F1/GAP-43 hybrid-ization were absent in the medioposterior mammillarynucleus (Fig. 5C,D), moderate in the dorsomedial andparaventricular hypothalamic nuclei, and very high in theventromedial hypothalamic nucleus (Figs. 4C,D; 5A,B).

Cerebellum

In cerebellar cortex, MARCKS hybridization wasmoder-ate in both the granule cell and Purkinje cell layers andabsent from the molecular layer whereas F1/GAP-43 hy-bridization was very high in the granule cell layer andabsent from the Purkinje and molecular layers (Figs. 7, 8).In the deep cerebellar nuclei, both MARCKS and F1/GAP-43 hybridization was low in the interposed nucleusand low-absent in the medial nucleus, whereas in thelateral nucleus both MARCKS and F1/GAP-43 hybridiza-tion was low-moderate (Fig. 7C,D).

Brainstem and spinal cord

Overall, MARCKS hybridization was low in the majorityof brainstem nuclei unlike F1/GAP-43 hybridization whichwas high inmany nuclei (Figs. 6, 7, 8). MARCKShybridiza-tion was largely absent from the substantia nigra unlikeF1/GAP-43 hybridization which was high in the paranigralnucleus and substantia nigra pars compacta, but absentfrom the substantia nigra reticular (Fig. 6A,B). Similarly,in the tegmentum, MARCKS hybridization was low-absent, whereas F1/GAP-43 hybridization was high inboth the laterodorsal tegmental nucleus and in the ventraltegmental area (Figs. 6A,B; 7A,B). In the superior collicu-lus, moderate levels of MARCKS hybridization were ob-served in the optic, superficial gray, and zonal layers,whereas F1/GAP-43 hybridization was low in each of theseregions (Fig. 6). Moderate MARCKS hybridization wasobserved in the inferior olive, locus coeruleus, lateralreticular nucleus, raphe pallidal, and nucleus of the soli-tary tract, whereas F1/GAP-43 hybridization was high ineach of these nuclei with the exception of the lateralreticular nucleus where hybridization was at backgroundlevels (Fig. 8D). MARCKS hybridization was low in thecentral gray, caudal linear raphe, dorsal raphe, giantocel-

lular reticular, interpeduncular, prepositus hypoglossal,raphe magnus nuclei, whereas F1/GAP-43 hybridizationwas pronounced in these nuclei. MARCKS hybridizationwas largely absent from the ambiguus, lateral superiorolive, parvocellular reticular, raphe obscurus, red, andspinal vestibular nuclei, whereas F1/GAP-43 hybridiza-tion was low in these nuclei with the exception of theambiguus and lateral superior olive where hybridizationwas high (Figs. 7B, 8B). In the facial motor nucleus,MARCKS hybridization was present at low levels, whereasF1/GAP-43 hybridization was largely absent (Fig. 7C,D).In the spinal cord, both MARCKS and F1/GAP-43 hybrid-ization was high in the caudal spinal trigeminal nucleus,whereas in the medullary reticular field and oral spinaltrigeminal nucleus, bothMARCKS and F1/GAP-43 hybrid-ization was low (Fig. 9). In the retroambiguus nucleus andinferior olive B subnucleus, MARCKS hybridization waslow and F1/GAP-43 hybridization was moderate.

DISCUSSION

The present results demonstrate the discrete and heter-ogeneous localization of MARCKS gene expression in theadult rat brain. MARCKS gene expression was highest inthe olfactory bulb, piriform cortex, subregions of theamygdala, medial habenular nucleus, select hypothalamicnuclei, hippocampal granule cells, neocortex, and cerebel-lar cortex; intermediate in hippocampal CA1, superiorcolliculus, and locus coeruleus; and low-absent in medialgeniculate nuclei, lateral habenular nucleus, hippocampalCA3 and hilar neurons, and the majority of the thalamusand brainstem. Comparison of the distribution ofMARCKSmRNAin rat brain to that recently described in chick brain(Meberg et al., 1996) indicates that in both speciesMARCKS mRNA is expressed in cerebellar granule cells,hippocampus, and in cells adjacent to the lateral ven-tricles. Moreover, a general pattern of higher expression ofMARCKS mRNA in medial structures relative to lateralstructures was noted in chick brain (Meberg et al., 1996)and observed in rat brain, particularly in the diencephalon(see Fig. 5A). Whereas the significance of this midlineexpression pattern is unclear, it is of interest that thecerebral hemispheres of mutant mice lacking MARCKSfail to fuse along the midline (Stumpo et al., 1995).Comparison ofMARCKSmRNAexpression withMARCKSimmunoreactivity (Ouimet et al., 1990) in rat brain revealsa high degree of complementarity. For example, bothMARCKS immunoreactivity and hybridization are high inthe amygdala, select hypothalamic nuclei, medial habenu-lar nucleus, and cerebellar cortex and low-absent in stria-tum, thalamus, and the majority of the brainstem exceptthe locus coeruleus. Within the hippocampus, MARCKSgene expression is highest in the granule cell layer of thedentate gyrus and MARCKS immunostaining is pro-nounced in the hilus region (Ouimet et al., 1990), theterminal field of a large portion of granule cell axons andaxon collaterals (Claiborne et al., 1986). The relativeabsence of MARCKS immunoreactivity from the dentategyrus molecular layer, composed primarily of granule celldendrites and perforant path axons originating primarilyfrom neurons in layers II of the entorhinal cortex (Stewardand Scoville, 1976), indicates that the MARCKS protein islocalized exclusively in granule cell axons but absent fromthe axons of layer II entorhinal cortex neurons, which also


Fig. 3. Autoradiographs illustrating MARCKS (A,C) and F1/GAP-43 (B,D) hybridization distribution at the level of Bregma 3.7mm (A,B) and Bregma 1.7 mm (C,D). AI, agranular insular cortex; Cg,cingulate cortex; CPu, caudate-putamen, IL, infralimic cortex, LV,

lateral ventricle, Pir, piriform cortex; SHi, septohippocampal nucleus;tu, olfactory tubercle; VP, ventral pallidum. Other abbreviations as inFigure 2. Erratic white spots outside of the tissue were eliminated inD to mask distracting background artifact. Scale bar 5 1 mm.

Fig. 4. Autoradiographs illustrating MARCKS (A,C) and F1/GAP-43 (B,D) hybridization distribution at the level of Bregma 1.0mm (A,B) and Bregma 22.3 mm (C,D). 3V, third ventricle; Acb,Accumbens nucleus; ACo, anterior cortical amygdaloid nucleus; AD,anterodorsal thalamic nucleus; AV, anteroventral thalamic nucleus; B,basal nucleus of Meynert; ca1, hippocampal CA1 field; ca3, hippocam-pal CA3 field; cc, corpus callosum; Ce, central amygdaloid nucleus; Cl,claustrum; DB, diagonal band of broca; DEn, dorsal endopiriform

nucleus; fi, fimbria/fornix; gc, granule cells; GP, globus pallidus; LD,laterodorsal thalamic nucleus; Lateral septal nucleus; MD, mediodor-sal thalamic nucleus; MHb, medial habenular nucleus, MS, medialseptum; ox, optic chiasm; pvh, paraventricular hypothalamic nucleus;VEn, ventral endopiriform nucleus; VMH, ventromedial hypothalamicnucleus. Other abbreviations as in preceding figures. Erratic whitespots outside of the tissue were eliminated in C to mask distractingbackground artifact. Scale bar 5 1 mm.

Fig. 5. Autoradiographs illustrating MARCKS (A,C) and F1/GAP-43 (B,D) hybridization distribution at the level of Bregma 23.7mm (A,B) and Bregma 24.9 mm (C,D). APT, anterior pretectalnucleus; BL, basolateral amygdaloid nucleus; BM, basomedial amyg-daloid nucleus; ca2, hippocampal CA2 field; CG, central gray; DMH,dorsomedial hypothalamic nucleus; Ent, entorhinal cortex; h, hilus;IMD, intermedio dorsal thalamic nucleus; LHb, lateral habenularnucleus; Me, medial amygdaloid nucleus; MePD, medial posterodorsalamygdaloid nucleus;MePV,medial posteroventral amygdaloid nucleus;

MGd, mediodorsal geniculate nucleus; MP, medioposterior mammil-lary nucleus; Op, optic nerve layer, superior colliculus; Par, parietalcortex; PMCo, posteromedial cortical amygdaloid nucleus; PN, para-nigral nucleus; PVP, paraventricular thalamic nucleus; Re, reuniensthalamic nucleus; SNC, substantia nigra pars compacta; Sug, superfi-cial gray layer; Te, temporal cortex; VM, ventromedial thalamicnucleus. Other abbreviations as in preceding figures. Erratic whitespots outside of the tissue were eliminated in C and D to maskdistracting background artifact. Scale bar 5 1 mm.

Fig. 6. Autoradiographs illustrating MARCKS (A,C) and F1/GAP-43 (B,D) hybridization distribution at the level of Bregma 26.1mm (A,B) and Bregma 26.3 mm (C,D). CLi, caudal linear raphenucleus; CG, central gray; DR, dorsal raphe nucleus; IP, interpeduncu-lar nucleus; MGv, medioventral geniculate nucleus; Ru, red nucleus;

SNR, substantia nigra reticular; SuG, superficial gray layer, superiorcolliculus; VTA, ventral tegmental nucleus; Zo, zonal layer, superiorcolliculus. Other abbreviations as in preceding figures. Erratic whitespots outside of the tissue were eliminated in C to mask distractingbackground artifact. Scale bar 5 1 mm.

Fig. 7. Autoradiographs illustrating MARCKS (A,C) and F1/GAP-43 (B,D) hybridization distribution at the level of Bregma 210.1mm (A,B) and Bregma 211.5 mm (C,D). 4V, fourth ventricle, 7, facialnucleus; GiA, gigantocellular reticular nucleus; Gr, granule cell layer,cerebellar cortex; Int, interposed cerebellar nucleus; Lat, lateralcerebellar nucleus; LC, locus coeruleus; LDT, laterodorsal tegmental

nucleus; LSO, lateral superior olive; Med, medial cerebellar nucleus;Ml, molecular layer; Pkj, purkinje layer, cerebellar cortex; PrH,prepossitus hypoglossal nucleus; RMg, raphe magnus nucleus; RPa,raphe pallidus nucleus; Sol, solitary tract nucleus; sp5, spinal trigemi-nal tract; Sp5O, spinal trigeminal nucleus, oral. Scale bar 5 1 mm.

Fig. 8. Autoradiographs illustrating MARCKS (A,C) and F1/GAP-43 (B,D ) hybridization distribution at the level of Bregma 212.3mm (A,B) and Bregma 213.3 mm (C,D). amb, ambiguus nucleus; IO,inferior olive nucleus; IOD, inferior olive dorsal nucleus; IODM,inferior olive doromedial nucleus; IOM, inferior olive medial nucleus;IOPr, inferior olive principle nucleus; LRt, lateral reticular nucleus;

MVe,medial vestibular nucleus; PCRt, parvocellular reticular nucleus;PrH, prepositus hypoglossal nucleus; py, pyramidal tract; ROb, rapheobscurus nucleus; SpVe, Spinal vestibular nucleus; Sp5I, spinal trigemi-nal nucleus, interpolar. Other abbreviations as in preceding figures.Scale bar 5 1 mm.

Fig. 9. Autoradiographs illustrating MARCKS (A) and F1/GAP-43(B) hybridization distribution at the level of Bregma 214.7 mm. IOB,inferior olive, subnu B med nucleus; Md, medullary reticular field;

pyx, pyramidal decussation; Ramb, retroambiguus nucleus; Sol, soli-tary tract nucleus; Sp5C, spinal trigeminal nucleus, caudal. Scalebar 5 1 mm.


exhibit a high level of MARCKS hybridization (Figs. 5C,6A,C).

MARCKS expression in the olfactory system

An interesting pattern of MARCKS hybridization is itshigh expression in the olfactory system. In the olfactorybulb, MARCKS is highly expressed in both mitral andgranular cell layers as well as in the accessory olfactorybulb. MARCKS hybridization is highly expressed in theglomerular layer of the olfactory bulb (Fig. 2A), which iscomposed primarily of the dendrites from mitral, periglo-merular, and tufted cells. Localization of mRNA to thedendritic domain is also shared with a number of cytoskel-etal-related protein mRNAs including RC3 (neurogranin)and MAP-2 (reviewed in Steward, 1995). Together theaxons originating from the olfactory bulb comprise themajor output from the olfactory bulb and project to theanterior olfactory nucleus, piriform cortex, olfactory tu-bercle, amygdala, mediodorsal thalamic nucleus, and ento-rhinal cortex. With the exception of the mediodorsalthalamic nucleus, MARCKS hybridization is also highly

expressed in these afferent regions as is MARCKS protein(Ouimet et al., 1990). Finally, MARCKS hybridization ishighly expressed in the medial habenular nucleus, a relaybetween limbic forebrain and midbrain and brainstemregions, which has been implicated in olfactory-guidedbehavior (Sutherland, 1982). Thus, while the high level ofMARCKSmRNAin the olfactory systemmay reflect a highdemand for synaptogenesis in this system, the precise roleof MARCKS in olfaction awaits further analysis.

Comparison of MARCKS and glial expression

A second interesting pattern of MARCKS hybridizationis its localization in ventricular walls and paraventricularstructures. This pattern is most apparent at the level ofthe thalamus where MARCKS hybridization, as well asMARCKS immunoreactivity (Ouimet et al., 1990), is promi-nent in the medial habenular nucleus, which borders thedorsal third ventricle (Fig. 10A), and in dorso- and vento-medial hypothalamic nuclei which border the ventral thirdventricle (Figs. 4C, 5A). This pattern is also observed inthe midbrain and hindbrain where MARCKS hybridiza-

Fig. 10. Photomicrographs of emulsion dipped slides illustratingMARCKS (A–C) and F1/GAP-43 (D–F) hybridization distribution inthe habenular complex (A,D), wall of the third ventricle (B,E, arrows),and superficial aspect of layer I of cerebral cortex (C,F, arrows). 3V,

third ventricle; DV3, dorsal third ventricle; LHb, lateral habenularnucleus; MHb, medial habenular nucleus; VMH, ventromedial hypo-thalamic nucleus. Scale bar 5 100 µm.


tion and immunoreactivity (Ouimet et al., 1990) is high inthe medial vestibular nucleus and locus coeruleus whichborder the fourth ventricle (Figs. 7A, 8C). That theMARCKS hybridization observed in the ventricle walls(Fig. 10A,B) is localized to glial cells is suggested by thepresence of MARCKS immunostaining in glial cells resem-bling microglia and astrocytes (Ouimet et al., 1990) as wellas the high level of glial fibrillary acidic protein (GFAP)immunoreactivity, an astroglia-specific protein, observedin the walls lining the olfactory, lateral, and third ven-tricles (Kalman and Hajos, 1989). Moreover, the MARCKShybridization present in the superficial layer of cerebralcortex (Fig. 10C) throughout the forebrain (Figs. 2–4) andmidbrain (Fig. 6C) may also be localized to astrocytesbecause GFAP-staining is also high in this layer (Kalmanand Hajos, 1989). Across the majority of the brain, how-ever, the regional distribution of MARCKS hybridizationand GFAP-staining are poorly correlated. For example, thehilus region of the dentate gyrus exhibits abundant GFAP-staining but low-absent level of MARCKS hybridization,whereas the granule cell layer exhibits weak GFAP-staining but intense MARCKS hybridization (Kalman andHajos, 1989). Furthermore, MARCKS expression, unlikethat of F1/GAP-43, does not correlate well with regionalmyelination patterns (Kapfhammer and Schwab, 1994).

Neurotransmitter systems and MARCKSexpression

The MARCKS protein has been localized to axonalprocesses and synaptosomes (Oiumet et al., 1990; Wang etal., 1989) and its phosphorylation by PKC is correlatedwith neurotransmitter release (Nichols et al., 1987). How-ever, unlike F1/GAP-43 hybridization which is highlyexpressed in the biogenic aminergic neurons of the locuscoeruleus (noradrenaline), dorsal and medial raphe nuclei(serotonin), and substantia nigra pars compacta (dopa-mine; present results; Bendotti et al., 1991; Kruger et al.,1993; Meberg and Routtenberg, 1991; Yao et al., 1993),MARCKS hybridization does not appear to be correlatedwith a specific category of neurotransmitters. MARCKShybridization is present at moderate levels in the locuscoeruleus (Fig. 7A), low levels in raphe nuclei (Fig. 6A),and largely absent from substantia nigral neurons (Fig.5C). Moreover, the major basal forebrain cholinergic cellgroups, nucleus basalis ofMeynert, nucleus of the diagonalband, and medial septum, are largely devoid of MARCKShybridization (Figs. 3C, 4A), yet in the medial habenula,the origin of cholinergic projection neurons to the interpe-duncular nucleus (Woolf, 1991), MARCKS hybridization isvery high (Figs. 4C, 5A). In the hippocampus, both CA3pyramidal cells and dentate gyrus granule cells synthesizeand release glutamate (Fonnum, 1984), yet the formershows only low levels of MARCKS hybridization, whereasthe latter shows high levels. Finally, a substantial numberof neurons in the hilus region of the dentate gyrus areGABAergic (Seress and Ribak, 1983), yetMARCKShybrid-ization is low-absent in this region (Figs. 5A, 6A). There-fore, any general statement regarding the association ofMARCKS with a particular neurotransmitter system iscurrently untenable. Indeed, PKC phosphorylation ofMARCKS (87-kDa) in rat brain synaptosomes is associ-ated with the release of a number of neurotransmitters,includingmonoamines, acetylcholine, and amino acid trans-mitters, and all brain regions tested, including cortex,

hippocampus, and striatum (Nichols et al., 1987), indicatinga more general role for MARCKS in neurotransmission.

F1/GAP-43 distribution

The distribution of F1/GAP-43 mRNA in rat brain islargely in agreement with previous in situ hybridizationstudies assessing the localization of F1/GAP-43 mRNA (Dela Monte et al., 1989; Kruger et al., 1992, 1993; Mahalik etal., 1992; Meberg and Routtenberg, 1991; Verhaagen et al.,1990; Yao et al., 1993). F1/GAP-43 hybridization waspronounced in olfactory bulb mitral cells, anterior olfac-tory nucleus, cerebral cortices, lateral habenular nucleus,select nuclei of the amygdala, thalamus, and hypothala-mus, hippocampal CA3 and hilar neurons, medial genicu-late nucleus, substantia nigra pars compacta, cerebellargranule cells, and many brainstem nuclei, including thedorsal and medial raphe nucleus and locus coeruleus.F1/GAP-43 hybridization was largely absent from thegranule layer of the olfactory bulb, basal ganglia, medialhabenula, hippocampal dentate gyrus granule cell layer,substantia nigra reticular, and cerebellar Purkinje cells. Itis important to note that some previous studies (Kruger etal., 1992, 1993) have observed F1/GAP-43 gene expressionat low levels in the hippocampal granule cell layer, whereasothers (present results; Meberg and Routtenberg, 1991;Yao et al., 1993) have not, and may reflect methodologicaldifferences such as the stringency of the posthybridizationwash. Nevertheless, in the adult rat brain, F1/GAP-43appears to be constitutively expressed at high levelsprimarily in regions associated with a high capacity forsynaptic plasticity.

Comparison of MARCKS and F1/GAP-43distribution

While the comparison of the absolute levels of MARCKSand F1/GAP-43 mRNAs within a given brain region mustbe made cautiously due to the differing kinetics of theMARCKS and F1/GAP-43 riboprobes, some comment canbe made as to their relative distribution in regions whereclear differences exist between their expression levels.Overall, F1/GAP-43 appears to be highly expressed in agreater number of regions than is MARCKS (see Table 1),and within a number of regions, including hippocampalCA3 region, medial geniculate, substantia nigra parscompacta, thalamus, and brainstem, F1/GAP-43 gene ex-pression clearly exceeds that of MARCKS. However,MARCKS hybridization also exceeds that of F1/GAP-43 ina small number of regions, most notably in the olfactorybulb, medial habenular nucleus, cerebellar Purkinje cells,and hippocampal granule cells, where F1/GAP-43 geneexpression is largely absent.As discussed, PKC activation prolongs the half-life of

F1/GAP-43 mRNA (Perrone-Bizzozero et al., 1991, 1993)but reduces the half-life of MARCKS mRNA (Brooks et al.,1991, 1992) in cultured cells without affecting transcrip-tion rates. If PKC isozymes also regulate MARCKS andF1/GAP-43 mRNA expression in the same manner inbrain, then regions with a high level of PKC expressionshould express low levels of MARCKS mRNA and highlevels of F1/GAP-43mRNA, whereas regions with low PKCexpression should express high levels of MARCKS mRNAand low levels of F1/GAP-43 mRNA. Consistent with thisprediction, in hippocampal granule cell bodies, MARCKSmRNA is present at high levels, whereas F1/GAP-43mRNA, as well as a-PKC, bI-PKC, bII-PKC, e-PKC, and


g-PKC immunoreactivity are largely absent. However,hippocampal CA3 pyramidal cells, MARCKS mRNA levelsare low, whereas F1/GAP-43 mRNA levels, e-PKC andg-PKC immunoreactivity is pronounced, and a-PKC andbII-PKC immunoreactivity is low-moderate (Brandt et al.,1987; Hosoda et al., 1989; Ito et al., 1990; Kose et al., 1990;Saito et al., 1993). However, since the inverse patternobserved between MARCKS and F1/GAP-43 gene expres-sion is not observed consistently throughout the brain, itappears unlikely that their expression is regulated solelyby PKC-mediated stabilization and more likely involvesthe coordination of both posttranscriptional and transcrip-tional events.

MARCKS, F1/GAP-43, and neuroplasticity

The high level of MARCKS and F1/GAP-43 gene expres-sion that persists in limbic and integrative cortical regionsfollowing development suggests an on going role in neuro-plastic events in these regions, possibly relating to informa-tion storage processes (Nelson et al., 1987; Sheu et al.,1993). Indeed, phosphorylation by PKC of F1/GAP-43 andan 80-kDa (pI 4.0) protein is elevated in the hippocampusfollowing the induction of long-term potentiation (Nelsonet al., 1989), an electrophysiological model of informationstorage processes (Bliss and Collingridge, 1993). In thisregard, phosphorylation of MARCKS by PKC prevents itscapacity to cross-link F-actin (Hartwig et al., 1992) whichmay alter the cytoskeletal rigidity at targeted sites withinthe plasmamembrane required for the structuralmodifica-tions in pre- and postsynaptic terminals observed afterLTP (Desmond and Levy, 1988). Collectively, these datasupport the notion thatMARCKS and F1/GAP-43 continueto mediate the actions of PKC in the regulation of neuro-plastic events in select neurons and regions of the maturebrain.

ACKNOWLEDGMENTS

The authors are grateful to Dr.A.Aderem for the murineMARCKS cDNA subclone, Dr. A. Routtenberg for theF1/GAP-43 cDNA subclone, and Dr. D.J. Stumpo for thepMG80K2.4 DNA. The authors also gratefully acknowl-edge Dr. D.G. Watson for his help in preparing the cDNAsubclones and B. Olarte and S. Sweeny for their technicalassistance. Research was supported in part by grants fromNIMH (MH-50105) and The Stanley Foundation to R.H.L.and a MRC/CIBA-GEIGY Fellowship to R.K.M.

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comparative distribution of myristoylated alanine-rich c kinase substrate (marcks) and f1/gap-43...

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