Post on 24-Apr-2023
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Sodium imaging of climbing fiber innervation fields in developing mouse
Purkinje cells
Bibiana Scelfo1, Piergiorgio Strata2 and Thomas Knöpfel1
¹ Laboratory for Neuronal Circuit Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa,
Wako-Shi, Saitama 351-0198, Japan, ² Department of Neuroscience and Rita Montalcini
Centre for Brain Repair, University of Turin, I-10125 Turin, Italy.
Abbreviated title:
Running Title: Climbing fiber territories in developing cerebellum
Numbers of words in the abstract: 210
Number of text pages: 21
Number of figures: 7
Number of tables : 1
Author for correspondence: Dr Thomas Knöpfel Brain Science Institute, RIKEN 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan Phone: ++ 81 48 467 9740 Fax: ++ 81 48 467 9739 Email: knopfel@brain.riken.go.jp
Copyright (c) 2003 by the American Physiological Society.
Articles in PresS. J Neurophysiol (January 29, 2003). 10.1152/jn.00884.2002
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Abstract
Maturation of specific neuronal connections in the mature nervous system includes
elimination of redundant synapses formed earlier during development. In the cerebellum of
adult animals, each Purkinje cell (PC) is innervated by a single climbing fiber (CF). In early
postnatal development each PC is innervated by multiple CFs and elimination of synapses
formed by supernumerary CFs occurs until monoinnervation is established at around post-
natal day 20 (P20) in mice. It is not clear whether multiple CFs, or only a single CF,
translocate from the cell body of immature PCs to the developing dendrite and, in case
several CFs translocate, if they share or segregate their innervation fields.
To localize CF innervation fields, we imaged changes in postsynaptic sodium
concentration resulting from CF-mediated postsynaptic currents.
We found that more than one CF translocates from an innervation field on the cell body of
the PC to the developing dendrite and that these CFs share rather than segregate their
innervation fields. We concluded that both the soma and the proximal dendrite of the PC
are territories of competition for the developing CFs and that the overlapping of their
termination fields may be the prerequisite for a local process of elimination of all but one
CF as previously demonstrated in the developing neuromuscular junction.
Key words: Imaging, cerebellum, development, multiple innervation, synapse elimination.
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Introduction
Establishment of specific neuronal connections involves elimination of redundant
synapses formed during early development (Goodman and Shatz 1993; Katz and Shatz
1996; Nguyen and Lichtman 1996). The contacts between climbing fibers (CFs) and
Purkinje cells (PCs) are a powerful model to study principles underlying such
developmental synapse elimination. In adult animals, each PC is innervated by a single
CF. However, in early postnatal development each PC is innervated by multiple CFs and
elimination of synapses formed by supernumerary CFs occurs until monoinnervation is
established. Morphological and electrophysiological data suggest that, in rats, the first
contacts between CF terminals and PCs are already established during embryonic life
(Morara et al. 2001) and functional synapses appear at P2 (Crepel et al. 1981). At P5,
virtually all PCs are multiply innervated with on the average 3.5 CFs impinging on each
PC (Crepel et al. 1981). At this developmental stage when PCs are morphologically
immature, with only rudimentary dendrites, the initial contacts between CFs and PCs are
made at the perisomatic level (Altman J. and Bayer S.A. 1997). Regression of multiple
innervation starts at P5 (Crepel et al. 1981) and it could be argued that confining inputs to
the limited space of the cell soma enhances a competitive process resulting in elimination
of supernumerary CFs (Hume and Purves 1981; Purves and Hume 1981). By P9 the
alignment of PCs into a monolayer is completed. At this stage the dendritic tree of the PC
starts showing its typical appearance: a single stem dendrite extends into the molecular
layer and divides into many branches from which tertiary and distal branches emerge. By
P13 PC morphology is reminiscent of that of adult animals (Mason et al. 1990). During
this time window CF branches gradually move to the growing dendrite and complete their
translocation to attain their final innervation at the proximal portion of the dendrite.
Parallel fibers (PFs), the second excitatory input to Purkinje cells, are required for normal
growth of the dendrite and they make contact with distal dendrites after P7 (Altman 1972;
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Sotelo 1978). In addition, PFs play a role in the regression of multiple innervation by CFs.
In fact, when granule cells are deleted, have degenerated (Crepel et al. 1981; Mariani et
al. 1990; Bravin et al. 1995), are impaired in their function (Rabacchi et al. 1992), or when
their transduction pathways are deficient (Conquet et al. 1994; Kano et al. 1995; Kano et
al. 1997; Kano et al. 1998; Hashimoto et al. 2001a,b) the regression of the CF multiple
innervation is hampered. It is known that in the cerebellum made hypogranular during
postnatal development, different CFs can translocate and occupy separate dendritic
territories of adult PCs (Bravin et al. 1995; Zagrebelsky and Rossi 1999). However, it is not
known whether in mice with normal development the presence of normal PFs prevents the
translocation of more than one CF from the soma to the dendrites, or whether competition
between CFs occurs, at least in part, at the dendritic level after more than one CF has
translocated. In the case of multiple translocation, a second question is whether their
innervation territory is separated or overlapping.
We addressed this question using sodium imaging techniques in slices prepared from
mice at developmental stages ranging from P6 to P20. In contrast to calcium imaging
techniques that relay on activation of voltage gated Ca2+ channels and hence reflect the
spread of voltage from the site of the synaptic contacts throughout the dendritic
arborization, changes in intracellular Na+ concentration ([Na+]i) represent, under the
conditions employed, the flux of Na+ through synaptic glutamate receptors and are
confined to the innervation fields.
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Methods
Slice preparation and patch-clamp recordings. Cerebellar slices were prepared from ICR
mice aged between P6 and P14 according to previously established techniques (Edwards
et al. 1989). Briefly, animals were anesthetized (in ice when aged between P6 and P7,
with ether from P8 on) and decapitated. The cerebellar vermis was removed and placed
in a ice cold artificial cerebrospinal fluid (ACSF) composed of (mM) NaCl, 118; KCl, 3;
MgCl2, 1; CaCl2, 1.5; NaH2PO4, 1; NaHCO3, 25; D-glucose, 10. The solution was
continuously gassed with 95% O2 and 5% CO2 resulting in a pH of 7.4. Parasagittal
cerebellar slices (300µm thick) were cut using a vibratome (Leica VT1000S) and
incubated in ACSF at 35°C for the first hour and then at 25°C for up to 6 h. After at least
one hour of incubation, one slice was transferred into a recording chamber and
continuously superfused with ACSF (24-26°C, 2ml/min). Whole-cell patch-clamp
recordings were obtained from PC somata with pipettes prepared from borosilicate glass
and having resistances of 2.5-3 MΩ when filled with intracellular solutions consisting of
(mM): CsCl, 131; TEA, 20; HEPES, 10; EGTA, 0.5; CaCl2, 0.1; Na-GTP, 0.4; Na-ATP, 4;
QX314, 5 and SBFI (tetraammonium salt, Molecular Probes, Eugene, OR, USA), 2;
(pH=7.3). Glass pipettes pulled from sodalime glass (tip diameter 3-10 µm) and filled with
extracellular solution were used for electrical stimulation (negative current pulses, 5 to
100 µA, 300 µs). Synaptic responses were recorded in ACSF containing (Bicuculline
Methiodide 20µM and D-2-amino-5-phosphono-pentanoic acid, D-APV, 50µM).
Imaging. Fluorescence of SBFI was excited by epi-illumination with light provided by a
monocromator (Polychrome II, Till Photonics, Germany) and detected by a cooled 12-bit
CCD at 4-5 Hz under control of Axon Imaging software (Axon Instruments, Forster City,
CA, USA) as previously described (Knöpfel et al. 2000). Fluorescence images were
corrected for background fluorescence (measured from image regions free of dye). At the
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excitation intensities employed, photo-bleaching of SBFI was small (< 0.05% / s) and was
corrected for by using control recordings without stimulation. Changes of [Na+]i were
expressed as relative fluorescence changes (∆F/F values) as described previously (Muri
and Knöpfel 1994). Color-coded maps of ∆F/F were obtained from the change in
fluorescence measured during the first second following onset of the CF stimulation using
custom-made macros under IDL 5.2 (Research Systems Inc.) and Image-Pro Plus (Media
Cybernetics, Inc.). ∆F/F values are unreliable in regions where the absolute baseline
fluorescence level (F) approaches zero (i.e. at the border of the cells) and ∆F/F values
are undefined when F reaches zero. Therefore a masking technique was employed in
which the brightness of each pixel of the ∆F/F maps was derived from the corresponding
F value. Therefore, regions exhibiting no dye fluorescence (F=0) are black and dim
structures such as very fine processes are in darkened colors. In images where the color
scale ranges from 0% to a maximal ∆F/F level (Fig. 3B, C&E; Fig. 5B-D&G-H; Fig. 6B-D;
Fig. 7B&C), nonresponsive areas of the cells are represented in the color corresponding
to zero ∆F/F values (i.e. bluish colors). In some images (Fig. 3G-I; Fig. 4A) the color scale
was limited to a range starting from a threshold value above zero. Pixels with ∆F/F values
below this threshold represent the corresponding F value in grey scale to indicate the
cells morphology.
Results
The morphology of postnatal development of rat and mouse PCs has been well described
(Eccles 1970; Altman 1972). SBFI-filled mouse PCs between P6 and P14 exhibits a
remarkable degree of variability within the same age, and the development of the
dendritic tree displays rapid maturation between P6 and P9 (Fig. 1).
To study responses mediated by individual CFs we first established a rigorous scheme for
their identification. CF-mediated excitatory postsynaptic currents (EPSCs) were first
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identified by their all-or-none nature in responses to a stimulus of graded intensity and by
their feature of paired-pulse depression (Eccles et al. 1966). Multiple CFs were recruited
in some initial experiments with one stimulation electrode, after establishing multiple all-
or-none thresholds of stimulation intensity (Crepel et al. 1976), but in the majority of
experiments two stimulating electrodes were used. When using two stimulation
electrodes, each stimulation pipette was moved systematically in the granule cell layer
until an isolated all-or-none CF response was recruited at minimal stimulation intensity.
Higher stimulation intensities often recruited additional CF responses but stimulation at
these intensities was not used. The use of two stimulating electrodes allowed us to
confirm that isolated individual CF responses, when activated together, sum up and do
not depress each other when stimulated sequentially within a short time window as
expected if the same CF was stimulated by the two stimulation electrodes (see Fig.
2C&D).
Previous experiments illustrated that CF activation induces relatively small changes in
[Na+]i (Lasser-Ross and Ross 1992; Callaway and Ross 1997), and, consequently, only a
small change in fluorescence of the sodium sensitive dye SBFI. In order to achieve clearly
detectable fluorescence signals, CFs were stimulated with a train of 5 stimuli at the
frequency of 10Hz. Figure 3 illustrates maps of increases in [Na+]i obtained from a P10
PC under these conditions. Suprathreshold, but not subthreshold CF stimulation induced
an elevation of [Na+]i that was confined to the proximal dendrites (Fig. 3 A-C). The time
course of changes in [Na+]i obtained from the responsive area showed that elevated [Na+]i
decays to resting levels with a time constant of about 1 s as described previously (Fig.3D,
Knöpfel et al. 2000). The CF-mediated change in [Na+]i was abolished by the AMPA
receptor blocker NBQX (Fig. 3E&F, n=29). In a set of experiments we also performed
sodium imaging with PF activation (5 stimuli at 10 Hz) in order to compare the spatial
location of the PF responses relative to that of CFs (Fig. 3G-I, n=3). PF-induced sodium
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changes were confined to a small portion of the distal dendrite of the PC in close vicinity
of the location of the stimulation electrode (Fig. 3H). As with the CF-mediated signals, PF-
mediated [Na+]i signals were completely abolished by NBQX (20µM) (Figs. 3I). In the
majority of the experiments Purkinje cells were loaded with QX314 (5mM), a blocker of
voltage gated sodium channels that was added to the pipette solution. QX314 abolished
induced activation fast action potentials by antidromic stimulation or direct depolarization
of the PC (not shown). The use of QX314 is indicated for each illustrated cell in the
corresponding figure legend and in the summarizing table 1. However, no difference was
seen in CF-induced [Na+]i responses between cells with and without QX314 indicating
that the present [Na+]i signals do not contain a component mediated by QX314-sensitive
voltage-gated ion channels.
It has been shown that Na+ diffuses essentially freely in the cytoplasm of Purkinje cells
(Callaway and Ross 1997; Knöpfel et al. 2000). Consistent with diffusion of Na+ entering
the cell via AMPA receptors, the monitored rise of the [Na+]i responses outlasted the
synaptic currents for up to 500 ms. Therefore our maps of ∆[Na+]i overestimate the actual
innervation field. However, the size of [Na+]i signals resulting from diffusion rapidly decline
as a function of distance from the source such that they turn out to be clearly sub-
threshold under the present conditions of generating the maps of ∆[Na+]i at distances
greater than 30 µm (Knöpfel et al. 2000). This upper limit of overestimation of innervation
field size is also consistent with the restricted PF-mediated [Na+]i signals (Fig 3H).
All the above control experiments indicate that the maps of ∆[Na+]i represent sodium flux
through postsynaptic AMPA receptors and represent the innervation field of the activated
presynaptic elements at a spatial resolution sufficient to detect segregated innervation
fields as described in hypogranular or some mutant mice (Hashimoto et al. 2001a; Bravin
et al. 1995).
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Translocation of climbing fibers
Using the above approach we characterized the developmental transition of CF
innervation fields. Maps of CF-induced increases in [Na+]i from 43 PCs at postnatal ages
between P6 and P20 were divided into four age groups and classified depending on the
localization of the [Na+]i signals at the cell body (soma), soma and proximal dendrite, and
proximal dendrite (Fig. 4). We never detected CF-induced elevations of [Na+]i at the distal
part of the PC dendrite in agreement with anatomic investigations reporting that CF
contacts are confined to the large dendritic branches (Palay S.L. and Chan-Palay V.
1974; Mason et al. 1990). The developmental profile of CF termination field shows that
CF translocation starts at P6 and is essentially completed by P11 in mice (Fig. 4B).
More than one CF translocates from the soma to the proximal dendrite
To investigate whether one or multiple CFs translocate to the dendritic target field we
performed imaging experiments in PCs in which we could isolate two independent CFs.
Figure 5 illustrates data obtained from P9 and P11 PCs. At this developmental stage the
bulk of translocation takes place (Fig.4). The P9 cell (Fig. 5 A-E) exhibits two CF
termination fields that are most prominent at the level of the soma but both extend to the
proximal dendrite. The two CFs of the P11 cell (Fig. 5 F-H) innervate the proximal
dendrite while only one of them appears to have a remaining weak contact on the cell
body (Fig. 5G). At P14, when translocation is completed in the majority of cells (Fig. 4B)
while innervation by more than one CF is still observed (Kano et al. 1995), two CFs are
fully translocated (Fig. 6).
Multiple translocated CFs share their innervation fields
The examples in Figs 5 and 6 not only show that more than one CF can translocate into
the dendrite but also that their innervation fields are overlapping or at least are
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intermingled. Figure 7 shows a particular illustrative example of a P11 PC with two
primary dendrites and dendritic innervation by two fully translocated CFs. One of the two
primary dendrites (upper dendrite in Fig. 7) is innervated by the first CF while the second
primary dendrite is innervated by both CFs. Thus, the first primary dendrite represents the
mature situation of a mono-innervated proximal dendrite while the second primary
dendrite illustrates translocation of two CF with intermingled innervation fields.
We divided the data into three age groups and with regard to the relative location of
different CFs innervation fields (see table 1). Our data demonstrate that multiple CFs
have intermingled or partially intermingled innervation fields either on the soma or on the
proximal dendrite of the PC. We never detected multiple innervation where CFs have
completely separate synaptic territories supporting the conclusion that multiple CFs
share rather than segregate their innervation fields on the PCs in the developing
cerebellum.
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Discussion
We employed electrophysiological and imaging techniques to investigate the CF synaptic
termination fields in cerebellar cortex of mice during the period in which developmental
synapse elimination converts multiple CF innervation to mono-innervation (Kano et al.
1995). We found that more than one CF can translocate from the cell body to the proximal
dendrite and that translocated dendritic CFs have intermingled terminal arbors.
Our data of the developmental profile of CF termination fields confirms that CF
translocation lags behind outgrowth of the dendrite (Mason et al. 1990). In fact, already at
P8 the PC dendrites display a significant degree of development (see Fig. 1) while at the
same time period CF innervation is mainly confined to the peri-somatic level (Fig.4;
(Mason et al. 1990). These findings are in agreement with the view that PC dendritic
development is not strictly dependent on CFs (Mason et al. 1990) but under the influence
of the PFs. In fact, in several experimental conditions where granule cells are deleted, PC
dendrites are atrophic and disoriented in space (Altman and Anderson 1972; Berry and
Bradley 1976; Bradley and Berry 1978; Woodward et al. 1975; Mariani et al. 1977;
Caviness, Jr. and Rakic 1978; Sotelo 1978; Crepel et al. 1980; Bravin et al. 1995).
Experiments aimed to test the influence of CFs on PC dendritic development have shown
that dendritic growth can occur in the absence of CF input (Calvet et al. 1976; Sotelo and
Arsenio-Nunes 1976).
Although it is clearly established that regression from poly- to monoinnervation is
hampered when granule cell function is abnormal this information does not answer the
question whether in normal conditions the presence of functionally active PFs conveys to
the dendrites signals to prevent the invasion of more than one CF. Indeed, we showed
that more than one CF invades the dendritic tree indicating that normal PFs do not
prevent a transient innervation of the PC dendrites by multiple CFs and that at least part
of the competition occurs on the dendritic territory.
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Imaging experiments performed on poly-innervated PCs allowed us not only to follow the
process of translocation but also to study the relative allocation of CF-innervation fields. In
all cases of multiple dendritic innervation we found that CF-termination territories are
overlapping. The situation is similar to that described at the neuromuscular junction that
exhibits a variety of similar features. Also, in the latter system, a transient phase of poly-
innervation is followed by a permanent state of mono-innervation, and perturbation of this
process is associated with functional deficits. Many studies, in vivo and in vitro, have
shown that regression of redundant connections is an activity-dependent process
involving pre- and post-synaptic mechanisms (Lo and Poo 1991; Dan and Poo 1992).
Morphological studies involving in situ imaging of poly-innervated muscle fiber revealed
that, at birth, terminal branches of different axons are completely intermingled. However,
during several weeks after birth, the termination fields progressively segregate before the
complete withdrawal of all but one motor axon. The axon branches that innervate
overlapping postsynaptic muscle cells retract first (Gan and Lichtman 1998; Keller-Peck
et al. 2001). In a cell culture system containing motoneurons and myocytes it was shown
that brief tetanic stimulation of one neuron resulted in immediate functional suppression of
un-stimulated axons innervating the same muscle cell only and only if innervation fields of
the interacting axons were spatially separated by less than 50 µm (Lo and Poo 1991).
Thus, the degree of separation of different innervation sites can be a determining factor in
the process of synapse elimination. Such a scheme would explain the finding that in
hypogranular cerebellum and mice lacking glutamate receptor δ2 subunit or metabotropic
glutamate receptor subtype 1 (mGluR1) where PC remain poly-innnervated in adults, CF
innervation territories are segregated (Bravin et al. 1995; Hashimoto et al. 2001) while, as
shown in this work, lack of CF segregation in normal cerebellar development facilitates
competition between developing CF terminal arbors and ultimately elimination of all but
one CF.
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Table 1
CF innervation territories in poly-innervated PCs.
Age (post-natal days)
number of cells with partially intermingled
innervation fields
number of cells with fully intermingled innervation fields
6-8 3 (2) 4 (2)
9-11 5 (1) 2 (2)
12-14 1 (1) 2 (1)
A total of 17 cells in which two CF responses could be independently
evoked, were analyzed. For three age intervals cells were classified
with respect to the overlap of their innervation fields. Values in
parenthesis indicate the number of cells that have been recorded
with QX314 containing patch pipettes.
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Figure legends
Figure 1.
Morphology of developing Purkinje cells as revealed by fluorescence microscopy. All
Purkinje cells were recorded in mid-sagittal slices. Note rapid maturation between P6 and
P9 and the variability of the state of maturation within the same age.
Figure 2.
(A): Experimental design. Two stimulating electrodes (S1 and S2) were placed in the
granule cell layer (GL) below the recorded Purkinje cell. Reponses to CF stimulation were
recorded from the soma of the Purkinje cell with a patch pipette (R).
(B): Identification of CF responses by their all-or-nothing nature at threshold stimulation
intensity and by paired pulse depression. (C, D): Identification of independent CF
responses evoked by first (S1) and second (S2) stimulation electrode. (C): Simultaneous
stimulation with both electrodes leads to summation of individual CF responses. (D): The
response induced by one electrode does not induce paired pulse depression of the
response induced by the other stimulation electrode.
Figure 3.
Sodium imaging reveals territory of climbing fiber innervation. (A-F): A P10 Purkinje cell
was patch-clamped with a pipette containing 2mM SBFI and was voltage clamped at -
40mV. (A): fluorescence image of the cell, calibration bar: 50µm; (B,C,E): Color-coded
maps of ∆[Na+]i obtained with subthreshold stimulation (B), and suprathreshold climbing
fiber stimulation before (C) and after (E) adding 20µM NBQX to the external solution.
Bluish color corresponds to 0 % change in SBFI fluorescence and green through red
colors indicate decrease in SBFI fluorescence (corresponding to an increase in [Na+]i; see
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color code in E). (D): Membrane current and time course of ∆[Na+]i monitored in the
responsive area indicated by red outline in (A) with sub- and suprathreshold stimulation
(open and closed symbols, respectively). Note different time scale of electrical and optical
signal. (F) Responses to stimulation with suprathreshold intensity after addition of NBQX.
(G, H, I): CF and PF fiber innervation territories. A P11 PC voltage-clamped at -65mV. One
stimulation electrode was placed in the granule cell layer and recruited a CF and a second
electrode was placed in the molecular layer in order to elicit parallel fiber responses. Color
coded maps of ∆[Na+]i induced by CF activation (G), and parallel fiber stimulation (H). (I):
PF stimulation in the presence of NBQX 20µM. Regions of the image in which there was
no significant change in SBFI fluorescence show the fluorescence image of the cell in gray
scale. Note that the two responsive areas are spatially separated and PF responses are
confined to the distal part of the dendrite. Both cells illustrated in this figure were recorded
with QX314 in the pipette. In this and the subsequent figures vertical arrows indicate time
points of climbing fiber stimulation. The single arrow associated with the ∆[Na+]i indicates
time point of the first of 5 CF stimuli delivered at 10 Hz.
Figure 4.
Translocation from somatic to dendritic innervation fields of CFs during development. (A):
Maps of CF-induced ∆[Na+]i in P7, P9 and P12 PCs. Responsive areas (defined as ∆[Na+]i
greater than the minimal value indicated at the color bars) are superimposed on the
fluorescence image of the cells; calibration bars: 20µm. Recordings from the P9 and the
P12 PCs were conducted with patch pipettes containing QX314. Regions of the image in
which the change in SBFI fluorescence was smaller than the range given by the color
scale show the fluorescence image of the cell in gray scale.
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(B): CF territories classified as soma only, soma and proximal dendrite and proximal
dendrite only at different developmental stages.
Figure 5.
Multiple CF innervation of P9 and P11 Purkinje cells. (A): Fluorescence image of a P9 PC,
calibration bar: 20µm; (B,C,D,E): Color-coded maps of ∆[Na+]i and time courses of
membrane currents and local ∆[Na+]i. Responses to stimulation of first, second and both
CFs are shown in B, C and D, respectively. Corresponding current and ∆[Na+]i traces
(obtained from selected regions of corresponding colors indicated in (A)) are shown below
each ∆[Na+]i map. In (E) responses to stimulation of first, second and both CFs are
superimposed. Note that the largest Na+ signals are observed at the level of the cell body
even though the dendritic tree was relatively developed.
(F,G,H): Morphology and ∆[Na+]i maps of a P11 PC where two different CFs have
translocated and share their innervation territories on the proximal dendrite. The patch
pipette contained QX314. Calibration bar is 20µm. Note some somatic innervation by first
CF but not by second CF. The ∆[Na+]i time courses shown below each ∆[Na+]i map were
obtained from the regions indicated with corresponding colors in (F). (I): Stimulation of first
and second CF demonstrating lack of depression of second CF response following
activation of first CF.
Figure 6.
Multiple dendritic CF innervation in a P14 PC. Two individual climbing fibers were
stimulated with two separated electrodes. (A): fluorescence image of the cell; calibration
bar: 30µm. (B,C,D): Color-coded maps of ∆[Na+]i and time courses of membrane currents
and ∆[Na+]i obtained with stimulation of the first CF (B), the second CF (C) and concurrent
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stimulation of both CFs (D). ∆[Na+]i traces were obtained from the responsive area
indicated in (A). (E): Lack of mutual paired pulse depression. Note that translocation has
been completed for both the climbing fibers and the territories of innervation are
intermingled on the proximal dendrite but are separated on the secondary dendrite.
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Figure 7.
Multiple dendritic CF responses in a bipolar P14 Purkinje cell. (A): two individual CFs were
stimulated with two separate electrodes. The CF-evoked currents (Vh=-40mV) were
additive on the individual responses. (B,C): fluorescence images of the cell taken at two
different focal planes. Calibration bar is 20µm. Note that the CCD was saturated at pixels
in the center of then cell body. (D): reconstruction of the morphology of the cell. The
pseudo color-coded maps of ∆[Na+]i revealed that the upper dendrite is innervated mainly
by the first CF whilst the lower dendrite is contacted by both CFs. The upper dendrite of
this bipolar cell illustrates features of a mono-innervated PC while the lower dendrite
shows multiple CF innervation with, at least, partially overlapping fields of innervation.
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Acknowledgments. We thank Ms Ayako Takada for expert administrative and secretarial assistance. This work
was supported by an intramural grant from the RIKEN Brain Science Institute.
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C
Fig. 2
S1
S2
1nA50ms
A
S2S1
R
GL
B
1nA50ms
All-or-nothing
DSummation Lack of mutual depression
50ms1nA
S1
S S
S1
S1
S2
S2
S2S1+S2 S1+S2
0.5% 3.5%
H
0.5% 3.5%
I
0.5% 1.3%
G
A B C
D
2nA
100ms
1s
- 2.5%
2.5%2.5% (- F/F)∆
(- F/F)∆ (- F/F)∆- 0 %
0 %0 %
F
2nA100ms
1s
E
Fig. 3
Fig. 4
B
A
100
1P7
0.1% 0.8%
P9
0.7% 1.7%
P12
0.5% 1.5%
soma
soma andprox. dend.
prox. dend.
80
60
4020
0
Age(postnatal days)
6-8(N=6)(N=16)(N=14)(N=7)> 1512-149-11
100pA200ms
1s
- 0.4%
- 0 %
(- F/F)∆ (- F/F)∆ (- F/F)∆
1nA200ms
B C D
E
20ms1nA
200ms 1nA
F G H
I
50ms
2nA
2s 2s
200ms200ms
2nA2nA
Fig. 5
1stCF
1stCF1stCF
1stCF
2ndCF
2ndCF2ndCF
2ndCF
bothCFs
bothCFs
A
F
1 %
(- F/F)∆
0 %
1 %
(- F/F)∆
0 %
1 %
0 %
0.4%
0 %
0.4%
0 %
0.4%
0 %
S1 S2
S2 S1
E
B
500pA
100ms
1s
C
500pA
100ms
1s
D
1s
500pA100ms
Figure 6
50ms500pA
A
1stCF 2ndCF both CFs
1stCF
1stCF
2ndCF
2ndCF
0 %
0.9%
(- F/F)∆
0 %
0.9%
(- F/F)∆
0 %
0.9%
(- F/F)∆
0 %
0.9%