www.elsevier.com/locate/brainres
Brain Research 1057
Research Report
Robust axonal sprouting and synaptogenesis in organotypic slice cultures
of rat cerebellum exposed to increased potassium chloride
Suzanne Chena,b, Kasunari Hiratab, Yuqin Rena,b, Mutsuyuki Sugimorib,
Rodolfo Llinasb, Dean E. Hillmana,b,*
aDepartment of Otolaryngology and Physiology and Neuroscience, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USAbDepartment of Physiology/Neuroscience, New York University School of Medicine, New York, NY 10016, USA
Accepted 19 July 2005
Available online 25 August 2005
Abstract
Organotypic slices of the rat cerebellum, cultured in physiological levels [K+]o (5 mM) for 14 days, loose the majority of granule cells in
the anterior lobe resulting in few axons and atypical Purkinje cell dendrites with vacant spines. When the culture medium was switched from
5 mM to 20, 30 or 40 mM [K+]o during the last 7 days of cultures, slices developed axons with numerous vesicle-filled boutons that made
synaptic contact with Purkinje cell spines. Most boutons had one or two spine profile contacts, while some were unusually large. Enlarged
boutons abutted Purkinje cell somata or their dendrites, causing intervening spines to invaginate terminals to form rosette synaptic
complexes. Calbindin immuno-labeling excluded Purkinje cell axonal collaterals as the source of rosette boutons and suggested a granule cell
origin. Quantification of vacant spines as compared to those on boutons revealed a threshold for potassium, between 10 and 20 mM, where
the number of synaptic spines increased and vacant spines decreased drastically. These findings suggest that elevated [K+]o triggers an
activity-dependent plasticity in rat cerebellar slice cultures by promoting axonal sprouting with formation of vesicle-filled boutons and
synaptogenesis on open receptor sites of Purkinje cell spines.
D 2005 Elsevier B.V. All rights reserved.
Theme: Development and regeneration
Topic: Formation and specificity of synapses
Keywords: Axogenesis; Synaptic vesicle production; Enlarged bouton; Granule cell; In vitro method; Purkinje cell; Rosette synaptic complexes; Vacant spine
1. Introduction
Organotypic cultures from various brain regions retain
much of the local morphological framework but show
changes in connectivity suggesting circuit plasticity in vitro
[4,11,39,49]. In rat cerebellar slice cultures using standard
media, there is a substantial loss of granule cells (GCs) and
their axons, thus removing a major input to Purkinje cells
(PCs) as well as to inhibitory interneurons. Because PCs in
organ culture are very resistant to cell death [1,17,21], the
0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2005.07.039
* Corresponding author. Department of Otolaryngology and Physiology
and Neuroscience, New York University School of Medicine, 550 First
Avenue, New York, NY 10016, USA.
E-mail address: [email protected] (D.E. Hillman).
neuropil is composed mostly of PC dendrites that are
studded with vacant spines (spines without presynaptic
contact) and a few spine synapses with PC axon collaterals
[42,43]. Physiological analysis showed that these neurons
have the expected resting and spike potential properties but
slowed membrane repolarization and a lack of spontaneous
synaptic activity [16,32].
While using high [K+]o (30 mM) in slice culture media to
investigate neurotoxicity of PCs [28,29], we were surprised
to find a neuropil with spine synapses and enlarged
presynaptic boutons, instead of numerous vacant spines as
observed in standard 5 mM [K+]o [12]. Previous studies
reported that membrane depolarization by elevated [K+]oreleased transmitter [14,51] as well as enhanced GC survival
in dissociated cell cultures (DCC) [5,19,20]. Here, we report
(2005) 88 – 97
S. Chen et al. / Brain Research 1057 (2005) 88–97 89
that [K+]o treatment engenders plasticity in circuitry by
axonal sprouting, formation of vesicle-filled boutons and
synaptogenesis yielding PC spine rosette complexes that are
not observed in the normal cerebellar cortex.
2. Materials and methods
The animal protocol utilized here was approved by
Institutional Animal Care and Use Committee at New York
University School of Medicine and followed the guidelines
issued by the National Institutes of Health. Sprague–
Dawley rat pups, accompanied by their mother, were
purchased from Taconic (Germantown, NY).
For in situ fixation, 12-day-old (P12) pups were
anesthetized with pentobarbital (40 mg/kg body weight,
i.p.) and then perfused through the heart with a wash
solution of 0.2 M phosphate buffer (PB) followed by a
fixative consisting of 1% paraformaldehyde and 1%
glutaraldehyde in 0.12 M PB. Midsagittal sections of the
cerebellum were made with a vibratome at 50 Am.
For slice cultures, P12 pups were anesthetized, and their
brains were aseptically removed and immersed in cold
sterile artificial CSF-like ringer [29]. The vermis of the
cerebellum was sectioned sagittally into 300 Am slices using
a Microslicer (Dosaka EM, Japan). Each slice was collected
in the CSF ringer and placed on a porous membrane insert
(Millicell CM, Millipore, MA) at the interface between air
and a standard culture medium. It consists of 50% Minimum
Essential Medium, 25% Earl’s Balanced Salt Solution
(EBSS), 25% heat-inactivated horse serum, 1 mM l-
glutamine and 36 mM d-glucose. All slices were incubated
at 35 -C in a humidified incubator with 7% CO2 (pH 7.3)
and fed every 2 or 3 day [46].
Control slices were cultured in standard medium (5 mM
KCl) for 14 days, whereas experimental slices were cultured
in the standard medium for 7 days and then switched to 10,
20, 30 or 40 mM [K+]o for 7 more days. This 7-day
preconditioning in 5 mM [K+]o stabilized the neuronal
population following marked reduction in the number of
GCs and a complete degeneration of parallel fibers.
At 14 days in vitro (14 DIV), slices of all groups were
fixed for 2 h, in 1% paraformaldehyde and 1% glutaraldehyde
in EBSS for ultrastructure quantification. They were post-
fixed in 1% OsO4 containing 1% potassium ferrocyanide for
1 h and left in 2% aqueous uranyl acetate overnight. Slices
were dehydrated gradually from 70% to 100% ethyl alcohol
and embedded in Durcupan (Sigma/Fluka, St. Louis, MO).
Ultrathin sections were cut and collected on Formvar-coated
grids. They were double stained with uranyl acetate and lead
citrate for examination in a JEOL 100C electron microscope.
Calbindin immuno-EM was used to determine the extent
of PC axon collaterals as a bouton source. Slices from all
groups were fixed in 4% paraformaldehyde and 0.15%
glutaraldehyde in EBSS for 2 h. Slices were immersed for 1
h in 5% normal goat serum with 1% bovine serum albumin
(minimizes background) and then reacted with monoclonal
calbindin antibodies (1:10,000 dilution, Swant, Switzerland)
overnight at 4 -C. On the following day, the slices were
incubated with a secondary antibody and then processed
using an ABC Elite Kit (Vector Lab, Burlingame, CA) and
DAB as the chromagen. The reacted slices were washed
several times with 0.1 M phosphate buffer (PB) and then
postfixed in 1% OsO4 in pH 8.0 PB for 30 min. Slices were
embedded in Durcupan, and ultrathin sections were pro-
cessed as described above. Some slices were counterstained
with cresyl violet (Nissl) before alcohol dehydration and
coverslipped with Permount for LM.
Ultrastructural quantification of spines and boutons was
done to assess the effect of [K+]o on bouton development
and synaptogenesis. Due to an anterior–posterior difference
in the number of surviving GCs and synapses (preliminary
report [13]), we used only lobules I–IV which consistently
loose the majority of GCs. Furthermore, areas exhibiting a
distinct molecular layer were sampled and photographed at
6600� for each [K+]o group. The EM films were digitally
scanned and printed using PhotoShop 5.0 software (Adobe,
San Jose, CA).
Spines in section profiles were identified at high
magnification using the zoom-in tool in Photoshop. Spine
necks in cross-section typically have one ER profile in the
center of the projection (see Figs. 1D–F). The identification
between spines and parallel fibers was possible by the single
ER profile in the spine neck and microtubules in parallel
fibers. Each quantified spine was characterized as being a
spine by its expanded head and one or more ER folds.
Active zones were not considered a criterion. Normally, in
vivo, one, and on occasion two, PC spine makes synaptic
contacts with each parallel fiber bouton. In control slices,
PC spines without a presynaptic partner were predominant,
while, in experimental preparations, multiple spines (three
or more) frequently contacted an enlarged bouton. These
spine/bouton relationships provided a natural classification
for four categories: 1) vacant- (without a presynaptic
bouton), 2) single-, 3) dual- or 4) multiple-spines per
bouton. For each category, the number of spine heads
appearing in a micrograph was recorded together with the
total number of spines on each film (approximately, 100
Am2). The mean and standard deviation for each spine/
bouton type were determined together with percent differ-
ences between control and experimental groups (Table 1).
Statistical significance was calculated using Student’s t test.
3. Results
3.1. P12-in-situ-fixed sagittal sections
Nissl-stained sagittal sections of P12 displayed a thick
external granule layer, a distinct molecular layer and
numerous GCs below a single layer of PCs (Fig. 1A).
Ultrastructural analysis of the molecular layer in the anterior
Fig. 1. LM and EM images of P12, 5 mM and 30 mM KCl slices at DIV 14. (A) Light micrograph of P12-in-situ-fixed Nissl-stained section displays a thick
external granule layer (EGL), a distinct molecular layer (ML), a single PC layer (PCL) and numerous GCs below the PCL. (B) EM analysis of the neuropil at
P12 revealed numerous parallel fibers (Pf) and PC dendrites (PCD) with primitive spines having growth cone appearances (s*) in synaptic contacts (arrows)
with parallel fiber boutons. (C) 1-Am sections of control (5 mM KCl) slices that were stained with toluidine blue show a cerebellar cortex with distinct laminae:
molecular (ML), PC, GC layers as well as a white matter (WM). Note that PC dendrites (arrows) in the ML run parallel to the pial surface. (D) EM analysis of
the neuropil from 5 mM [K+]o consists mostly of vacant spines without parallel fibers or boutons. Note that there is a striking difference in the neuropil
composition as compared with that of P12. (E) A bouton (B*), likely from stellate interneurons, has a direct apposition to a medium-sized PC dendrite as is
seen, typically, in vivo. (1F) A 30 mM [K+]o preparation has numerous parallel fibers (Pf) and boutons (B) forming synaptic contacts with PC spines. The spine
neck can be identified by a narrow tube of ER that extends into the spine head where it elaborates into the spine apparatus (D–F, arrowheads). Scale bar equals
to 10 Am (A and C) and 1 Am (B, D–F).
S. Chen et al. / Brain Research 1057 (2005) 88–9790
lobe revealed masses of parallel fibers crossing through PC
dendritic branches, but only a few boutons appeared in the
field. Primitive spines without narrowing of the neck
protruded from the PC dendrites to contact one or two
small boutons of parallel fibers (Fig. 1B).
3.2. 14 DIV control slices (5 mM KCl)
Toluidine-blue-stained 1-Am plastic sections from the
anterior lobe of control slices displayed three laminae,
i.e., molecular, PC and GC layers (Fig. 1C). In these
slices, the majority of GCs that were present in P12
donor tissue were lost (compare to Fig. 1A). The
narrowed GC layer stained faintly and was composed of
a few GCs along with astrocytes and scattered Golgi cell
somata. The external granular layer was absent (Fig. 1C).
The PC somata aligned in a single layer, but their
dendritic arbors were flattened and projected almost
parallel to the PC layer rather than toward the molecular
layer surface (Fig. 1C).
Table 1
Effect of high [K+]o on synaptogenesis in the anterior lobe
K5-A K10-A K20-A K30-A K40-A
No. of films 21 16 13 10 30
Vacant spine type, M T SD 67.1 T 21.0 73.0 T 26.2 20.0 T 17.3 8.3 T 4.5 4.33 T 2.4
% change from control +9% �70% �88% �94%
P value and significant level ns <0.001*** <0.001*** <0.001***
Mono-spine type, M T SD 5.8 T 3.0 4.3 T 3.0 10.1 T 6.0 6.6 T 2.7 2.0 T 1.5
% change from control �26% +74% +13% �66%
P value and significant level ns <0.01** <0.05* <0.001***
Dual spines type, M T SD 1.5 T 0.9 1.9 T 0.8 3.0 T 2.4 3.0 T 2.4 1.8 T 0.9
% change from control �33% +100% +100% +20%
P value and significant level ns ns ns ns
Multiple spines type, M T SD 0.57 T 1.2 0.38 T 1.0 5.0 T 7.4 3.5 T 3.3 5.6 T 7.0
% change from control �30% +780% +510% +880%
P value and significant level ns <0.001*** <0.001*** <0.001***
K5-A: 5 mM KCl from the anterior lobe, M T SD: mean T standard deviation, ns: not significant.
* P < 0.05.
** P < 0.01.
*** Very significant P < 0.001.
S. Chen et al. / Brain Research 1057 (2005) 88–97 91
Ultrastructural analysis of control slices revealed very
few parallel fibers and bouton profiles. Nevertheless,
various-sized PC dendrites with elongated small spines
Fig. 2. EM micrographs of 30 mM KCl-treated slices at 14 DIV. (A) Boutons (B)
scattered at sites along the parallel fiber (arrowheads). (C and D) Enlarged bouton
without interposed glial projections. Scale bar equals to 1 Am (B–D) and 0.5 Am
formed the bulk of the molecular layer (Fig. 1D). These
vacant spines invaginated the surface of surrounding
astrocytic projections. Occasional isolated boutons were
are seen in continuation with parallel fibers. (B) A few synaptic vesicles are
s (B) contacted the plasma membranes of PC dendrites and somata directly
(A).
S. Chen et al. / Brain Research 1057 (2005) 88–9792
opposed to the plasma membrane of PC dendrites without
spine contact. These boutons appeared to be inhibitory
terminals from stellate neurons that are seen normally in
vivo (Fig. 1E).
3.3. 14 DIV experimental slices (7-day exposure to 10, 20,
30 or 40 mM KCl)
In LM analysis, the molecular and GC layers were
similar between control and all experimental groups.
Nonetheless, the Nissl staining of GCs appeared stronger
when the KCl was increased to 20, 30 or 40 mM as
compared to control or 10 mM groups.
Ultrastructural analysis of the molecular layer in the 10
mM group was indistinguishable from control prepara-
tions. In contrast, preparations from the 20, 30 or 40 mM
[K+]o groups all had a neuropil composed of large and
Fig. 3. EM micrographs from 40 mM KCl-treated slices show enlarged vesicle-fill
presence of PC dendrites (D) and parallel fibers in the neuropil. Enlarged boutons
made direct contact on the surface of dendrites, and these boutons were larger in di
than 20 PC spines in this sectional plane. Scale bar equals to 1 Am (A–C).
small dendrites separated by axons, boutons and spines.
Small axonal bundles were oriented in various directions
(Fig. 1F). In fortunate sections, these axons were seen
continuing into vesicle-filled boutons (Fig. 2A). Further-
more, scattered or clustered synaptic vesicles were found,
atypically, in axonal shafts (Fig. 2B). The density of
synaptic vesicles was essentially the same in large or
small boutons.
3.3.1. Bouton/spine synapses
Many of the presynaptic boutons contacted one or more
PC spines. These spine heads were larger in diameter than
those of vacant spines and contained a prominent spine
apparatus that was connected by a single central ER
projection through a narrow spine neck (Figs. 1D–F,
arrowheads). At spine/bouton appositions, distinct active
zones were apposed to presynaptic vesicles.
ed boutons making synaptic contact with numerous PC spines. (A) Note the
(B) had many PC spines invaginating the bouton surface. (B) Some boutons
ameter than the postsynaptic dendrite. (C) A giant bouton (B) contacts more
Fig. 4. Calbindin immuno-EM of slices treated with 30 mM KCl (A) revealed calbindin-IR in PC spines and dendrite (D), while an enlarged bouton (B) showed
no IR. (B) Calbindin-IR in PC dendrite and opposed bouton implies that this labeled bouton is derived from a PC collateral axon and synapses on a PC
dendrite. Scale bar equals to 0.5 Am (A and B).
S. Chen et al. / Brain Research 1057 (2005) 88–97 93
3.3.2. Rosette synaptic complexes
Following 7-day exposure to 20 to 40 mM [K+]o,
rosette complexes with multiple spine contacts appeared
among normal sized boutons. The number of spines and
the amount of synaptic vesicles increased, proportionally,
with the size of rosette complexes. Most often, profiles
of these large boutons were apposed to PC dendrites
(Fig. 2C) or somata (Fig. 2D) without interposed
Fig. 5. Histogram distribution of four spine/bouton types sampled from the anterio
are vacant along with a very few mono-spine/bouton type in control and 10 mM
spines and a progressive increase in synaptic spines with each increment of [K+]o f
types toward the right bins and also a marked increase in multiple spines/bouton i
0.001, **P < 0.01 or *P < 0.05.
astrocytic glia. Remarkably, enlarged boutons exceeded
PC dendritic diameter and wrapped the dendritic surface
(Figs. 3A and B). Numerous spine heads were sand-
wiched within the apposed area and invaginated the
bouton surface (Figs. 2 and 3). Up to 20 spine heads
could be observed in synaptic contact on a single
enlarged bouton, forming rosette synaptic complexes
(Fig. 3C).
r lobe of control, 10, 20, 30 and 40 mM KCl groups. The majority of spines
KCl groups. In contrast, there is a large reduction in the number of vacant
rom 20 to 40 mM. Note a shift in the frequency distribution of spine/bouton
n the 40 mM KCl group. The significance levels are represented as ***P <
S. Chen et al. / Brain Research 1057 (2005) 88–9794
3.4. EM calbindin immunoreactivity for PC axon
identification
Enlarged boutons failed to show calbindin immunoreac-
tivity (IR) (Fig. 4A). PC dendrites, spines and axons had
strong calbindin-IR (Figs. 4A and B). Calbindin-IR boutons
of PC recurrent axon collaterals contacted IR positive PC
dendrites and somata, directly (Fig. 4B).
3.5. Spine/bouton quantification and data analysis
Quantification of four spine/bouton types in the anterior
lobe showed no difference between the control and 10 mM
[K+]o groups (Table 1). Remarkable, the number of vacant
spines was reduced by 70% in 20 mM, 88% in 30 mM and
94% in 40 mM [K+]o groups as compared to the control
(Table 1). In parallel, 20–40 mM [K+]o groups had a 4- to 8-
fold increase in the number of spines that formed multiple
contacts on large boutons. Both comparisons were highly
significant (P < 0.001). Although the number of mono- and
dual-spine/bouton types appeared to increase, the exper-
imental group differences from control were not statistically
significant (see Table 1). The histogram (Fig. 5) illustrates
similar distribution patterns between control and 10 mM
[K+]o for vacant spines and types of spine contacting
boutons. In contrast, the 20 mM [K+]o or higher groups had
sharp reductions in vacant spine number. Furthermore, with
each raise in [K+]o, there was a progressive increase in
bouton size and number of spine synapses.
4. Discussion
Numerous in vitro studies have applied various compo-
sitions of culture media to investigate neuronal survival
[2,3,38] and synaptic plasticity [18,47]. Increased [K+]olevels have been shown to release glutamate transmitter from
GCs of DCCs [14,51] and induce cell death by over-
stimulation [22,48].
Our finding shows that elevated [K+]o enhances neuronal
connectivity in cerebellar slice cultures by inducing axonal
sprouting, synaptic vesicle production and synaptogenesis.
GC survival did not have any noticeable improvement in
our delayed [K+]o treatment model, however, these neurons
do survive at near normal number when the [K+]oconcentration was increased on the first day rather than
after 7 days (our preliminary result [27]).
4.1. Axonal growth and rosette formation
Quantitative data revealed extensive rosette formations in
20–40 mM [K+]o groups in contrast to few in standard and
10 mM [K+]o. The dose response of [K+]o is seen by the
distinct shift in histogram bins from vacant spines in 5 or 10
mM [K+]o preparations to bins of increasing number of
spines on boutons with each increment of [K+]o between 20
and 40 mM (see Fig. 5). Secondly, there was a threshold for
the [K+]o response that occurred between 10 and 20 mM,
where newly formed boutons begin to make robust synapses
on PC spines.
When sagittal slices of the rat are cultured in standard
[K+]o media, the molecular layer of anterior lobes resembles
the agranular cerebellum of various models [44]. This may
be because all parallel fibers were transected and incapable
of regenerating in standard culture media. Nevertheless,
after the initial 7 days in 5 mM [K+]o and then treatment for
7 days with 20 to 40 mM [K+]o, the circuitry was rescued
through axonal sprouting by the few remaining GCs.
Frequently, small clusters of vesicles were observed along
axonal shafts in regions without boutons. Elongation of axons
provides a route for newly generated vesicles to reach target
sites on PC spines where rosette complexes could form.
These axons generated various-sized boutons filled with
synaptic vesicles that targeted vacant spines. In 20–40 mM
KCl slices, enlarged boutons were frequently opposed to the
plasmalemma of PC dendrites and soma. The interposed
spines invaginated the bouton within the opposition. Since
more than 20 spines could be found on a rosette synaptic
formation of each section profile, the total number of spine
heads per rosette could be several times larger in three
dimensions.
4.2. GC axons are likely the source of increased number of
boutons
In cultures, there is a total absence of cerebellar
afferents, and therefore any axonal terminals must be of
local origin. In our study, calbindin-IR was used as a
specific marker for PC recurrent collateral axons. We
found calbindin-IR boutons, synapsing directly on PC
dendrites and somata. However, enlarged boutons that
formed multiple synaptic contacts on PC spines lacked
calbindin-IR. Furthermore, these boutons contained round
vesicles, rather than irregular and flattened vesicles, and
occasionally were observed in continuity with parallel
fiber-like axons. These results suggest that GC axons are
the source of reactive boutons.
Other studies in explant cultures show sprouting of PC
recurrent axon collaterals and formation of inhibitory
synapses on PC somata as well as, heterotypically, on PC
spines when there is a deficit in the number of GCs
[16,41,43]. In roller-tube organ cultures, large boutons
resembling mossy fibers were suggested to arise from deep
cerebellar nuclei and make synaptic contact on dendrites of
granule and Golgi cells, as well as on PC spines [31]. In
mutant mice having granuloprival cerebella, large presy-
naptic boutons were proposed to be from both climbing and
mossy fibers [34]. More recently, hyperspiny PCs and
nodding mutants were found to have large boutons
contacted by multiple PC spines. Sotelo suggested that
these axons arise from GCs in both mutants as well as from
stellate axonal terminals in nodding mice [44].
S. Chen et al. / Brain Research 1057 (2005) 88–97 95
4.3. Neuronal survival
Elevation of [K+]o from normal physiological levels of
3–5 mM up to 25 mM in DCCs was reported to improve
GC survival of rat cerebella [5,19,20]. The same level of GC
survival in DCCs could be achieved by glutaminergic
agonists [5–7,24,30]. In our organotypic slices, both control
and 10 mM [K+]o groups had good survival of PCs but
exhibited only a narrow band of GC layer throughout the
cerebellar vermis except in lobules 9 and 10 (preliminary
report [13]). Following 7-day exposure to 20, 30 or 40 mM
[K+]o, the GC layer remained thin but had an increased
intensity of Nissl staining. This finding indicates that the
few remaining GCs acquired ribosomes (indicated by
increased Nissl staining) for protein synthesis serving
neurite outgrowth and synaptic vesicle production.
In DCCs of the mouse, GCs flourish equally well in
either low or high [K+]o [35–37]. This mouse to rat species
difference has been attributed to slower AMPA receptor
desensitization in mice allowing more Ca++ entry to sustain
the mouse GCs [35]. Likewise, we found no massive GC
loss in slice cultures of the mouse using our standard
medium (unpublished data). The apparent inability of rat
GCs to sustain themselves in lobules 1–8 following trans-
section of parallel fibers raises important questions on
conditions controlling the survival of specific neuronal types
in vitro.
There was increased risk for PC excitotoxicity in 20–40
mM KCl. Hirata et al. reported a 30% reduction in PC
number of slice cultures at 30 mM KCl as compared to
standard media [28,29]. Concurrently, the remaining PC
dendritic trees were shorter and there were fewer branch
points. Ultrastructural study confirmed PC atrophy showing
pathological changes in ER and mitochondria, with the most
severe alterations occurring at 40 mM KCl.
4.4. Mechanism of circuitry enhancement by increased
[K+]o
Presumably, there is a chronic membrane depolarization
by 20–40 mM [K+]o that opens voltage-gated calcium
channels [10,14,20,33] and, possibly, even ligand-depend-
ent NMDA types [50]. This calcium flux is believed to
activate signal transduction cascades [19,23] leading to de
novo protein synthesis [10,20,33]. The importance of Ca++
for neuronal survival is supported by the failure of enhanced
GC survival following the depolarizing agent, veratridine,
which acts on sodium channels without Ca++ influx [15].
Such plasticity is consistent with studies showing that
chronic electrical stimulation of denervated cortex produces
significant axonal re-growth and circuit remodeling [40].
Remarkably, static electric fields facilitate axonal growth
and apparently restore some function in the adult spinal cord
[8,9].
The availability of PC spines appears to play a key role in
robust synaptogenesis leading to formation of rosette
boutons. Since the number of presynaptic boutons was
inadequate for postsynaptic targets, multiple PC spines
contacted boutons and thus formed rosette complexes. One
reason for this disproportional relationship is a constancy
principle for the total contact area of active zones that
sustains large numbers of spines following deafferentation
[25,26]. Regardless of increases or decreases in the number
of afferent fibers, total contact area of postsynaptic densities
on each PC remains constant [25,26]. These findings
suggest that individual postsynaptic areas shift dynamically
between contact sites facilitating plastic changes such as the
formation of rosette synaptic complexes found in this study.
The constancy of postsynaptic contacts is supported by
studies showing proliferation of spines over the entire PC
dendritic arbor that follows climbing fiber deafferentation
[45] and in agranular cerebella where spines elongate to
reach available presynaptic sites when preterminals are
scarce [34].
5. Conclusions
This study shows that rat cerebellar slices, cultured with
3–8� higher [K+]o, have marked axonal sprouting, synaptic
vesicle production, rosette bouton formations and synapto-
genesis. The results suggest that the elevated K+ environ-
ment is responsible for an activity-dependent plasticity of
neuronal circuitry that may have implications for study of
stroke and spinal cord injury in the adult.
Acknowledgments
This work was supported by NIH-NINCDS NS-13742
and NYS Spinal Cord Injury Research Program. We thank
Dr. Eric Lang for his critical reading with the manuscript.
References
[1] K.H. Adcock, F. Metzger, J.P. Kapfhammer, Purkinje cell dendritic
tree development in the absence of excitatory neurotransmission and
brain-derived neurotrophic factor in organotypic slice cultures, Neuro-
science 127 (2004) 137–145.
[2] S. Alavez, D. Pedroza, J. Moran, Role of heat shock proteins in the
effect of NMDA and KCl on cerebellar granule cells survival,
Neurochem. Res. 25 (2000) 341–347.
[3] R. Anelli, E. Mugnaini, Enrichment of unipolar brush cell-like
neurons in primary rat cerebellar cultures, Anat. Embryol. 203
(2001) 283–292.
[4] E. Audinat, T. Knopfel, B.H. Gahwiler, Responses to excitatory amino
acids of Purkinje cells’ and neurones of the deep nuclei in cerebellar
slice cultures, J. Physiol. 430 (1990) 297–313.
[5] R. Balazs, V. Gallo, A. Kingsbury, Effect of depolarization on the
maturation of cerebellar granule cells in culture, Brain Res. 468 (1988)
269–276.
[6] R. Balazs, O.S. Jorgensen, N. Hack, N-methyl-d-aspartate promotes
the survival of cerebellar granule cells in culture, Neuroscience 27
(1988) 437–451.
S. Chen et al. / Brain Research 1057 (2005) 88–9796
[7] R. Balazs, N. Hack, O.S. Jorgensen, C.W. Cotman, N-methyl-d-
aspartate promotes the survival of cerebellar granule cells: pharmaco-
logical characterization, Neurosci. Lett. 101 (1989) 241–246.
[8] R.B. Borgens, Stimulation of neuronal regeneration and development
by steady electrical fields, Adv. Neurol. 47 (1988) 547–564.
[9] R.B. Borgens, Electrically mediated regeneration and guidance of
adult mammalian spinal axons into polymeric channels, Neuroscience
91 (1999) 251–264.
[10] L.N. Borodinsky, O.A. Coso, M.L. Fiszman, Contribution of Ca2+
calmodulin-dependent protein kinase II and mitogen-activated
protein kinase kinase to neural activity-induced neurite outgrowth
and survival of cerebellar granule cells, J. Neurochem. 80 (2002)
1062–1070.
[11] P. Cavelier, F. Pouille, T. Desplantez, H. Beekenkamp, J.L. Bossu,
Control of the propagation of dendritic low-threshold Ca(2+) spikes in
Purkinje cells from rat cerebellar slice cultures, J. Physiol. 540 (2002)
57–72.
[12] S. Chen, K. Hirata, Y. Ren, M. Sugimori, R. Llinas, D. Hillman,
Robust synaptic reorganization in cerebellar organotypic cultures
exposed to increased potassium concentrations, Abstr.-Soc. Neurosci.
29 (2003).
[13] S. Chen, K. Hirata, Y. Ren, M. Sugimori, R. Llinas, D. Hillman,
Unipolar brush cells underlying regional difference of synaptic
organization in organotypic slice cultures, Abstr.-Soc. Neurosci. 30
(2004).
[14] M.A. Cousin, H. Hurst, D.G. Nicholls, Presynaptic calcium channels
and field-evoked transmitter exocytosis from cultured cerebellar
granule cells, Neuroscience 81 (1997) 151–161.
[15] R. Diaz-Trelles, A. Novelli, G. Puia, M. Baraldi, M.T. Fernandez-
Sanchez, NMDA receptor dependent and independent components of
veratridine toxicity in cultured cerebellar neurons are prevented by
nanomolar concentrations of terfenadine, Amino Acids 19 (2000)
263–272.
[16] R. Drake-Baumann, F.J. Seil, Electrophysiological differences
between Purkinje cells in organotypic and granuloprival cerebellar
cultures, Neuroscience 69 (1995) 467–476.
[17] I. Dusart, M.P. Morel, R. Wehrle, C. Sotelo, Late axonal sprouting
of injured Purkinje cells and its temporal correlation with
permissive changes in the glial scar, J. Comp. Neurol. 408 (1999)
399–418.
[18] J.C. Fiala, S.A. Kirov, M.D. Feinberg, L.J. Petrak, P. George, C.A.
Goddard, K.M. Harris, Timing of neuronal and glial ultrastructure
disruption during brain slice preparation and recovery in vitro,
J. Comp. Neurol. 465 (2003) 90–103.
[19] C. Galli, O. Meucci, A. Scorziello, T.M. Werge, P. Calissano, G.
Schettini, Apoptosis in cerebellar granule cells is blocked by high KCl,
forskolin, and IGF-1 through distinct mechanisms of action: the
involvement of intracellular calcium and RNA synthesis, J. Neurosci.
15 (1995) 1172–1179.
[20] V. Gallo, A. Kingsbury, R. Balazs, O.S. Jorgensen, The role of
depolarization in the survival and differentiation of cerebellar granule
cells in culture, J. Neurosci. 7 (1987) 2203–2213.
[21] A.M. Ghoumari, R. Wehrle, O. Bernard, C. Sotelo, I. Dusart,
Implication of Bcl-2 and Caspase-3 in age-related Purkinje cell death
in murine organotypic culture: an in vitro model to study apoptosis,
Eur. J. Neurosci. 12 (2000) 2935–2949.
[22] R. Griffiths, L. Ritchie, K. Lidwell, A. Grieve, C.S. Malcolm, M.
Scott, C. Meredith, Calcium influx via L-type voltage-gated channels
mediates the delayed, elevated increases in steady-state c-fos mRNA
levels in cerebellar granule cells exposed to excitotoxic levels of
glutamate, J. Neurosci. Res. 52 (1998) 641–652.
[23] D. Guerini, E. Garcia-Martin, A. Gerber, C. Volbracht, M. Leist, C.G.
Merino, E. Carafoli, The expression of plasma membrane Ca2+ pump
isoforms in cerebellar granule neurons is modulated by Ca2+, J. Biol.
Chem. 274 (1999) 1176–1667.
[24] N. Hack, H. Hidaka, M.J. Wakefield, R. Balazs, Promotion of granule
cell survival by high K+ or excitatory amino acid treatment and
Ca2+/calmodulin-dependent protein kinase activity, Neuroscience 57
(1993) 9–20.
[25] D.E. Hillman, S. Chen, Reciprocal relationship between size of
postsynaptic densities and their number: constancy in contact area,
Brain Res. 295 (1984) 325–343.
[26] D.E. Hillman, S. Chen, Compensation in the number of
presynaptic dense projections and synaptic vesicles in remaining
parallel fibres following cerebellar lesions, J. Neurocytol. 14
(1985) 673–687.
[27] D. Hillman, S. Chen, R. Bing, M. Sugimori, R. Llinas, Activity
dependence of long-term slice cultures on cerebellar granule cell
survival, Abstr.-Soc. Neurosci. 31 (2005).
[28] K. Hirata, Y. Takamura, S. Chen, D. Hillman, M. Sugimori, R. Llinas,
T5-88 and T-817, neuroprotective agents, decrease high K+-induced
necrotic damages of Purkinje cells in cerebellar organotypic slice
culture, Abstr.-Soc. Neurosci. 29 (2003).
[29] K. Hirata, S. Chen, D. Hillman, M. Sugimori, R. Llinas, T-588, a
neuroprotective agent, decreases high extracellular potassium induced
necrotic damage of Purkinje cells in cerebellum organotypic slice
culture, Neuroscience (submitted for publication).
[30] S. Ikonomovic, E. Kharlamov, H. Manev, M.D. Ikonomovic, D.R.
Grayson, GABA and NMDA in the prevention of apoptotic-like cell
death in vitro, Neurochem. Int. 31 (1997) 283–290.
[31] C.B. Jaeger, R. Kapoor, R. Llinas, Cytology and organization of rat
cerebellar organ cultures, Neuroscience 26 (1988) 509–538.
[32] R. Kapoor, C.B. Jaeger, R. Llinas, Electrophysiology of the
mammalian cerebellar cortex in organ culture, Neuroscience 26
(1988) 493–507.
[33] A. Kingsbury, R. Balazs, Effect of calcium agonists and antago-
nists on cerebellar granule cells, Eur. J. Pharmacol. 140 (1987)
275–283.
[34] R. Llinas, D.E. Hillman, W. Precht, Neuronal circuit reorganization
in mammalian agranular cerebellar cortex, J. Neurobiol. 4 (1973)
69–94.
[35] H.S. Mogensen, O.S. Jorgensen, AMPA receptor subunit mRNAs and
intracellular [Ca(2+)] in cultured mouse and rat cerebellar granule
cells, Int. J. Dev. Neurosci. 18 (2000) 61–68.
[36] H.S. Mogensen, N. Hack, R. Balazs, O.S. Jorgensen, The survival
of cultured mouse cerebellar granule cells is not dependent on
elevated potassium-ion concentration, Int. J. Dev. Neurosci. 12
(1994) 451–460.
[37] L.A. Peng, B.H. Juurlink, L. Hertz, Differences in transmitter release,
morphology, and ischemia-induced cell injury between cerebellar
granule cell cultures developing in the presence and in the absence of a
depolarizing potassium concentration, Brain Res. Dev. Brain Res. 63
(1991) 1–12.
[38] J.L. Pongrac, R.J. Rylett, Optimization of serum-free culture con-
ditions for growth of embryonic rat cholinergic basal forebrain
neurons, J. Neurosci. Methods 84 (1998) 69–76.
[39] F. Pouille, P. Cavelier, T. Desplantez, H. Beekenkamp, P.J. Craig, R.E.
Beattie, S.G. Volsen, J.L. Bossu, Dendro-somatic distribution of
calcium-mediated electrogenesis in purkinje cells from rat cerebellar
slice cultures, J. Physiol. 527 (Pt. 2) (2000) 265–282.
[40] L.T. Rutledge, The effects of denervation and stimulation upon
synaptic ultrastructure, J. Comp. Neurol. 178 (1978) 117–128.
[41] F.J. Seil, R. Drake-Baumann, Reduced cortical inhibitory synapto-
genesis in organotypic cerebellar cultures developing in the absence of
neuronal activity, J. Comp. Neurol. 342 (1994) 366–377.
[42] F.J. Seil, R. Drake-Baumann, Circuit reorganization in granuloprival
cerebellar cultures in the absence of neuronal activity, J. Comp.
Neurol. 356 (1995) 552–562.
[43] F.J. Seil, R.M. Herndon, K.L. Tiekotter, N.K. Blank, Reorganization
of organotypic cultures of mouse cerebellum exposed to cytosine
arabinoside: a timed ultrastructural study, J. Comp. Neurol. 313 (1991)
193–212.
[44] C. Sotelo, Cerebellar synaptogenesis: what we can learn from mutant
mice, J. Exp. Biol. 153 (1990) 225–249.
S. Chen et al. / Brain Research 1057 (2005) 88–97 97
[45] C. Sotelo, D.E. Hillman, A.J. Zamora, R. Llinas, Climbing fiber
deafferentation: its action on Purkinje cell dendritic spines, Brain Res.
98 (1975) 574–581.
[46] L. Stoppini, P.A. Buchs, D. Muller, A simple method for organo-
typic cultures of nervous tissue, J. Neurosci. Methods 37 (1991)
173–182.
[47] L. Stoppini, P.A. Buchs, D. Muller, Lesion-induced neurite sprouting
and synapse formation in hippocampal organotypic cultures, Neuro-
science 57 (1993) 985–994.
[48] M. Tanaka, M. Sawada, M. Miura, T. Marunouchi, Insulin-like
growth factor-I analogue prevents apoptosis mediated through an
interleukin-1 beta converting enzyme (caspase-1)-like protease of
cerebellar external granular layer neurons: developmental stage-
specific mechanisms of neuronal cell death, Neuroscience 84
(1998) 89–100.
[49] M. Tanaka, N. Maeda, M. Noda, T. Marunouchi, A chondroitin sulfate
proteoglycan PTPzeta/RPTPbeta regulates the morphogenesis of
Purkinje cell dendrites in the developing cerebellum, J. Neurosci. 23
(2003) 2804–2814.
[50] M.L. Vallano, B. Lambolez, E. Audinat, J. Rossier, Neuronal activity
differentially regulates NMDA receptor subunit expression in cer-
ebellar granule cells, J. Neurosci. 16 (1996) 631–639.
[51] T. Varming, P. Christopherson, A. Schousboe, J. Drejer, Pharmaco-
logical characterisation of voltage-sensitive calcium channels and
neurotransmitter release from mouse cerebellar granule cells in culture,
J. Neurosci. Res. 48 (1997) 43–52.
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