Purkinje cell activity during learning a new timing in classical eyeblink conditioning

www.elsevier.com/locate/brainres

Brain Research 994 (2003) 193–202

Research report

Purkinje cell activity during learning a new timing in

classical eyeblink conditioning

Sadaharu Kotania, Shigenori Kawaharaa,b,*, Yutaka Kirinoa

aLaboratory of Neurobiophysics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, JapanbCore Research for Evolution Science and Technology (CREST), Japan Science and Technology Corporation (JST), Kawaguchi, Saitama 332-0012, Japan

Accepted 16 September 2003

Abstract

During classical eyeblink conditioning, animals acquire adaptive timing of the conditioned response (CR) to the interstimulus interval

(ISI) between the conditioned stimulus (CS) and the unconditioned stimulus (US). To investigate this coding of the timing by the cerebellum,

we analyzed Purkinje cell activities during acquisition of new timing after we shifted the ISI. Decerebrate guinea pigs were conditioned to an

asymptotic level of learning using a delay paradigm with a 250-ms ISI. A 350-ms tone and a 100-ms electrical shock were used as the CS and

US, respectively. As reported previously in other species, Purkinje cells in the simplex lobe exhibited three types of responses to the CS:

excitatory, inhibitory, or a combination of the two. After we increased the ISI to 400 ms, the frequency of the CR stayed at an asymptotic

level, but the latency of the CR peak became gradually longer. Two types of cells were observed, based on changes in the nature of their

response to the CS; one changed its type of response in parallel with learning the new timing, while the other did not. There was no

correlation between the type of response before and after we changed the ISI. In some cells, the peak latency of activities became longer or

shorter, while the type of response did not change. These results suggest that some Purkinje cells code the timing of the CR, but do not play a

consistent role in shaping the CR over a range of ISIs.

D 2003 Elsevier B.V. All rights reserved.

Theme: Neural basis of behavior

Topic: Learning and memory: systems and functions

Keywords: Cerebellum; Classical conditioning; Decerebrate; Eyeblink; Guinea pig; Purkinje cell; Timing

1. Introduction timing between two discrete stimuli, the conditioned stim-

Since the early theoretical studies of Marr [22] and Albus

[1], the cerebellum has been hypothesized to be the site

responsible for motor learning. Various experiments have

provided details of the function of the cerebellum in motor

learning [14]. One important cerebellar function is related to

well-timed movement during various tasks: multi-joint

movement [32], adaptation of the vestibuloocular reflex

[27], and smooth pursuing eye movements [21]. Pavlovian

eyeblink conditioning is a relatively simple motor learning

task in the sense that the animal learns only one kind of

0006-8993/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2003.09.036

* Corresponding author. Laboratory of Neurobiophysics, Graduate

School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo,

Bunkyo, Tokyo 113-0033, Japan. Tel.: +81-3-5841-4801; fax: +81-3-5841-

4805.

E-mail address: [email protected] (S. Kawahara).

ulus (CS) and the unconditioned stimulus (US). This feature

is useful for investigating the relationship between the

operation of the well-known cerebellar neural network and

timing.

The essential neural circuits for eyeblink conditioning are

known to reside in the cerebellum and brainstem [24,33].

With repeated presentations of the CS paired with the US,

the CS comes to elicit an eyeblink, i.e., the conditioned

response (CR). The acquired CR has a clear peak around the

expected time of US arrival, and its latency becomes longer

with an increase in the interstimulus interval (ISI) [7,13].

During eyeblink conditioning, animals learn not only the

predictive value of the CS but also the ISI, to express the CR

with an adaptive timing.

Studies using local lesions [11,19,20,36], pharmacolog-

ical blockade [2], and neural recording [4,10,12,16,29,34]

have revealed the critical involvement of the ipsilateral

S. Kotani et al. / Brain Research 994 (2003) 193–202194

lobule HVI (simplex lobe) in this learning, although there

are some reports of no effects of the HVI lesion [9,26].

However, these lesion and pharmacological studies did not

dissociate the issue of the CR timing from that of CR

acquisition and/or expression. For example, lesion of the

lobule HVI after sufficient conditioning reduced the fre-

quency of CR expression and disrupted accurate timing of

residual responses [2,11]. One recent pharmacological study

with sequential application of an agonist and an antagonist

of GABA receptors to the interpositus nucleus did succeed

in separating these issues and the results clearly indicate that

CR timing and expression are encoded by different mech-

anisms [3]. In electrophysiological studies, Purkinje cells in

lobule HVI showed a variety of responses to the CS after

delay conditioning had been established [4,10,12,16,34].

Continuous recording of a single Purkinje cell during

conditioning may be preferable when investigating the cell’s

role in learning. However, many of the previous studies

investigated the responses of Purkinje cells in a static state

after acquisition of the CR, except for one study in intact

rabbits [10] and another in decerebrate ferrets [12]. Gould

and Steinmetz [10] analyzed changes in Purkinje cell

activity during alternating extinction-reacquisition sessions

in animals that had been trained. And they reported that

Purkinje cells in lobule HVI code a variety of aspects of the

extinction and reacquisition phases of conditioning. How-

ever, the issue of CR timing still has not been clarified. To

understand the operation of the cerebellar circuit during

learning, it is important to clarify the role of Purkinje cells in

the timing of the CR, which is very likely to be distinct from

their role in CR acquisition.

In the present study, we analyzed responses of Purkinje

cells to an ISI change from 250 to 400 ms in animals that

had learned both the CS-US timing and the predictive value

of the CS in a delay conditioning paradigm with an ISI of

250 ms. We used a decerebrate guinea pig preparation, in

which all of the brain was removed except the cerebellum

and the brainstem [17]. There are two reasons for this

choice: (1) this preparation permits successive conditioning

for several hours in a fixed condition, a useful feature for

long-lasting single-unit recording; and (2) there are some

reports that the forebrain affects the CR timing [6,8] and

others that it does not [28,31], thus this preparation is useful

for eliminating such potential interference.

Portions of this study previously have been reported in

abstract form [18].

2. Materials and methods

2.1. Subjects

We used male guinea pigs (Hartley, Japan, SLC, Hama-

matsu, Japan) weighing 250–300 g. They were housed in

clean cages on a 12-h light/dark cycle (with light onset at

0700 h) with free access to water and food. During the course

of the present study, care of the animals conformed to the

guidelines established by the Institutional Animal Investiga-

tion Committee at the University of Tokyo. All efforts were

made to optimize the comfort and to minimize the use of

animals.

2.2. Surgery

Surgery was performed as described previously [17].

Thirty minutes after administration of atropine (0.1 mg/kg

i.m.; Tanabe Pharmaceutical, Osaka, Japan), the animal was

deeply anesthetized with isoflurane (1.5–2.5%; Dainippon

Pharmaceutical, Osaka, Japan) in a mixture of O2 and N2O

(1:1). After the right carotid artery was fastened and a

tracheotomy was performed, anesthetic gas was introduced

directly into a tracheal tube with a respirator. The head of

the animal was firmly fixed by a clamp (Type SH-15;

Narishige, Tokyo, Japan). A window was opened in the

skull between the bregma and the lambda suture, and the

cerebral cortex and hippocampus overlaying the brainstem

were aspirated up to the level of the thalamus. Decerebration

was then performed by a section between the thalamus and

the superior colliculus. All of the brain tissues superior to

the section were removed by aspiration. HistOmer (HistO-

tech, Arhus C, Denmark) mixed with saline was placed in

the empty space and paraffin oil was poured in to prevent

drying.

Two pairs of Teflon-coated stainless-steel wires (no.

7910; A-M systems, Carlsborg, WA, USA) were used for

conditioning. One pair was implanted in the left upper

eyelid for recording electromyograms (EMGs) and the other

was implanted in the left lower eyelid for delivering the US.

After implantation of the electrodes, the anesthesia was

discontinued. Artificial ventilation was provided for the

duration of the experiments. The rectal temperature was

continuously monitored and kept at 37.5 jC. Most of the

decerebrate animals were maintained for about 24 h after

decerebration.

2.3. Conditioning and neural recording procedures

Two hours after decerebration, conditioning was started

with a 350-ms tone CS (5 kHz, 85 dB) and a 100-ms

electrical shock US (100-Hz square pulses, 0.5-2 mA). The

US intensity was carefully determined to be sufficient to

elicit an eyeblink, and had a tendency to increase progres-

sively during the experiment. Conditioning included a CS-

alone trial at every 10th trial in addition to the CS-US trials,

and was performed successively for more than 6 h. The

inter-trial interval was pseudorandomized between 20 and

40 s, with a mean of 30 s. These successive trials were

grouped into sessions for analysis, each of which contained

five blocks of 10 trials (9 CS-US paired trials and 1 CS-

alone trial). There were two conditioning groups: a pair-

conditioned group (38 animals) and a pseudoconditioned

group (16 animals). In the pair-conditioned group, the US

S. Kotani et al. / Brain Research 994 (2003) 193–202 195

started 250 ms after the onset of the CS (ISI = 250 ms), and

coterminated with the CS in the CS-US paired trials (Delay

250). In the pseudoconditioned group, the CS and the US

were delivered with pseudorandomized ISIs (1–20 s) in the

CS-US trials. To investigate the response to the tone before

any conditioning started, 10 of the 16 pseudoconditioned

animals received 50 CS-alone trials before pseudocondition-

ing, with Purkinje cell recording as described below. Be-

cause the types and their ratios of Purkinje cell responses

after pseudoconditioning were similar to that of the other six

animals, the data from all 16 pseudoconditioned animals

were combined for analysis.

After 16 sessions (800 trials) of paired conditioning or

pseudoconditioning, the decerebrate animals in both groups

were re-anesthetized with isoflurane. The left cerebellar

hemisphere around the simplex lobe (Lobule HVI), ipsilat-

eral to the trained eye, was exposed by craniotomy. Thirty

minutes after termination of the anesthesia, one or two

glass-insulated tungsten microelectrodes were inserted into

the left simplex lobe using a dissecting microscope. After

identifying the Purkinje cells by the presence of complex

spikes (Fig. 3A), CS-US paired presentation started again in

the case of the pair-conditioned animals (or pseudorandom-

ized ISI presentation in the case of the pseudoconditioned

animals). Cells were recorded continuously for at least 10

trials and for a mean number of 69.1F 3.3 trials (meanFS.E.M., n = 300).

After the Purkinje cells of an animal in the pair-condi-

tioned group had been recorded successfully for more than

50 trials, the ISI was increased from 250 to 400 ms. The

duration of the CS also was extended from 350 to 500 ms so

that it coterminated with the US (Delay 400). Conditioning

was performed as long as the Purkinje cells continued to

permit recording. After the last session, the recording site

was marked by passing a negative current through the

recording electrode (� 50 AA, 30 s) under anesthesia.

2.4. Data analysis

Behavioral data were analyzed in the same way as

described previously [17]. The EMG activity was band-pass

filtered between 0.15 and 1.0 kHz and fed into a computer

with a sampling rate of 10 kHz. The maximum amplitude of

EMG signals during a time period of tF 1 ms was calcu-

lated and designated the EMG amplitude at t. We defined

the threshold as the average + S.D. of the EMG amplitudes

during the pre-CS period (0–300 ms before CS onset) of a

session containing 50 trials. If the average EMG amplitudes

above the threshold during the pre-CS period exceeded 10%

of this threshold, the trial was regarded as a hyperactivity

trial and excluded from further analysis. A trial was

regarded to contain the CR if the average EMG amplitude

above the threshold over any of the 100-ms period from 50

ms after CS onset to US onset exceeded 10% of the

threshold and exceeded 10 times the average EMG ampli-

tude of the pre-CS period. In the CS-alone trials, the period

of average EMG was extended to the CS end. The frequency

of CRs (CR%) was expressed as a percentage for a session

and presented as the meanF S.E.M. In the case of pseudo-

conditioning, the CR% was calculated as in the CS-alone

trials.

The latency of the CR onset from the CS onset was

calculated in the trials that were judged to contain a CR. The

time of CR onset was defined as the time when the average

EMG amplitude above the threshold over a 10-ms period

met the above criterion for the first time between 50 ms after

the CS onset and the US onset. The latency of the CR onset

was averaged over each session and denoted the ‘‘onset

latency’’ of the session. The latency of the CR peak from the

CS onset was calculated in those CS-alone trials which were

judged to contain the CR, to avoid the US artifacts. The data

were averaged over a session and denoted the ‘‘peak

latency’’ of the session. To show the temporal pattern of

the CR, the EMG amplitude data were averaged over the

CS-alone trials in that session. This averaged EMG pattern

therefore does not depend on the criterion for detecting the

CR.

Neural activity was filtered between 0.15 and 3 kHz and

recorded with a sampling rate of 40 kHz using Experimen-

ter’s Workbench (DataWave Technologies, Longmont, CO,

USA). Off-line analysis was also performed using this

system. A single Purkinje cell was classified using ‘‘cluster

analysis’’ and only the simple spike was separated and

analyzed. The details of the criterion to discriminate the

simple spikes from the complex spikes are similar to those

described by Welsh and Schwarz [35]. The analysis was

performed session by session. For analysis of the response

type of a cell, the 300-ms period before the CS onset was

divided into six 50-ms bins. The spike frequency in each

pre-CS bin during a session (50 ms� 50 trials) was calcu-

lated and averaged over six bins; this was then defined as

the spontaneous spike frequency for that session. The S.D.

was also calculated. The neural activities during a Delay 250

session were regarded to contain a response to the CS if the

five 50-ms bins during the 250-ms ISI contained at least two

such bins that showed a 2 or more standard deviations

increase or decrease in spike frequency from the spontane-

ous spike frequency. To examine changes in the Purkinje

cell response during the learning of new timing, the data in

each session in the Delay 400 conditioning, in which the

mean peak latency of the CR increased by at least 100 ms

from that of the first session of the Delay 400 conditioning,

were compared with those in the first Delay 400 session.

The response type in the Delay 400 session was determined

by using the data of eight 50-ms bins after the CS onset, in

the same way as in the Delay 250 session.

The latency from the CS onset to the peak of an

excitatory or inhibitory response was calculated after

smoothing the response by averaging the spike frequency

over 50 ms. Namely, the spike frequency of neural activity,

calculated from the peri-stimulus time histogram for a

session (50 trials), was averaged over a time period of

Fig. 1. CS-US paired and pseudoconditioning in decerebrate guinea pigs.

(A) Average percentage of conditioned response (CR%) for pair-conditioned

group (open circle, n= 38) and for pseudoconditioned group (filled circle,

n= 16). Abscissa, session: a session consisted of 50 trials. Error bar indicates

S.E.M. (B) Representative temporal patterns of EMG amplitude for pair- and

pseudoconditioned groups. For pair-conditioned subjects, data were

averaged for CS-alone trials in the 16th session. The horizontal lines

indicate the timing of the CS and the expected timing of US that was not

delivered in the CS-alone trials. For pseudoconditioned subjects, data were

averaged for all trials in the 16th session. The horizontal lines indicate the

timing of the CS.


tF 25 ms. The t that showed the maximum smoothed spike

frequency in excitatory response was defined as the excit-

atory peak latency, and that showed the minimum spike

frequency in inhibitory response was defined as the inhib-

itory peak latency. To analyze the topographical relation

between the neural response and behavioral response, a real-

time correlation was calculated for the spike frequency and

the EMG in each 3-ms time period, i.e. the ‘‘cross-correla-

tion’’ analysis introduced by Hoehler and Thompson [13]

for the analysis of the hippocampal neural activity during

eyeblink conditioning.

2.5. Histology

At the end of the experiment, the decerebrate animals

were injected intraperitoneally with sodium pentobarbital

(>80 mg/kg; Schering-Plough, Kenilworth, NJ, USA). The

anesthetized animals were next perfused intracardially with

0.9% saline, and then with 10% formalin. The brain was

removed from the skull and stored in 10% formalin for

several days. After infiltration with 30% sucrose, the brain

was frozen, sectioned sagittally at a thickness of 60 Am, and

stained with cresyl violet (Sigma). The marking lesion of the

recording site was confirmed microscopically.

3. Results

3.1. Purkinje cell activities after learning a delay paradigm

with a 250-ms interstimulus interval

As already noted in our previous report [17], all

decerebrate guinea pigs that received the CS-US paired

presentation with a 250-ms ISI (Delay 250) acquired the

CR successfully within 800 trials (Fig. 1). The CR% just

before the start of neural recording, i.e. after 16 sessions

(800 trials), was 94.8F 2.1% (n = 38). The temporal pat-

tern of the EMG amplitude indicated that they had ac-

quired an adaptive timing to the 250-ms ISI (Fig. 1B). The

latency of the CR peak from the CS onset in the CS-alone

trials in the last Delay 250 session was 307.2F 15.2 ms.

On the other hand, decerebrate guinea pigs that received

pseudoconditioning (n = 16) showed neither any sign of

increase in the CR frequency (Fig. 1A) nor any meaningful

response in the EMG temporal pattern (Fig. 1B). The CR%

after 16 sessions was 4.5F 1.1%. A two-way repeated

measures ANOVA on the CR% of the pair-conditioned

group and the pseudoconditioned group revealed signifi-

cant effects of interaction between the groups and sessions

(F15, 780 = 13.464, P < 0.001).

After 16 sessions, Purkinje cell activity was recorded

from the simplex lobe ipsilateral to the trained eye. Histo-

logical analysis of the lesion mark (Fig. 2B and C) con-

firmed that all of the recording sites were in the left simplex

lobe (Fig. 2A). We recorded 147 cells in the pair-condi-

tioned animals and 153 cells in the pseudoconditioned

animals. Fig. 3B shows Purkinje cell activity in a CS-US

paired trial. This cell exhibits a clear inhibitory response to

the CS. We found three types of responding patterns in the

Purkinje cells: an excitatory response (Fig. 3C), an inhibi-

tory response (Fig. 3D), or a complex response, which had

an inhibitory response, and an excitatory response during

the CS period (Fig. 3E,F). Complex response types included

those cells that exhibited an inhibitory response in the early

phase and an excitatory response in the late phase during the

CS period (Fig. 3E), and those that exhibited excitation in

the early phase and inhibition in the late phase during the

CS period (Fig. 3F).

Table 1 summarizes the types of response to the CS

across all Purkinje cells. Consistent with the previous

studies in intact rabbits [4,10,16,34], the ratio of the number

of the excitatory response cells to inhibitory response cells

in the pair-conditioned group was about 2:1. Comparison of

the distribution of response types between the pair-condi-

tioned group and the pseudoconditioned group revealed a

significant difference (v2(3) = 22.8; P < 0.001). The test of

difference in the population ratios revealed a significant

difference in the percentage of no-response cells (v2(1) =16.9; P < 0.001), inhibitory response cells (v2(1) = 8.0; P <

0.01), and complex response cells (v2(1) = 6.2; P < 0.05)

between the pair-conditioned group and the pseudocondi-

tioned group. However, the percentage of excitatory re-

sponse cells did not differ significantly between the two

groups (v2(1) = 0.25; P>0.05).

We also examined the Purkinje cell response to the CS

before any conditioning started. Ten naıve decerebrate

animals received 50 CS-alone presentations. Twenty-eight

cells were recorded and consisted of 10 excitatory, 2

inhibitory, 1 complex and 15 no-response cells (Table 1).

The distribution of response types during CS-alone trials in

Fig. 3. Activities of representative Purkinje cells. (A) Example of the simple

spike (arrow) and complex spike (arrowhead). (B) Example of raw unit

activity during the delay paradigm with a 250-ms ISI. The cell included in

this unit shows an inhibitory response to the CS. The horizontal solid line

indicates the timing of the CS and the dotted line indicates the US. The large

signal at the US is an artifact due to the electrical shock. (C–F) Peri-stimulus

time histogram (PSTH) of four representative cells after learning delay

paradigm with a 250-ms ISI. (C) Excitatory response cell. (D) Inhibitory

response cell. (E) A cell that shows an inhibitory response in the early phase

and an excitatory response in the late phase during the CS period. (F) A cell

that shows an excitatory response in the early phase and an inhibitory

response in the late phase during the CS period. Each histogram represents

average activity for 50 trials. Time bin: 5 ms. Activity prior to the US onset is

shown. The arrow indicates CS onset.

Table 1

Types of Purkinje cell responses to the CS after learning in the Delay 250

paradigm

Pair-conditioned Pseudoconditioned CS-alone

No response 41 (28.0%) 78 (51.0%) 15 (53.6%)

Excitatory 53 (36.0%) 51 (33.3%) 10 (35.7%)

Inhibitory 29 (19.7%) 13 (8.5%) 2 (7.1%)

Complex 24 (16.3%) 11 (7.2%) 1 (3.6%)

Total 147 (100%) 153 (100%) 28 (100%)

The numbers of Purkinje cells that showed each type of response to the CS

after CS-US pair- and pseudoconditioning, and during CS-alone trials are

shown.

Fig. 2. Histological analysis of recording site. (A) Dorsal view of the left

cerebellar cortex. All recording sites were included within the filled region.

(B and C) An example of a recording site. Sagittal section of the cerebellar

hemisphere is shown. A recording site is indicated by an arrow. (C)

Magnified sagittal section of simplex lobe in (B).


naıve animals (i.e., before any conditioning) differed

significantly from that in animals that received CS-US

paired conditioning (v2(3) = 10.4; P < 0.05), but not from

that in animals with pseudoconditioning (v2(3) = 0.67;

P>0.05).

To compare the temporal pattern of the neural response

between the pair-conditioned group and the pseudocondi-

tioned group, the latency (from the CS onset) of the peak of

the excitatory or inhibitory response was calculated. In all

four types of responses (namely, excitatory response, inhib-

itory response, excitatory response in complex response cell

and inhibitory response in complex response cell), the

distribution of the peak latency of the neural responses

did not differ significantly between the groups. In addition,

the distribution of the peak latency of excitatory and

inhibitory response did not differ significantly between

before any conditioning and after paired conditioning or

pseudoconditioning.

Table 2

Changes in response type of Purkinje cells accompanying a 100-ms

increase in the peak latency of the CR

Purkinje cell response after 100-ms increase in peak

latency was achieved with change in conditioning from

Delay 250 to Delay 400

No

response

Excitatory Inhibitory Complex

Purkinje cell No response 2 1 1 1

response Excitatory 11 1

during the Inhibitory 2 1 1

first Delay

400 session

Complex 2 1 3

The numbers of response-type changes observed in Purkinje cells after a

100-ms increase in the peak latency of the CR was achieved after a 150-ms

increase in the ISI by switching from a Delay 250 to a Delay 400 paradigm

are shown.


To compare the temporal pattern of the behavioral

response with the neural response in the pair-conditioned

group, correlation between the peak latency of the CR and

the peak latency of the neural response was analyzed. There

was a weak but significant correlation between the peak

latency of the CR and that of the neural response in the

excitatory response cells (Spearman rank correlation;

r = 0.29, P < 0.05). However, there was no such correlation

in other response cell types (Spearman rank correlations:

r =� 0.12, P>0.05 for inhibitory response cells; r =� 0.02,

P>0.05 for excitatory responses in complex response cells;

r = 0.10, P>0.05 for inhibitory responses in complex re-

sponse cells). To further investigate their temporal relation-

ship, the correlation between the topography of the neural

response and the CR was calculated for individual cells.

Twenty-one cells out of 53 excitatory response cells, 13 out

of 29 inhibitory response cells, and 11 out of 24 complex

response cells showed a significant correlation with the

behavioral response. Among them, five excitatory, six

inhibitory and five complex response cells showed a sig-

nificant negative correlation with the behavioral response.

Because Purkinje cells form inhibitory synapses on the

interpositus nucleus, the neural activities that showed a

negative correlation with the CR topography are considered

to be the CR-related activities. Although such CR-related

cells were not numerous within each response type, the ratio

of the CR-related cells in inhibitory response cells and that

in complex response cells was larger than that in excitatory

response cells.

3.2. Changes in Purkinje cell response during learning of a

new timing

The pair-conditioned animals, in which the Purkinje cell

activity had been successfully recorded for more than 50

trials in the Delay 250 sessions, received subsequent delay

conditioning with a 400-ms ISI (Delay 400). Forty cells

were continuously recorded successfully during the first 50

trials of Delay 400 conditioning. Among them, 27 cells in

18 animals were kept recorded even longer, until the CR

peak latency extended an additional 100 ms. The CR peak

latency of these animals in the first Delay 400 session was

243.0F 9.2 ms. The number of trials required for the

additional 100-ms increase in CR peak latency was

172.2F 24.3 trials. Therefore, the animal received about

220 trials with a 400-ms ISI. During this 100-ms increase,

any increase or decrease in the CR onset latency was

observed, but there was little change (15.0F 4.2 ms) from

85.6F 9.0 ms.

Table 2 shows a summary of the changes in response

type during acquisition of new timing. Among the 27 cells,

10 cells changed their type of response to the CS after the

100-ms increase in CR peak latency. Three of five non-

responding cells in the first Delay 400 session later began to

respond to the CS and showed excitatory, inhibitory, or

complex responses. The remaining two non-responding

cells stayed non-responding. Among 12 excitatory response

cells in the first Delay 400 session, only one cell changed its

response and exhibited a complex response: an early inhib-

itory response was added before the excitatory response.

Two of four inhibitory response cells in the first Delay 400

session eventually exhibited an excitatory response, and one

inhibitory response cell did a complex response in which a

late excitatory response was added after the inhibitory

response. The remaining one inhibitory response cell did

not change its response. One of the six complex response

cells in the first Delay 400 session lost its excitatory

component and two cells lost their inhibitory component;

the remaining three cells did not show any change. Fig. 4

shows the change from a complex response cell to an

excitatory response cell during the acquisition of a new

timing. This subject required 200 trials for the 100-ms

increase in CR peak latency. Just after the ISI was shifted

to 400 ms, the cell exhibited an early inhibitory response

and a late excitatory response to the CS. However, as the CR

peak shifted toward the new US onset, the inhibitory

response disappeared, while the excitatory response became

larger.

3.3. Changes in the temporal pattern of Purkinje cell

responses during the learning of new timing

We analyzed changes in the temporal patterning of

Purkinje cell responses during the Delay 400 sessions that

followed the Delay 250 sessions. We calculated the differ-

ence between the peak latencies of the neural responses in

the first session and those in which the peak latency of the

CR increased by 100 ms (Fig. 5; empty columns). The

excitatory responses in the excitatory response cells and the

responses in the complex response cells were grouped

together (Fig. 5A; empty columns). During the Delay 400

sessions, the peak latency of the excitatory response became

longer in six cells and shorter in 11 cells. The change in the

peak latency of the response in these Purkinje cells was

84.2F 18.2 and � 86.1F18.0 ms, respectively. The

Fig. 4. Representative changes in the activity of a Purkinje cell and the

temporal pattern of EMG amplitude accompanying the learning of new

timing. (A) Changes in Purkinje cell activity per 50 trials. The arrow in each

histogram indicates CS onset, and the activity until the US onset is shown.

Time bin: 5 ms. (B) Changes in temporal pattern of EMG amplitude per 50

trials. The data were averaged over 50 trials for the CS-alone trials. The

horizontal lines indicate the timing of the CS and the expected timing of the

US that was not delivered in the CS-alone trials. (A) and (B) are in

correspondence with each other.

Fig. 5. Shift in the peak latency of a Purkinje cell response. (A) Shift in the

peak of an excitatory response of an excitatory response cell and a complex

response cell. (B) Shift in the peak of the inhibitory response of an

inhibitory response cell and a complex response cell. Empty columns

represent the shift during conditioning with a new ISI. The shift was

calculated by subtracting the peak latency of the response at the first Delay

400 session from that at the session in which the peak latency of the CR

increased by 100 ms by conditioning with Delay 400 for each cell. Filled

columns represent the shift during successive conditioning with an ISI of

250 ms, which had been learned sufficiently. The shift was calculated by

subtracting the peak latency of the response in the first session from that in

the last session of Delay 250 for each cell.


change for the cell in Fig. 4, in which the peak of the

excitatory response became more delayed as the acquisition

of new timing progressed, was 92 ms. The inhibitory

response was analyzed in the same way as the excitatory

response (Fig. 5B; empty columns). The peak latency of the

inhibitory response in the inhibitory response cells and the

complex response cells became longer in two cells and

shorter in four cells. The changes in peak latency in these

cells were 48 and 4 ms for the former two cells, and

� 19.7F 17.7 ms for the latter cells. These results suggest

that the large changes in temporal pattern that accompanied

the learning of new timing were mainly observed in cells

that exhibited an excitatory response to the CS.

Analysis of the correlation between neural and behavior-

al responses revealed only three CR-related cells out of 27

cells during the Delay 400 sessions. A CR-related inhibitory

response cell during the first Delay 400 session changed to a

non-correlated excitatory response cell along with the 100-

ms shift of the CR timing. On the other hand, a non-

correlated complex response cell changed to a CR-related

excitatory response cell. There was also one CR-related

complex response cell that remained so during the Delay


400 sessions. Many of the excitatory response cells that

showed a large shift in excitatory peak were not CR-related.

3.4. Stability of the response of Purkinje cell to the tone

To eliminate the possibility that the change in response

type or temporal pattern is not due to the change of the ISI

but instead to a spontaneous change just observed in a

lengthy successive conditioning period, we checked the

stability of the response during conditioning with the

identical-ISI paradigm that the subjects had already learned

sufficiently. Purkinje cell recording started after condition-

ing with 800 trials of the Delay 250 paradigm. Twenty-three

Purkinje cells, consisting of six excitatory response cells,

seven inhibitory response cells, eight no-response cells and

two complex response cells, were continuously recorded for

more than 150 trials in the same paradigm (228.3F 17.2

trials). None of these cells showed any change in response

type during the recording. We also checked for changes in

the peak of the excitatory response (Fig. 5A; filled columns)

and of the inhibitory response (Fig. 5B; filled columns)

during the lengthy Delay 250 sessions. Although shifts to a

shorter latency or to a longer latency were observed for both

response types, these shifts were small.

We performed a one-way ANOVA among the peak-

latency shifts in four groups: the excitatory and inhibitory

responses recorded in subjects conditioned with the new ISI

(empty columns in Fig. 5A and B, respectively) and those

responses in subjects conditioned with an identical ISI

(filled columns in Fig. 5A and B, respectively). We found

a significant difference in the extent of peak-latency shift

across the four groups (F3, 36 = 5.968, P < 0.01). A post hoc

Tukey test revealed a significant difference between the

excitatory response in the ISI-shift paradigm and the excit-

atory response (P < 0.05) and the inhibitory response

(P < 0.01) in the identical-ISI paradigm. There were no

other significant differences. These results demonstrate that

the shift in the excitatory-response peak during the learning

of new timing is larger than the shift during the lengthy

successive conditioning after establishment of a response.

Analysis of the cross-correlation between the neural and

behavioral responses during the identical-ISI paradigm

revealed CR-related activities in one inhibitory response

cell and one complex response cell, but none in excitatory

response cells.

4. Discussion

The present study confirmed the findings of previous

reports that the responses of Purkinje cells to the CS include

a wide variety of types of response [4,10,12,16,34]. This

study also revealed that these response patterns are not

fixed, including the polarity of the responses after change

in the ISI. In contrast, changes in the responding type and

the temporal pattern were rarely observed during the long

conditioning sessions as long as the ISI was constant. Since

the animals had already learned the association between the

CS and the US before the ISI was changed, these changes in

the responding pattern are mainly due to learning of a new

timing, rather than to learning of the CS-US association.

Previous studies of intact rabbits that analyzed the

response of Purkinje cells after sufficient CS-US paired

conditioning have revealed that Purkinje cells located in the

ipsilateral simplex lobe exhibit excitatory or inhibitory

responses to the CS. The major response to the CS was

excitatory, and the ratio of the number of excitatory

response cells to inhibitory response cells was 2:1

[4,10,16,34]. Our results in the first half of the present

experiments were consistent with these findings. Purkinje

cells in the pair-conditioned animals showed excitatory,

inhibitory, or both types of responses during the CS period.

The ratio of the number of excitatory response cells to

inhibitory response cells was also about 2:1, as reported in

the intact rabbits.

Purkinje cell activities also were recorded in the pseu-

doconditioned animals. Although about a half of the

recorded Purkinje cells showed no response to the CS, the

other half showed an excitatory or inhibitory response. This

is also consistent with the intact rabbit studies, in which

many Purkinje cells responded to the CS after backward or

unpaired conditioning [10,34]. Compared to the cells in the

pair-conditioned group, fewer Purkinje cells in the pseudo-

conditioned group showed an inhibitory response or a two-

phase complex response. One reason why we found fewer

inhibitory response cells might be that cerebellar LTD was

unlikely to occur by the unpaired presentation of the CS and

the US [30].

The ratio of excitatory response cells to all recorded cells

did not differ between the pair- and pseudoconditioned

groups. Therefore, the responses of Purkinje cells after

CS-US paired conditioning probably contain non-associa-

tive activities. In fact, the proportion of excitatory response

cells found in 50 CS-alone trials in naıve animals was

similar to that after pair- or pseudoconditioning. This is

reasonable because the information about the tone stimulus

reaches the Purkinje cells via parallel fiber as an excitatory

input. These results suggest that some of the excitatory

responses observed after the conditioning might already be

present in the naıve state. However, our results do not rule

out the possibility that the excitatory responses contain

plastic changes due to conditioning.

As mentioned above, the Purkinje cell responses to the

CS in the pair-conditioned subjects are likely to contain

non-associative responses like those observed in the pseu-

doconditioned subjects. It is difficult to distinguish an

associative response from a non-associative one, since the

distribution of the peaks of neural responses did not differ

between the pair-conditioned and the pseudoconditioned

groups, although the peak of the neural response was

weakly correlated with the peak of the CR in the excitatory

response cells of the pair-conditioned group. Our protocol


using an ISI shift enabled us to classify Purkinje cells into

two groups; one changed the type of its response to the CS

with the acquisition of new timing, while the other main-

tained the same type of response. In fact, one third of

recorded cells changed their response to the CS. In these

cells, changing response type after the CR timing shift is

likely to be a timing-related associative component. For

example, only one cell out of 12 excitatory response cells

changed to a complex response cell, and conversely, five

other response type cells came to elicit an excitatory

response after a change in the behavioral response. This

means that we succeeded in separating the learning-related

excitatory response cells from the non-associative response

cells by tracking a single cell activity during the learning of

new timing. This cannot be achieved by the simple obser-

vation of only one stage of the learning process. On the

other hand, the cells that responded to the CS but did not

show any change in response with an ISI shift could be

related to either a non-associative or an associative compo-

nent. The latter probably contributes to modulation of the

level of excitability of interpositus nucleus neurons during

the CS input, and might affect the expression of the CR [3].

To express the adaptive CR, the eyelid response must be

suppressed in the early phase during the CS period, but be

amplified in the late phase. This implies that Purkinje cells

must eventually exhibit an excitatory response in the early

phase and an inhibitory response in the late phase during

learning of the timing of the CR. Recent theoretical studies

based on the well-known cerebellar neural circuit have

proposed a mechanism for this schema [23,25]. However,

we found only a few such cells among our many recorded

cells in sufficiently conditioned decerebrate guinea pigs. In

previous studies, a small proportion of the cells with an

expected response pattern were found in decerebrate ferrets

[12], and cells with an inhibitory response timed to the CR

were found in intact rabbits [4,10,16,34]. Although these

cells seem to contribute to the well-timed CR, it is not clear

whether these responses depend on the ISI. According to the

above hypothesis, when the ISI is lengthened, the cells that

contribute to shaping the adaptive CR should show exten-

sion of the excitatory phase and a shift of the inhibitory

phase toward the new timing of the US, along with an

increase in CR latency. However, none of our cells showed

such simple behavior after the CR timing shift. In excitatory

responses, forward or backward shifts of the peak-latency

were observed after the CR timing shift. In addition, our

cross-correlation analysis revealed that almost none of the

excitatory response cells that showed a peak latency shift

were CR-related. Therefore, the individual shift of each cell

response might not be directly related to the shift in CR peak

latency. However, the possibility remains that the population

peak of the excitatory response of Purkinje cells shows a

shift that is consistent with the shift of the CR to suppress

the eyelid response in the early phase of the CS period.

Against the ideal simple shift, changes in the response

during learning of the new timing were rich in their variety.

Some of them changed the polarity of the response in

addition to the time course of the response. We are currently

unable to predict the topography of the adaptive CR from

the changes in response we observed in the timing-related

cells. On the other hand, the interpositus nucleus is known

to exhibit activities that correspond to the well-timed CR

after sufficient paired CS-US presentations [5,10,24]. These

suggest that the information about CS-US timing is not fully

converged at the Purkinje cell level, but is converged at the

interpositus level in order to express an adaptive CR. As

suggested in previous reports [10,16], the response pattern

in interpositus neurons is formed by the summation of

activities of many types of Purkinje cells, which have a

diversity of response types. In fact, each interpositus neuron

anatomically receives many Purkinje cell axons.

In the present study, about one third of the cells changed

their type of response with the learning of new timing, while

the other two thirds did not. Among the former type of cells,

some cells gained the response. This type of cell seems to

become to respond only when the animal learns a specific

ISI, and might correspond to the cell type assumed in the

cerebellar timing model of Ivry [15], in which modular units

for a specific ISI are hypothesized to be similar to orienta-

tion columns in the visual cortex. Since we examined only

two kinds of ISIs, it is not clear whether the behavior of

cells with no response during both paradigms is related to

CR timing or not. In addition to these single-ISI type cells,

there were some cells which changed their response from

one type to another during acquisition of new timing, e.g.

from an excitatory to a complex response, or the reverse. In

the process of learning a new ISI, these two types of cells

may have switched their roles in timing the CR. Moreover, a

change in the temporal pattern of the excitatory response

also was observed, in which the peak of this response

shifted either forward or backward in time. These results

indicate that there are a variety of possible changes in the

responses of Purkinje cells, as well as in the response itself;

they also suggest that a more complex mechanism than the

hypothesis proposed previously for the cerebellar cortex

may mediate the timing of the CR. To resolve the timing

mechanism in the cerebellum, future experiments using

simultaneous multi-electrodes recording from Purkinje cells

and interpositus nucleus neurons combined with the ISI-

shift will be useful.

Acknowledgements

This work was supported by grants from the Ministry of

Education, Science, Sports and Culture of Japan (#13210036

and #13680734), Core Research for Evolution Science and

Technology (CREST) of Japan Science and Technology

(JST), and Kato Memorial Bioscience Foundation. We are

grateful to Dr. E. Tabuchi, Dr. E. Hori and Dr. H. Nishijo, of

the Department of Physiology, Faculty of Medicine, at

Toyama Medical and Pharmaceutical University, for intro-


ducing us to the experimental procedure of single unit

recording.

References

[1] J.S. Albus, A theory of cerebellar function, Math. Biosci. 10 (1971)

25–61.

[2] P.J. Attwell, S. Rahman, C.H. Yeo, Acquisition of eyeblink conditio-

ning is critically dependent on normal function in cerebellar cortical

lobule HVI, J. Neurosci. 21 (2001) 5715–5722.

[3] S. Bao, L. Chen, J.J. Kim,R.F. Thompson,Cerebellar cortical inhibition

and classical eyeblink conditioning, Proc. Natl. Acad. Sci. U. S. A. 99

(2002) 1592–1597.

[4] N.E. Berthier, J.W. Moore, Cerebellar Purkinje cell activity related to

the classically conditioned nictitating membrane response, Exp. Brain

Res. 63 (1986) 341–350.

[5] N.E. Berthier, J.W. Moore, Activity of deep cerebellar nuclear cells

during classical conditioning of nictitating membrane extension in

rabbits, Exp. Brain Res. 83 (1990) 44–54.

[6] B.A. Christiansen, N.A. Schmajuk, Hippocampectomy disrupts the

topography of the rat eyeblink response during acquisition and ex-

tinction of classical conditioning, Brain Res. 595 (1992) 206–214.

[7] S.R. Coleman, I. Gormezano, Classical conditioning of the rabbit’s

(Oryctolagus cuniculus) nictitating membrane response under sym-

metrical CS-US interval shifts, J. Comp. Physiol. Psychol. 77 (1971)

447–455.

[8] H. Eichenbaum, H. Potter, J. Papsdorf, C.M. Butter, Effects of frontal

cortex lesions on differentiation and extinction of the classically con-

ditioned nictitating membrane response in rabbits, J. Comp. Physiol.

Psychol. 86 (1974) 179–186.

[9] K.S. Garcia, P.M. Steele, M.D. Mauk, Cerebellar cortex lesions pre-

vent acquisition of conditioned eyelid responses, J. Neurosci. 19

(1999) 10940–10947.

[10] T.J. Gould, J.E. Steinmetz, Changes in rabbit cerebellar cortical and

interpositus nucleus activity during acquisition, extinction, and back-

ward classical eyelid conditioning, Neurobiol. Learn. Mem. 65 (1996)

17–34.

[11] A. Gruart, C.H. Yeo, Cerebellar cortex and eyeblink conditioning:

bilateral regulation of conditioned responses, Exp. Brain Res. 104

(1995) 431–448.

[12] G. Hesslow, M. Ivarsson, Suppression of cerebellar Purkinje cells

during conditioned responses in ferrets, NeuroReport 5 (1994)

649–652.

[13] F.K. Hoehler, R.F. Thompson, Effect of the interstimulus (CS-UCS)

interval on hippocampal unit activity during classical conditioning of

the nictitating membrane response of the rabbit (Oryctolagus cunicu-

lus), J. Comp. Physiol. Psychol. 94 (1980) 201–215.

[14] M. Ito, The Cerebellum and Neural Control, Raven Press, New York,

1984.

[15] R.B. Ivry, The representation of temporal information in perception

and motor control, Curr. Opin. Neurobiol. 6 (1996) 851–857.

[16] D.B. Katz, J.E. Steinmetz, Single-unit evidence for eye-blink condi-

tioning in cerebellar cortex is altered, but not eliminated, by interpo-

situs nucleus lesions, Learn. Mem. 3 (1997) 88–104.

[17] S. Kotani, S. Kawahara, Y. Kirino, Classical eyeblink conditioning in

decerebrate guinea pigs, Eur. J. Neurosci. 15 (2002) 1267–1270.

[18] S. Kotani, S. Kawahara, Y. Kirino, Purkinje cell activities during

classical eyeblink conditioning with different CS-US intervals in de-

cerebrate guinea pigs, Neurosci. Res., Suppl. 26 (2002) S47.

[19] D.G. Lavond, J.E. Steinmetz, Acquisition of classical conditioning

without cerebellar cortex, Behav. Brain Res. 33 (1989) 113–164.

[20] D.G. Lavond, J.E. Steinmetz, M.H. Yokaitis, R.F. Thompson, Reac-

quisition of classical conditioning after removal of cerebellar cortex,

Exp. Brain Res. 67 (1987) 569–593.

[21] S.G. Lisberger, E.J. Morris, L. Tychsen, Visual motion processing and

sensory-motor integration for smooth pursuit eye movements, Annu.

Rev. Neurosci. 10 (1987) 97–129.

[22] D. Marr, A theory of cerebellar cortex, J. Physiol. (Lond.) 202 (1969)

437–470.

[23] M.D. Mauk, N.H. Donegan, A model of Pavlovian eyelid condition-

ing based on the synaptic organization of the cerebellum, Learn.

Mem. 3 (1997) 130–158.

[24] D.A. McCormick, R.F. Thompson, Cerebellum: essential involvement

in the classically conditioned eyelid response, Science 223 (1984)

296–299.

[25] J.F. Medina, K.S. Garcia, W.L. Nores, N.M. Taylor, M.D. Mauk,

Timing mechanisms in the cerebellum: testing predictions of a

large-scale computer simulation, J. Neurosci. 20 (2000) 5516–5525.

[26] S.P. Perrett, B.P. Ruiz, M.D. Mauk, Cerebellar cortex lesions disrupt

learning-dependent timing of conditioned eyelid responses, J. Neuro-

sci. 13 (1993) 1708–1718.

[27] J.L. Raymond, S.G. Lisberger, M.D. Mauk, The cerebellum: a neuro-

nal learning machine? Science 272 (1996) 1126–1131.

[28] L.W. Schmaltz, J. Theios, Acquisition and extinction of a classically

conditioned response in hippocampectomized rabbits (Oryctolagus

cuniculus), J. Comp. Physiol. Psychol. 79 (1972) 328–333.

[29] B.G. Schreurs, M.M. Oh, D.L. Alkon, Pairing-specific long-term de-

pression of Purkinje cell excitatory postsynaptic potentials results

from a classical conditioning procedure in the rabbit cerebellar slice,

J. Neurophysiol. 75 (1996) 1051–1060.

[30] B.G. Schreurs, P.A. Gusev, D. Tomsic, D.L. Alkon, T. Shi, Intracel-

lular correlates of acquisition and long-term memory of classical con-

ditioning in Purkinje cell dendrites in slices of rabbit cerebellar lobule

HVI, J. Neurosci. 18 (1998) 5498–5507.

[31] P.R. Solomon, E.R. Vander Schaaf, R.F. Thompson, D.J. Weisz, Hip-

pocampus and trace conditioning of the rabbit’s classically condi-

tioned nictitating membrane response, Behav. Neurosci. 100 (1986)

729–744.

[32] W.T. Thach, H.P. Goodkin, J.G. Keating, The cerebellum and the

adaptive coordination of movement, Annu. Rev. Neurosci. 15 (1992)

403–442.

[33] R.F. Thompson, S. Bao, L. Chen, B.D. Cipriano, J.S. Grethe, J.J.

Kim, J.K. Thompson, J.A. Tracy, M.S. Weninger, D.J. Krupa, Asso-

ciative learning, Int. Rev. Neurobiol. 41 (1997) 151–189.

[34] J.A. Tracy, J.E. Steinmetz, Purkinje cell responses to pontine stimu-

lation CS during rabbit eyeblink conditioning, Physiol. Behav. 65

(1998) 381–386.

[35] J.P. Welsh, C. Schwarz, Multielectrode recording from the cerebel-

lum, in: M.A.L. Nicolelis (Ed.), Methods for Neural Ensemble Re-

cordings, CRC Press, Boca Raton, FL, 1999, pp. 79–100.

[36] C.H. Yeo, M.J. Hardiman, M. Glickstein, Classical conditioning of

the nictitating membrane response of the rabbit: II. Lesions of the

cerebellar cortex, Exp. Brain Res. 60 (1985) 99–113.

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