astroglial ciliary neurotrophic factor mrna expression is increased in fields of axonal sprouting in...

Astroglial Ciliary NeurotrophicFactor mRNA Expression Is Increased

in Fields of Axonal Sproutingin Deafferented Hippocampus

KATHLEEN M. GUTHRIE,1* ALISA G. WOODS,2 THOMAS NGUYEN,1

AND CHRISTINE M. GALL1,2

1Department of Anatomy and Neurobiology, University of California at Irvine,Irvine, California 92967

2Department of Psychobiology, University of California at Irvine, Irvine, California 92967

ABSTRACTEvidence that ciliary neurotrophic factor promotes axonal sprouting and regeneration in

the periphery raises the possibility that this factor is involved in reactive axonal growth in thebrain. In situ hybridization was used in the present study to determine whether ciliaryneurotrophic factor mRNA expression is increased in association with axonal sprouting indeafferented adult rat hippocampus. In untreated rats, ciliary neurotrophic factor cRNAlabeling density was high in the olfactory nerve, pia mater, and aspects of the ventricularependyma and was relatively low within areas of white matter (fimbria, internal capsule) andselect neuronal fields (hippocampal cell layers, habenula). After an entorhinal cortex lesion,hybridization was markedly increased in fields of anterograde degeneration, including mostprominently the ipsilateral dentate gyrus outer molecular layer and hippocampal stratumlacunosum moleculare. Labeling in these fields was increased by 3 days postlesion, wasmaximal at 5 days, and returned to normal levels by 14 days. Double labeling demonstratedthat, in both control and experimental tissue, ciliary neurotrophic factor mRNA wascolocalized with glial fibrillary acidic protein immunoreactivity in astroglia, but it was notcolocalized with markers for oligodendrocytes or microglia. These results demonstrate thatastroglial ciliary neurotrophic factor expression is increased in fields of axonal and terminaldegeneration and that increased expression is coincident with 1) increased insulin-like growthfactor-1 and basic fibroblast growth factor expression and 2) the onset of reactive axonalgrowth. The synchronous expression of these glial factors in fields of deafferentation suggeststhe possibility of additive or synergistic interactions in the coordination of central axonalgrowth. J. Comp. Neurol. 386:137–148, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: gliosis; perforant path; astrocytes; insulin-like growth factor-1; basic fibroblast

growth factor; ciliary neurotrophic factor

The adult nervous system exhibits a wide range ofplastic responses to traumatic injury, including the sprout-ing of intact axons in regions of deafferentation. A well-characterized example of this is the collateral sprouting ofcommissural/associational afferents into hippocampal den-dritic fields deafferented by ablation of the primary inner-vation from the entorhinal cortex (Zimmer, 1973; Lynch etal., 1975; Gall et al., 1986). The afferents to the dentategyrus granule cells are normally distinctly laminatedwithin the stratum moleculare; the outer two-thirds isdensely innervated by perforant path axons arising fromipsilateral entorhinal cortex, whereas the proximal one-

third is innervated by commissural and associationalafferents arising from polymorph neurons of the contralat-eral and ipsilateral hilar regions, respectively (Gall, 1990;

Grant sponsor: NIA; Grant number: AG00538; Grant sponsor: NIMH;Grant numbers: MH00974, MH11279; Grant sponsor: NSF; Grant number:BNS 9024143.K.M.G. andA.G.W. contributed equally to the work presented here.*Correspondence to: K.M. Guthrie, Department ofAnatomy andNeurobi-

ology, University of California at Irvine, Irvine, CA92697-1275.E-mail: [email protected] 4 October 1996; Revised 3April 1997; Accepted 25April 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 386:137–148 (1997)

r 1997 WILEY-LISS, INC.

Amaral and Witter, 1995). In the adult rat, removal ofentorhinal input results in sprouting of the commissural/associational projection within the denervated middle andouter molecular layers, with the most robust phase ofgrowth occurring from 5 to 14 days postlesion (Lee et al.,1977; Lynch et al., 1977; Gall and Lynch, 1981; Stewardand Vinsant, 1983; Gall et al., 1986).The specific processes that initiate the sprouting re-

sponse are not known, but possible mechanisms are sug-gested by the cascade of cellular events triggered bydeafferentation, including the proliferation, migration,and hypertrophy of resident glial cells; the removal ofdegenerating axons and terminals; and the subsequentappearance of vacant synaptic space. Microglia and astro-cytes undergo dramatic morphological and histochemicalchanges just prior to the onset of sprouting and accumu-late in laminae within which sprouting occurs (Gall et al.,1979, 1986; Gehrmann et al., 1991; Jensen et al., 1994).This has given rise to hypotheses that glial activitiesfacilitate or stimulate reactive axonal growth (Lynch et al.,1975; Gall et al., 1979, 1986; Goldowitz and Cotman, 1980;Fagan and Gage, 1994). Evidence that glial-derived tro-phic substances can promote axonal growth has come fromstudies of sprouting and regeneration in the peripheralnervous system. Candidate factors include the insulin-likegrowth factors (IGFs), basic fibroblast growth factor(bFGF), nerve growth factor (NGF), and ciliary neuro-trophic factor (CNTF). Application of exogenous IGFs(Caroni and Grandes, 1990), CNTF (Kwon and Gurney,1994), and bFGF (Gurney et al., 1992) promotes paratermi-nal sprouting of intact motor axons in vivo and canfacilitate axonal regeneration following sciatic nerve tran-section or crush (Kanje et al., 1989; Ishii et al., 1994;Laquierriere et al., 1994; Sahenk et al., 1994). This type ofinjury produces an up-regulation of endogenous IGF-1 andNGF expression by Schwann cells during the period ofaxon regeneration (Heumann et al., 1987; Johnson et al.,1988; Glazner et al., 1994; Ishii et al., 1994). In contrast,Schwann cell CNTF expression declines after nerve tran-section/crush, although CNTF immunoreactivity becomesredistributed to extracellular sites in the vicinity of theinjury, which may increase its availability to regeneratingaxons (Friedman et al., 1992; Sendtner et al., 1992).Agoal of studies in this laboratory has been to determine

whether trophic factors implicated in peripheral nerveregeneration are expressed in association with axonalsprouting in the brain. In a previous report, we demon-strated that entorhinal cortex ablation induces a markedincrease in the expression of IGF-1 by reactive microglia indeafferented rat hippocampus (Guthrie et al., 1995). Theincrease in IGF-1mRNAexpression occurs just prior to theonset of sprouting and is restricted to fields into whichsprouting afferents grow. Expression of bFGF by astro-cytes in the denervated hippocampus is also increased,althoughwith a different time course and broader distribu-tion (Gomez-Pinilla et al., 1992; Guthrie et al., 1995). Thepresent study evaluated CNTF expression in the deaffer-ented hippocampus during the period of axon sprouting. Insitu hybridization of [35S]-labeled CNTF cRNAwas used todescribe the normal distribution of CNTF mRNA in adultrat forebrain and to characterize changes in this expres-sion following lesion of the entorhinal cortex.

MATERIALS AND METHODS

Animal treatments

Adult male Sprague-Dawley rats were used (SimonsenLabs, Gilroy, CA; 250–350 g). Experimental rats wereanesthetized with a mixture of ketamine (50 mg/kg) andxylazine (10 mg/kg), and an electrolytic lesion was placedin the right entorhinal cortex with a stainless steel wireand anodal current, as described previously (Guthrie et al.,1995). For analysis of the time course of changes in CNTFmRNAexpression, rats with entorhinal lesions were killed1 (n 5 5), 3 (n 5 5), 5 (n 5 5), 7 (n 5 5), 10 (n 5 4), and 14(n 5 5) days postlesion (dpl). Control rats were anesthe-tized as above and then killed 3 (n 5 2), 5 (n 5 2), and 14(n 5 1) days later. To evaluate the cellular localization ofCNTF mRNA relative to glial markers, rats receivedlesions as described above and were killed 3 (n 5 2) and 5(n 5 7) dpl. Finally, to control for the possible contributionof epileptiform activity, which can arise with stainlesssteel wire lesions, rats received entorhinal cortex lesionswith a platinum-iridium wire (Pico and Gall, 1994; n 5 4).All rats were killed by an overdose of sodium pentobarbital(100 mg/kg) and transcardially perfused with 0.9% NaCl,followed by 4% paraformaldehyde in 0.1 M phosphatebuffer, pH 7.4 (PB). Brains were dissected out of thecranium, postfixed in 4% paraformaldehyde in PB over-night at 4°C, and then cryoprotected in 20% sucrose in 4%paraformaldehyde/PB for 2 days at 4°C. All animal treat-ments were carried out according to protocols approved bythe Institutional Animal Care and Use Committee at theUniversity of California, Irvine.

In situ hybridization

The [35S]-labeled or digoxigenin (DIG)-labeled cRNAprobes for rat IGF-1, rat CNTF, and mouse proteolipidprotein (PLP) were prepared from cDNA templates, asdescribed previously (Gall et al., 1994, 1995; Bizon et al.,1996). The IGF-1 cRNA was transcribed from the EcoR1digested rat cDNA construct by using T7 RNA polymeraseand contains 322 bases complementary to positions 14–336 of the sequence published by Shimatsu and Rotwein(1987). The CNTF cRNA was generated from PvuII di-gested cDNA construct (kindly provided by Dr. D. Lind-holm) by using T3 RNA polymerase; this generates a600-base probe complementary to the full coding sequenceof rat CNTF (Stockli et al., 1989). The PLP cRNA wastranscribed from a Bgl II digested cDNA construct (kindlyprovided by Dr. L. Hudson) by using T3 polymerase; thisgenerates a 980-base antisense sequence corresponding tothe full coding sequence of mouse PLP (Hudson et al.,1987). Sense probes were generated from the IGF-1 andCNTF cDNAs by using SP6 and T7 RNA polymerases,respectively.Free-floating tissue sections were processed for single-

label in situ hybridization of [35S]-labeled cRNAs, asdescribed in detail elsewhere (Gall et al., 1995), withhybridization carried out at 60°C for 36–44 hours and theradiolabeled cRNA added to a final concentration of 1 3107 cpm/ml. Following hybridization, slide-mounted tissuewas processed for film (b-Max Hyperfilm; Amersham,Arlington Heights, IL) and emulsion (Kodak NTB2, Roch-ester, NY) autoradiography with exposure times of 3–5days and 3–4 weeks, respectively. Following developmentof emulsion autoradiography (Kodak D19 developer 1:1

138 K.M. GUTHRIE ET AL.

with H20), the tissue was stained with cresyl violet andcoverslipped with Permount.Levels of radiolabeled cRNA hybridization were quanti-

fied by densitometric analysis of film autoradiograms byusing the MCID imaging system (Imaging Research, St.Catherines, Ontario, Canada) and were calibrated relativeto radiolabeled brain paste standards exposed to eachsheet of film (for details, see Gall et al., 1994). For eachregion evaluated, measurements were taken from sixsections from each animal. Hybridization densities wereevaluated for the dentate gyrus granule cell layer, innerone-third of the molecular layer, outer half of the molecu-lar layer, and hilus; the stratum lacunosum moleculareand stratum radiatum of region CA1; and the alveus. Withthe exception of the hilus, measurements were collectedfrom a continuous, 1-mm-wide field aligned with thecenter of the internal blade of the stratum granulosum.For the hilus, measurements were collected from multiplesmaller zones centered on the deeper portion of the hilusand avoiding region CA3c. For experimental animals,separate measurements were collected from hippocampiipsilateral and contralateral to the lesion. In each case,measurements from individual tissue sections were aver-aged to provide animal means; these values were thenaveraged to provide group means 6 standard error. Thesignificance of the effect of treatment was evaluated byusing the one-way analysis of variance (ANOVA). Becausesignificant differences in the standard deviations betweengroups precluded the use of standard post-hoc texts,Welch’s two-tailed t test was used for individual post-hoccomparisons (Motulsky, 1995). Although some measuresare illustrated as percent of measures from control rats, allstatistical analyses used raw group mean values; differ-ences considered to be significant had P values of 0.05 orless.

Colocalization with glial markers

Tissue sections were processed for the combined localiza-tion of CNTF mRNA and glial markers, including glialfibrillary acidic protein (GFAP) immunoreactivity(GFAP-IR) to identify astrocytes, Bandeiraea simplicifoliaB4 isolectin (BS-1) binding to identifymicroglia (Streit andKreutzberg, 1987), and PLP cRNA hybridization to labeloligodendroglia (Shiota et al., 1989). Tissue processed forastroglial and microglial labeling was first processed forCNTF cRNAhybridization (described above), rinsed for 30minutes in PB, and then incubated overnight at 4°C inrabbit anti-GFAP (1:500; DAKO, Carpenteria, CA) or BS-1lectin (15 µg/ml; Sigma, St. Louis, MO) in PB containing0.3% Triton X-100 and 3% normal goat serum. Tissueincubated with anti-GFAP was then rinsed in PB, incu-bated in biotinylated goat anti-rabbit IgG, and processedfor the localization of antibody binding with the avidin-biotin technique and diaminobenzidine (DAB) as the chro-mogen, using kit reagents according to the manufacturer’sinstructions (Vector Laboratories, Burlingame, CA). Tis-sue incubated with lectin was processed identically, exceptthat the incubation in secondary antibody was omitted.Following the DAB reaction, tissue was processed foremulsion (Kodak NTB2) autoradiography, as describedabove. The relative localization of autoradiographic grains,indicative of [35S]-CNTF cRNA hybridization, and thebrown DAB reaction product, indicative of GFAP-IR orBS-1 binding, was assessed by microscopic examinationwith brightfield and darkfield microscopy.

Double in situ hybridization with DIG-labeled PLPcRNA to identify oligodendroglia and [35S]-labeled CNTFcRNA to identify CNTF mRNA expression was conductedas described in detail elsewhere (Bizon et al., 1996), withprobes added to the hybridization incubation at a concen-tration of 1 3 107 cpm/ml for CNTF cRNA and a 1:1,000dilution of DIG-labeled PLP cRNA. The DIG cRNA waslocalized by using alkaline phosphatase-conjugated anti-DIG IgG (Boehringer Mannheim, Indianapolis, IN) andnitroblue tetrazolium as chromagen, which yields a bluereaction product in the DIG-cRNA-labeled cells. Followingthe color reaction, the tissue sections were processed foremulsion autoradiography by using Ilford K5.d emulsionand a 5-week exposure interval.

RESULTS

Distribution of CNTF mRNAin untreated adult rat brain

In agreement with previous reports, CNTFmRNAlevelsappeared to be fairly low throughout the forebrain (Fig. 1)with the exception of a few areas enriched in nonneuronalcells. Labeling was dense within the olfactory nerve layer(Fig. 1B), the pineal gland and pia mater (Fig. 1C), andalong limited aspects of the ventricular ependyma (Fig.1E). With regard to the latter, the superior aspect of thethird ventricle was densely labeled, whereas the inferiorportion of more densely Nissl-stained cells was not (Fig.1F).CNTF mRNA did not appear to be expressed at particu-

larly high levels in the optic chiasm or the optic tract, but itwas localized to a few scattered cells in these regions aswell as within the fimbria, alveus, and internal capsule(Fig. 1D). There was low-density labeling in some regionsof neuronal cell bodies, including the lateral habenula(Fig. 1E), the pyramidal and granule cell layers of hippo-campus (Figs. 1A, 2A), and layer II of the entorhinal cortex(Fig. 2A). Finally, in addition to discrete cellular labeling,hybridization of the CNTF cRNA resulted in higher thanbackground levels of autoradiographic grains diffuselydistributed over most regions of neuropil compared withtissue processed with the sense riboprobe.

CNTF cRNA hybridization followingentorhinal cortex lesions

The horizontal tissue sections in Figure 2 show that theelectrolytic lesion generally ablated the medial entorhinalcortex and, to a variable extent, portions of the lateralentorhinal cortex, presubiculum, and parasubiculum. Le-sion-induced changes in hybridization were first observedat 3 dpl. Figure 2B,E shows that, at this time point,labelingwas increasedmost prominently within the ipsilat-eral dentate gyrus middle molecular layer, the stratumlacunosum moleculare, and alveus and, in these regions,appeared as clusters of autoradiographic grains. Fewergrain clusters were evident within the ipsilateral dentategyrus inner molecular layer. Very small increases inhybridization were also evident within these same lami-nae in the hippocampus contralateral to the lesion. Inaddition, at 3 dpl, labeling was elevated in other areasexpected to contain axonal degeneration, including thefimbria and the subcortical white matter; in these regions,hybridization was increased bilaterally, although labelingdensities were greater on the ipsilateral side. Finally, the

DEAFFERENTATION INCREASES ASTROGLIAL CNTF mRNA 139

granule and pyramidal cell layers of the hippocampus,which were labeled by very low densities of autoradio-graphic grains in control tissue, appeared to be moredensely labeled in tissue from rats killed at 3 dpl (Fig. 2B).Labeling was not elevated around the lesion cavity at thistime point.In tissue from rats killed at 5 dpl, CNTF cRNAhybridiza-

tion was further increased within the ipsilateral dentategyrus outer molecular layer and stratum lacunosum mo-leculare (Figs. 2C,F, 3), but grain clusters were observedless frequently in the inner molecular layer. In the contra-lateral hippocampal molecular layers, labeling appearedto be less dense at 5 dpl than at 3 dpl, but it was stillgreater than in tissue from control rats. Labeling patternsin the ipsilateral alveus, internal capsule, fimbria andhippocampal neuronal layers appeared comparable intissue from rats killed at 3 and 5 dpl. At the 5-day timepoint, a few very densely labeled cells were scattered in theproximity of the lesion cavity (Fig. 2C). Rats killed 5 daysafter lesion with platinum wire exhibited similar patternsof CNTF cRNA labeling (data not shown).

By 7 dpl, there were greater numbers of densely labeledcells in the region surrounding the lesion cavity. Theselabeled cells were distributed near the wound, but not atits very edges. By 10 dpl, labeling appeared to havereturned to control levels in all regions except the areaaround the lesion (Fig. 2D, arrows). At 14 dpl, labeled cellswere still seen along the edges of the lesion cavity.Densitometric measures of film autoradiograms corrobo-

rated our qualitative observations. Hybridization densi-ties were measured for the dentate gyrus inner molecularlayer, outer molecular layer, and granule cell layer as wellas for the hilus, CA1 stratum radiatum, and alveus. Figure4 (top graph) shows the time course of changes in labelingdensity for the ipsilateral dentate gyrus and stratumradiatum. The lesion induced significant changes in hybrid-ization in the outer molecular layer (P , 0.0001), the innermolecular layer (P , 0.05), and the granule cell layer (P ,0.05) but not in the stratum radiatum (ANOVA; comparedwith control rat values). Among the areas measured, thelargest increase occurred in the outer molecular layer. Inthis lamina, mean hybridization density was increased

Fig. 1. Darkfield photomicrographs illustrating the distribution of[35S]-labeled ciliary neurotrophic factor (CNTF) cRNAhybridization inuntreated rat brain. A: Coronal section showing labeling of thehabenula (hb) and the hippocampal pyramidal cell layer (p). B: Sagittalsection showing dense labeling of the olfactory nerve layer (arrow)encapsulating the olfactory bulb. C: Horizontal section showing densecRNA hybridization in the pineal gland (arrow) and pia matter(arrowhead) overlying the superior colliculus. D: CNTF mRNA is

expressed by cells (arrows) within the internal capsule (cortex is to theright of the arrow). E,F: Dense hybridization occurs in aspects of theventricular ependyma (arrowheads in E) including the span betweenthe habenulae (hb) and the dorsal aspect of the third ventricle (arrowin F). In contrast, the lining of the inferior aspect of the third ventricle(F, arrowhead) is not labeled. Scale bar 5 1.5 mm in A, 1.3 mm in B,750 µm in C, 440 µm in D–F.


Fig. 2. A–F: CNTF cRNA hybridization is increased followingentorhinal cortex lesion. Darkfield photomicrographs showing CNTFcRNA labeling in horizontal tissue sections through the hippocampusand retrohippocampal area of an untreated rat (A) and of rats killed 3days (B,E), 5 days (C,F), and 14 days (D) after an ipsilateral entorhinalcortex lesion. B–D show the field of entorhinal cortex ablation. A: Inthe control hippocampus, CNTF cRNA labeling appears in the granule(g) and pyramidal (p) cell layers of hippocampus, as well as in layer IIof entorhinal cortex (ec); the asterisk indicates the location of therhinal fissure separating neocortex and entorhinal cortex. B,E: At 3

days postlesion (dpl), labeling is elevated in the alveus (a), stratumlacunosum moleculare (arrow in B), and the molecular (ml) andgranule cell layers of the dentate gyrus. C,F: At 5 dpl, hybridization isfurther elevated in stratum lacunosum moleculare (arrow in C) andwithin the outer molecular layer (ml). Arrowheads in E and F indicateindividual labeled cells in the molecular layer. D: By 14 dpl, labelinghas returned to control levels with the exception of some denselylabeled cells located along the edge of the wound cavity (arrows). Scalebar 5 660 µm inA–D, 200 µm in E,F.


significantly at 3 dpl, peaked at about threefold controlvalues at 5 dpl, and began to decline by 7 dpl. In contrast,granule cell labeling was elevated modestly but signifi-cantly at both 1 dpl and 3 dpl and declined thereafter.Lesion-induced changes in hybridization within the

ipsilateral stratum lacunosum moleculare were similar inmagnitude and time course to those within the dentategyrus outer molecular layer, with densities significantlygreater than values from control rats at 3, 5, and 7 dpl (P,0.05; Welch’s two-tailed t test). Within the hilar region,labeling was not increased significantly above controlvalues at any time point examined.Figure 4 (bottom graph) illustrates the increases in

hybridization densities seen across the principle hippocam-pal laminae at 5 dpl. In this graph, measures fromexperimental tissue are expressed as percent of measuresof these same laminae in tissue from paired control rats.Hybridization was significantly increased within the ipsi-lateral dentate gyrus outer molecular layer and stratumlacunosum moleculare (P , 0.05 and P , 0.01, respec-tively; Welch’s two-tailed t test using raw group meanvalues). Lesser increases were evident in the alveus andgranule cell layer. At this time point, the only significantcontralateral effects were small increases in labelingwithinstratum granulosum and the outer molecular layer (P ,0.05). Significant differences from control levels (not shown)also occurred at 3 dpl in the ipsilateral outer molecularlayer, stratum lacunosum moleculare, stratum granulo-sum (P, 0.05), and alveus (P, 0.005) and at 7 dpl in thesesame areas as well as in the inner molecular layer (P ,0.05).

Comparison to IGF-1 mRNA expression

Previous work has demonstrated that entorhinal deaffer-entation induces a large increase in the expression of

IGF-1 mRNA in the ipsilateral hippocampus (Guthrie etal., 1995). To directly compare changes in IGF-1 and CNTFmRNAcontent, alternate tissue sections from 25 rats wereprocessed for in situ hybridization to IGF-1 cRNA. Figure5 shows photomicrographs comparing the distribution ofCNTF and IGF-1 mRNAs in the hippocampus of controlrats (Fig. 5A,C) and rats killed at 5 dpl (Fig. 5B,D). Figure6 shows the quantitative analysis of changes in IGF-1 andCNTF cRNA hybridization within the outer molecularlayer. Increases in IGF-1 and CNTF mRNA levels, asshown, follow a similar time course, with peak valuesattained at 5 dpl and labeling declining rapidly thereafter.Expression of these two factors in the wound area followeddifferent time courses. IGF-1 mRNA was increased asearly as 3 dpl and was no longer detectable by 10 dpl,whereas CNTF mRNA was not increased until 5 dpl andremained elevated through 14 dpl. There were also differ-ences in the distribution of maximal increases in IGF-1and CNTF mRNA levels in experimental lesion rats. Thelargest increases in IGF-1 mRNAwere localized to fields ofterminal degeneration in the ipsilateral outer molecularlayer. CNTFmRNAwasmarkedly increased in this laminaas well, but equivalent increases were observed in regionsknown to contain damaged fibers of passage, including thestratum lacunosum moleculare, the alveus, and the fim-bria (Fig. 5B,D).

Colocalization with glial markers

The distribution of lesion-induced increases in CNTFmRNA within deafferented hippocampal laminae sug-gested that these changes were localized to reactive glialcells. To evaluate this possibility, tissue sections fromcontrol rats and rats killed at 5 dpl were processed for theautoradiographic localization of 35S-labeled CNTF cRNAhybridization in combination with the histochemical iden-

Fig. 3. Laminar distribution of CNTF cRNA hybridization inhippocampus. Photomicrographs of comparable hippocampal fieldsshowing Nissl staining (A) and CNTF cRNA labeling (B,C). A and Bshow Nissl staining and CNTF hybridization, respectively, in thehippocampus of a control rat. C shows CNTF cRNA labeling inhippocampus ipsilateral to entorhinal cortex ablation at 5 dpl. Withinthe ipsilateral hippocampus (C), CNTF cRNA labeling is substantially

increased in the outer molecular layer (oml), the stratum lacunosummoleculare (slm), and the alveus (a). In addition, small increases canbe seen in the pyramidal cell layer (p) of the deafferented hippocampus(C). sr, CA1 stratum radiatum; g, granule cell layer; h, hilus. Arrow-head indicates the position of the hippocampal fissure. Scale bar 5250 µm.


tification of 1) GFAP-IR astrocytes, 1) BS-1-labeledmicrog-lial cells, or 3) PLPmRNA-containing oligodendroglia.In tissue from both control and experimental rats, the

CNTF cRNA labeled GFAP-IR astrocytes. Double-labeledcells were observed in most regions of neuropil containingmoderate levels of hybridization, including the optic tract(Fig. 7A), the glial limitans (Fig. 7B), the choroid fissure(Fig. 7C), the fimbria (Fig. 7D), and the internal capsule.In tissue from experimental rats, CNTF cRNA labeledGFAP-IR astrocytes in the ipsilateral outer molecularlayer (Fig. 7E,F), stratum lacunosum moleculare (Fig.7G,H), and neocortex (Fig. 7I). In contrast, CNTF mRNA

was never localized to BS-1-labeled microglial cells (Fig.7K). Similarly, in sections processed for autoradiographiclocalization of CNTFmRNAand colormetric localization ofPLPmRNA, clustered autoradiographic grains were neverobserved overlying darkly stained oligodendrocytes(Fig. 7J).

DISCUSSION

The present results describe the distribution of CNTFmRNA in adult rat forebrain and demonstrate that CNTFexpression is increased in regions of a direct traumaticwound and anterograde degeneration. In the untreatedadult rat, dense hybridization of CNTF cRNA was local-ized to the olfactory nerve, pineal gland, pia mater, andportions of the ventricular ependyma. Lower hybridizationdensities labeled cells in the glial limitans; in areas ofwhite matter, including the optic nerve, fimbria, internalcapsule, and alveus; and in neuronal fields, including themedial habenula, hippocampal pyramidal and granule celllayers, and layer II of the olfactory cortex. This labelingpattern is similar to that described by Thoenen andcolleagues for transgenic mice in which the CNTF codingregion was replaced by a b-galactosidase reporter geneunder control of CNTF promoter elements (Carroll et al.,1995). These data indicate that both neuronal and glialpopulations express CNTF mRNA but, in most instances,at very low levels.Previous Northern blot studies have shown low levels of

CNTF mRNA throughout much of the rat brain, withparticularly high levels in the olfactory bulb and opticnerve (Stockli et al., 1991). These results are generallyconsistent with our data, although only low levels of CNTFmRNA were detected in optic nerve by in situ hybridiza-tion. The higher levels of expression localized to the pialsurface of the nerve and at the choroid fissure mightaccount for some of the message detected in blot studies.Some areas in which CNTF mRNA is detectable by North-ern blot analysis (e.g., striatum and septum; Stockli et al.,1991) did not exhibit distinct cellular labeling with in situhybridization. However, in these and most other areas ofgray matter, there was diffuse, low-density autoradio-graphic labeling similar to that seenwith in situ hybridiza-tion of bFGF cRNA (Gall et al., 1994). In both instances,this would be consistent with low levels of expression byglial cells.Although CNTF mRNA is normally expressed at very

low levels in adult rat brain, we found that injury leads to adramatic increase in its expression in regions that containneuronal degeneration. After unilateral entorhinal cortexablation, CNTF cRNA hybridization was prominently in-creased in the ipsilateral dentate gyrus molecular layer,stratum lacunosum moleculare, and alveus, all regionsdensely occupied by aspects of the degenerating perforant-path and temperoammonic systems (Hjorth-Simonsen,1972; Gall, 1990), as well as in fields expected to containdegenerating cortical axons due to damage from electrodeplacement. Smaller increases in CNTF mRNA withincontralateral hippocampal stratum lacunosummolecularecorrespond with the lower density of crossed temperoam-monic projections to this lamina (Goldowitz et al., 1975;Gall, 1990). Within the ipsilateral hippocampus, CNTFexpression was clearly increased at 3 dpl, reached maxi-mal levels at 5 dpl, and returned to control levels by 10 dpl.In contrast, increased cRNA labeling in the region of the

Fig. 4. Quantification of lesion-induced changes in CNTF cRNAhybridization. Top: Graph showing changes in the density of CNTFcRNA labeling within major laminae of hippocampus ipsilateral toentorhinal cortex lesion over a range of postlesion time points. Values(group means 6 standard error) are shown for the granule cell layer(gcl), outer third of molecular layer (oml), inner third of molecularlayer (iml), and stratum radiatum (sr). Significant changes in hybrid-ization density occurred in the outer molecular layer (P , 0.0001),inner molecular layer (P , 0.05), and granule cell layer (P , 0.05) butnot in stratum radiatum (ANOVA; compared with control rat values).Bottom: Bar graph showing the distribution of hybridization acrossmajor hippocampal laminae ipsilateral (ipsi.) and contralateral (con-tra.) to an entorhinal cortex lesion placed 5 days before sacrifice. In thetop graph, asterisks indicate P , 0.05 compared with measures fromcontrol rats (Welch’s two-tailed t test using raw group mean values).


lesion cavity was first apparent at 5 dpl and was stillevident at 14 dpl.Regions containing the largest increases in CNTF cRNA

hybridization are populated primarily by reactive glialcells, making these a likely source of CNTF expression.With the exception of neuronal populations, our double-labeling preparations demonstrated that, in both controland experimental tissue, cells labeled by CNTF cRNAwerealso GFAP-IR, indicating that astrocytes normally expressthis neurocytokine and increase levels of expression inregions of axonal and terminal degeneration 1. Microglialcells and oligodendrocytes did not express CNTF after thelesion, as demonstrated by the failure to double labelCNTF mRNA-expressing cells with BS-1 lectin or withPLP cRNA. These results corroborate the observations ofIp et al. (1993b), who found coincident distributions of

1After submission of this paper, a study by Lee et al. (1997) was publishedreporting that CNTF immunoreactivity was elevated within astroglial cellsin the deafferented dentate gyrus molecular layer; this increase was firstevident at 3 days and was maximal at 7 days after an entorhinal cortexlesion. These results complement very nicely our own and indicate thatlesion-induced increases in astroglial CNTF mRNA (reported here) arerapidly translated and give rise to increases in CNTF protein. Lee et al. alsoreported that CNTFa receptor mRNA levels are increased in hippocampalastroglia during the same interval. This finding is consistent with the ideathat the newly expressed CNTF has, at least in part, autocrine or paracrineeffects on astroglial cells within the deafferented field.

Fig. 5. Increases in CNTF mRNA and insulin-like growth factor(IGF-1) mRNA overlap in deafferented hippocampus. Darkfield photo-micrographs showing the distribution of CNTF cRNA (A,B) and IGF-1cRNA(C,D) hybridization in the hippocampus of control rats (A,C) andpaired experimental rats killed at 5 dpl (B,D; coronal sections). In thecontrol rat, CNTF cRNA (A) labels the granule (g) and pyramidal celllayers (p), whereas IGF-1 cRNA (C) labels scattered cells, primarilylocated in the alveus (a) and subgranular hilus (h). At 5 dpl, both

CNTF cRNA (B) and IGF-1 cRNA (D) hybridization are increased inthe outer molecular layer (arrows) and stratum lacunosummoleculare(slm); one can see that IGF-1 cRNAhybridization density is greatest inthe outer molecular layer, whereas the density of CNTF cRNA labelingis comparably increased in these two laminae. Slight increases inexpression of both mRNAs are evident in the overlying neocortex(B,D). Scale bar 5 750 µm.

Fig. 6. Graph comparing the postlesion time courses of increasedCNTF and IGF-1 cRNA labeling in the outer molecular layer ofdeafferented hippocampus. Alternate tissue sections from the sameratswere processed for in situ hybridization and quantitative densitom-etry; values are given as group means 6 standard error. Note thatincreases in the expression of these two factors follow a similarpostlesion time course, with peak levels attained at 5 dpl (*, P , 0.05;Welch’s two-tailed t test).


CNTF mRNA and GFAP-IR in hippocampal and corticalareas damaged by aspirative lesion. Previous studies havealso colocalized CNTF and GFAP-IR in the optic andolfactory nerves in vivo (Stockli et al., 1991; Dobrea et al.,1992), and cultured astrocytes have been shown to express

high levels of CNTF mRNA and protein (Stockli et al.,1991; Rudge et al., 1992). The observation that CNTFcRNA labeling is slightly increased in neuronal layerswithin the hippocampus ipsilateral to the lesion indicatesthat either deafferentation or trophic interactions within

Fig. 7. CNTF mRNA is expressed by astrocytes. In A–I, [35S]-labeled CNTF cRNA hybridization is indicated by autoradiographicgrains, and glial fibrillary acidic protein immunoreactivity (GFAP-IR)appears as brown staining. Cells double labeled by CNTF riboprobeand GFAP antibody are seen in the optic tract (A, arrowheads), theglial limitans (B), and at the choroid fissure (C). At 5 dpl, the CNTFcRNA labels GFAP1 astrocytes (arrowheads) in the fimbria (D), outermolecular layer (E,F), stratum lacunosum moleculare (G,H), andneocortex (I). G and H show the same astrocyte in two different focalplanes to optimize visualization of the autoradiographic labeling (G)

and GFAP immunostaining (H) of a single cell. J: Oligodendrocyteslabeled with digoxigenin (DIG)-labeled mouse proteolipid protein(PLP) cRNA (purple staining, arrowheads) are not labeled by CNTFcRNA, as indicated by the absence of overlying silver grains. Clustersof silver grains indicate CNTF mRNA expression by nearby cells(arrows) that are not labeled by DIG-PLP cRNA. K:Microglia labeledby Bandeiraea simplicifolia B4 isolectin (BS-1; brown staining, arrow-heads) are not autoradiographically labeled by CNTF cRNA. Arrowsindicate cells labeled by CNTF cRNA that are not labeled by BS-1lectin. Scale bar 5 30 µm inA–C, 22 µm in D–H, 20.5 µm in I–K.


the deafferented field induce a modest increase in neuro-nal CNTF expression as well.CNTF, bFGF, and IGF-1, as described above, are of

particular interest with regard to reactive axonal growthdue to demonstrated effects on axonal sprouting andregeneration in the periphery. Local or systemic treatmentwith exogenous IGF-1, CNTF, and bFGF stimulates sprout-ing of intact somatic motor axons (Caroni and Grandes,1990; Gurney et al., 1992; Kwon and Gurney, 1994).Sensory neurons injured by sciatic nerve crush can retro-gradely transport exogenously supplied CNTF (Curtis etal., 1993), and systemic treatment of rats with CNTF aftersciatic nerve transection promotes regenerative growth ofaxon tips into the distal nerve stump (Sahenk et al., 1994).These results and the present demonstration of increasedCNTF expression in deafferented hippocampus suggestthat CNTF may play a role in reactive sprouting. Intactcommissural/associational and septal afferents sprout intothe deafferented dentate gyrus molecular layer with arapid phase of axonal growth and synaptogenesis thatoccurs from 5 to 14 dpl (Lee et al., 1977; Lynch et al., 1977;Steward and Vinsant, 1983) and continued slower reinner-vation for several weeks thereafter (Matthews et al., 1976;Gall et al., 1986). Our results show that increased CNTFexpression coincideswith the period of hippocampal sprout-ing, occurring just prior to and during the phase ofmaximal axonal growth, and is localized to cells that, tolarge extent, are distributed within the zones that receivesprouting afferents. In situ hybridization studies havedemonstrated that hippocampal neurons express theCNTFa receptor subunit, which binds and confers respon-siveness to CNTF (Ip et al., 1993a; Rudge et al., 1994),indicating their potential stimulation by CNTF underconditions of increased availability.There is evidence to suggest that IGF-1 and bFGF

promote axonal growth in the brain as well as in theperiphery (Bondy, 1991; Miyagawa et al., 1993; Miyamotoet al., 1993). Entorhinal lesion induces significant in-creases in IGF-1 and bFGF mRNA levels in the deaffer-ented molecular layer, with maximal values attained at 5dpl (Guthrie et al., 1995). The overlapping distribution andsynchronous expression of CNTF, bFGF, and IGF-1mRNAsby reactive glial cells in deafferented zones suggest thepossibility of sequential or coordinated actions that collec-tively facilitate axonal growth. Such interactions havebeen demonstrated for motor neuron sprouting in vivo.Coadministration of bFGF with CNTF significantly poten-tates terminal sprouting at motor end plates, whereasapplication of bFGF alone has no effect (Gurney et al.,1992). Other trophic interactions are also possible due tothe fact that hippocampal neurons normally express anumber of trophic factors, including the neurotrophins(Gall and Lauterborn, 1992). Neurotrophin expression isnot altered appreciably in the deafferented hippocampusduring periods of sprouting (Beck et al., 1993; Lapchak etal., 1993), but there could be a contribution by normallyexpressed factors. Following entorhinal deafferentation,NGF immunoreactivity is redistributed to the molecularlayer during the period of axonal growth (Conner et al.,1994), and there is evidence that NGF selectively promotesgrowth of septal, but not commissural/associational, affer-ents in this paradigm (Van Der Zee et al., 1992). Moreover,potential trophic interactions between CNTF and theneurotrophins are suggested by demonstrations that intra-ventricular CNTF infusion up-regulates expression of the

low-affinity NGF receptor (Hagg et al., 1992) and thatbrain-derived neurotrophic factor is synergistic with CNTFin support of neuronal survival (Kato and Lindsay, 1994;Mitsumoto et al., 1994).Whereas glial-derived factors induce neuronal differen-

tiation and axonal growth in a variety of systems and mayact directly on neurons to stimulate the sprouting re-sponse, it is also possible that they indirectly facilitateaxonal growth by producing local cellular changes thatprovide a favorable sprouting environment. Microglia andastrocytes produce a variety of factors that might haveparacrine or autocrine actions in regions of degeneration.For example, following entorhinal lesion, interleukin-1band IGF-1 are expressed by reactive microglia (Fagan andGage, 1990; Guthrie et al., 1995); both factors stimulateastrocyte proliferation and, in the case of IGF-1, hypertro-phy in vitro (Guilian and Lachman, 1985; Han et al., 1987,1992). This suggests that activated microglial cells, whichrespond rapidly to axonal degeneration, trigger subse-quent reactive changes in neighboring astrocytes. Reactiveastrocytes in denervated hippocampus express increasedlevels of bFGF and CNTF. bFGF promotes astrocyteproliferation (Perraud et al., 1988) and neurotrophin pro-duction (Yoshida and Gage, 1991; Rivera et al., 1994), andCNTF can induce reactive astrogliosis and microglialactivation (Hagg et al., 1993; Winter et al., 1995; Levinsonet al., 1996)2.The above findings suggest a variety of trophic interac-

tions between glial cells that could essentially ‘‘recruit’’reactive glia to deafferented hippocampal laminae. Theglia could, in turn, facilitate axon sprouting by 1) removingcellular debris and thereby clearing synaptic space, 2)providing growing axons with substrates that supportgrowth (Liesi et al., 1984; Tuszynski and Gage, 1995), or 3)secreting factors that act on neuronal elements to initiateor direct growth. Using transgenic or expression-suppres-sion systems, it should be possible to test the roles of theglial factors discussed here in coordinating the variousglial and neuronal responses induced by deafferentation.

2See footnote 1.

ACKNOWLEDGMENTS

This work was supported by NIA grantAG00538, NIMHResearch Scientist Development Award MH00974, NSFaward BNS 9024143 to C.M.G., and NIMH PredoctoralFellowship MH11279 toA.G.W.

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