Table Of ContentZOOLOGICAL SCIENCE 10: 547-555 (1993) © 1993 Zoological SocietyofJapan
REVIEW
Purkinje-Granule-cell Interaction in theV Cerebellar Development
of Hyperbilirubinemic Murant
Hiroomi Keino1 and Shigeo Kashiwamata2
lDepartment of Perinatology, Institute for Developmental Research,
Aichi Prefecture Colony, 713-8 Kamiya-cho, Kasugai, Aichi 480-03,
and 'Aichi Prefecture/Chita District Public Health Center,
88-2 Yawata Aza Arakonochi, Chita, Aichi 478, Japan
—
ABSTRACT The importance of Purkinje-granule cell interaction for cerebellar development was
repeatedly emphasized by many authors. Mutant animals such as weaver and pcd mouse were useful
instrumentstospeculate the mechanismsofthe interaction. In weavermice, almost all the population
of postmitotic granule cells fails to migrate and dies. Purkinje cells, however, exist in the
underdeveloped cerebellum. The death of Purkinje cells occurs after synaptogenesis in pcd mutant
mouse. Developed granule cells remain unaffected in the mouse. Gunn rat isanothertype ofmutant
animal whose Purkinje cells are severely damaged at initial stage ofsynaptogenesis. The Gunn rat is
devoid of hepatic UDP-glucuronosyltransferase activity toward bilirubin and as a consequence shows
hyperbilirubinemia. Apreferential deposition ofbilirubin on Purkinje cellsoccurs in Gunn rat infants
at postnatal day 7. then the cells are severely damaged. Postmitotic granule cells lost companions for
synaptogenesis and faile to migrate. Our recent studies show that some lectins temporarily bind to
Purkinje and/or granule cells in the initial stage of synaptogenesis. The Purkinje-granule cell
communications may be mediated by various glycoproteins.
One ofmajorproblems in developmentalneuro- the cerebellar cortex, Purkinje and granule cells
biology is to elucidate the process in which a high (Fig. 1, p and g, respectively) and two afferent
degree of specificity that characterizes neuronal (climbing, cf, and mossy, mf, fibers) and one
connectivity in the mature brain is established. efferent (axon of the Purkinje cell, a) systems.
Such a complex organization must result from The granule cell stretches its axon toward the
progressive and sequential processes in which the molecular layer and spreads two branches that
interaction between a growing neuron and other make synapses with Purkinje cell dendrites. The
neurons including its cellular environment mod- Purkinje cell stretches one or two primary de-
ulates the expression of a genetic program. ndrites toward the surface of the folium. The
Neuronal connectivity in adult animals is better primary dendrites branch into two or more secon-
understood in the cerebellar cortex than in any dary dendrites that tend to run horizontally.
other part ofthe brain [26] because ofan apparent Forked branches continue to divide again in the
simplicity of the cerebellar circuitry. Thus, the same manner. The terminal branchlets are fitted
genetic malformation hasbecome avaluable mate- with numerous spines or thorns, which project
rial to analyze the role of cellular interactions in from all sides, like bristles on a bottlebrush. The
the formation of neuronal circuitry in the central dendrites form a characteristic and extraordinary
nervous system. There are two main neurons in arborization that extend through the whole thick-
nessofthe molecularlayer. It issuggestedthat the
Received Januarv 29. 1993 Purkinje-granule-cell interaction accomplishes an
548 H. Keino and S. Kashiwamata
isolatedfrom rat [30] and human [27] hepatocytes.
BR:UDPGT
In the j/j rat the genetic defect of
has been also proved to be a —1 frameshift
mutationcausedbyonebasedeletioninthecDNA
sequence [31]. This creats a new termination
codon resulting in a failure in acomplete synthesis
mI ofthe BR:UDPGTprotein. Thus, thej/j ratfails
to excrete bilirubin into the bile as a form of
glucuronides and develops severe hyperbilir-
ubinemia.
The concentration ofbilirubin in the circulating
r A
P I 4' < blood of the j/j rat has been reported to be
A
reduced with photoirradiation [28]. single 24-hr
photoirradiationin aperiod ofpostnataldaysfrom
3 to 11 revealed that the most effective day of
a!!^!?^IBff it1 irradiation is centered on postnatal day 7 [21], the
day being probably most critical in the bilirubin-
inducedcerebellarhypoplasia. Theseobservations
V**** indicatethatthecerebellarhypoplasiainthej/j rat
^w*****'***
is not directly related to the genetic abnormality
but secondary to hyperbilirubinemia.
Fig. 1. Cortical layers in a midsagittal section of the Although cerebellar weight in the j/j rat is
adult rat cerebellum. Left, hematoxylin-stained markedly reduced as a whole, the cerebellar
section; right, schematic representation ofneuronal
circuitry, m, molecular layer: P, Purkinje cell: g, medulla progresses normally. In fact, our histolo-
granule cell: a, axon of Purkinje cell: cf, climbing gical observations showed a well developed and
fiber, d, v, r, c, Is and rs indicate ventral, rostral, fully myelinated medulla [17]. Enzyme histoche-
caudal, left and right sides, respectively. mical examinations also showed strong acetylcho-
line esterase activity (a marker of mossy fibers) in
the hypoplastic cerebellum. The activity of cere-
essential function for the establishment of these bellar 2',3'-cyclic nucleotide 3'-phosphohydrolase
characteristic appearances. For the purpose of (a markerofmyelin) in the j/j rat were almostthe
analysing the Purkinje-granule-cell interaction we same as that in the j/+ rat at all developmental
have developed a mutant strain of rats (Sprague- ages [2]. These findings support the hypothesis
Dawley strain Gunn rat) from the original jaun- that afferent axons are not an attack point of
diced strain [18]. Homozygous (j/j) Sprague- bilirubin and impairment by the pigment occurs in
Dawley strain Gunn rats show a severe cerebellar interneurons or glial cells.
hypoplasia in lobules I to VIII but not in lobule X Cerebellar wet weight, dry weight and DNA
(Fig. 2B). The cerebellum of heterozygous (j/+) content have been shown to be very low in the j/j
ratsdevelops normally (Fig. 2A). Thej/j rat lacks rat [15], which is, for example, one fourth in wet
DNA
the ability ofbilirubin disposal in the liver andas a weight of the j+ rat at postnatal day 30.
consequence shows unconjugated hyperbilir- content, the rate of incorporation of
ubinemia, which develops soon after birth and 3H)thymidine into DNA, and protein concentra-
(
persists throughout life. The jaundiced condition tion in the j/j rat cerebellum are significantly
has been characterized as an autosomal recessive reduced on and after postnatal day 10 [16]. The
inheritance. The j/j rat is evidenced to be devoid majority of cerebellar neurons are granule cells.
DNA
of hepatic UDP-glucuronosyltransferase activity Therefore, decrease in content and the rate
toward bilirubin (BR:UDPGT, EC2.4.1.17) [32]. of thymidine incorporation into DNA indicates
Recently, cDNAs for BR:UDPGT have been diminution in the number ofgranule cells. Histo-
Cerebellar Development in Mutant Rats 549
VII
Vill
' *
;-•
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•
• -'
. '.V~ «. *••%•'•
- \'4 . :-* / £
- »
V. i <
6"
* • «•- '•.*•.itsj*.'".«-»z.*.- .-••. •• • .40i -
* • ••
- VII
' S
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-- <1 • • V *. • T. \ \ *? ®4
•"•VY"".-.-'.': ' . *** * •m »
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Fig. 2. Light-microscopic photographs of sagittal sections of the cerebellar vermis in heterozygous (j/+) and
homozygous (j/j) Gunn rats. A, j/+ rat with well-developed lobules at postnatal day 30. B, j/j rat. Lobules
I-VIII andthedorsalpartoflobule IXareseverelydamaged,whiletheventralpartoflobuleIXandlobuleXare
nearly normal. C. j/j rat at postnatal day 30with severely damaged lobules VII, where no Purkinje cells and a
reducednumberofinternal granulecellsareobserved. D, LobuleVII ofjI] ratatpostnatalday 10. Developing
external granule cells (ex) and many internal granule cells (in) are recognized. Purkinje cells (p) are severely
damaged.
logical observations revealed that the hypoplastic ly up to postnatal day 10 (Fig. 2D), the time being
cerebellum hadapoorgranulecelllayerandonlya included in the critical period of bilirubin toxicity
few granule cells remained in the layer (Fig. 2C). (postnatal day7 [21]), and alotofgranulecellsare
Granule cellsare severelyaffected in the hypoplas- observed in the granule cell layer at postnatal day
ticcerebellum. However, theymay not be the first 10. Itwas also true that much bilirubin deposition
attack point of bilirubin, since in the j/j rat cere- occurred on the granule cell layer at postnatal day
bellum the numberofgranule cellsincreasesrapid- 15 [7], but the effort to reduce the bilirubin
550 H. Keino and S. Kashiwamata
concentration at that day failed to protect the j/j followingthe impairmentbybilirubinofcerebellar
rat from cerebellar hypoplasia [18]. neuronstodigestthesedamagedneuronsandtheir
The amounts of/9-S100 and glial fibrillary acidic constituents by lysosomal heterophagous func-
proteins, markerproteins ofastrocytes, have been tions.
reported to be markedly increased in the j/j rat Cerebellar glutamate decarboxylase (GAD)
cerebellum [5]. The hypertrophy of Bergmann's activity is reported to be significantly lower in the
glial cells and astrocytes is also observed in the j/j rat than that in the j/+ rat [2]. The lower
GAD
cerebellum by the immunohistochemical method activity was observed at postnatal day 8 to
with anti-glial fibrillary acidic protein antiserum 20. This indicates that the bilirubin-induced im-
[6]. In particular, somata and apparently thick- pairment of synapse formation occurs mainly in
ened glial fibers of Bergmann's cells are strongly GABAergic neurons during the suckling period.
immunostained. These findings suggest that astro- Purkinje, Golgi, basket and Lugaro cells are
cytes are lessvulnerable to bilirubin thanneuronal knowntobe GABAergic. Severe abnormalitiesof
cells. Purkinje cells are seen even in newborn j/j rats;
During the early postnatal period the activities vacuolation ofthe cytoplasm is pointed outbefore
oflysosomalenzymesin the cerebellumincreasein 24hr of life [3]. The degree of impairment of
a linear manner, reach maximum at postnatal day Purkinje cells become severe and the cell popula-
20, and then decrease. It has been demonstrated tion diminishes with age in the j/j rat. It has been
thatthe maximumlevel ishigherin thej/j rat than also shown that little or no abnormalities are
in the j/+ rat [2]. However, the increase of recognized in Golgi and basket cells in the j/j rat
lysosomal enzyme activities in the j/j rat shows a cerebellum. These cells may be resistant to bilir-
big difference among enzymes [29]. The most ubintoxicity. Amongcerebellarneurons, Purkinje
striking rise has been recognized in /?- cellsseem toshowthe earliest [3] andseverest [33]
glucuronidase whose activity is elevated 8 times as manifestation ofabnormalities as characterized by
high as thatin thej/+ rat. Otherenzymessuch as the presence of bizarre membranous bodies, en-
arylsulfatase, cathepsin and acid phosphatase have larged mitochondria, and vacuoles. Biochemical
been found to be 2.0, 3.1 and 1.3, respectively, in data also suggest serious damages of Purkinje
the maximum activity ratio of the j/j to j/+ rat. cells. Aphosphoprotein, which is characteristicof
Enzyme histochemicalobservationsin the hypo- Purkinje cells, hasbeen extensivelyinvestigatedin
plastic j/j rat cerebellum have shown that the rat and mouse cerebella (P40o [24], GR-250 [13],
accumulation of acid phosphatase and /?- InsP3-binding protein [11]). The protein increases
glucuronidase reaction products is particularly rapidly from postnatal day 3 to 21 in the rat
marked in microglia-like cells whose cytoplasm is cerebellum coinciding with the development of
filled with lysosomes [17, 19, 22]. Lysosomes in Purkinje cell dendrites [13]. In the jj rat cerebel-
these cells are very large in size and often contain lum, reduction of the protein is prominent [4],
electron-lucent vacuoles. The age-related change suggestingstrongly the destructionofPurkinje cell
in the numberofmicroglia-like cells resembles the dendrites. These results may indicate that bilir-
pattern of biochemically detectable lysosomal en- ubin attacks Purkinje cells at the stage of synap-
zyme activities in the j/j rat. Microglia-like cells togenesis.
appear in the cerebellum at postnatal day 5 and This is also supported by the following findings.
reaches maximum in number at postnatal days 15 The severity of cerebella hypoplasia in the j/j rat
to 20. Thus, the enhancement of lysosomal en- differed considerably among lobules [16, 17].
zyme activities in the j/j rat cerebellum is consi- Histological observations in the j/j rat cerebellum
dered to be associated with the increase in the showed the lobular difference of Purkinje cells in
numberofmicroglia-likecells. Microglia-like cells vulnerabilitytobilirubin toxicity: Lobule I to VIII
are never observed in a slightly damaged cerebel- were greatly reduced in cell number, while no
lumwhosewetweightishalfofthatinthej/+ rat. significant difference was observed in lobule X
It would appear that microglia-like cells emerged between the j/j andj/+ rats at anypostnatal day.
Cerebellar Development in Mutant Rats 551
Disappearance of Purkinje cells in lobule I-VIII blue at pH3.8 were rich in number in lobule VII,
amounted to more than 90%. butonlyafewin lobuleX(Fig. 3AandB). On the
Some morphological differences of Purkinje other hand, azocarmin-stained Purkinje cells were
cells between lobule I-VIII and IX-X have been abundantin lobule X andpoorin lobule VII atthe
reported with hamsters [23] and rats [12]. This same pH. These findings suggest that the intra-
encouraged us to pursue histochemically the back- cellular pH of Purkinje cells in lobule VII is
ground of cellular vulnerability to bilirubin. Pur- more acidic than that in lobule X, resulting in a
kinje cells showing dark blue staining by toluidine larger amount of bilirubin deposition on Purkinje
9*
*
9
<&
Fig. 3. Light microscopicphotographsofPurkinje cellsin lobule VII (Aand C) and lobule X (B and D). A and B,
toluidine blue-stained Purkinje cells at pH3.8. In A, most of Purkinje cells (p) and some Golgi cells (g) are
stained dark blue, while granule cells are light blue. In B, Golgi cells (g) are stained blue but Purkinje cells (p)
light-bule. C and D. photographs ofasection incubated with bilirubin at pH8.0. In C, a marked deposition of
bilirubinisobservedinthecytoplasmofPurkinje(p)andGolgi(g)cells. In D,bilirubindepositioninonlyasmall
in amount on Purkinje cells but large on Golgi cells.
552 H. K.EINO AND S. KASHIWAMATA
cellsin lobule VII thanin lobuleX. Such a lobular cells have been discussed with cerebellar mutant
difference in bilirubin deposition on Purkinje cells animals [9]. 1) Weaver mice are classified as a
was also observed at pH8.0. Purkinje cells in mutant strain whose granule cellsdegenerate at an
lobule VII and X were stained deep and light early developmental stage [36]. 2) No mutant
yellow, respectively (Fig. 3C and D). Golgi cells animals whose granule cells are lost at a late stage
also stained yellow, but there was no difference in of development have been found. 3) Pcd is a
bilirubin deposition between lobule VII and X. mutantstrainofmice, inwhichthedegenerationof
Only a small amount ofbilirubin was deposited in Purkinje cells occurs at a late developmental stage
the granule cell and molecular layers and white [34]. 4) In the j/j Gunn rat, Purkinje cells de-
matter in both lobules. generate at an early stage of development due to
The affinity of Purkinje cells for bilirubin has the secondary effect of inherited deficiency of
been also evidenced in vivo. A single sub- BR UDPGTactivity. Amongthese mutant anim-
:
cutaneous injection ofbucolome, a potent displac- als, the j/j rat is considered to be more important
er of bilirubin from the bilirubin-serum albumin to study the interaction between Purkinje and
complex, increasedthe cerebellarbilirubin level in granule cells in an early cerebellar development.
the j/j rat [7]. At 7 days of life, the most critical In weaver mice with an autosomal incomplete
day of the bilirubin-induced cerebellar hypoplasia dominantmutation,despitenormalmitoticactivity
[14], Purkinje cells were stained yellow with bilir- ofgerminal cells in the external granule cell layer,
ubin in the bucolome-treated j/j rat. Although almost all the population of postmitotic granule
bilirubin lost toxicity to impair the cerebellar de- cellsfailstomigrate anddiesatthe interfaceofthe
velopmentat 15days[18], localizedyellowstaining external granule cell and molecular layers before
in the bucolome-treated rat cerebellum was most emitting their axons. Purkinje cells, however,
apparent in the granule cell layer with no staining exist in the underdeveloped cerebellum with no
of Purkinje cells [7]. The results suggest that the obvious abnormalities either in the size of cell
severity ofhypoplasia in lobule I to VIII ofthe j/j bodies or in the shape ofdendritic trees. Purkinje
ratiscausallyrelated to thecytochemical tendency cells are able to form postsynaptic apparatus (de-
of Purkinje cells to deposit bilirubin. Thus, it ndritic spines) in an apparent absence of the
seems likely that a preferential deposition ofbilir- presynaptic induction [9]. In fact, the electron
ubin on Purkinje cells in lobule I to VIII occurs in microscopic feature that characterizes the weaver
j/j rat infants, through which Purkinje cells are mouse cerebellum is the presence of innumerable
damaged eventually leading to degeneration. dendritic spines similar to those normally arising
The critical period of bilirubin-induced cerebel- from 'spinebranchlets' atthetipofdendritictrees.
lar hypoplasia is centered on postnatal day 7 [20, Purkinje cells form synapses with two afferent
21]. Takagishi and Yamamura [35] reported that systems, climbing and mossy fibers. Mossy fibers
Purkinje cells in rats, to which hyperbilirubinemia not only make synapses on their specific targets,
was experimentally induced by hemolysis, were but extend their terminal fields and establish
destroyed by bilirubin at around postnatal day 7. synaptic connections with branchlet spines. A
The postnatalday7 iscomparable tothestage that transient phase ofmultiple climbing fiber innerva-
Purkinje cells begin to form synapses with parallel tions on a single Purkinje cell, which occur during
fibers [1]. The developmental increase ofP40o has an early stage of development in a one-to-one
not been observed in the j/j rat cerebellum [4]. It climbing fiber-Purkinje cell ratio, remains
is suggested that bilirubin affects directly or in- throughout life. The numerical mismatch in the
directly the synapse formation between Purkinje weaver mouse suggests that the establishment of
and granule cells, followed by failure of the synaptic contacts between parallel fibers and Pur-
differentiation/translocation of external to inter- kinje dendrites is necessary for maturation of the
nal granule cells, and then by eventual death and climbing fiber-Purkinje cell synapse. Although
reduction in number of granule cells. mossy fibers can not reach their normal targets,
The interaction between Purkinje and granule they are able to come into contact not only with
Cerebellar Development in Mutant Rats 553
the branchlet spines ofPurkinje cell dendrites, but ofthe central nervous system. The chemo-affinity
also with basket and stellate cells. It is clearly hypothesis on intercellular recognition generally
shown that in the weaver mouse Purkinje cells involves membrane-bound glycans, since they are
survived and developed to a certain extent into the mostperipheralcomponentofcells. Zanettaet
common forms without interaction with granule al. [38] show the emergence of concanavalin A-
cells. These results indicate that the intrinsic binding glycoproteins on newly formed parallel
specificity of afferent-efferent coupling in the de- fibers. Initial observations have reported that the
velopment of cerebellar circuitry can be overrid- membraneofnewlyformedparallelfibersisrichin
den by a local cellular milieu. a specific class of glycoproteins that binds to con-
Pcd is an autosomal recessive mutation produc- canavalin A, and such glycoproteins disappear
ingdeath ofPurkinje cells. The death occurs after when synaptogenesisiscompletedinthecerebellar
synaptogenesis. There is a general agreement that molecularlayer. Dontenwill etal. [10] suggestthat
cerebellar neurons initially affected are Purkinje glycans located on the membrane ofparallel fibers
cells though the mechanism is unknown [25]. De- are enteredinto anddigestedin Purkinje cellswith
veloped granule cells remain unaffected regardless a concomitant and transient increase of a-D-
of the absence of Purkinje cells throughout life. mannosidase activity. Furthermore, immunohis-
Studies on adult pcd mice have made it possible to tochemical studies have shown that the enzyme is
analyze the fate ofpresynaptic elementsbelonging notlocated inthe extracellularspacebutexclusive-
to parallel fibers, which are deprived of their lyin Purkinjecells, where lectin-like moleculesare
postsynaptic elements shortly after normal synap- also detected. Our recent studies show that some
togenesis. In the pcd cerebellum, the molecular lectins temporarily bind to Purkinje and/or gra-
layer contains bundles of parallel fibers bearing nule cells in the developmental stage (in prepara-
numerous presynapticvaricositiesbut free ofpost- tion). For instance, peanut agglutinin-binding
synaptic partners. These observations indicate glycans appear near the nucleus of migrating gra-
that developed granule cells are able to be alive nule cells and developing parallel fibers (Fig. 4A).
and to maintain theirmorphologywithout connec- Zanetta et al. [39] have hypothesized that both
tion to Purkinje cells. lectin-like molecules and glycans may be recogni-
No model animals whose granule cells degener- tion molecules allowing a specific contact between
ate at a postsynaptic stage have been investigated parallel fibers and Purkinje cells at the period of
yet. In such animals, Purkinje cells may probably synaptogenesis. The lectin-like molecules are re-
keeptheircharacteristicdendritictreesnormaland portedly localized not only in the plasma mem-
the innervationofclimbing and mossy fiberswould brane and the endoplasmic reticulum, but also in
newly appear. In this context the j/j Gunn rat, in endocytoticfigures, coatedvesicles andlysosomes.
which the degeneration of most Purkinje cells is These molecules are likely tobe membrane-bound
observed just before synaptogenesis, may be a receptors participating in the internalization of
suitable modeltostudytheinteraction, theprocess extracellular compounds that are digested in lyso-
of membrane recognition, and the regression of somes. Thishypothesisarepartlysupportedbythe
transient synapses between Purkinje and granule experiments [38] using chloroquine, a drug known
cells, and serve for understanding the mechanism to induce lysosomal dysfunctions. Mice treated by
of cerebellar development. chloroquine show remarkable changes ofPurkinje
During development of the cerebellum, cell-to- cells such as accumulation of grains consisting of
cell communications are mediated by various gly- membranous whorls in lysosomes at the time of
coproteins. Many reviews which discussed the synaptogenesis. These grains are stained with
cerebellar development from a view point of pro- HRP-labeled Concanavalin A, but not with mar-
tein chemistry have been recently presented [37], kers of membrane of Purkinje cells.
but only a little attention is paid as to the develop- In-vitro experiments indicate that bilirubin
mental change of glycans. It is well known that affects the hepatic lysosomal membrane, and re-
many kinds ofglycans appear during development leases enzymes from the lysosomes [8]. Our pre-
554 H. Keino and S. Kashiwamata
Fig. 4. Light microscopic photographs of sagittal seactions of the j/+ rat cerebellum at postnatal day 7. A,
histochemical demonstration of peanut agglutinin-binding activity. The agglutinin binds near the nulceus of
external granule cells (arrowhead) and developing parallel fibers (arrow). B; enzyme histochemistry of acid
phosphatase activity. A large amount ofreaction products is accumulated in Purkinje cells (p).
liminary experiments also showed the release of (1985) J Neurochem 44: 1877-1884
acid phosphatase from lysosomes separated from 6 AonoS, SatoH, SembaR,KashiwamataS(1988) J
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