Rapid growth of parallel fibers in the cerebella of normal andStaggerer mutant mice

Rapid Growth of Parallel Fibers in theCerebella of Normal and Staggerer

Mutant Mice

JAMES M. SOHA,1* SUGENE KIM,1 JAMES E. CRANDALL,2 AND MICHAEL W. VOGEL3

1Department of Surgery, Yale University School of Medicine,New Haven, Connecticut 06520-8062

2E.K. Shriver Center, Waltham, Massachusetts 022543Maryland Psychiatric Research Center, Catonsville, Maryland 21228

ABSTRACTThe growth of cerebellar granule cell axons was examined by placing focal implants of

1,18,dioctadecyl-3,3,38,38-tetramethyl-indocarbocyanine perchlorate (DiI) in the cerebella ofnormal and staggerer mutant mice at a series of developmental ages between postnatal day 2(P2) and P30. Parallel fibers contacting the implant site were brightly labeled by thefluorescent dye, as were the associated granule cell bodies located principally in the internalgranule layer. The extent of parallel fiber labeling in the molecular layer and the distance fromthe implant to the most extreme labeled granule cells were measured in sectioned material.Two additional measures describing the distribution of labeled granule cells about the implantsite suggest length bounds for most parallel fibers. Parallel fiber growth is surprisingly rapid;all measures approached peak values at P3–P5, only a few days after the earliest postmitoticgranule cells differentiate and migrate. At intermediate ages (P8 and P10), parallel fiberlengths appeared to decrease transiently. At later ages (P15 and beyond), the measures of fiberlength increased to their mature values. These values differed little from lengths measured atP3–P5, suggesting that most parallel fiber growth occurs within a few days of cell birth. Atearly and intermediate ages, parallel fiber lengths in staggerer mice were comparable tocontrols, suggesting that an interaction with normal healthy Purkinje cells is not essential forparallel fiber outgrowth. J. Comp. Neurol. 389:642–654, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: axon growth; axon retraction; cerebellar granule cell development; Purkinje cell;

1,18,dioctadecyl-3,3,38,38-tetramethyl-indocarbocyanine perchlorate

In the mouse cerebellum, granule cells experience anumber of significant developmental milestones postna-tally, including proliferation and differentiation, migra-tion, extension of axons along appropriate pathways,elaboration of dendrites, synaptogenesis, and the targetand afferent interactions that regulate cell death. Granulecells are generated in a proliferative zone, the externalgranule layer (EGL), that forms the superficial layer ofcerebellar cortex between birth and postnatal day 15 (P15;Miale and Sidman, 1961; Fujita et al., 1966; Swisher andWilson, 1977). Newly differentiated granule cells accumu-late along the deep boundary of the EGL, where theyextend opposing bipolar processes (nascent parallel fibers)mediolaterally along the long axes of the cerebellar folia.Soon after final mitosis (Fujita, 1967), the cells migratethrough a radial process (Rakic and Sidman, 1973; Ed-mondson and Hatten, 1987) to the internal granule layer(IGL). After migration, the granule cell axon consists of aninitial ascending segment that divides in a ‘‘T’’ pattern in

the molecular layer to produce oppositely directed parallelfiber branches. Postmigratory granule cells experience anepisode of cell death that is regulated by their Purkinje celltargets (Caddy and Biscoe, 1979).

Little is known about the rate and extent of earlyparallel fiber outgrowth. Understanding this process isimportant for several reasons. Failure of granule cells tomigrate in the weaver mutant mouse is accompanied by anapparent failure to extend bipolar processes (Rakic andSidman, 1973), suggesting that migration may be coupledto parallel fiber growth. The granule cell-Purkinje cell

Grant sponsor: NIH; Grant numbers: NS32163, NS24386, and NS29277.*Correspondence to: James M. Soha, Department of Surgery, Yale

University School of Medicine, P.O. Box 208062, New Haven, CT 06520-8062. E-mail: [email protected]

Received 27 August 1996; Revised 27 June 1997; Accepted 6 August 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 389:642–654 (1997)

r 1997 WILEY-LISS, INC.

circuit is a useful model system for the study of target-dependent neuronal death during development (Wetts andHerrup, 1983; Chen and Hillman, 1989; Vogel et al., 1991).Parallel fibers are likely to play a role in mediating trophicinteractions with Purkinje cells. The onset of normalapoptotic death and the spatial extent of Purkinje celltrophic influence may depend on the rate of parallel fibergrowth. A better understanding of parallel fiber growth isalso relevant to the study of synaptogenesis in the molecu-lar layer. The sequence of parallel fiber deposition suggeststhat progressively deeper fibers arise from granule cells ofincreasing maturity. If this is true, then rapid parallel fibergrowth might lead to a sharp vertical gradient of synapticmaturation in the molecular layer.

We used implants of the fluorescent dye 1,18,dioctadecyl-3,3,38,38-tetramethyl-indocarbocyanine perchlorate (DiI)to label parallel fibers and granule cell bodies in neonatalmouse cerebellum. The implants label sizeable popula-tions of fibers and cells, so that individual fibers cannot betraced in their entirety. Nevertheless, the pattern oflabeling in the implanted cerebella provides considerableinsight into the nature of parallel fiber growth. To assessthe initial rate of parallel fiber growth, we measured theextent of labeled cells and fibers in very young animals(P2–P5), in which postmitotic, postmigratory granule cellshave been present for only a few days. Implants in olderneonates were used to determine whether parallel fiberscontinue to grow slowly over an extended interval duringdevelopment.

Parallel fibers grow orthogonally across sets of parasag-ittally organized Purkinje cell compartments defined byhistochemical markers and afferent connections (Hawkesand Gravel, 1991). Activation of parallel fiber ‘‘beams’’ bymossy fiber afferents may play a role in coordinating theactivity of Purkinje cells in the successive longitudinalcompartments (Thach et al., 1992). Little is known, how-ever, about the mechanisms that regulate the growth ofparallel fibers across their Purkinje cell targets: Whatimpels parallel fibers to grow across cerebellar lobules;what tells them to stop; and what role, if any, do Purkinjecells play in regulating this growth?

Axons in situ often accomplish much of their growthwith little apparent guidance from their targets, particu-larly when these targets are distant. However, as theynear or enter their target region, the target often plays asignificant role in regulating axon growth. For example,even after deletion of all muscle precursors by unilateralextirpation of somites in chicks, motor axons follow normalpathways through a nerve plexus and along the correctperipheral trunks into the muscleless wings, and musclenerves form in appropriate places, despite the completeabsence of muscle tissue (Phelan and Hollyday, 1990).Once axons enter a muscle in an intact limb, however, thetarget exerts significant influence, as evidenced by differen-tial axonal growth and branching behavior in slow vs. fastmuscle (Dahm and Landmesser, 1988). Initial growth andpathway selection by corticopontine efferents also appearto be target independent, because these axons continue togrow past their target and into the spinal cord (O’Learyand Terashima, 1988). Subsequently, the target exerts itsinfluence by inducing and attracting collateral sproutsthrough a process that is likely to involve a chemotropicagent (Heffner et al., 1990). Sympathetic axons thatnormally innervate sweat glands in the mouse footpadsuccessfully grow to the presumptive target region even in

mutant mice in which the glands are totally absent(Guidry and Landis, 1995). However, the axons then fail toelaborate the extensive plexus that normally develops inassociation with a maturing gland. In contrast to theseexamples, parallel fibers grow in close proximity to theirtarget from the outset and, hence, are potentially affectedby target influences throughout their maturation. Whenthey are cultured under appropriate conditions, granulecells elaborate axonal processes that extend hundreds ofmicrons in the absence of Purkinje cell targets (Powell etal., 1997), although it should be noted that these granulecells have been exposed to possible target influences priorto their dissociation and purification from intact cerebella.Although this behavior suggests a significant intrinsiccomponent to parallel fiber growth, the rate, orientationand full extent of growth in situ might depend upon targetinteractions.

To investigate whether Purkinje cells, the principalsynaptic target of granule cells, might play a role inregulating the growth of parallel fibers, we placed im-plants at corresponding ages in the cerebella of staggerermice, a mutant strain whose few remaining Purkinje cellsexhibit severe developmental defects. The staggerer muta-tion affects Purkinje cell development in a direct, cell-autonomous manner (Herrup and Mullen, 1979b; Herrupand Mullen, 1981; Soha and Herrup, 1995). Purkinje cellnumbers in staggerer mice are substantially lower thannormal (Herrup and Mullen, 1979a), and the remainingcells are small and ectopically positioned. Their dendritesare atrophic, are very sparsely branched, and fail toexpress the dendritic spines that normally accept synapsesfrom parallel fibers (Landis and Sidman, 1978). Diverseobservations suggest that Purkinje cell development isblocked at an early stage in staggerer mice. StaggererPurkinje cells abnormally retain their immature polyinner-vation by climbing fibers (Crepel et al., 1980; Mariani andChangeux, 1980), they fail to express certain early postna-tal proteins (Messer et al., 1990; Sotelo and Wassef, 1991),and staggerer-lurcher double mutants never express thelurcher phenotype that appears by about P7 (Messer et al.,1991). The staggerer mutation also affects granule cells,although indirectly. These are generated at a slower rate(Yoon, 1972) over a somewhat longer interval (Sidman etal., 1962) than in wild type animals and migrate success-fully. But, soon after reaching the IGL, granule cells beginto die in large numbers, so that, by maturity, staggerercerebella are virtually agranular (Sidman et al., 1962).Studies in staggerer&wild type chimeras have shown thatgranule cell death in staggerer mice is not a cell-autonomous effect of the staggerer gene but, instead, is anindirect consequence of the absence of a viable targetpopulation of Purkinje cells (Herrup, 1983). The strikinglylinear relationship between the number of surviving gran-ule cells and the number of genetically wild type Purkinjecells in staggerer mouse mutants and staggerer&wild typechimeras provides a compelling example of target-depen-dent cell death during development (Herrup and Sunter,1987). If granule cells require some interaction withPurkinje cells, perhaps involving Purkinje cell dendrites inthe molecular layer, in order to elaborate normal parallelfibers, then this activity might reveal itself in a similarmanner by its absence in staggerer mutants.

PARALLEL FIBER GROWTH 643

MATERIALS AND METHODS

C57BL/6 mice obtained from Jackson Laboratories (BarHarbor, ME) were bred in a colony at the MarylandPsychiatric Research Center to generate wild type mice forthis study. Heterozygous staggerer mice (1/sg) on a B6C3hybrid background were obtained in pairs from JacksonLaboratories and were bred at Yale Medical School in theAnimal Care Facility. Litters from these pairs providedexperimental staggerer pups (sg/sg) and a mixed group of1/sg and 1/1 controls. All animal procedures were ap-proved by the appropriate animal care and use commit-tees. Mice were deeply anesthetized with ether and werekilled by intracardial perfusion with 0.1 M phosphatebuffer, pH 7.4, followed by 4% paraformaldehyde in phos-phate buffer. Brains were postfixed, dissected out, andstored indefinitely in fixative.

Parallel fibers and granule cells in the fixed tissue werelabeled by using DiI (Molecular Probes, Eugene, OR)adhered to the tips of finely drawn glass micropipettes.Drops of a saturated solution of DiI in ethanol were spreadon a glass surface, and the tips of micropipettes werecoated by dredging them in viscous deposits of DiI thatappeared around the edges as the ethanol evaporated.Implants were accomplished by inserting a DiI-coatedpipette just beneath the pial surface of an intact cerebel-lum and cutting it with microscissors, so that the tipremained in the tissue. Ideally, implants would have beenrestricted to the molecular layer (50–100 µm deep), but, inpractice, such shallow implants seldom remained in place.Thus, implants generally reached into the underlying IGLand beyond, typically to a depth of about 200–300 µm.Implants in wild type and control brains were placed in thefolium pyramis vermis at or near the midline. Implants instaggerer brains were placed at a roughly correspondingposition near the midline. Implanted tissue was incubatedat 37°C in phosphate-buffered 4% paraformaldehyde inthe dark for varying intervals to allow diffusion of thelipophilic dye throughout the external membranes of gran-ule cells contacting the implant site. Labeled tissue wassectioned on a Vibratome at 50–100 µm in a plane parallelto the long axis of the folium and roughly perpendicular tothe cerebellar surface at the implant site.

Labeling in the Vibratome sections was assessed byusing a fluorescence microscope, and sections containinglabeled parallel fibers or granule cell bodies were selectedfor analysis. The mediolateral character of the labelingfollowing the parallel fiber projection is illustrated sche-matically in Figure 1 and photographically in Figures 2Aand 3. Overlapping photographs of selected sections wereacquired at low magnification (35 and 310 objectives) toprovide complete coverage of labeled granule cells. The35-mm photographic negatives of these images were digi-tized (at 550 dpi) by using a Microtek scanner (MicrotekLab, Redondo Beach, CA) driven by Adobe Photoshop(Adobe Systems, Mountain View, CA) on a Macintoshcomputer (Apple Computer, Cupertino, CA). The NIHImage program (NIH, Bethesda, MD) was used to obtainfour measurements, each consisting of a distance along thearc of parallel fiber travel, beginning at the center of theimplant site and continuing to a defined point in thedistribution of label (Figs. 2A, 3). For each hemisphere, theextent of the labeled pathway defined by fluorescentparallel fibers was measured laterally from the implantsite along the molecular layer (measure 1), and the dis-

tance from the implant site to the most lateral labeledgranule cell body was measured along an arc just deep tothe molecular layer (measure 2). The latter measureprovides an estimate of the maximal length of mediallydirected parallel fiber branches for granule cells locatednear the extreme of the labeled zone. Initial observationssuggested that these often isolated, labeled granule cellsmay have highly asymmetrical parallel fiber branches incontrast to more typical granule cells, which are likely tobe more symmetric. To explore this possibility and tobetter assess the range of typical parallel fiber lengths, weacquired two additional but more subjective measures.These include the distance from the implant site to thelateral boundary of the most distant cluster of labeledgranule cell bodies (measure 3) and the distance from theimplant site to the approximate lateral boundary of adensely labeled zone (in the IGL, centered on the implantsite) where nearly all granule cells (i.e., greater than 95%by visual estimate) appear to be labeled (measure 4). Bothdistances were measured along arcs that paralleled thesuperficial boundary of the IGL. Variations in the qualityof labeling and plane of section sometimes made it difficultto reliably identify these subjective boundaries. Hence, weacquired the latter two measures only in that material inwhich labeling was favorable and our confidence was high.Measurements from normal and mutant animals of vari-ous ages were compared by using Student’s t-test (usingSYSTAT on a Macintosh). In addition, normalized versionsof the latter three measures were computed by dividingeach by the first measure. Thus, the normalized measuresexpress the extent of granule cell labeling in a particularhemicerebellum as a fraction of the extent of parallel fiberlabeling in that same hemicerebellum.

To determine the appropriate diffusion time, we incu-bated implanted brains at 37°C for varying lengths of timebetween 2 and 6 days and assessed the spread of dye inVibratome sections. Figure 4 plots the unilateral extent oflabeled parallel fibers from the implant site vs. incubationtime in control and staggerer mice for two of the age groupsstudied. The curves are biphasic. The initial rate of dyespread in the fixed parallel fibers is at least as great as theslope of the line connecting the origin and the day 2 mean,roughly 0.7 mm/day at P8–P10 and 1 mm/day at P3–P5.Beyond 2 days of incubation, the extent of labeling contin-ued to increase in three of four cases, although much moreslowly (about 0.06 mm/day). This slower increase mayarise from extracellular diffusion of dye around the im-plant site to label parallel fibers that approach but do notquite reach the implant. Based on this analysis, we believethat a 2-day incubation is adequate to obtain completegranule cell labeling. To expand our data set, we pooleddata obtained from 2-, 3-, and 6-day incubations. Althoughthe diffusion rate of DiI in our material seems unusuallyhigh, elevated diffusion rates appear to be characteristic ofyoung axons (Snider and Palavali, 1990; Elberger, 1993)and may reflect differences in membrane composition(Elberger, 1993, 1994). We considered the possibility thatonly the very youngest axons in our mixed population werebeing completely labeled. We did this by analyzing mea-sures 3 and 4 (see above) vs. incubation time. Thesemeasures better represent the majority of parallel fiberbranch lengths present in the population. No increasingtrend was apparent in these measures for incubations upto 10 days (data not shown), indicating that our incubationtimes were adequate.

644 J.M. SOHA ET AL.

Two sources of error affect our measurements. Varia-tions in the size of the implant site arising from differencesin the thickness and DiI loading of implanted pipettesintroduce bias and variability. Measurement of the extentof intense florescence surrounding the implant in 25randomly selected sections yielded a half-width (center toedge) of 0.045 6 0.012 mm (mean 6 S.D.). Fibers enteringwithin this distance of the center of the implant sitepresumably acquire label, so our measures likely include apositive bias of this magnitude. Pooling of data from arange of incubation times may produce an additionalpositive bias of about 0.12 6 0.12 mm in our lengthmeasures, reflecting the slow component of dye spread(0.06 mm/day). Combining both sources of bias suggeststhat our measures may overstate actual length by up to0.16 6 0.13 mm.

In some sections, a permanent marker was substitutedfor the fluorescent label (Maranto, 1982; Sandell andMasland, 1988) by using the fluorescent light emitted bythe DiI to photooxidize 3,38-diaminobenzidine (DAB). Sec-tions were incubated in 1.5 mg/ml DAB in 0.1 M Tris, pH

8.2, and the appropriate regions were illuminated by usinga rhodamine filter set and a 35 or 310 objective untildevelopment was judged complete. Photoconverted sec-tions were lightly counterstained with cresyl violet, thenmounted, and coverslipped.

The staggerer mouse behavioral phenotype is not readilyapparent until the end of the second postnatal week. Afterperfusion, staggerer mice at P5 or older were easily distin-guished from control litter mates by the reduced size oftheir cerebella. At P3, however, the immature cerebellacould not be typed reliably by gross inspection, so animmunohistochemical assay was used. Vibratome sectionsthat did not contain fluorescent label were selected andincubated for 60 minutes with an anti-calbindin monoclo-nal antibody (Sigma, St. Louis, MO) diluted 1:200 inTris-buffered saline (TBS) containing 0.5% Tween 20. Afterrinsing with TBS, sections were incubated for 30 minuteswith a peroxidase-conjugated goat anti-mouse antibodydiluted 1:200 in TBS-Tween 20, then washed, and devel-oped in 0.5 mg/ml DAB in 0.05 M Tris, pH 7.6, containing0.01% H2O2. Developed sections were lightly counter-

Fig. 1. Schematic illustrating the organization of 1,18,dioctadecyl-3,3,38,38-tetramethyl-indocarbocyanine perchlorate (DiI)-labeled cellsand processes in cerebellar sections. Glass micropipettes coated withDiI were implanted normal to the exposed pial surface near themidline in folium pyramis vermis. Implants usually extended throughthe external granule layer (EGL) and molecular layer (ML) and intothe internal granule layer (IGL) deep to the Purkinje cells (PC).Premigratory (1) and probably migrating (2) granule cells were labeledon occasion, but the bulk of labeled granule cells were located in theIGL (3–5). Some granule cells may have acquired label via cell bodiesthat contacted the implant site (4). In most cases, labeling wasacquired by parallel fibers that entered (3) or traversed (5) the implant

site, which was considered to include the pipette itself and a surround-ing zone of dye diffusion (lightly shaded). The unilateral extent oflabeled fibers might provide a reasonable estimate of the total(bipolar) branch length for some cells. Such a cell might logically befound near a lateral extreme of labeling in the IGL (3); its centrallydirected parallel fiber branch would just reach the implant, and itslaterally directed process would extend to the lateral edge of labelingin the ML. It is possible, albeit unlikely, that a cell might extend aparallel fiber branch to each extreme of ML labeling (4). Our datasuggest that most labeled granule cells exhibit an intermediateconfiguration (5).


Fig. 2. Examples of labeling in a photoconverted coronal sectionfrom a postnatal day 5 (P5) wild type implant. Photographic printswere scanned, and their contrast and color balance were adjustedindividually prior to compositing in Adobe Photoshop. A: Overview oflabeling in the ML and the IGL. Four unilateral distance parameterswere measured, starting from the implant site (arrowhead) andcontinuing laterally along the ML to defined points (arrows). Theseinclude the extent of labeled parallel fibers (open arrow), the greatestlateral extent of labeled granule cell bodies (thin arrow), the extent oflabeled granule cell clusters (medium arrow), and the extent of a zoneof dense labeling in which almost all granule cells appear to be labeled(thick arrow). On the right side, labeled fibers extend beyond the plane

of section. B: In lightly labeled regions near the lateral extremes,parallel fibers can sometimes be seen to terminate in swellingssuggestive of growth cones (arrow). C: Labeled premigratory granulecells (arrows) in the EGL just superficial to the ML. D,E: Left and rightlateral extremes of labeling in the IGL illustrating representativelabeled granule cells (open arrows) and ‘‘T’’ junctions, where ascendinggranule cell axons (from other granule cells) bifurcate in the deep ML(solid arrows). The orientation of the focal plane prevents ascendingaxons from remaining visible over their full course from cell body to Tjunction, although this continuity was generally clearly visible nearthe lateral extremes in fluorescent material. Scale bars 5 200 µm in A,20 µm in B–E.


stained with cresyl violet and permanently mounted. Wildtype and heterozygous controls exhibit a layer that isdensely populated with calbindin-positive Purkinje cells,whereas staggerer mice lack an organized Purkinje celllayer except in a narrow band near the midline (Ji et al.,1997).

RESULTS

Focal implants of the fluorescent lipophilic dye DiI wereplaced in the cerebellar cortex of neonatal mice to label arestricted set of parallel fibers and to study their growth.Implants consisted of the cut tips of glass micropipettescoated with small DiI crystals (see Materials and Meth-ods). Implants were placed in fixed brains from animals

ages P2–P30 near the midline in the folium pyramisvermis (or a roughly corresponding position in staggerermutants), perpendicular to the surface, so that they pen-etrated the superficial EGL, the molecular layer, thePurkinje cell layer, and the underlying IGL. The objectivewas to label in their entirety all granule cells whoseparallel fiber axons entered or traversed the implant site.Labeling of individual granule cells and their processescan arise in a number of ways and at varying stages oftheir maturation, as summarized in the schematic draw-ing of Figure 1. Granule cell bodies in the IGL that areadjacent to the implant may be labeled directly. Mostlabeled granule cells acquire their label through theircentrally directed parallel fiber branch, which contacts theimplant in the molecular layer. The centrally directed

Fig. 3. Fluorescence photomicrographs of sections from P3 control(A) and staggerer (B) implants. Extensive labeling of parallel fibersand granule cell bodies is apparent in both genotypes at this early age,indicating rapid parallel fiber growth. Extent of labeling in staggerermice is similar to controls, suggesting that Purkinje cells do notregulate early growth. Plane of section is intermediate between

coronal and horizontal. Asterisk denotes implant site. Lateral limits ofmeasured distance parameters are indicated by arrows, as describedin Figure 2 legend. Our fourth measure, the extent of the zone of denselabeling, was not acquired in B because of uncertainty in identifyingthe boundary in this section. Scale bar 5 500 µm.


branch may terminate within the implant site or, morelikely, may continue an indeterminant distance beyond. Afew implants placed up to 1 mm off the midline (not shown)established that parallel fibers cross the midline in substan-tial numbers. In addition, other cell and fiber types wereoccasionally labeled by the implants, including Purkinjecells and their processes, mossy fibers, climbing fibers,Golgi neurons, and interneurons of the molecular layer.

Clearly defined labeling of parallel fibers and granulecell bodies was frequently obtained. The pattern of label-ing in a representative photoconverted section from a P5wild type implant is illustrated in Figure 2. In sectionscontaining or adjacent to the implant site, parallel fibersare labeled at all depths in the molecular layer (Fig. 2A).Labeled fibers travel laterally for considerable distances,about 1.2 mm in the illustrated section, sometimes exceed-ing 2 mm in other cerebella. Labeled fibers often continuedinto adjacent sections due to curvature of the cerebellumrelative to the plane of section. The dense mass of labeledfibers makes it impractical to follow individual fibers overtheir entire length, particularly when viewing them underfluorescence in thick sections. In less densely labeledregions at the lateral or rostrocaudal extremes of thelabeled zone, fibers terminating in swellings suggestive ofgrowth cones are seen (Fig. 2B), and these are probablyalso present throughout the densely labeled zone. Labeledpremigratory granule cells are apparent in the deep half ofthe EGL in this section (Fig. 2C). Such cells were seen onlyrarely, perhaps because they must be distinctly superficialto the molecular layer to be distinguished reliably from the

heavy parallel fiber labeling. Deep to the molecular layerand centered on the implant site is a zone populated bylabeled granule cell bodies (Fig. 2A). Labeled cells aredensely packed in the medial half of this zone. Labelingdensity declines as the lateral margins of the labeled zoneare approached. Labeled granule cells are most clearlydelineated near this lateral margin where, generally, onlya few isolated cells are labeled (Fig. 2D,E). Labeledgranule cells radiate short dendrites and a single axonthat rises to the molecular layer and bifurcates in theclassical ‘‘T’’ junction, with parallel fibers continuing inopposing directions (Fig. 2D,E). Additional examples oflabeled granule cells and their ascending axons in fluores-cent material are shown in Figure 5.

The dense labeling inherent in our paradigm precludeddirect measurement of individual parallel fibers. Instead,we examined the spatial distribution of labeling by usingfour measures to assess the extent and rate of growth ofparallel fibers (Figs. 2, 3; see also Materials and Methods).Measure 1 is simply the unilateral extent of labeledparallel fibers from the implant site. Although it is difficultto relate this measure to the lengths of individual parallelfiber branches, it seems likely to bear a consistent relation-ship to maximal parallel fiber growth. Thus, the measureis useful for comparing different ages to determine whenmost parallel fiber growth occurs and for comparing geno-types to assess the role of target in this growth. Theremaining measures relate to the distribution of labeledgranule cell bodies. How labeled granule cell bodies aredistributed about the implant site reveals much about thedistribution of parallel fiber branch lengths. The distribu-tions are mathematically related; if we could clearlyidentify the position of every labeled cell, then we coulddeduce the distribution of parallel fiber lengths by numeri-cal integration. Although such a comprehensive descrip-tion is not feasible with our material, we take advantage ofthis relationship by using measures that describe partiallyhow labeled granule cell bodies are distributed about theimplant site. Measure 2, the mediolateral distance fromthe implant site to the farthest removed of the labeledgranule cell bodies, provides a reasonable length estimatefor the longest parallel fiber branches. If all parallel fiberswere of equal length, then the somata of cells whose fibersbarely reach the implant site would lie at the lateralextremes of the zone of labeled cell bodies. In reality, theneonatal molecular layer contains a mix of fibers ofvarying maturities, and even the oldest fibers are likely tovary in length. At greater lateral distances from theimplant, only cells with exceptionally long, centrally di-rected processes will reach the implant and acquire label.Two additional measures of the extent of granule celllabeling offer further insight into the distribution of paral-lel fiber branch lengths. Progressing medially from therelatively isolated labeled cells near the lateral margin ofthe labeled zone, a boundary is often apparent, where thedensity of labeled granule cells increases and clusters oflabeled cells predominate. The distance of this boundaryfrom the implant site, measure 3, defines a threshold forparallel fiber branch length that appears to be exceededonly rarely. Continuing medially, a second boundary isoften visible, medial to which a substantial majority (i.e.,greater than 95%) of all granule cells appear to be labeled.The distance to this boundary from the implant site,measure 4, appears to define an approximate lower boundfor parallel fiber branch lengths. Together, these final two

Fig. 4. Unilateral extent of parallel fiber labeling plotted as afunction of incubation time in control and staggerer mice of younger(P3–P5) and intermediate age (P8–P10) reveals a biphasic relation-ship. Diffusion of DiI within parallel fiber membranes occurs at leastas fast as the slope of the first line segment (0–2 days; dashed line).Beyond 2 days of incubation, labeling continued to spread in three ofthe four groups but at a much reduced rate (solid line). This slowincrease in the extent of labeling vs. incubation time may reflectextracellular diffusion of dye from the implanted pipette to reachprocesses that approach but do not quite reach the implant. Weconclude that 2 days is sufficient to achieve complete labeling ofgranule cells whose processes enter the implant site.


measures suggest a range of variability in the length ofcentrally directed parallel fiber branches that appears toencompass most of the more mature granule cells.

To obtain an overview of parallel fiber growth, we placedimplants in the fixed cerebella of C57BL/6 wild type miceat ages ranging from P0 to P30. Data from these experi-ments are summarized in Figure 6. Implants at P0 and P1yielded no clear evidence of parallel fibers in the nascentmolecular layer. Implants at P2 provided only marginallyacceptable labeling of parallel fibers, and labeling in theIGL was diffuse, so that no labeled granule cells could beclearly discerned. Implants in older animals producedclear labeling of parallel fibers and granule cell bodies. At

P2, the unilateral extent of labeled fibers in the molecularlayer was relatively limited (Fig. 6), suggesting that theseearly fibers were still in the process of growing. By P5–P6,unilateral extent had increased considerably. Any furtherincreases in parallel fiber extent were slow and lesspronounced; the extent of unilateral labeling seen at P30does not differ significantly from the extent at P6. Curi-ously, the extent of fiber labeling was reduced at P8 by justover 20%. The extent of fiber labeling at P8 is significantlyless than at P30 (P , 0.02) but is not significantly differentthan at P6 because of a relatively high standard error inthe P6 data. The lateral extent of granule cell labelingexhibited a similar variation with age, but, for this param-eter, the value at P8 was significantly less than that at P6(P , 0.02). These measurements suggest a complex se-quence of parallel fiber development, with initial rapidoutgrowth between birth and P6; a transient retractioncirca P8; and, finally, a resumption of growth within thelabeled population.

DiI implants were also placed in the fixed cerebella ofpups from B6C3 heterozygous staggerer crosses. Theseimplants serve three objectives: 1) to examine more closelythe initial rate of outgrowth as defined by the youngestanimals, 2) to further examine the apparent transientdecline in parallel fiber branch lengths seen at P8, and 3)to analyze the role of target in regulating the outgrowth ofparallel fibers. Homozygous staggerer mice were identifiedas described in Materials and Methods. The earliest age atwhich mutants could be identified reliably was P3. Wildtype and heterozygous staggerer litter mates could not bedistinguished and were pooled as controls. Results aresummarized in Tables 1 and 2 and in Figure 7.

Analysis of the control mice confirmed both the rapidinitial outgrowth of parallel fibers and the transientdecrease in extent at intermediate ages seen in the firstexperimental group of wild type mice. An example of aDiI-labeled section from a P3 control cerebellum (Fig. 3A)illustrates the extensive parallel fiber growth evident atthis age. The unilateral extent of labeled parallel fibers inP3 controls, the youngest mice in this experimental group,exceeded 2 mm, whereas the most distant labeled cell

Fig. 6. Unilateral extent of labeled parallel fibers (PF extent) andlabeled granule cell bodies (furthest GC) in wild type C57BL/6 mice ata series of postnatal ages. Plotted points for the two measures areoffset slightly to improve clarity. Ages examined (number of hemicer-ebella): P2 (8), P5 (6), P6 (4), P8 (6), P15 (6), P30 (4).

Fig. 5. Fluorescent image of DiI-labeled granule cells in the IGL of a P5 control cerebellum. Labeledaxons (solid arrows) arising from several granule cell bodies (open arrows) can be seen ascending to theML. Leftmost marked cell is approximately 800 µm from the implant site. Scale bar 5 100 µm.


bodies were located about 1.9 mm lateral to the implantsite (Table 1), reflecting extensive parallel fiber growthduring the first few days of postnatal life. Figure 7A,Billustrates that, except for the transient decrease aroundP10, there is little change in these two measures duringthe subsequent 4 weeks. To compare fiber extent atdifferent ages, experimental mice were combined intoyounger (P3–P5), intermediate (P8–P10), and older (P15–P17) groups. Group means and statistical comparisonsbetween groups are summarized in Table 1. Both of thesemeasures of parallel fiber length were significantly de-creased in intermediate-age controls compared with theyounger age group, consistent with our observations inC57BL/6 wild type mice. These observations suggest thepossibility of exuberant outgrowth of parallel fibers that isfollowed within days by a partial retraction. To be consis-tent with our data, exuberant growth and partial retrac-tion could involve only early generated granule cells;otherwise, exuberant axons should be present in each agegroup and would obscure any retraction. In older controls,both measures regained the values seen in the youngergroup. The unilateral extent of labeled parallel fibers inolder controls differed significantly from the extent atintermediate ages but did not differ from the extent atyounger ages. Similarly, the lateralmost labeled cell bodieswere significantly farther from the implant site in oldercontrols than in the intermediate group. There was nosignificant difference between older and younger controlsin this parameter.

Our two additional measures of the distribution oflabeled granule cell bodies, the unilateral extent of labeledcell clusters and the zone of dense labeling (Fig. 7C,D),provide more inclusive estimates of the range of parallel

fiber branch lengths at the ages studied. These measuressuggest that, at P3, most centrally directed branchesextend between 1.0 mm and 1.5 mm (Table 2), arguing thatrapid outgrowth of parallel fibers is prevalent and is notlimited to a few exceptional granule cells. The distributionof branch lengths was reduced somewhat in intermediate-aged mice compared with the younger age group, rangingbetween 0.8 mm and 1.2 mm, although the lower bound(extent of the zone of dense labeling) did not differ signifi-cantly between the two groups. The distribution of branchlengths increased modestly in the older age group, rangingbetween 1.2 mm and 1.6 mm. These values are signifi-cantly greater than those observed in the younger group,suggesting that a second, but slower, phase of growth mayfollow the initial episode of rapid outgrowth.

Cerebella of staggerer mice were noticeably smaller thanthose of the pooled controls by P5 and were substantiallysmaller at older ages. The quality of labeling obtained inthe staggerer mice was comparable to controls. Consistentwith their smaller size, it appeared that both fewer cellsand fewer fibers were labeled in staggerer cerebella, al-though we did not attempt to quantify this difference.Initial outgrowth of parallel fibers is also rapid andextensive in staggerer mice. The mean unilateral extent oflabeled parallel fibers and the unilateral extent of labeledcells at P3 were both modestly lower than control values(Table 1, Fig. 3B), but the differences are not statisticallysignificant at this earliest age examined. The extensivelabeling observed at P3 strongly suggests that a normal,healthy target population of Purkinje cells is not necessaryfor the initial phase of parallel fiber growth. At later ages,the unilateral extents of both parallel fiber and granulecell labeling were consistently smaller in staggerer mice

TABLE 1. Maximal Extent of Labeled Parallel Fibers and Granule Cells in B6C3 Staggerer Mice (sg/sg) and Pooled Controls: Mean 6 S.E.M. (N)1

Age group (days)

Parallel fiber extent (mm) Farthest granule cell (mm)

1/?2 sg/sg 1/? vs. sg/sg3 1/?2 sg/sg 1/? vs. sg/sg3

3 2.08 6 0.08 (12) 1.95 6 0.16 (6) * 1.92 6 0.07 (12) 1.78 6 0.15 (6) *5 1.98 6 0.06 (12) 1.63 6 0.08 (7) P , 0.01 1.75 6 0.10 (11) 1.44 6 0.09 (7) P , 0.058 1.68 6 0.08 (14) 1.32 6 0.05 (12) P , 0.001 1.41 6 0.08 (14) 1.07 6 0.06 (12) P , 0.01

10 1.46 6 0.05 (20) 1.49 6 0.10 (12) * 1.31 6 0.04 (20) 1.36 6 0.11 (12) *15 2.09 6 0.03 (4) 1.70 6 0.18 (4) * 1.90 6 0.08 (4) 1.48 6 0.15 (4) *17 2.12 6 0.13 (4) 1.72 6 0.06 (8) P , 0.05 2.01 6 0.05 (4) 1.60 6 0.07 (8) P , 0.00130 2.21 6 0.05 (4) 1.72 6 0.15 (8) P , 0.05 2.09 6 0.05 (4) 1.53 6 0.13 (8) P , 0.01Younger (3–5) 2.03 6 0.05 (24) 1.78 6 0.09 (13) P , 0.05 1.84 6 0.06 (23) 1.60 6 0.09 (13) P , 0.05Intermediate (8–10) 1.55 6 0.05 (34) 1.40 6 0.06 (24) P , 0.05 1.35 6 0.04 (34) 1.22 6 0.07 (21) *Older (15–17) 2.10 6 0.06 (8) 1.72 6 0.07 (12) P , 0.001 1.96 6 0.05 (8) 1.56 6 0.07 (12) P , 0.0013–5 vs. 8–102 P , 0.001 P , 0.001 P , 0.001 P , 0.018–10 vs. 15–172 P , 0.001 P , 0.01 P , 0.001 P , 0.0013–5 vs. 15–172 * * * *

1Number of hemicerebella.2Pooled 1/1 and 1/sg.3Student’s t-test.*P . 0.05.

TABLE 2. Extent of Labeled Granule Cell Clusters and Dense Cell Labeling in Staggerer Mice (sg/sg) and Pooled Controls: Mean 6 S.E.M. (N)1

Age group (days)

Farthest cell cluster (mm) Zone of dense labeling (mm)

1/?2 sg/sg 1/? vs. sg/sg3 1/?2 sg/sg 1/? vs. sg/sg3

Younger (3–5) 1.43 6 0.06 (17) 1.29 6 0.10 (10) * 0.95 6 0.08 (9) 0.90 6 0.13 (4) *Intermediate (8–10) 1.16 6 0.04 (33) 1.01 6 0.06 (22) P , 0.05 0.84 6 0.05 (21) 0.70 6 0.02 (19) P , 0.05Older (15–17) 1.63 6 0.05 (8) 1.28 6 0.05 (12) P , 0.001 1.24 6 0.06 (5) 0.89 6 0.06 (10) P , 0.013–5 vs. 8–102 P , 0.001 P , 0.05 * *8–10 vs. 15–172 P , 0.001 P , 0.01 P , 0.01 P , 0.013–5 vs. 15–172 P , 0.05 * P , 0.05 *

1Number of hemicerebella.2Pooled 1/1 and 1/sg.3Student’s t-test.*P . 0.05.


than in controls (except at P10; see Fig. 7). Although theyare small, the differences are often statistically significant,particularly when ages are grouped (Table 1), and mayreflect the exaggerated granule cell death in staggerermice. This cell loss may preferentially remove more ma-ture granule cells that have longer parallel fibers. Therange between the extent of dense labeling and the extentof labeled cell clusters, which likely includes the bulk ofparallel fiber branch lengths, varied with age in a mannersimilar to controls (Fig. 7C,D), except that the range didnot increase in the older group of staggerer mice over thatof the younger age group. If a second, slower phase ofgrowth normally occurs, it may be lacking in staggerermice or be masked by the selective loss of older granulecells. The transient declines in the measures of parallelfiber length that were observed in control mice were alsofound in staggerer (Fig. 7), but they appear to occur earlier,

beginning perhaps at P5 and attaining minimum valueson P8 (vs. P10 in controls). When grouped by age forstatistical comparison, all measures of parallel fiber extentwere smaller at intermediate ages than in either theyounger or the older age groups; this difference wassignificant in all but one instance (Tables 1, 2). Measuresfor the younger and older groups did not differ signifi-cantly.

To further assess possible changes in the distributions oflabeled granule cell bodies with age or genotype, wenormalized the three measures of the extent of cell bodylabeling, expressing each as a fraction of the unilateralextent of labeled parallel fibers in that hemicerebellum.The results of this analysis are summarized in Figure 8.Staggerer and control mice (averaged over all ages) do notdiffer significantly for any of the three normalized mea-sures, suggesting a close similarity in the distribution of

Fig. 7. A–D: Four measures of parallel fiber extent in B6C3 staggerer mice and litter mate controls(pooled wild type and heterozygous staggerer mice) during postnatal development.


parallel fiber branch lengths. For each genotype, theextent of labeled cells was about 90% of labeled fiberextent. Labeled granule cell clusters reached about 70% oflabeled fiber extent, whereas the zone of dense labelingcovered roughly 50% of labeled fiber extent. These percent-ages provide some insight into the degree of symmetryexhibited by opposing parallel fiber branches (see Discus-sion). It is also interesting that, despite the significanttransient decline in the raw measures at intermediateages, the normalized measures show little systematicvariation with age when averaged across genotype.

DISCUSSION

Our observations show that parallel fibers grow rapidly.The best evidence for this comes from younger material, inwhich we can place an upper bound on the age of allpostmitotic granule cells. At P3, the zone of dense granulecell labeling in the hybrid (B6C3) mice extended about 1mm from the implant site, suggesting that most parallelfiber branches of postmigratory granule cells are at leastthis long. It is tempting to infer that parallel fiber branchlengths generally reach this threshold before granule cellbodies complete their migration to the IGL. However, theheavy labeling in this zone impaired the search for isolatedunlabeled cells, so we cannot conclude that they were notpresent. Clusters of labeled cells continued laterally toabout 1.5 mm from the implant, indicating that parallelfiber branches of this length are not uncommon in P3 mice.A few parallel fiber branches extend unilaterally as far as1.9 mm. Because postmitotic granule cells first appear onthe date of birth (Fujita, 1967), this extensive growth musthave occurred within only a few days.

A second implication of our data is that parallel fiberscomplete most of their growth within a few days of cellbirth. The branch length of a labeled granule cell located inthe zone of labeled cell clusters increased by less than 25%from younger (P3–P5) to older (P15–P17) ages. Our twomore precise measures, labeled fiber extent and mostdistant labeled granule cell, did not change significantlyfrom younger to older ages. The values of these measures,about 2 mm, resemble the parallel fiber branch lengthsreported by Pichitpornchai et al. (1994) in mature rats.These findings appear to be at variance with those ofLauder (1978), who reported a steady, almost threefoldincrease in average parallel fiber length in rats from P10 toP30 using lesion studies and degeneration staining. Follow-ing the reduction in the extent of labeling at intermediateages (P8–P10), we observed a significant increase inparallel fiber length at older ages. The magnitude of thisincrease, depending on which of the four measures is used,ranged between 30% and 50%, considerably less than thatseen by Lauder in rats.

We labeled nonhomogeneous populations of granulecells that include a wider range of maturities in olderanimals. The slow increase of parallel fiber length with ageargues that older cells cannot have grown much over thepreceding interval. We cannot infer that older cells areactually continuing to grow; perhaps younger cells contrib-ute all of the longer parallel fibers seen at later ages.Similarly, with the present technique, we cannot be certainthat later generated cells duplicate the rapid outgrowthexhibited by the earliest born cells. It is possible, forexample, that an early population fulfills a pioneering roleby growing faster and more exuberantly to provide guid-ance for subsequent, slower growing parallel fibers.

A third important aspect of our observations is theapparent transient decline in parallel fiber extent fromyounger to intermediate ages. The greater number ofanimals in the intermediate age group reflects our effortsto confirm this finding. The observations suggest that atleast the longer parallel fiber axons present at P5 undergoretraction or disappear altogether. It is surprising thatretraction should be apparent in our experiment. Whydon’t granule cells generated at P5–P7 grow equallyrapidly and attain the same lengths by P8–P10 that theearliest cells reach by P3–P5? This paradox suggests aunique role for early generated cells, perhaps laying downa preliminary network of longer fibers that facilitatessubsequent parallel fiber growth. Support for this hypoth-esis can be found in experiments that interrupt thesequence of granule cell development. Aberrantly orientedparallel fibers have been reported in rodents receivingx-irradiation at dosages sufficient to remove early gener-ated granule cells without totally destroying the EGL(Altman, 1973). Cytotoxic lesions of proliferating granulecells induced by early postnatal injection of methylazoxy-methanol (MAM) lead to disoriented growth of parallelfibers following reconstitution of the EGL a few days later(Lovell et al., 1980). Serious disruption of both foliationand parallel fiber orientation are produced by MAM injec-tions on P0, whereas delaying MAM treatment until P5reduces the severity of these effects (Bejar et al., 1985;Hillman et al., 1988). Such findings are consistent with apossible pioneering role for early generated parallel fibers.

If all granule cells were symmetrical, that is, if both oftheir parallel fiber branches extended equal distancesfrom the T junction, then labeled granule cell bodies

Fig. 8. Distribution of labeled granule cell bodies in staggerer andcontrol implants. Measures of the lateral extent of labeled cells fromthe implant site were normalized by dividing by the unilateral extentof labeled fibers in the same hemicerebellum and were then averagedover genotype or age group. None of the normalized measures differssignificantly between genotypes. Similarly, there are few differencesbetween age groups (genotypes pooled); the only significant differencesare extreme labeled granule cells (intermediate vs. late; P , 0.02) andthe extent of labeled clusters (early vs. late; P , 0.05), and themagnitude of these differences is small.


should be restricted to roughly the central half of the zonetraversed by labeled parallel fibers. Instead, we findlabeled granule cells extending laterally to about 90% oflabeled parallel fiber extent. This percentage did not varysystematically with incubation time, suggesting that theobserved pattern of labeling was not caused by incompletediffusion of dye. A more likely explanation is that not allgranule cells are symmetric; indeed, other investigatorshave reported observing granule cells with unequal paral-lel fiber branches (Palay and Chan-Palay, 1974; Quesadaand Genis-Galvez, 1983). In this case, our experimentalprotocol would selectively label a subpopulation of granulecells at the lateral extremes of the labeled zones, namely,those that are highly asymmetric and have a longer,centrally directed parallel fiber process. Other laterallysituated cells that are more symmetrical, or have theopposite asymmetry, would not be labeled, because theircentrally directed processes are not long enough to reachthe implant site. The zone of dense granule cell labeling inour experiments typically occupied only 50% of the medio-lateral extent of labeled parallel fibers (Fig. 8), suggestingthat many granule cells are approximately symmetrical.

Cerebellar Purkinje cells are known to affect theirgranule cell afferents during development. Most notable istheir role in rescuing granule cells from target dependentcell death. There is also evidence that Purkinje cellsinfluence granule cell proliferation in the EGL. The EGL isthinner than normal in both staggerer (Yoon, 1972) andlurcher mice (Dumesnil-Bousez and Sotelo, 1992), suggest-ing a reduction in proliferation. Both mutant strainsexhibit an early reduction in Purkinje cell number. Smeyneet al. (1995) provide further evidence of a mitogenicactivity by analyzing a line of transgenic mice that expressan attenuated diptheria toxin under the control of aPurkinje cell-specific promoter. The toxin causes Purkinjecell loss in neonates that occurs initially in parasagittalbands. Proliferative activity in the EGL is attenuated inbands that are superficial to and correlate with the para-sagittal zones of Purkinje cell ablation. Thyroid hormonesalso affect granule cell genesis, and Messer and colleagues(Messer et al., 1985; Messer, 1988) have provided two linesof evidence suggesting that this influence is mediated byPurkinje cells. Thyroid hormones also affect the rate ofparallel fiber growth (Lauder, 1978).

To determine whether Purkinje cells might influence theelaboration of parallel fiber axons, we examined parallelfiber branch length in staggerer mice at various ages. Thismutant is appropriate, because its Purkinje cells, whichare few in number and highly atrophic, fail to providetarget support for granule cells and may have less influ-ence on granule cell proliferation. The staggerer mutationaffects Purkinje cells in a direct, cell-autonomous manner(Herrup and Mullen, 1979b, 1981; Soha and Herrup,1995). Studies in chimeras show that staggerer granulecells are intrinsically normal: The wholesale death of thesecells in mutants is an indirect consequence of the Purkinjecell lesion (Herrup, 1983; Herrup and Sunter, 1987). Wefound that, at P3, all measures of parallel fiber length instaggerer mice are indistinguishable from control mice. Atlater ages, parallel fiber lengths are consistently less instaggerer mice, but the magnitude of the difference is smalland is easily explained by the continuing loss of moremature granule cells. Conduction velocities of staggererparallel fibers approximate control values (Crepel andMariani, 1975), providing further evidence that these

fibers develop normally. The similarity of parallel fibers instaggerer mutants and controls places clear limits on therole that Purkinje cells might play in stimulating orinhibiting parallel fiber growth. A dense, growing web ofhealthy Purkinje cell dendrites providing a growth sub-strate or local trophic stimulus is apparently not required,but what about a more diffuse trophic influence? StaggererPurkinje cells do not merely lack healthy dendrites withspines to support parallel fiber synaptogenesis, but theyalso fail to mature by numerous other criteria. StaggererPurkinje cells fail to express some early postnatal pro-teins, including calmodulin (Messer et al., 1990) andzebrin (Sotelo and Wassef, 1991), and they retain theirimmature polyinnervation by climbing fibers (Crepel et al.,1980; Mariani and Changeux, 1980). Staggerer-lurcherdouble mutants express the staggerer but not the lurchermutant phenotype that affects Purkinje cells by the end ofthe first postnatal week (Messer et al., 1991). This appar-ently early block in the development of staggerer Purkinjecells argues that any trophic contribution to parallel fibergrowth must appear early and must be strong enough to beunaffected by the reduction in Purkinje cell numbers.

ACKNOWLEDGMENTS

The authors thank Karen Sunter-Mann, Aron Soha, andJennifer Prittie for expert technical assistance. This workwas supported by NIH grants NS32163 (J.M.S.), NS24386(J.E.C.), and NS29277 (M.W.V.).

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Rapid growth of parallel fibers in the cerebella of normal andStaggerer mutant mice