SYNAPSE-INDEPENDENT AND SYNAPSE-DEPENDENT APOPTOSIS OFCEREBELLAR GRANULE CELLS IN POSTNATAL RABBITS OCCUR ATTWO SUBSEQUENT BUT PARTLY OVERLAPPING DEVELOPMENTAL

STAGES

L. LOSSI,a S. MIOLETTIa and A. MERIGHIa;b�

aDepartment of Veterinary Morphophysiology, Via Leonardo da Vinci 44, I-10095 Grugliasco, ItalybRita Levi Montalcini Center for Brain Repair, Via Leonardo da Vinci 44, I-10095 Grugliasco, Italy

Abstract3It has long been known that cells in the external granular layer die during postnatal development of thecerebellum. More recent ¢ndings indicate that at certain developmental stages, cell death occurs upon activation of anapoptotic program. We show that cerebellar granule cells in rabbits undergo programmed cell death at two di¡erentstages of maturation. At postnatal day 5 (P5), granule cell precursors and pre-migratory granule cells in the externalgranular layer incorporate the S-phase markers 5-bromo-2P-deoxyuridine and 5-iodo-2P-deoxyuridine with a pattern thatis dependent upon the interval between the administration of the two tracers. Within 12^24 h after proliferation asigni¢cant number of labeled cells show typical ultrastructural alterations of apoptosis. DNA electrophoresis and cleav-age of poly-ADP-ribose polymerase con¢rm the activation of the apoptotic machinery. After Southern blotting andimmunodetection, incorporated 5-bromo-2P-deoxyuridine is present at the level of low size DNA oligomers as soon as 12 hafter cell division. Therefore, this apoptotic phase is intrinsic to external granular layer neurons and independent ofsynaptic interactions with targets.

Apoptotic cells, although fewer in number, are detected also in the internal granular layer and tend to increase from P5to P10. It seems unlikely that these cells undergo DNA fragmentation in the external granular layer and subsequentlymigrate to their ¢nal destination, considering the data on cell cycle kinetics and the rapid tissue clearance by the glia.Parallel ¢ber^Purkinje spine synapses are already present in the molecular layer at P5. Therefore, the post-migratorygranule cells likely undergo apoptosis as a failure to make proper synaptic contacts in the forming molecular layer.

We conclude that the massive apoptosis of pre-migratory cells likely has a role in regulating the size of this rapidlyexpanding population of pre-mitotic neurons. The less tumultuous cell death of post-mitotic granule cells in the internalgranular layer appears to be linked to the formation of the mature synaptic circuitry of the developing cerebellarcortex. @ 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Key words: cerebellum, proliferating cells, programmed cell death, cell cycle kinetics, light and electron microscopy, DNA gelelectrophoresis.

Apoptosis is a gene-regulated process of programmedcell death (PCD) which is essential for development ofthe nervous system and also plays a role in neurodegen-erative diseases and aging (Oppenheim, 1991; Sastry andRao, 2000). In this form of ‘cell suicide’ typical morpho-logical and biochemical changes have been described,

such as cell shrinkage, membrane blebbing, chromatincondensation, and DNA fragmentation (Kerr et al.,1972; Clarke, 1990). The process eventually leads to for-mation of apoptotic bodies which are rapidly clearedfrom tissues by phagocytic cells (Ellis et al., 1991). Arelatively large loss of di¡erent types of neurons (andglial cells) occurs during vertebrate development, andthe general explanation for this phenomenon is that thesurvival of nerve cells depends on speci¢c neurotrophicfactors which are synthesized by their targets. Additionalsignals from neighboring neurons and glial cells are alsorequired for the developing neurons to survive (Ra¡ etal., 1994), but ultimately the survival of neurons dependson a complex interaction of several factors that uponimbalance lead to cell death. Therefore, although manyneuronal types are produced in excess, only a portion ofthem get su⁄cient support for survival, and the othersdie, facilitating appropriate neuron-target innervation.How the signals for survival/death actually operate isstill a matter of debate. It is possible that cells areinstructed to die by switching on the apoptotic machin-

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*Corresponding author. Tel. : +39-11-670-9118; fax: +39-11-670-9138.E-mail address: adalberto.merighi@unito.it (A. Merighi).Abbreviations: ABC, avidin^biotin complex; BrdU, 5-bromo-2P-

deoxyuridine; BSA, bovine serum albumin; DAB, 3,3P-diamino-benzidine; EGL, external granular layer; EDTA, ethylenediami-netetra-acetate; GCP, granule cell precursor; IdU, 5-iodo-2P-deoxyuridine; IGL, internal granular layer; ML, molecularlayer; P, postnatal day; PARP, poly-ADP-ribose polymerase;PBS, phosphate-bu¡ered saline; PCD, programmed cell death;PCNA, proliferating cell nuclear antigen; SDS, sodium dodecyl-sulfate; TBST, Tris-bu¡ered saline with Tween 20; Tc, total cellcycle time; Ts, duration of S-phase; TUNEL, terminal deoxynu-cleotidyl transferase-mediated X-dUTP nick end labeling.

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ery, or cells intrinsically committed to death are rescuedby switching o¡ their suicide program (Bredesen andRabizadeh, 1997).

In altricial animals such as rats, mice, rabbits andhumans, much of the cerebellar development occurspostnatally (for review see Altman and Bayer, 1997).Extensive proliferation of the external granular layer(EGL) during the ¢rst postnatal weeks gives rise to sev-eral hundred million granule cells that migrate across themolecular and Purkinje cell layers to reach the internalgranular layer (IGL; Altman, 1972a,b; Rakic, 1971;Hatten, 1990). Starting in the 1970s, numerous studieshave shown that the EGL cells of several species dieduring postnatal development. These data have beenmainly obtained by counting pyknotic nuclei in normaland experimental animals (see Altman and Bayer, 1997for review). Pyknosis indicates the stereotyped nucleardegeneration with chromatin condensation and nuclearsize reduction that occurs at late stages of cell death,irrespective of the mechanism(s) which is (are) at thebasis of the death process (see Clarke, 1990). Recentwork in vivo led to the demonstration in di¡erent speciesof mammals, including humans, of extremely high num-bers of apoptotic cells in the postnatal cerebellum (Woodet al., 1993; Krueger et al., 1995; Lossi et al., 1998)compared to the ¢gures usually observed in other areasof the developing brain. Cells undergoing PCD in theEGL have been identi¢ed as mostly immature granulecells and their precursors (GCPs) and correlated withthe previously demonstrated pyknotic elements in thesame location. The cerebellar granule cells are excitatoryinterneurons that make axo-dendritic synapses onto thePurkinje cells through the parallel ¢bers. Consideringthat the temporal window in which apoptosis occursduring postnatal cerebellar development in mice doesnot coincide with the formation of synapses betweenthe parallel ¢bers and the Purkinje cell dendrites, it hasbeen excluded that granule cells undergo apoptosis as aconsequence of failure to make proper synaptic contactswith the Purkinje cells (Wood et al., 1993). In addition,the same authors suggested that a non-apoptotic mech-anism of cell death occurs at later stages of development,in parallel with the formation of synapses between thePurkinje and the granule cells.

In this paper we have devised a series of experimentsto obtain novel information about the relationshipbetween cell proliferation and death in the postnataldevelopment of cerebellar granule cells in vivo. Ourmajor goals were to establish the temporal links betweengenesis and death of individual cells in the course of theirmaturation and migration in the forming cerebellar cor-tex, and to clarify the relationship, if any, between syn-aptogenesis and apoptotic cell death.

EXPERIMENTAL PROCEDURES

Animals

Studies were performed on 50 New Zealand rabbits (Morini,Italy). Animals ranged in age from birth (P0) to postnatal day

30 (P30). All experiments were carried out according to Italianand EU regulations on animal welfare and were authorized bythe Italian Ministry of Health. The number of animals used waskept to a minimum and all e¡orts were made to minimize theirsu¡ering.

Time-window labeling of proliferating cells in vivo

P5 rabbits (n=26) received sequential i.p. injections (0.1 mg/gbody weight) of 5-iodo-2P-deoxyuridine (IdU) and 5-bromo-2P-deoxyuridine (BrdU) and were allowed to survive up to 48 hfollowing IdU administration. BrdU was administered 1, 3 or12 h before killing.

Analysis of cell cycle kinetics

Cell cycle kinetics were analyzed as described elsewhere(Yanik et al., 1992; Lossi et al., 2002). In brief, the method isbased on the fact that monospeci¢c anti-BrdU mouse monoclo-nal antibody recognizes only BrdU, whereas bispeci¢c anti-BrdU/IdU monoclonal antibody reacts with both IdU andBrdU. However, the anti-BrdU/IdU bispeci¢c antibody doesnot bind to BrdU after incubation with the monospeci¢cBrdU antibody at optimal titer so that all BrdU epitopes inthe sections are saturated. After dual labeling with colloidalgold particles of di¡erent sizes (see below), cells that incorpo-rated BrdU only were labeled with large gold particles only,while those that incorporated IdU only were singularly labeledby small size gold. Cells that incorporated both BrdU and IdUwere dual labeled. The speci¢city of labeling was assessed by:(i) absence of labeling when the immunogold procedure wasperformed on animals that were not previously injected withBrdU or IdU; (ii) observation of selective labeling of the nuclearcompartment of the cell ; (iii) subtraction of background label-ing. In this case we have calculated the number of gold particles/unit area over empty resin or cytoplasmic structures (n=3) andsubtracted it from the number of particles over labeled nuclei. Anucleus was considered to be positively labeled when it con-tained at least six gold particles after background subtraction.As the unit area the value of 12 Wm2 was chosen as an estima-tion of the mean nuclear area of GCPs.

To measure the percentage of cells in S-phase (labelingindex=LI), the duration of the S-phase (Ts), and the total cellcycle time (Tc) the following formulas were used:

LI ¼ BrdU only cellsþ double labeled cellsTotal cells counted

U100

Ts ¼ BrdU only cellsþ double labeled cellsIdU only cells

Ut

(where t is the time interval between the administration of thetwo tracers)

Tc ¼ TsLI

Ugrowth fraction

Our use of a 50% growth fraction is based upon the estima-tion of the percentage of proliferating cell nuclear antigen(PCNA) positive GCPs in the rabbit EGL (Lossi et al., 1995).

Histology and ultrastructural analysis

After the animals were killed with sodium pentobarbital, theywere perfused with 4% paraformaldehyde in phosphate bu¡er0.1 M pH 7.6 (light microscopy) or 2% glutaraldehyde+1%paraformaldehyde in So«rensen bu¡er 0.1 M pH 7.4 (electronmicroscopy). For light microscopy the cerebella were subse-quently cryoprotected in 2 M sucrose in phosphate-bu¡ered sa-line (PBS) and cut with a cryostat, or dehydrated and embeddedin para⁄n wax. Cryostat and para⁄n sections were cut in aparasagittal plane. For electron microscopy 200 Wm thick para-sagittal vibratome slices of the vermis were osmicated, staineden bloc with uranyl acetate, dehydrated and £at embedded inAraldite. After ultrathin sectioning and counterstaining, grids

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were observed with a Philips CM10 transmission electron micro-scope.

Immunocytochemistry

Light microscopic immunocytochemistry was carried outusing the avidin^biotin complex (ABC) peroxidase method. Inbrief, slides were pre-treated for 30 min with a solution of 2.5%methanolic hydrogen peroxide (35%) to block endogenous per-oxidase, followed by primary antisera overnight at room tem-perature and immunostained using the ABC peroxidase method(Vector, Burlingame, CA, USA). The peroxidase reaction wasdeveloped with the glucose oxidase^nickel^3,3P-diaminobenzi-dine (DAB) procedure. Electron microscopic immunocyto-chemistry for the visualization of BrdU/IdU was performedusing a post-embedding immunoglobulin^gold labeling methodwith anti-mouse IgG^gold conjugates [10 nm (single labelings)or 20 nm and 30 nm (double labelings)]. In brief, sections ontouncoated nickel grids were incubated with a 5% solution ofsodium metaperiodate, followed by 10% normal goat serum inTris^HCl 0.5 M, pH 7.4 containing 1% bovine serum albumin(Sigma, St. Louis, MO, USA) (Tris^HCl^BSA). After extensivewashes in bu¡er, grids were incubated with the monospeci¢canti-BrdU antibody (1:100), followed by anti-mouse IgG^goldconjugates (British BioCell, UK) of large size diluted 1:15 inTris^HCl^BSA. Grids were then incubated with the bispeci¢cBrdU/IdU antibody (1:200) and the immunocytochemical reac-tion was revealed with the anti-mouse IgG^gold conjugate (Brit-ish BioCell) of smaller size diluted 1:15 in Tris^HCl^BSA.Technical details on the procedures have been published else-where (Lossi et al., 2002).

In situ detection of apoptotic cells

Detection of apoptotic cells was performed in situ at bothlight and electron microscopy levels using a modi¢ed terminaldeoxynucleotidyl transferase-mediated dUTP-digoxigenin nickend labeling (TUNEL) method. For light microscopy the reac-tion was subsequently visualized using an anti-digoxigenin Fabfragment conjugated with alkaline phosphatase (Boehringer,Mannheim, Germany) and the Nitroblue Tetrazolium/5-bromo-3-indolylphosphate p-toluidine salt substrate. For ultrastructuralanalysis an anti-digoxigenin antibody conjugated with 10-nmgold particles was employed (British BioCell).

Genomic DNA electrophoresis

Genomic DNA was extracted using routine protocols fromP0, P5, P10, P15 and P30 cerebella (three animals for eachgroup). In brief, tissue was dropped into liquid nitrogen andhomogenized until ground to a powder. Extraction bu¡er(Tris^HCl pH 8.0, 10 mM; EDTA pH 8.0, 0.1 M; RNase 20Wg/ml; sodium dodecylsulfate (SDS) 0.5%) was then added andthe DNA isolated by phenol extraction in the presence of 100Wg/ml proteinase K. Low molecular weight contaminants wereremoved by dialysis. Recovered DNA was washed in 70‡C etha-nol, air dried, and resuspended in TE bu¡er pH 8.0 at a con-centration of 1 Wg/ml. Electrophoretic separation of DNAladders was done in 2% agarose gels. Following electrophoresisgels were stained in 0.01% ethidium bromide for 45 min.

Southern blotting and immunodetection of incorporated BrdU/IdU

Electrophoresed DNA was transferred overnight onto anitrocellulose membrane by capillary transfer. BrdU and IdUincorporated into the DNA fragments were detected usingmonospeci¢c or bispeci¢c antibodies (see below) and the ABCperoxidase method. In brief, membranes were soaked in PBScontaining 0.2% BSA and Tween 20; following overnight incu-bation in the primary antibody, they were washed in bu¡er,incubated for 60 min in biotinylated anti-mouse IgGs, rinsedin bu¡er and transferred for 30 min in the ABC mixture. Theperoxidase reaction was developed with DAB.

Preparation of whole cerebellar extracts

Cerebella from P0, P5, P15 and P30 rabbits (three animals foreach group) were suspended in 2.5 ml cold general lysis bu¡ercontaining protease inhibitors (1 mM phenylmethylsulfonyl £u-oride, 1 mg/ml leupeptin and 5 mg/ml aprotinin), gently homog-enized and centrifuged at 105Ug at 4‡C for 1 h. The pellet wasthen resuspended in lysis bu¡er.

SDS^polyacrylamide gel electrophoresis and western blotting

After sonication, protein concentration in whole cerebellarextracts was measured using the Coomassie protein assayreagent (Pierce, Rockford, IL, USA). Samples containing 15Wg of proteins were electrophoresed at 180 V on 15% acrylamidegel using a Tris^glycine running bu¡er. Separated proteins werethen transferred onto a nitrocellulose membrane using a Tris^glycine bu¡er at 100 V for 1 h. Blot was blocked with 5% BSAin TBST (20 mM Tris, pH 7.5, 150 mM NaCl, 0.2% Tween 20)overnight at 4‡C, and then probed for 1 h at room temperaturewith appropriate primary antibodies at optimal titer in TBST/1% BSA. After three washes in TBST, the membrane was incu-bated with peroxidase-labeled anti-rabbit secondary antibodies(Amersham, UK) diluted 1:10,000 in TBST/1% BSA for 1 h atroom temperature. Following washing in TBST, proteins were¢nally detected by enhanced chemiluminescence (ECL; Amer-sham).

Antibodies

The following antibodies have been used: monospeci¢c anti-BrdU mouse monoclonal (Amersham), bispeci¢c anti-BrdU/IdUmouse monoclonal (Caltag, USA), anti-poly-ADP-ribose poly-merase (PARP) rabbit polyclonal (Santa Cruz Biotechnology,Santa Cruz, CA, USA), anti-PCNA mouse monoclonal (Dako-patts, Copenhagen, Denmark), anti-synaptophysin mousemonoclonal (Dakopatts).

RESULTS

Apoptosis is a massive phenomenon in the P5^P10 rabbitcerebellum, in parallel with the intense proliferation of theEGL

After labeling of proliferating cells with a single injec-tion of BrdU and 1 h survival, positive cells weredetected in the P0^P30 rabbit cerebellum with a peakat P5. As expected these cells were concentrated in theouter proliferative zone of the EGL (Fig. 1A) and theirnumber decreased in parallel with the decline of thethickness of the EGL. After 1 h survival only a fewscattered positive nuclei could be observed in the IGL.However, if animals were injected at birth and allowed tosurvive until P5 the pattern of labeling changed in paral-lel with the well known migratory phenomena a¡ectingthe immature granule cells. In this case, labeling in theEGL was more disperse and numerous positive cells weredetected in the IGL (Fig. 1B), and, to a lesser extent, themolecular layer (ML). When proliferating cells were vi-sualized by immunocytochemical labeling with the anti-PCNA antibody, the pattern of staining was similar butcomparatively more intense (Fig. 1C). In this case virtu-ally all cells in the outer proliferative zone of the EGLwere stained, and numerous positive nuclei were clearlylabeled in the more super¢cial part of the IGL.

Conventional histology of semithin plastic sections

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Fig. 1.

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L. Lossi et al.512

from P5 rabbits revealed a number of cells with highlycondensed nuclear chromatin in the outer EGL (Fig.1D). In situ visualization of apoptotic cells by theTUNEL method showed the existence of comparablenumbers of positive nuclei in the same location (Fig.1E). Fewer positive nuclei were also detected in theinner pre-migratory part of the EGL and in the IGL.Scattered cells showing DNA fragmentation were alsovisible in the white matter. In P10 rabbits TUNEL pos-itive nuclei were reduced in the EGL, but they were moreeasily spotted in IGL (Fig. 1F).

Further con¢rmation of the true apoptotic nature ofcell death and the massive extent of apoptosis wasobtained by observation of DNA laddering directlyafter gel staining with ethidium bromide in P5^P10 rab-bits (Fig. 2A), and detection of the active p85 fragmentof PARP in western blottings from whole cerebellarextracts (Fig. 2B).

Granule cells and their precursors in the EGL undergoapoptosis within 24^36 h after their generation

After sequential injections of IdU and BrdU in P5rabbits and di¡erent survival times up to 48 h after theadministration of the ¢rst tracer, we have been able toanalyze cell proliferation within the EGL in a pre-deter-mined window of time according to the interval betweenthe administration of the two labels (Yanik et al., 1992).In addition, using electron microscopy and ultrastruc-tural immunocytochemistry, we were enabled to easilyidentify the tracer(s) incorporated by positive cells, thetype(s) of labeled cells, and the presence of apoptotic celldeath. Colloidal gold particles indicative of S-phasemarker incorporation were consistently found over thecell nucleus. There was no aspeci¢c staining over othercellular compartments. Background staining was negli-gible (see Experimental procedures). After a survival

Fig. 2. (A) Electrophoresis of genomic DNA reveals the presence of low size oligomers between P0 and P10. Note theabsence of DNA laddering at P15. (B) Cleavage of PARP is particularly evident at P5. Note that the enzyme is not cleaved

at P0 and P30.

Fig. 1. (A, B) Labeling of proliferating cells in the rabbit cerebellum following BrdU administration. After 1 h survival label-ing at P5 is concentrated in the outer EGL (A); if animals are injected at P0 and allowed to survive until P5 labeling ismore disperse in the EGL and positive cells are more easily spotted in the IGL (B). (C) PCNA immunostaining of the P5rabbit cerebellar cortex. Note that immunoreactivity is detected in virtually all cells in the outer EGL, and that numerouspositive nuclei are clearly visible at the interface between the Purkinje cells and the IGL. (D) Nissl staining of the P5 EGLreveals the presence of numerous pyknotic cells with intense nuclear staining (arrows). (E) TUNEL labeling of the P5 cerebel-lum shows numerous cells with fragmented DNA in the EGL and IGL, some of which are indicated by arrows. (F) Numer-

ous TUNEL labeled nuclei are present in the IGL of P10 rabbits. Scale bars = 20 Wm.

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Postnatal cerebellar apoptosis in rabbits 513

time of 6 h (IdU administration: t=0, BrdU administra-tion: t=3 h), we counted cells in the EGL (n=3345) andcalculated the following parameters: LI, 31.3% ; Ts, 18.8h; Tc, 30 h. All labeled cells were identi¢ed as GCP and/or pre-migratory granule cells in the electron microscope(Fig. 3A). Cells with a typical apoptotic morphology, i.e.chromatin condensation/blebbing and fragmentation intoapoptotic bodies, did not contain gold particles indica-tive of BrdU or IdU incorporation. However, if animalswere allowed to survive for at least 24 h with an intervalof 12 h between the administration of the two tracers,86% of the cells with an apoptotic morphology (n=157)within the EGL appeared to be labeled for either BrdUor IdU, or both, at the level of their condensed chroma-

tin (Fig. 3B). Using the same experimental paradigmthere were no labeled apoptotic cells (out of 131) withinthe IGL (Fig. 3C). After in situ TUNEL staining, label-ing of fragmented DNA was constantly observed in cellswith the typical ultrastructural alterations of apoptosis(Fig. 4A). However, granule cells and/or GCPs with anintact morphology were also stained as soon as 6 h afterBrdU administration (Fig. 4B).

The temporal links between proliferation and apopto-sis have been further analyzed after in vivo IdU/BrdUadministration, Southern blotting and immunodetection(Fig. 5). Within the ¢rst 6 h after incorporation of IdU/BrdU, both labels were localized at the level of highmolecular weight DNA. Conversely, when animals were

Fig. 3. Dual labeling of the P5 cerebellar cortex with IdU (t=0; small gold ^ arrowheads) and BrdU (t=12 h; large gold ^arrows). Survival : 24 h. (A) A double-labeled cell in the outer EGL is marked by the asterisk (see inset). (B) Apoptotic cell(ap) in the outer EGL. The area marked by the asterisk is shown at higher magni¢cation in the inset. The cell is singularlylabeled with large gold particles indicative of incorporation of BrdU, which was administered 12 h before killing. (C) Apo-ptotic cell in the IGL. The area marked by the asterisk is shown at higher magni¢cation in the inset. The cell is unlabeled.

mfr =mossy ¢ber rosette. Scale bars = 1 Wm (insets : 0.1 Wm).

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Fig. 4. (A) The nucleus of an early apoptotic cell (ap) in the outer EGL at P5 is labeled by the TUNEL assay for DNAfragmentation. The area marked by the asterisk is shown at higher magni¢cation in the inset. (B) A morphologically intactGCP (gcp) in the P5 EGL is labeled for DNA fragmentation (inset : small size gold ^ arrowheads). This cell has also incorpo-

rated the BrdU administered 6 h before killing (inset: large size gold ^ arrows). Scale bars = 1 Wm (insets: 0.1 Wm).

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Postnatal cerebellar apoptosis in rabbits 515

allowed to survive for longer periods (24^36 h), stainingof the ¢rst S-phase marker was detected at the level oflow molecular weight DNA oligomers. Following longersurvivals (48 h and more) there was no staining of lowmolecular weight DNA (not shown).

Apoptotic cells are cleared from tissue by the glia

Cells undergoing PCD in the outer EGL frequentlydisplayed the characteristic ultrastructural features ofearly apoptotic degeneration: they showed an intactnuclear envelope and initial chromatin condensationwith mild alterations of their cytoplasm (Fig. 4A). Inthe inner EGL these cells co-existed with those display-ing the ultrastructure of late apoptosis, i.e. advancednuclear condensation and general increase of cytoplasmicelectron-density with initial organelle disruption (Fig.6A). Interestingly, apoptotic cells were often in closeapposition to dark glial elements showing a nucleuswith ¢nely dispersed chromatin and abundant organellessuch as mitochondria, Golgi and rough endoplasmicreticulum. The nucleus of these glial elements was oftenbent around the apoptotic granule cell (Fig. 6A). Theapoptotic cells spotted in the IGL displayed similar spa-tial relationships with the dark glia. Interestingly, as onemoved towards the white matter, it was quite easy toobserve dark glial cells engulfed with apoptotic bodies.Frequently, the phagocyte completely enwrapped anentire apoptotic cell with its cytoplasmic expansions(Fig. 6B). This pattern likely corresponds to the initialstages of apoptotic cell clearance. At later stages thephagocyte contained several fragments of apoptoticbodies (phagosomes) and was often observed in closeapposition to blood capillaries (Fig. 7A). After BrdU/

IdU labeling it was common to observe labeling of apo-ptotic bodies within the phagosomes and phagocyticmonocytes in blood capillaries with heterophagosomesconsisting of highly condensed chromatin bodies show-ing BrdU/IdU labeling (Fig. 7B).

Synapses in the postnatal cerebellar cortex

From the histological and biochemical observationsreported above it appeared clear that there is a peak ofapoptotic cell death at P5. Therefore, we thought thatultrastructural analysis of the cerebellar cortex at thisdevelopmental stage would be very helpful to ascertainwhether or not PCD was related to synaptogenesis.

Previous observations on the immunocytochemicaldistribution of synaptophysin showed that the patternof immunoreactivity changed in parallel with theongoing maturation of the cerebellar cortex (Lossi etal., 1995). At P5 there was a neat staining within theML with a decreasing gradient from the Purkinje celllayer to the EGL. Staining in the ML took the form ofan extremely ¢ne punctate reaction surrounding the den-dritic tree of the Purkinje neurons (Fig. 8A). At thisstage, there was also staining of the IGL in the formof coarse clumps of immunoreactivity scattered amongthe unreactive granule cell bodies. In P10 animals theintensity of staining increased in both layers.

Ultrastructural examination of the P5 cerebellum con-¢rmed that the composition of the formative ML, sec-tioned in the translobular plane, was predominantlyconstituted by transversely cut parallel ¢bers containinga variable number of microtubules and occasional mito-chondria (Fig. 8B, C). The parallel ¢bers were more orless densely packed with a limited extension of the extra-cellular space. The dendritic shafts (Fig. 8C) and spines(Fig. 8B) of the Purkinje neurons were easily recogniz-able. Presumptive synapses between the parallel ¢bervaricosities and the Purkinje dendritic spines were alsoclearly visible (Fig. 8B). In addition, desmoid junctionsand concentric pro¢les of parallel ¢ber varicosities/Pur-kinje spines were frequently observed (Fig. 8B-C).

Initial formation of synapses was also clearly discern-ible in the IGL (Fig. 3C), but we have been unable toobserve synapses between pre-migratory horizontally ori-ented bipolar granule cells and Purkinje cell dendrites atthe interface between the inner EGL and ML (see Fujitaet al., 1996).

DISCUSSION

Apoptosis is a widely characterized phenomenon inthe normal development of the nervous system (Sastryand Rao, 2000). However, while there is a wealth of dataon the cellular and molecular mechanisms governing cer-ebellar granule cell apoptosis in vitro, only a few studieshave recently demonstrated the existence of a period ofmassive PCD during normal postnatal development ofthe cerebellum in altricial mammals (Wood et al.,1993; Krueger et al., 1995; Lossi et al., 1998). Theseobservations are somehow intriguing if one considers

Fig. 5. Southern blotting and immunodetection of S-phase labelsin P5 cerebellar extracts. Detection of BrdU (monospeci¢c anti-body) after 1 (a), 3 (b) and 12 (d) h survival. Detection of IdU(bispeci¢c antibody) after 6 (c) and 24 (e) h survival. The DNA inpanels b and c was extracted from the same group of experimentalanimals (n=4) following IdU (t=0) and BrdU (t=3 h) adminis-tration and 6 h survival. The DNA in panels d and e wasextracted from another group of experimental animals (n=4) fol-lowing IdU (t=0) and BrdU (t=12 h) administration and 24 hsurvival. Note in panel e the incorporation of IdU in low sizeoligomers demonstrating that cells undergoing PCD were in the

S-phase of their cycle 24 h before.

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Fig. 6. Clearance of apoptotic cells by the glia. (A) An apoptotic cell (ap) in the EGL showing typical nuclear and cytoplas-mic condensation is in close contact with a dark glial element. A thin rim of glial cytoplasm surrounds the dead cell at thebottom of the picture (arrowheads). The highly electron-dense nuclear chromatin (asterisk) of the apoptotic cell is shown athigher magni¢cation in the inset. Inset : Chromatin is dual labeled with gold particles of di¡erent sizes indicative of IdU(t=0; small gold ^ arrowheads) and BrdU (t=12 h; large gold ^ arrows) incorporation. Survival : 24 h. (B) A dark glial cell(gl) within the IGL has ingested an apoptotic granule cell at early stages of degeneration. Scale bars = 1 Wm (Inset: 0.1 Wm).

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Fig. 7. Clearance of apoptotic cells by the glia. (A) A phagocytic cell containing late heterophagosomes (pha) is in closeapposition with a blood capillary. (B) A blood monocyte engulfed with heterophagosomes. The phagosome marked by theasterisk contains gold particles indicative of IdU incorporation (inset, arrows). The S-phase marker was administered 24 h

before killing. ec = endothelial cell. Scale bars = 1 Wm (inset: 0.1 Wm).

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Fig. 8 (Caption overleaf).

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that: (i) the apoptotic index in the cerebellar EGL(Wood et al., 1993; Lossi et al., 1998) is surprisinglyhigh compared to other areas of the developing brain(Wood et al., 1993; Ra¡ et al., 1994), and (ii) it isunclear what the relationship is between proliferationand apoptosis that occur in overlapping temporal win-dows and a¡ect the same neuronal type.

We show here the existence of a massive apoptoticpeak at P5^P10 (the apoptotic index in the P5 rabbitEGL ranges from 5 to 8% : Lossi and Merighi, unpub-lished observations) in parallel with the intense prolifer-ation of the rabbit EGL (Lossi et al., 1995). Our lightand electron microscopic observations are con¢rmed bythe detection of DNA fragmentation and the speci¢ccleavage of PARP, a 113-kDa protein that binds atDNA strand breaks and is a substrate for certain cas-pases, including caspase-3 and -7, which are activatedduring early triggering of the apoptotic pathway(Alnemri et al., 1996). The visualization of the DNAnucleosomal ladders directly after ethidium bromidestaining con¢rms that apoptosis occurs synchronouslyin a very large population relative to the majority ofsurviving cells (for discussion see Nicholson et al.,1995; Chun, 1998). Finally, the notably high extent ofPCD can be deduced from the visualization in the elec-tron microscope of the clearance of dying cells, which, toour knowledge, has so far been directly observed only inCaenorhabditis elegans (Ellis et al., 1991). Apoptotic cellsare in fact cleared from tissue in about 3 h in vivo (Perryet al., 1983) rendering their detection in situ quite di⁄cultunless apoptosis a¡ects extremely high numbers of cells.

Previous reports on granule cell death in rodents showthat signi¢cant cell loss also occurs in the P15^P30 inter-val (see Caddy and Biscoe, 1979). Our observations indi-cate that the apoptotic interval in rabbits ends at P10^P15. Not considering species-speci¢c di¡erences, it ispossible that, at later stages, apoptosis is diluted inspace and time and, hence, it might have been di⁄cultto detect. Alternatively, or in addition, other mechanismsof cell death may operate in late postnatal stages.

An unequivocal con¢rmation of the true apoptoticnature of cell death in our experimental model is ofnotable importance, considering that in other species ithas been indicated that both apoptotic and non-apopto-tic cell death a¡ect the granule cells in di¡erent temporalwindows during their maturation (Wood et al., 1993).

Temporal links of proliferation and apoptosis in the EGL

Our experiments demonstrate the existence of a 24^

36-h interval between the generation and death of pro-genitor cells/GCPs in the EGL. After IdU/BrdU incor-poration the Tc of GCPs results in approximately 30 h.It must be noted that the calculation of this parameter isbased upon the assumption that 50% of the cells in theEGL are in cycle (growth fraction). As already men-tioned, the method employed here does not allow for adirect determination of growth fraction values (Yanik etal., 1992), and the value of 50% is based upon the per-centage of PCNA labeling in the EGL of P5 rabbits(Lossi et al., 1995; see also Experimental procedures).In this context, it should be noted that PCNA labelinglikely gives an overestimation of the growth fraction (asit stands by comparing Fig. 1A and C), because immu-noreactivity can be detected in G0 cells and may persistfor a certain period of time in daughter cells (Bravo andMacdonald-Bravo, 1987; Takahasi and Caviness, 1993;Beppu et al., 1994). This implies that, in actual terms, theTc of GCP may be even shorter than above.

After a DNA fragmentation assay, we observed thatwithin 24 h upon completion of their S-phase the major-ity of these cells had terminated PCD, displaying all thetypical ultrastructural features of apoptosis and di¡erentpatterns of incorporation of the S-phase labels accordingto the interval between the administration of the twotracers. This observation and estimation of Ts and Tcdemonstrate that the apoptotic machinery in GCPs isactivated upon completion of the S-phase and/or imme-diately thereafter. These data are substantiated by theidea that a cellular clock regulates the death of certainneuronal populations during development, and an intrin-sic time limit exists between genesis and death of theseneurons (Galli-Resta and Ensini, 1996).

Work in several strains of mutant mice, with impairedcerebellar development and increased apoptosis of granulecells (Migheli et al., 1995; Murtoma«ki et al., 1995; Wu«llneret al., 1995), indicates that augmentation of cell deathinvolves an apparent loss of cell cycle control in GCPs(Herrup and Busser, 1995; Migheli et al., 1999). Also,apoptotic cell death in Alzheimer diseased brain is relatedto expression of ectopic cell cycle proteins (Busser et al.,1998). The idea that apoptosis is closely linked to cell cycleparameters is reinforced by the present observation of asharp temporal sequence in the appearance of BrdU/IdUlabeling in low molecular weight DNA oligomers.

Clearance of apoptotic cells

Extensive cell death in the postnatal cerebellumaccounts for the possibility of directly observing the

Fig. 8. Synaptogenesis in the P5 rabbit cerebellum. (A) Synaptophysin immunoreactivity is detected in the form of a ¢nepunctate reaction in the ML and coarser clumps of immunopositive material within the deep IGL, which is populated byolder post-migratory granule cells. Note that there is no immunoreactivity at the interface with the Purkinje cells (double-headed arrow). (B) Ultrastructure of the ML. Three synapses between the parallel ¢ber (pf) varicosities (pfv) and the Purkinjedendritic spines (sp) are clearly recognizable. Desmoid junctions (arrows) and one concentric pro¢le of parallel ¢ber varicos-ity/Purkinje spine (asterisk) are also evident. Note that, although the presence of desmoid junctions and concentric pro¢lesindicates that the forming ML still shows some degree of immaturity, the parallel ¢ber varicosity^Purkinje spine synapsesshow the features of the typical mature axo-dendritic (spinous) asymmetrical contacts. They have few mitochondria and alarge concentration of tightly packed round synaptic vesicles. The presynaptic membrane is thin but densely stained, the post-synaptic membrane is much denser. (C) Three desmoid junctions (arrows) over a Purkinje cell dendrite (Pcd). On the left,

note the high degree of packaging of the parallel ¢bers (pf). Scale bars = 50 Wm (A); 0.5 Wm (B); 0.25 Wm (C).

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clearance of apoptotic cells in situ. Electron microscopyshows that dark glial elements remove the entire apopto-tic cell rather than its fragments. Although an unequiv-ocal identi¢cation of microglia is quite di⁄cult inconventional preparations, this type of clearance is sim-ilar to that observed in the mouse cerebellum after lectinlabeling of microglial cells (Ashwell, 1990), but di¡ersfrom classical phagocytosis (Van den Eijnde et al.,1999). From the present observations one can proposethat the dark glial cells (microglia) : (i) get in close appo-sition with the apoptotic neuron; (ii) enwrap the entirecell, which is subsequently broken up into apoptoticbodies and sequestered within heterophagosomes;(iii) approach the blood capillaries; and (iv) eventuallyenter their lumen. The ¢rst two phases take place mainlyin the EGL (and ML), while the others are more com-monly observed in the IGL and white matter. It is thuspossible that some glial cells engulfed with apoptoticbodies migrate from the EGL to the blood capillaries,which are by far more abundant as one moves towardsthe IGL and the white matter. It is of interest to notethat Ashwell has failed to ¢nd a correlation of microgliaconcentration and pyknotic ¢gures in the P10 and P14EGL. The discrepancy with our observations may beeasily explained considering that: (i) simple examinationof pyknotic nuclei in semithin sections is likely to give anunderestimate of the number of apoptotic cells, and/or(ii) glial cells in the ¢rst two phases of the clearanceprocess have not yet acquired all the features of activatedmicroglia, i.e. they are not yet labeled by the Gri¡oniasimplicifolia lectin.

Synapse-independent and synapse-dependent apoptosis ofcerebellar granule cells

Granule cell migration, formation of parallel ¢bersand their synapses with the Purkinje cell dendrites inthe ML are major events in cerebellar development (seeAltman and Bayer, 1997; Sotelo, 1990). In mice, it hasbeen indicated that the temporal window of apoptosis(between P5 and P9) does not coincide with the periodof synaptogenesis (from P10), and it was concluded that,during the temporal window of synaptogenesis, granulecells do not die following apoptosis (Wood et al., 1993).However, initial formation of parallel ¢ber^Purkinjespine synapses, although showing some features ofimmaturity, is clearly evident in P7 mice (Kurihara etal., 1997).

Our ultrastructural analysis demonstrates the presenceof well developed presumptive parallel ¢ber^Purkinjespine synapses at P5. Although it is highly unlikelythat the parallel ¢ber^Purkinje spine synapses are thesole synaptic type at this developmental stage, thepresent observations lead us to conclude that in rabbitsthere is a wide temporal overlap, albeit not a perfectcoincidence, of apoptosis and synaptogenesis.

We propose here that both synapse-independent andsynapse-dependent PCD a¡ect the granule cells duringpostnatal development: the former occurs when pre-migratory GCP are still populating the EGL, while thelatter concerns the post-migratory neurons in the IGL.

Apoptosis in the EGL could not be regarded as theconsequence of a neuronal selection according to theclassical neurotrophic paradigm (Lewin and Barde,1996) and/or to the synaptotrophic hypothesis of trophicfactor action (Snider and Lichtman, 1996), consideringthat numerous GCPs, for unknown reasons, are not res-cued from death by switching o¡ their intrinsic suicideprogram (see Bredesen and Rabizadeh, 1997). Since thereis a short temporal gap between proliferation and apo-ptosis, it might be di⁄cult to obtain a demonstration ofthe signals from neighboring cells which may be requiredfor the survival of GCPs in vivo (Ra¡ et al., 1994). Sincecell-to-cell signaling regulates the division of GCPs, asappears to be the case with the Sonic hedgehog made bythe Purkinje cells (Wechsler-Reya and Scott, 1999), onecan reasonably hypothesize that a cross-talk between dif-ferent cellular types regulates apoptosis in the EGL.

In mice and humans apoptotic cells in the IGL havebeen identi¢ed as post-migratory granule cells, but thesite where PCD of these cells occurred remained unclear(Wood et al., 1993; Lossi et al., 1998). From the presentwork, we believe that apoptotic cells in the IGL are post-migratory granule cells that failed to make proper syn-aptic contacts in the forming ML on the grounds thatthey (i) could be identi¢ed as granule cells on ultrastruc-tural basis, (ii) were not labeled by S-phase markers insitu, in keeping with the observation that BrdU/IdUlabeling of low size oligomers disappeared in Southernblots following a survival of 48 h or more, and (iii)increased in number with age, paralleling the maturationof synapses in the ML.

Several lines of evidence now converge to indicate thatPCD of post-mitotic granule cells is an indirect extrinsicphenomenon. Studies in mutant mice and chimeras haveshown that the activation/inhibition of di¡erent signalingpathways eventually results in apoptosis of granule cells(and other cerebellar neurons) (see for example Normanet al., 1995; Roberts et al., 2000; Selimi et al., 2000;Mitchell et al., 2001). Therefore, synapse-dependent ap-optosis of post-mitotic granule cells likely representsbut one of the several possible mechanisms triggeringPCD.

CONCLUSION

As for other neuronal populations (Jacobson, 1991),the curve of cell death in granule cells consists mainly oftwo phases. However, both these phases appear to be ofthe apoptotic type, and they are partially overlapping ona temporal sequence. The ¢rst, corresponding to earlyneurogenesis in the EGL, is characterized by a rapid,synapse-independent decrease of the number of cells.This tumultuous phase of PCD may have a role in thegross regulation of the otherwise rapidly expanding pop-ulation of pre-mitotic EGL neurons. In the second, amuch more limited decrease is achieved during a longerinterval of time, corresponding to the period of targetinnervation, and the less intense cell death likely allowsfor sculpting the circuitry of the mature cerebellar cortex(Frade and Barde, 1998).

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Acknowledgements4We thank Prof. Piergiorgio Strata and Dr.Stefano Gustincich for comments and critical reviewing of themanuscript. Grant sponsor: Italian MIUR (Co¢n2000); Minis-

try of Health (Progetto Finalizzato Alzheimer), and local grantsfrom the University of Torino to LL and AM.

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(Accepted 25 February 2002)

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