Brain Research, 245 (1982) 131-135

131

Elsevier Biomedical Press

Marked reorganization of Purkinje cell dendrites and spines in

adult rat following vacating of synapses due to deafferentation
S. CHEN and D. E. HILLMAN*
Department of Physiology and Biophysics, New York University Medical Center, 550 First Avenue,
New York, N Y 10016 (U.S.A.)

(Accepted April 22nd, 1982)

Key words: plasticity - - Purkinje cells - - deafferentation -- dendrites - - spines

Removal of climbing fiber afferents to Purkinje

cells in the adult rat and during development resulted in a shift in dendritic spines to the main dendritic
tree of these cells 11,1z. These newly developed spines
made synaptic contact with parallel fibers. Numerous reports on agranular cerebella from a variety of
developmental conditions (e.g. genetic mutants 9,10,
13,14, X_irradiation 1, virusa,8, chemicalsZ,7) have
shown that Purkinje cells develop spines with postsynaptic membrane thickenings in the absence of
presynaptic contacts. Typically, Purkinje cells had
dendrites with large diameters that were studded
with numerous naked spines each having a postsynaptic density. In this study a marked plasticity of
Purkinje cell dendrites in adult animals occurred
following a combination of granule cell reduction
and parallel fiber sectioning in order to achieve a
complete deafferentation of these large cerebellar
neurons. Modified Purkinje cell dendritic trees with
high densities of vacated spines on terminal branchlets as well as on large dendrites were located in a
thin molecular layer.
Sixty-day-old white rats were anesthetized with
2.5 mg of sodium pentobarbital and maintained with
inhalation of ether. The cranium was opened over
the vermis and the dura was left intact. Undercutting of the molecular layer in a vermal folium was
carried out with the aid of a dissecting microscope
by inserting a microknife (made from a flattened 21gauge injection needle) at one end of a vermal
* To whom correspondence should be addressed.
0006-8993/82/0000-0000/$02.75 Elsevier Biomedical Press

folium so that it penetrated the molecular layer and

followed the longitudinal axis of the folium as it
passed within the granular layer to cross the midline.
The blade was retracted and the wound was closed
for survivals from 14 to 21 days. One percent paraformaldehyde and 1 ~ glutaraldehyde in a 0.09 M
phosphate buffer was perfused through the aorta for
15-20 min and the brains were removed from the
skull for careful dissection of the lesion site and
removal of control specimens from remote folia.
Thin sagittal slices were postfixed in an osmium tetroxide and potassium ferrocyanide mixture for 1 h
followed by block staining overnight in 2 ~ uranyl
acetate. The dehydrated samples were embedded in
D E R epoxy and semithin sections were cut from
each block to localize the lesion site. Thin sections
were stained with uranyl acetate and lead citrate
prior to examination with a JEOL 100C electron
microscope.
One micron plastic sections that were stained with
toluidine blue, revealed a thin molecular layer and a
uniform layer of intact Purkinje cell somata. The
underlying granular layer showed marked degeneration and on many occasions cavitation (Fig. 1). The
large Purkinje cell dendrites traversed the molecular
layer to almost reach the surface of the cerebellum
which was about two-thirds the original cortical
thickness.
Ultramicroscopical examination revealed an absence of parallel fibers in the undercut molecular

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Fig. 1. Light micrograph showing cerebellar molecular layer and granule layer following longitudinal undercutting of the folium.
Note granule layer is destroyed and partially replaced by a huge cavity while the Purkinje cells (arrow heads) remain intact.
Fig. 2. Electron micrograph showing many vacated spines with postsynaptic thickenings embedded in the glial processes. Large
Purkinje cell spine (GS) is surrounded by a large bouton which has characteristics of a climbing fiber. The postsynaptic thickening
also is present on the dendritic shaft as indicated by arrows,

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Fig. 3. Low power electron micrograph of the cerebellar cortex in a region where parallel fibers have been nearly completely removed. Numerous spines with small heads and very small postsynaptic thickenings evaginate the surrounding glial processes which
separate the almost opposed dendritic processes (D). Main shafts of Bergmann glial (BG) processes which are filled with filament
bundles extend between the Purkinje cell layer and the pial surface.

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layer nearest the penetration of cerebellar surface
and a marked reduction of these afferents occurred
in boundary zones. Parallel fibers and their synapses
on Purkinje cell spines gradually increased in occurrence in segmental sections toward the midline.
The altered neuropile was composed of closely
packed large dendrites of Purkinje cells with prominent spine profiles without presynaptic contacts.
These dendrites had diameters that were about twice
the size of spiny branchlets yet had marked numbers
of spines. Each profile was encased in a glial encapsulation which separated these processes (Figs. 2
and 3). Spines from adjacent dendrites interdigitated
and were even enclosed by the same glial process. A
small postsynaptic density on the head of the spines
faced the glial shroud. On occasion, postsynaptic
densities were found on the plasma membrane of the
dendritic shafts (Fig. 2).
A few isolated presynaptic elements contacting
spines and dendritic shafts were found in the zone
where parallel fibers were absent. Many of these
boutons had flat vesicles and presumably represented stellate and basket cell processes while others
had pleomorphic round vesicles with occasional
dense cores. Mossy fibers were not found to penetrate the molecular layer nor were climbing fibers a
marked feature; both were presumably sectioned
along with Purkinje cell axons. Evidence of climbing
fiber sprouting to the region could not be determined at this time; although occasional giant spines
were found in contact with large boutons that had
characteristics of climbing fibers (Fig. 2). In many
preparations filamentous shafts of Bergmann glial
processes could be seen extending through the neuropile towards the surface of the molecular layer
(Fig. 3).
The number of spines per unit length of dendritic
process was greatly increased. This was evident by
the fact that the perimeter of a vast majority of these
dendritic profiles was larger than normal and spines
emerged in all directions from cross-sections (Fig.
3), Serial reconstructions of dendritic segments in
electron micrographs from zones without parallel
fibers revealed that the number of spines emerging
from dendrites of comparable size between control
and lesioned regions was increased 5-fold. The
spines were usually long and slender and occasional-

ly had multiple heads. Each head was about half of

the diameter that occurred normally. In addition the
single postsynaptic density on each spine head was
much smaller than normal synaptic contacts.
In transitional zones, parallel fiber synapses could
be found along with vacated spines. Here giant
spines made contact with enlarged boutons of parallel fibers. Near the end of the probe site vacated
spines were absent and only a slight enlargement of
the spines was noted.
The morphological observations in this study
were similar to those produced in developmental
agranular cerebella with certain differences. First of
all the Purkinje cells remained in a uniform layer in
contrast to the stratified organization of Purkinje
cell somata with inverted dendritic trees found in
many developing agranular preparationsl3,14.
Secondly, mossy fibers did not invade the molecular
layer to make contact with spines on Purkinje cellss,
9. The large dendritic processes with numerous emerging spines were encased in glia and each had a very
small postsynaptic density similar to that found in
agranular cerebella. This apparent shifting of spines
seemed to occur by withdrawing of the dendritic tree
into larger processes presumably due in part to the
reduction in the thickness of the molecular layer.
The mechanism for this alteration in dendritic trees
in the adult may occur as a process similar to that
proposed by Herndon and Oster-Granite as the
'slippery fiber' hypothesis4.
This study points out the extreme capability for
plasticity of the Purkinje cell in sustaining itself.
Furthermore, the maintenance of small postsynaptic
densities on what appears to be an increase in total
number of spines on Purkinje cells supports the contention that the Purkinje cell intrinsically determines
the total areas of synaptic contactS, 6 (numbers of
macromolecules inserted in the postsynaptic membrane) while the distribution of this membrane
thickening to dendritic trees and spines is interactively determined through specific afferent systems if
they are present. However, in the absence of afterents this distribution seems to be largely through the
neuron itself.
Supported by USPHS Grants HD-10934 and NS13742.

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