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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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Fig. 1

50 µm

50 µm

50 µm

P6 P7 P8 P9

P10 P11 P12 P13

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%

2nd CF both CFs1st CF

50 ms

3 nA

A

B

1st CF 2nd CF both CFs

D

C

region 1 region 3region 2

1st CF 2ndCF both CFs

Fig. 7

1stCF2ndCFbothCFs 2%

2%

0%

0%

(−∆F/F)

2 s

1stCF2ndCFbothCFs

1stCF2ndCFbothCFs