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The effects of lesions of the cerebellum on the acquisition and retention of aversive Pavlovian conditioned bradycardia were examined in rabbits. Lesions of the anterior cerebellar vermis severely attenuated the acquisition of simple conditioned bradycardia without disrupting baseline heart rate (HR), or unconditioned HR responses. Also, lesions of the vermis performed after the acquisition of conditioned bradycardia eliminated evidence of prior conditioning. Bilateral lesions of the cerebellar hemispheres did not affect conditioned or unconditioned HR responses. These results were interpreted to indicate that anterior vermis lesions specifically disrupted part of an essential conditioned response pathway without interfering with the neural circuits that mediate unconditioned HR responding. These lesion data, coupled with recent electrophysiological evidence of learning-related changes in neuronal activity within the anterior vermis of the fear-conditioned rabbit, suggest that the anterior cerebellar vermis is critically involved in the acquisition and retention of this rapidly learned autonomic conditioned response.
The Journal of Neuroscience, September 1993, 13(g): 37053711
The Anterior Cerebellar Vermis: Essential Involvement in Classically
Conditioned Bradycardia in the Rabbit
William F. Supple,
Jr., and Bruce
Department of Psychology, The University of Vermont, Burlington, Vermont 05405
The effects of lesions of the cerebellum on the acquisition
and retention of aversive Pavlovian conditioned bradycardia
were examined in rabbits. Lesions of the anterior cerebellar
vermis severely attenuated the acquisition of simple con-
ditioned bradycardia without disrupting baseline heart rate
(HR), or unconditioned HR responses. Also, lesions of the
vermis performed after the acquisition of conditioned brady-
cardia eliminated evidence of prior conditioning. Bilateral
lesions of the cerebellar hemispheres did not affect condi-
tioned or unconditioned HR responses. These results were
interpreted to indicate that anterior vermis lesions specifi-
cally disrupted part of an essential conditioned response
pathway without interfering with the neural circuits that me-
diate unconditioned HR responding. These lesion data, cou-
pled with recent electrophysiological evidence of learning-
related changes in neuronal activity within the anterior
vermis of the fear-conditioned rabbit, suggest that the an-
terior cerebellar vermis is critically involved in the acquisition
and retention of this rapidly learned autonomic conditioned
[Key words: cerebellar vermis, conditioned bradycardia,
lesions, unconditioned response, cerebellar hemisphere,
learning and memory]
The cerebellum contributes to a variety of complex behavioral
processes in addition to its well-documented role in skeletal
motor regulation and coordination (Dow and Moruzzi, 1958).
The midline cerebellum, which includes the vermal cortex and
fastigial nuclei, has a functional contribution to a variety of
affective and fear-related behaviors (Berman et al., 1974; Bernt-
son and Torello, 1982; Supple et al., 1987). Decreased behav-
ioral reactivity followed lesions of the vermis in cats and mon-
keys (Peters and Monjan, 197 1; Berman et al., 1974), and several
fear-related behaviors are attenuated after vermis or fastigial
nucleus lesions in rats (Berntson and Torello, 1982; Supple et
al., 1987, 1988). Furthermore, stimulation of the midline cer-
ebellum evokes affective behavior that resembles fear-related
responses (Asdourian and Frerichs, 1970; Reis et al., 1973; Al-
bert et al., 1985) and also results in many of the autonomic
responses that accompany these affective states (Maimer, 1975).
For example, midline cerebellar stimulation resulted in dra-
Received Nov. 12, 1992; revised Jan. 29, 1993; accepted Mar. 11, 1993.
We acknowledge the technical assistance of Lauren Archer. This work was
supported by NIMH Grant MH09549 and NIMH FIRST MH47307-0 1 to W.F.S.
Correspondence should be addressed to William F. Supple, Department of
Psychology, John Dewey Hall, The University ofvermont, Burlington, VT 05405-
Copyright 0 1993 Society for Neuroscience 0270-6474/93/133705-07$05.00/O
matic changes in heart rate (HR) (Hockman et al., 1970; Hoffer
et al., 1972; Nisimaru and Watanabe, 1985) arterial blood pres-
sure (Miura and Reis, 1969; Chida et al., 1986) and respiration
rate (Snider, 1972).
This correspondence between the effects of midline cerebellar
lesions and stimulation on fear-related behaviors and autonom-
ic responses prompted an examination of the contributions of
the vermis to a classically conditioned fear-related autonomic
response. Large lesions of the vermis severely attenuated the
acquisition of conditioned bradycardia in the restrained rat
(Supple and Leaton, 1990a,b). Importantly, the lesions specif-
ically disrupted the conditioned response (CR) without altering
unconditioned HR (UCR) responses. Furthermore, within the
cerebellum the vermis seems particularly critical as bilateral
lesions ofthe cerebellar hemispheres did not disrupt conditioned
HR responding (Supple and Leaton, 1990b).
The present experiment examined the contributions of the
cerebellum to the acquisition and retention of conditioned bra-
dycardia in the rabbit, a species considered by many as an ideal
intact preparation for the study of neural mechanisms of leam-
ing and memory, and in particular, cardiovascular conditioning
(see Schneiderman et al., 1986; Pascoe et al., 199 1). This ex-
periment examined the effects of cerebellar lesions on condi-
tioned bradycardia using a simple, nondiscriminative condi-
tioning paradigm. HR was recorded during several different
phases of the conditioning procedure to determine the effects
of the lesions on unconditioned and conditioned HR changes.
HR was recorded during baseline periods and unreinforced tone
conditioned stimulus (CS) presentations to determine if lesions
altered resting HR, the HR orienting response (HR OR), or its
habituation. During acquisition training the unconditioned HR
response following the aversive unconditioned stimulus (UCS)
was also examined. Measurement of these unconditioned HR
responses during the various phases of training should indicate
if the lesions have disrupted unconditioned features of the HR
response that might complicate the interpretation of any ob-
served conditioning deficit.
Two distinct regions of the cerebellum were lesioned in sep-
arate groups. The anterior cerebellar vermis (ACV) was targeted
because it was included in the effective lesions in the rat (Supple
and Leaton, 1990a,b) and because subsequent electrophysio-
logical data in the rabbit indicated learning-related alterations
in neuronal activity within the anterior vermis (Supple and
Kapp, 1988). Another group with bilateral cerebellar hemi-
spheric lesions (HEM) was included to serve as a surgical control
for any general debilitating effects of cerebellar damage per se,
and because previous research (Lavond et al., 1984; Supple and
Leaton, 1990b) has shown similar lesions to be ineffectual on
3706 Supple and Kapp * Cerebellar Vermis and Conditioned Bradycardia
autonomic conditioning yet devastating on somatomotor CRs
like the nictitating membrane response in the rabbit (McCor-
mick and Thompson, 1984; Yeo et al., 1985).
Materials and Methods
Experiments were performed on 20 adult New Zealand White rabbits
(Oryctolagus cuniculus) weighing between 2.2 and 2.5 kg and obtained
from a local licensed supplier. Animals were maintained on a 12 hr: 12
hr light : dark cycle with food and water available ad libitum. Each rabbit
was handled extensively prior to the start of the experiment. Principles
for the care and use of laboratory animals as outlined by the U.S. Public
Health Service were strictly followed.
At least 2 weeks before the initiation of behavioral training, the animals
were surgically prepared. After application of topical lidocaine to the
region of the marginal ear vein, the rabbits were pretreated with chlor-
promazine HCl (20 mg in 0.8 ml of saline, iv.) and anesthetized with
sodium pentobarbital (60 m&kg, i.v.). The rabbits were mounted in a
Kopf stereotaxic instrument and the scalp was incised. One group (n =
5) received lesions of the vermis (VER). A section of skull beginning
approximately 1 .O mm posterior to lambda and extending 3.0 mm on
both sides of the midline and 5.0 mm in length was removed by drilling.
The anterior lobules of the vermis were aspirated under visual guidance
with the assistance of a dissecting microscope. A second group (n = 5)
received bilateral cerebellar hemispheric aspiration lesions (HEM), made
by removing bilateral sections of interparietal bone starting 3.0 mm
lateral to the midline and extending to the external suture, and extending
4.0 mm posterior to lambda. These lesions were intended to remove
the cortex of the anterior lobules of the cerebellar hemispheres without
extending into the midline vermis or underlying dentate-interpositus
nuclear complex. A third group (n = 10) served as an unoperated control
(UNOP). Each animal’s recovery was closely monitored until it regained
sternal position and then was returned to its home cage.
Experimental apparatus and procedure
This experiment consisted of three phases: orienting response habitu-
ation, acquisition, and retention.
Habituation. Each rabbit was habituated to restraint by placement in
a standard rabbit restrainer with adjustable head stock and backplate
for five daily 30 min sessions. During the last two of these sessions, the
rabbits were habituated to the sound-attenuating experimental chamber.
This chamber was equipped with a ventilating fan that provided broad-
range masking noise of -70 dB SPL and a speaker, and was located
within a larger electrically shielded, soundproof chamber. Recording
leads located within the chamber led to a Grass Instruments model 78
polygraph that recorded the heart signal as well as event markers de-
noting stimulus presentations. All recording and programming equip-
was located outside the larger soundproof chamber.
Orienting response habituation. On the next day following the last
restraint habituation session. each rabbit was fitted with ECG leads
consisting of stainless steel 26 gauge wire loops positioned subcutane-
ously on the right front shoulder and left flank. Stainless steel 26 gauge
wire loops for delivery of the UCS were inserted into the left pinna 5
mm medial to the marginal ear vein under local anesthesia (4.0% topical
Xylocaine). All recording leads were implanted 2 hr prior to each train-
ing session.
Approximately 10 min after placement into the chamber, each rabbit
received 20 unreinforced presentations of the tone stimulus (5.0 set,
1000 Hz, 90 dB SPL) presented on a 90 set variable interval (VI)
intertrial schedule. These tone-alone trials were given prior to acqui-
sition training to habituate the unconditioned HR OR that rabbits show
to novel auditory stimuli. Without habituation, any bradycardiac re-
sponse subsequently observed during training would be confounded
with conditioned bradycardiac responses.
Acuuisition training. Immediately after completion of habituation,
the first acquisition phase consisting of 40 paired trials commenced.
Each nresentation of the CS was followed at offset by a 1 .O mA, 60 Hz,
500 msec pinna shock UCS; trials were presented on a 90 set VI sched-
ule. The next day each animal received an additional identical 40 paired
training trials, generating a total of 80 acquisition trials across 2 d of
Retention. To assess the effects of VER lesions on retention of the
conditioned bradvcardiac resnonse. the UNOP groun
= 10) from the
acquisition phase was randomly split into two equal groups:‘a SHAM
group and a VER group. The SHAM group was anesthetized and the
scalp was incised and sutured, while the VER group received aspiration
lesions of the anterior vermis as described above. Five days later 20
CS-alone test (retention) trials were presented on a 90 set VI intertrial
interval to assess retention of conditioned bradycardia.
Histological procedures
At the conclusion of behavioral testing, the lesioned rabbits were given
a lethal dose of sodium pentobarbital (120 mg/kg, i.v.) and perfused
intracardially with 0.9% saline followed by 10% buffered neutral for-
malin. The brains were removed and stored in 10% formalin for at least
48 hr. Frozen 80 pm sections were taken throughout the extent of the
lesions. Every fourth section was mounted and stained with thionin.
The extent of the lesions was determined by camera lucida drawings.
Data analysis
HR responses were calculated by measuring the interbeat interval as
indexed by successive R-waves of the cardiac cycle. HR changes during
the CS (expressed in beats per minute) were computed by comparing
HR during the 5.0 set CS from that during the 5.0 set pre-CS baseline
period using a microcomputer system that measured the interbeat in-
tervals with millisecond resolution. Responses following the UCS were
computed by comparing HR during a 5.0 set period following UCS
offset with the pre-CS baseline HR. The initial 1.0 set block after CS
offset was not analyzed as shock artifact blocked amplification of the
heart signal. These responses were computed by measuring successive
R-R intervals in the aforementioned periods. These intervals were read
to the nearest 0.5 mm from the polygraph chart, which traced the heart
signal on a paper speed of 25 mm/set. To examine the topography of
the HR responses during the various phases of training, the following
procedures were used. The topography of the response to the tone or
CS was determined by comparing the mean pre-CS HR (in beats/min)
with each 1 set interval during the 5.0 set tone. The topography of the
response to pinna shock was determined by comparing the mean pre-
CS HR with each 1 set interval for 5.0 set commencing after the initial
1 .O set shock artifact period described above. Repeated-measures anal-
yses of variance (ANOVA) were performed on the beats per minute
values. Post hoc Neuman-Keuls pairwise comparisons between means
were performed where appropriate.
The typical postoperative recovery of the cerebellar-lesioned
rabbits was rapid and unremarkable. There were no persistant
postural or obvious motor impairments, other than several VER
animals showing a mild ataxia that dissipated in 1 or 2 d post-
Histological analysis
Serial reconstructions of representative VER and HEM lesions
are presented in Figure 1. Lesions of the vermis were restricted
to the anterior midline of the cerebellum primarily involving
the culmen (lobules IV, V, and VI according to the terminology
of Brodal, 1940) with some damage to the more anteriorly lo-
cated central lobule (lobule III). The underlying fastigial nuclei
were not invaded or damaged. Lesions of the cerebellar hemi-
spheres were bilaterally symmetrical, with damage confined to
the lateral ansiform and paramedian lobules (Crus 1). There was
no damage to either the interpositus or dentate nucleus. There
was no extracerebellar damage in either group. These two lesion
groups are essentially complementary in that there was no over-
lap of involved tissue between the VER and HEM groups.
Heart rate
Baseline heart rate. All three groups showed similar and con-
sistent patterns of baseline HR during acquisition training. The
The Journal of Neuroscience, September 1993, 13(9) 3707
mean baseline HRs for the UNOP, VER, and HEM groups were
232.6 (SD = 22.3) 230 (SD = 25.6) and 221.9 (SD = 17.7)
beats/min, respectively. There were no significant group differ-
ences in baseline HR assessed in five-trial blocks across the 2
d of acquisition training [F(2,17) = 0.78, p > 0.271, suggesting
no effect of these lesions on resting or baseline HR.
HR OR and habituation. The initial HR response to the tone
in all the animals consisted ofbradycardia that habituated across
repeated tone presentations. The magnitude and subsequent
habituation of this OR were similar in all groups. Figure 2A
presents each group’s mean HR response to the tone during this
phase of training. The initial response was a deceleration that
habituated over trial blocks [F(3,51) = 45.17, p < 0.011. The
main effect of groups was not significant [F(2,17) = 0.491, nor
was the groups x blocks interaction [F(6,5 1) = 1.22, p > 0.321.
No group differences emerged when the topography of the HR
OR during the 5 set tone was examined (F values < 1). These
results indicate that VER or HEM lesions did not disrupt the
initial magnitude, topography, or habituation characteristics of
the bradycardiac HR OR.
Acquisition of conditioned bradycardia. Figure 2B presents the
data for each group across the 2 d of acquisition training. In-
spection of Figure 2B shows that both the UNOP and HEM
groups developed robust bradycardiac responses during the fear
conditioning procedure. In contrast, lesions of the ACV severely
impaired the acquisition of conditioned bradycardia. This pat-
tern of responding resulted in a significant main effect of groups
[F(2,17) = 8.47, p < 0.011, trial blocks [F(7,119) = 7.59, p <
Figure I. Serial reconstructions
showing representative examples of the
extent of damage
(darkly shaded area)
following cerebellar vermis and bilat-
era1 cerebellar hemispheric lesions. Note
that these two lesion groups are essen-
tially complementary with no overlap
of involved cerebellar tissue between
the two groups.
0.0 11, and a groups x trial blocks interaction [F( 14,119) = 4.32,
p < 0.011, on the first day of acquisition training. This pattern
continued on the second day of training as reflected by a sig-
nificant main effect for groups [F(2,17) = 11.23, p < 0.011.
Separate Newman-Keuls comparisons showed that the VER
group demonstrated significantly less bradycardia during the CS
compared to either the UNOP or HEM group on both days (all
p values < 0.05).
The topography of the HR response during the CS was also
examined. Figure 3 presents the second-by-second topography
of the HR response of each group collapsed across trials and
days. The response of both the UNOP and HEM group was
markedly different than that ofthe VER group. An overall ANO-
VA found a significant groups x CS periods interaction [F(8,136)
= 3.79, p < 0.051. This interaction reflected the development
of greater bradycardia during the latter seconds of the CS for
the UNOP and HEM groups, while the VER group demonstrat-
ed minimal bradycardiac responses during the CS. This finding
illustrates that the VER group, in addition to showing minimal
bradycardia during the CS, also showed little evidence of a
within-CS pattern that resembled that of the two other groups.
Retention of conditioned bradycardia. To assess the contri-
bution of the anterior vermis to the retention of conditioned
bradycardia, the UNOP control group from the acquisition phase
was randomly divided into two groups. Half received VER le-
sions; half served as surgical controls (SHAM operations). Fig-
ure 2C shows the effects of lesions of the anterior vermis per-
formed after the acquisition of conditioned bradycardia
3708 Supple and Kapp - Cerebellar Vermis and Conditioned Bradycardia
6. C.
Day 1 Day 2
I I , I
1 2 3 4
Figure 2.
The effects of cerebellar VER and HEM lesions on the HR OR, acquisition of the HR CR, and retention of the CR.
HR OR. Shown
is the mean HR change in beats/min during the 5.0 set tone period compared to the 5.0 set baseline period in blocks of five trials. Deceleration
is indicated by negative numbers along the vertical axis. Note that the initial response to the tone was bradycardiac that habituated across trials.
Cerebellar lesions had no effect on this response. B, Acquisition of conditioned bradycardia. UNOP (Control) and HEM rabbits acquired conditioned
bradycardia while the VER group did not across the 2 d of acquisition training. C, Retention of conditioned bradycardia. The control group from
the acquisition phase was split into two groups, one group received VER lesions the other SHAM lesions. After a 5 d recovery period, the retention
of conditioned bradycardia was assessed under extinction conditions. Initially, the SHAM animals demonstrated bradycardia to the CS that
subsequently extinguished. The VER group did not show bradycardia, suggesting that the lesion eliminated evidence of prior conditioning.
compared to SHAM-operated controls. The SHAM group
showed an initial bradycardia during the CS-alone retention
trials that subsequently extinguished. These data suggest that
intact rabbits retain conditioned bradycardia across this time
interval and that surgery alone did not disrupt conditioned bra-
dycardia established during the acquisition phase of training. In
contrast, lesions of the vermis severely reduced evidence of prior
conditioned responding and attenuated the magnitude of con-
ditioned bradycardia to a level observed in rabbits with lesions
performed prior to acquisition training (compare VER group,
Fig. 2B, with VER group, Fig. 2C). This pattern of responding
generated significant main effects for groups [F(1,8) = 9.86, p
< 0.051, trial blocks [F(3,24) = 6.97, p < 0.051, and a groups
x trial blocks interaction [F(3,24) = 3.71, p < 0.051, for the
retention phase of training.
Unconditioned HR response to pinna UCS. The HR UCR
consisted of a monophasic tachycardiac response. The mean
tachycardiac response of each group in beats/min was UNOP
9.33 (SD = 16.45), 13.78 (SD = 13.24); HEM 13.49 (SD =
12.79) 14.87 (SD = 14.22); and VER 13.88 (SD = 19.34) 16.23
(SD = 18.20) for days 1 and 2 of acquisition training, respec-
tively. Importantly, neither the magnitude of the UCR nor the
pattern over acquisition trials was affected by these cerebellar
lesions. Analysis of blocks of five acquisition trials across both
days of training resulted in no significant main effects for groups
[F(2,17) = 0.42, p > 0.651, trial blocks [F( 15,255) = 1.294, p
> 0.201, or groups x trials blocks interaction [F(30,255) = 0.47,
p > 0.801. Also, examination ofthe topographical characteristics
of the HR response following the pinna shock revealed a similar
pattern of responding between the three groups. As shown in
Figure 4, the second-by-second pattern consisted of a mono-
phasic tachycardia peaking at the second measurement interval.
An overall ANOVA of these data revealed a significant main
effect for measurement periods [F(4,316) = 3.87, p < 0.051,
reflecting the change across measurement periods; however, no
main effects or interactions involving groups were significant
(all p values > 0.23). Overall, these results indicated that VER
lesions, which disrupted conditioned HR responses, did not
disrupt unconditioned HR responses to the pinna shock UCS
used in conditioning.
This study demonstrated that lesions of the ACV severely dis-
rupted the acquisition and retention of simple Pavlovian fear-
conditioned bradycardiac responses in the rabbit. The aversive
HR conditioning paradigm is a useful model system for the
analysis of the neural substrates of Pavlovian fear conditioning
because it allows for the assessment of unconditioned HR re-
sponsiveness after brain lesions. An advantage of studying a
response system like conditioned bradycardia is that it is rela-
tively simple to detect manipulation-induced sensory or motor
impairments that could interfere with the detection of the stim-
uli used in training or interruption of the efferent pathway for
the CR (e.g., Schneiderman et al., 1986; Pascoe et al., 1991).
The unconditioned HR responses of rabbits with ACV lesions
were not disrupted. Baseline or resting HR was not altered, nor
was the magnitude, topography, or habituation characteristics
of the HR OR to the unreinforced tone presentations. These
data suggest that the observed conditioning deficit following
ACV lesions was probably not secondary to brain lesion-in-
duced auditory impairments or to a disruption of the rabbit’s
ability to demonstrate stimulus-evoked bradycardia. The bra-
The Journal of Neuroscience, September 1993, 73(9) 3709
5: -VW
j !
-30 ,
2 3 4 5
Figure 3. Topography of the conditioned HR response. Shown is the
mean change in HR (beatsimin) during each 1 .O set period of the 5.0
set CS collapsed across days and trials for the UNOP, HEM, and VER
groups. Note the difference in the topography of the response of the
VER group compared to the UNOP and HEM groups. The HEM and
UNOP groups demonstrated progressively greater bradycardia through
the CS period, while the VER group showed minimal bradycardia during
all periods of the CS.
dycardiac responses to the novel auditory stimulus indicated
that the animal heard the stimulus and could unconditionally
decelerate the heart. ACV lesions also did not disrupt the mag-
nitude or topographical features of the unconditioned tachy-
cardiac responses to the pinna shock UCS. The results indicating
intact UCRs following vermis lesions in rabbits are consistent
with previous data in rats (Peters et al., 1973; Supple and Leaton,
1990a,b). Overall, these results suggest that the ACV lesions did
not produce nonspecific primary sensory or cardiomotor im-
pairments that could interfere with the development of the con-
ditioned bradycardiac response. Rabbits with ACV lesions can
hear the tone CS, respond similarly to the shock UCS, and
unconditionally decelerate the heart. Given the selective elim-
ination of conditioned but not unconditioned HR responses,
these lesion results are consistent with the hypothesis that the
ACV is a component of a neural circuit importantly involved
in classically conditioned bradycardiac responses in the rabbit.
Rabbits with ACV lesions showed minimal CRs during both
acquisition and retention. This minimal response consisted of
a 3-4 beats/min bradycardia, which was in contrast to the typical
18-20 beats/min bradycardia shown by the UNOP and HEM
groups. This minimal bradycardia in the VER group was very
similar in magnitude to that shown by explicitly unpaired or
pseudoconditioned control groups from previous studies con-
ducted in this laboratory and others (e.g., Gallagher et al., 198 1;
Jarrell et al., 1986). In addition to the greatly reduced overall
conditioned bradycardia was the relative lack of
topographical features characteristic of conditioned responding
for this autonomic response in the VER group. In summary,
ACV lesions severely attenuated the overall magnitude of the
CR and blocked the appearance of the topographical features
of the CR that were present in the control animals.
The demonstrated role of the cerebellum in motor coordi-
nation and regulation raises the possibility that a subtle motor
impairment could somehow be responsible
for the observed
autonomic conditioning deficit. The lesion results from the sur-
gical control group (HEM) provided evidence against this pos-
20 -
--o- H&4
4- *VW
0 I I
0 1 2 3 4 5
Figure 4. Topography of the HR response following application of the
pinna shock UCS during acquisition training. Shown is the mean change
in HR (beats/min) during each 1 .O set period following the offset of the
UCS collapsed across days and trials. Note the monophasic acceleration
in HR after the UCS, and the similarity in response profiles among the
three groups.
sibility as HEM lesions did not disrupt conditioned bradycardia
despite any subtle, undetectable motor disruptions that may
have resulted from these cerebellar lesions. Therefore, these
that cerebellar damage per se does not disrupt
conditioned bradycardia and generate support regarding the an-
atomical specificity for the effect consistent with the hypothesis
that the ACV is importantly involved in this learned autonomic
The present results implicating the cerebellar vermis in HR
conditioning are not without precedent, and are consistent with
those obtained with rats (Supple, 1986; Supple and Leaton,
1990a,b). Large lesions of the vermis blocked the acquisition of
simple and discriminatively conditioned HR responses without
disrupting unconditioned HR responses in rats. Furthermore,
bilateral cerebellar hemispheric lesions also did not affect
CRs, which is consistent with the data in the rabbit. The present
results extend those previous data on the following points: (1)
replicating the effect ofvermis lesions on HR CRs in the rabbit-
a species with a wealth of accumulated data regarding the neural
substrates of classical conditioning (e.g., Schneiderman et al.,
1986) (2) providing partial anatomical localization for the be-
havioral effect to the ACV, and (3) demonstrating that the ACV
is importantly involved in retention of the HR CR as well as
acquisition. Regarding the last point, it is notable that ACV
lesions blocked the retention of the bradycardiac CR. Disruption
of a previously acquired response following brain damage, cou-
pled with the selective abolition of CRs but not UCRs in ac-
quisition training, may be convincing behavioral evidence that
the ACV is critically important for this CR. Furthermore, single-
unit electrophysiological recordings of Purkinje cell activity
during retention trials allowed us to identify several distinct
subpopulations of ACV Purkinje cells that show short-latency
differential responses to tone stimuli trained as CS+ and CS-
(Supple and Kapp, 1988). Moreover, a group of Purkinje cells
showed significant correlations between discharges during the
CS+ and the magnitude of the concomitant conditioned bra-
dycardiac response (Supple and Kapp, 1988), suggesting possible
ACV mediation of the amplitude-time course of this autonomic
3710 Supple and Kapp + Cerebellar Vermis and Conditioned Bradycardia
CR. These electrophysiological results, along with the lesion
data from the present study, provide compelling evidence in
support of the hypothesis that the ACV has an important func-
tional contribution to the acquisition and expression of the clas-
sically conditioned bradycardiac response in the rabbit.
A related question concerns the location of the ACV in a CR
circuit for conditioned bradycardia; is it located on the sensory
or motor side of a CR circuit, efferent to a site of essential
plasticity or a candidate site of stimulus convergence-a possible
site of primary neural plasticity? Although answers to these
questions cannot be derived from lesion experiments alone,
there are findings from the present study that may be useful
when considering these issues. In light of the finding of intact
UCRs following ACV lesions in the present study, it is probable
that the ACV is not part of a primary or fundamental response
system for the auditory CS or pinna shock UCS. However, there
are several reasons to consider the ACV as a site for stimulus
convergence in this response system. First, the ACV receives
abundant sensory input (e.g., Fadiga and Pupilli, 1964; Aitkin
and Boyd, 1975; Ito, 1984) and has extensive output to both
brainstem and forebrain regions involved in cardiovascular reg-
ulation (e.g., Ito, 1984; Supple and Kapp, 1993). Second, the
cerebellar cortex has well-defined and orderly sensory input and
motor output pathways. The two major types of input to the
cortex via mossy and climbing fibers may provide the anatom-
ical substrate for stimulus convergence necessary for some forms
of associative learning. This anatomical parcellation has sug-
gested to others that the cerebellum may be a promising site for
sensorimotor learning (e.g., Marr, 1969; Gilbert, 1975), and has
recently been extensively studied in the context of the contri-
butions of the lateral cerebellar hemisphere to aversively con-
ditioned nictitating membrane responses in the rabbit (Thomp-
son, 1986; Steinmetz and Sengelaub, 1992). Finally, we have
obtained electrophysiological evidence of convergence onto sin-
gle ACV Purkinje cells ofthe two exact stimulus modalities used
to condition HR in our preparation. Single-unit recordings of
Purkinje cells showed short-latency UCRs to the prospective
CSs prior to training, and these responses subsequently habit-
uated over repeated presentations of the tones. Also, some Pur-
kinje cells demonstrated short-latency UCRs to brief presen-
tations of pinna shock stimulation prior to training. Studies in
progress are examining the relationships between these neuronal
responses and the conditioned bradycardiac response in the rab-
The present results, and a number of previous observations,
are consistent with the hypothesis that the ACV is part of a
more extensive neuroanatomical system involved in the acqui-
sition and expression of conditioned bradycardia and possibly
other fear-conditioned autonomic responses as well (discussed
at length in Supple and Leaton, 1990b). This suggestion is con-
sistent with the growing appreciation of the neuroanatomical
overlap the ACV shares with other forebrain regions impor-
tantly involved in conditioned bradycardia, most notably the
amygdaloid central nucleus (ACE). Converging lines of evidence
implicate the ACE in conditioned bradycardia in the rabbit.
Lesions or pharmacological manipulations of the ACE severely
attenuated the acquisition of conditioned bradycardia without
disrupting HR UCRs (Kapp et al., 1982). Recordings from ACE
neurons revealed the presence of associative responses to a fear-
conditioned CS (Applegate et al., 1982; Pascoe and Kapp, 1985).
The ACE has extensive anatomical connections with cardioreg-
ulatory nuclei, thereby suggesting pathways through which al-
tered ACE neuronal activity could affect HR (Schwaber et al.,
1980). In addition to involvement in this autonomic CR, the
ACE also importantly contributes to some somatomotor re-
sponses that are acquired as a result of fear conditioning pro-
cedures. Lesions of the ACE severely impair conditioned freez-
ing (Iwata et al., 1986) and potentiated acoustic startle responses
(Hitchcock and Davis, 1986), thus suggesting a wider role of
the ACE in the generation of a conditioned central state of fear.
It is interesting that experimental manipulations of two seem-
ingly disparate neuroanatomical systems, the ACV and ACE,
result in strikingly similar effects on conditioned bradycardia in
the rabbit. These similarities suggest the possibility that these
two regions are components of a common, more extensive neu-
roanatomical system involved in this response. Recent anatom-
ical observations lend support to this hypothesis. Both the ACE
and ACV receive afferents from common sources, the pontine
parabrachial nucleus (Dietrichs, 1985) and the lateral hypo-
thalamus (Dietrichs and Haines, 1986, 1989), both of which
have been implicated in cardiovascular function. Furthermore,
we have recently demonstrated that single-pulse electrical mi-
crostimulation of either the lateral parabrachial nucleus or the
lateral hypothalamus modulates Purkinje cell discharges in the
rabbit ACV (Supple and Kapp, 1993; Supple, in press), sug-
gesting the possibility that these structures may serve as an
interface between the ACV and other brain regions importantly
involved in conditioned bradycardia. Further experiments will
be required to examine the nature of the information relayed
by these afferents into the ACV and their functional relevance
to conditioned bradycardia, and the specific contributions of
the ACV within the context of this larger neuroanatomical sys-
tem to aversively conditioned bradycardiac responses.
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... Depending on stimulus intensity, participants may withdraw their hand or at least prepare a hand movement. In fact, early animal, but also human cerebellar lesion studies highlight the involvement of the cerebellar vermis in the conditioning of autonomic fear responses (Apps and Strata, 2015;Apps et al., 2018 for reviews;Sacchetti et al., 2002;Supple and Leaton, 1990a;Supple and Kapp, 1993). For example, Maschke et al. (2002) found that fear-conditioned bradycardia was impaired in patients with lesions of the cerebellar midline but not the lateral cerebellar hemispheres. ...
... This allows performance of event-related fMRI studies, which can separate between CSrelated activation, US-related activation, and activation related to the omission of the US. Both animal (Lavond et al., 1984;Sacchetti et al., 2002;Supple and Leaton, 1990a;Supple and Leaton, 1990b;Supple and Kapp, 1993) and human studies have shown that the cerebellar vermis is involved in fear conditioning -despite these long CS-US time intervals. The contribution of the cerebellum to discrete somatic motor behavior (such as eyeblink responses) may be different compared to slowly reacting autonomic responses. ...
Full-text available
Prediction errors are thought to drive associative fear learning. Surprisingly little is known about the possible contribution of the cerebellum. To address this question, healthy participants underwent a differential fear conditioning paradigm during 7T magnetic resonance imaging. An event-related design allowed us to separate cerebellar fMRI signals related to the visual conditioned stimulus (CS) from signals related to the subsequent unconditioned stimulus (US; an aversive electric shock). We found significant activation of cerebellar lobules Crus I and VI bilaterally related to the CS+ compared to the CS-. Most importantly, significant activation of lobules Crus I and VI was also present during the unexpected omission of the US in unreinforced CS+ acquisition trials. This activation disappeared during extinction when US omission became expected. These findings provide evidence that the cerebellum has to be added to the neural network processing predictions and prediction errors in the emotional domain.
... Growing evidence indicates that the cerebellum has non-motor functions and is critical for associative fear conditioning, in particular the consolidation of fear memory [11][12][13] . Fear conditioning enhances excitatory postsynaptic responses at the parallel fiber-Purkinje cell synapse and presynaptic GABA release from molecular layer interneurons (MLIs, basket and stellate cells), as well as feed-forward inhibitory connectivity in the cerebellum [14][15][16] . ...
... These approaches allowed us to disrupt consolidation without affecting learning and so avoided the complications that can be present when using knockout mice 56 . Furthermore, inactivation of the cerebellar vermis after memory acquisition disrupts fear memories, assessed by conditioned freezing, bradycardia, and inhibitory avoidance tasks in animals and humans 12,13,15,[57][58][59][60][61][62] . Therefore, the cerebellum is also critical for the consolidation of associative fear memory. ...
Full-text available
Endocannabinoids retrogradely regulate synaptic transmission and their abundance is controlled by the fine balance between endocannabinoid synthesis and degradation. While the common assumption is that “on-demand” release determines endocannabinoid signaling, their rapid degradation is expected to control the temporal profile of endocannabinoid action and may impact neuronal signaling. Here we show that memory formation through fear conditioning selectively accelerates the degradation of endocannabinoids in the cerebellum. Learning induced a lasting increase in GABA release and this was responsible for driving the change in endocannabinoid degradation. Conversely, Gq-DREADD activation of cerebellar Purkinje cells enhanced endocannabinoid signaling and impaired memory consolidation. Our findings identify a previously unappreciated reciprocal interaction between GABA and the endocannabinoid system in which GABA signaling accelerates endocannabinoid degradation, and triggers a form of learning-induced metaplasticity.
... Motor learning, including both adaptation of existing movement sequences, such as the vestibuloocular reflex (VOR) (Robinson, 1976) (Ito, 1982b), and acquisition of novel ones, such as the conditioned eyeblink (Lincoln et al., 1982), has been shown to rely upon an intact cerebellar cortex. The cortex has additionally been found to be important in non-motor forms of associative learning such as autonomic conditioning (Supple, Jr. and Leaton, 1990) (Supple, Jr. and Kapp, 1993). Thus it is presently unclear whether the cortex is primarily motor in function, as has often been suggested, or is really a more general form of associative module within the brain. ...
The cerebellar cortex is known to be central to motor learning. However, the question of whether it is a motor memory store or just a crucial element of the circuitry involved in encoding such memory has yet to be resolved. It is also not clear how the deep nuclei, the other major cerebellar component, function in motor learning. For the last quarter of a century much of the research into these problems has used eyeblink conditioning as a model to investigate these problems. Most recently, in an attempt to identify the site or sites of memory encoding and storage, pharmacological intervention during this classical conditioning procedure has been used to reversibly inactivate restricted elements of the cerebellum and associated brain structures. The delivery of such inactivating infusions, and the means by which their spread is assessed, are becoming increasingly accurate. Reversible inactivation has enabled the development of a progressively more detailed picture of the circuitry essential for eyeblink conditioning. Such studies have not, however, conclusively revealed the site, time-course or mechanism of memory storage. It has recently become clear that crucial elements of the conditioned eyeblink circuitry involve a dynamically connected cerebellar/brainstem loop. This feature of eyeblink circuitry means that reversible inactivation of one component may have consequences for activity in the other components. Conclusive identification of a locus of memory storage has therefore been impossible. This thesis details the results of a series of experiments that address this problem by limiting experimental manipulations to the processes commonly known as consolidation, and thereby the mechanism by which memories are stored long-term, without interfering with the initial process of memory encoding.
... stimulation of the cerebellar vermis elicits a repertoire of animal behavior, such as sham-rage, predatory attack, and freezing-like behavior (Asdourian and Frerichs, 1970;Reis et al., 1973;Zanchetti and Zoccolini, 1954). In conjunction with the CEA (see Section 2), conditioned heart rate responding is also regulated by the medial cerebellum (Supple and Kapp, 1993), likely due to synaptic plasticity in cerebellar Purkinje cells (Kotajima-Murakami et al., 2016;Yoshida and Kondo, 2012). In eyeblink conditioned rats, Supple and Leaton (1990) reported a double dissociation of cerebellar function-lesions of the cerebellar hemispheres impaired EBC acquisition while vermis lesions disrupted conditioned bradycardia and tachycardia, without affecting the baseline heart rate. ...
... Others have reported in both mice and rats that cerebellum also participates in fear learning and memory 30,31 . Besides that, the cerebellum also plays an important role in the classical fear conditioning in both mammals and non -mammals [32][33][34][35] . In another study, it has been observed that disruption of reticular-limbic central auditory pathway resulted in an impairment of noise-cued fear conditioning 36 . ...
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Long-term operations carried out at high altitude (HA) by military personnel, pilots, and astronauts may trigger health complications. In particular, chronic exposure to high altitude (CEHA) has been associated with deficits in cognitive function. In this study, we found that mice exposed to chronic HA (5000 m for 12 weeks) exhibited deficits in learning and memory associated with hippocampal function and were linked with changes in the expression of synaptic proteins across various regions of the brain. Specifically, we found decreased levels of synaptophysin (SYP) (p < 0.05) and spinophilin (SPH) (p < 0.05) in the olfactory cortex, post synaptic density−95 (PSD-95) (p < 0.05), growth associated protein 43 (GAP43) (p < 0.05), glial fibrillary acidic protein (GFAP) (p < 0.05) in the cerebellum, and SYP (p < 0.05) and PSD-95 (p < 0.05) in the brainstem. Ultrastructural analyses of synaptic density and morphology in the hippocampus did not reveal any differences in CEHA mice compared to SL mice. Our data are novel and suggest that CEHA exposure leads to cognitive impairment in conjunction with neuroanatomically-based molecular changes in synaptic protein levels and astroglial cell marker in a region specific manner. We hypothesize that these new findings are part of highly complex molecular and neuroplasticity mechanisms underlying neuroadaptation response that occurs in brains when chronically exposed to HA.
... The cerebellum is the principal regulator of motor coordination, timing, and adaptation (Ito, 2008;De Zeeuw and Ten Brinke, 2015;Boyden et al., 2004;Inoshita and Hirano, 2018). The vestibular cerebellum had been linked to eye movements and reflections, whereas the anterior vermis of the cerebellum had been thought to relate to autonomic nervous system (Reis et al., 1973;Supple and Kapp, 1993). Although our acute inflammation model did not show severe motor deficits ( Figures S5A and S5B), 20-mM apamin-injected rats showed ataxia (4 rats of 5 tested animals, data not shown), suggesting the inflammatory effects do not achieve full suppression of SK channels in the injected region during inflammation. ...
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Cerebellar dysfunction relates to various psychiatric disorders, including autism spectrum and depressive disorders. However, the physiological aspect is less advanced. Here, we investigate the immune-triggered hyperexcitability in the cerebellum on a wider scope. Activated microglia via exposure to bacterial endotoxin lipopolysaccharide or heat-killed Gram-negative bacteria induce a potentiation of the intrinsic excitability in Purkinje neurons, which is suppressed by microglia-activity inhibitor and microglia depletion. An inflammatory cytokine, tumor necrosis factor alpha (TNF-α), released from microglia via toll-like receptor 4, triggers this plasticity. Our two-photon FRET ATP imaging shows an increase in ATP concentration following endotoxin exposure. Both TNF-α and ATP secretion facilitate synaptic transmission. Region-specific inflammation in the cerebellum in vivo shows depression- and autistic-like behaviors. Furthermore, both TNF-α inhibition and microglia depletion revert such behavioral abnormality. Resting-state functional MRI reveals overconnectivity between the inflamed cerebellum and the prefrontal neocortical regions. Thus, immune activity in the cerebellum induces neuronal hyperexcitability and disruption of psychomotor behaviors in animals.
... Moreover, structures that were not even considered in early models of conditioned fear, namely, the zona incerta as well as the cerebellum and its brainstem targets, are also critical for the formation and consolidation of conditioned fear (Strata et al., 2011;Supple and Kapp, 1993). Also unexpected, the recall of conditioned fear shows a changing dependence on different brain structures depending on time since conditioning. ...
The neural basis of defensive behaviors continues to attract much interest, not only because they are important for survival but also because their dysregulation may be at the origin of anxiety disorders. Recently, a dominant approach in the field has been the optogenetic manipulation of specific circuits or cell types within these circuits to dissect their role in different defensive behaviors. While the usefulness of optogenetics is unquestionable, we argue that this method, as currently applied, fosters an atomistic conceptualization of defensive behaviors, which hinders progress in understanding the integrated responses of nervous systems to threats. Instead, we advocate for a holistic approach to the problem, including observational study of natural behaviors and their neuronal correlates at multiple sites, coupled to the use of optogenetics, not to globally turn on or off neurons of interest, but to manipulate specific activity patterns hypothesized to regulate defensive behaviors.
Fear is an important emotion for survival, and the cerebellum has been found to contribute not only to innate affective and defensive behavior, but also to learned fear responses. Acquisition and retention of fear conditioned bradycardia and freezing have been shown to depend on the integrity of the cerebellar vermis in rodents. There is a considerable number of brain imaging studies, which observe activation of the human cerebellum in fear conditioning paradigms. Different to what one may expect based on the initial cerebellar lesion studies, activations related to the learned prediction of threat go well beyond the vermis, and are most prominent in the lateral cerebellum. Different parts of the cerebellum likely contribute to learning of autonomic, motor, emotional and cognitive responses involved in classical fear conditioning. The neural operation which is performed in the various parts of the cerebellum is frequently assumed to be the same. One hypothesis is that the cerebellum acts as, or is part of, a predictive device. More recent findings will be discussed that the cerebellum may not only be involved in the processing of sensory prediction errors, but also in the processing of reward and reward prediction errors, which may play a central role in emotions and emotional learning. Current knowledge about the intrinsic learning mechanisms underlying fear memory in the cerebellum, and its connections with subcortical and cortical fear circuitry will be presented. The chapter will conclude with a discussion on how disordered cerebellar fear learning may contribute to affective disorders.
When animals repeatedly receive a combination of neutral conditional stimulus (CS) and aversive unconditional stimulus (US), they learn the relationship between CS and US, and show conditioned fear responses after CS. They show passive responses such as freezing or panic movements (classical or Pavlovian fear conditioning), or active behavioral responses to avoid aversive stimuli (active avoidance). Previous studies suggested the roles of the cerebellum in classical fear conditioning but it remains elusive whether the cerebellum is involved in active avoidance conditioning. In this study, we analyzed the roles of cerebellar neural circuits during active avoidance in adult zebrafish. When pairs of CS (light) and US (electric shock) were administered to wild-type zebrafish, about half of them displayed active avoidance. The expression of botulinum toxin, which inhibits the release of neurotransmitters, in cerebellar granule cells (GCs) or Purkinje cells (PCs) did not affect conditioning-independent swimming behaviors, but did inhibit active avoidance conditioning. Nitroreductase (NTR)-mediated ablation of PCs in adult zebrafish also impaired active avoidance. Furthermore, the inhibited transmission of GCs or PCs resulted in reduced fear-conditioned Pavlovian fear responses. Our findings suggest that the zebrafish cerebellum plays an active role in active avoidance conditioning.
The aim of the present study was to ascertain possible roles for the posterior cerebellar vermis in cardiovascular control using the anaesthetised, paralysed and artificially ventilated rabbit. The cortex of lobule IXb was stimulated both electrically and chemically and the effects of removal of lobules VI, VII and IX on the cardiorespiratory responses evoked from defensive behaviour related structures were observed. Arterial blood pressure, heart rate, femoral and renal vascular blood flow, and phrenic and renal nerve activities were routinely measured. Removal of lobule IX resulted in an increase in the sensitivity of the baroreceptor reflex response to a pressor challenge induced by intraluminal balloon inflations in the descending aorta. The increase in baroreflex gain was still evident when the experiments were carried out under β1-receptor blockade, the cell bodies in only lobule IXb were lesioned and whether the gain was calculated using R-R intervals derived from the heart rate or absolute R-R intervals. Stimulation of the HDA or PAG and ACe results in cardiorespiratory responses that are synonymous with those which occur in "fight or flight" and "playing dead" behaviours, respectively. Removal of lobule IX, but not lobules VI and VII, resulted in attenuated HDA, PAG and ACe evoked cardiovascular responses. On the other hand, simultaneous stimulation of lobule IXb with either of these structures resulted in facilitated "cardiovascular defence responses". Indeed, chemical activation of neurons in the HDA, PAG, ACe and lobule IXb identified the structure related nature of these cerebellar-midbrain/forebrain interactions. The cardiovascular effects elicited from the HDA or ACe and lobule IXb were vastly attenuated when cell bodies in the ipsilateral lateral parabrachial nucleus (LPBN) were lesioned with the excitotoxin kainic acid. Neurons in lobule IX demonstrated their ability to receive baroreceptor and hypothalamic inputs upon single or paired-pulse stimulation of the ipsilateral aortic nerve and hypothalamic defence area (HDA). A possible role for lobule IX of the posterior vermis in cardiovascular control is discussed in relation to published physiological and neuroanatomical studies and the results gained in the present study.
To achieve a more complete understanding of the neural substrates of learning and memory, several goals must be attained. Clearly, identifying critical brain areas that participate in the acquisition, storage, and retrieval of information would be necessary. Having identified these areas, it would then be necessary to determine the exact manner in which each contributes to learning and memory processes. This chapter reviews selected examples of the current research efforts of neuroscientists who use vertebrate models to investigate the neural substrates of associative learning and memory in the intact, behaving animal. Our discussion emphasizes research that incorporates the integrated use of a variety of methods and research strategies and begins with a discussion of the desirable characteristics of a vertebrate model with which to study the neural substrates of learning and memory. The chapter focuses primarily on a discussion of nonspecific CRs, although two models featuring specific CRs are described in a later section. A discussion of interactions between specific and nonspecific CRs is included at the end of the chapter.
Although the relationship between limbic system structures and emotionality is well known, the role of the cerebellum in the control of affective behavior is not usually appreciated (Berman, 1970a, b; 1971). Involvement of the limbic system in the elaboration of emotional behavior has been demonstrated by studies such as those of Kluver and Bucy (1939), Pribram and Bagshaw (1953) and Weiskrantz (1956), in which a taming effect was reported following amygdaloidectomy in the monkey. Reduced emotionality in the monkey has also been reported following cingulectomy (Glees, Cole, Whitty, & Cairns, 1950) and postero-medial orbital frontal cortex ablations (Butter, Snyder, & McDonald, 1970).
The research described represents an attempt to determine the exact amygdala components which contribute to the acquisition of conditioned responding during aversive conditioning. Our initial analysis focuses on the amygdala central nucleus and its contribution to the acquisition of conditioned bradycardia during Pavlovian fear conditioning in the rabbit. The results demonstrate that (a) lesions of the central nucleus attenuate the magnitude of the conditioned bradycardia response, (b) the administration of ß-adrenergic antagonists and opiate agonists into the region of the central nucleus also attenuate the magnitude of the conditioned bradycardia response, (c) the medial component of the central nucleus projects directly to cardioregulatory nuclei in the dorsal medulla including the site of origin of cardioinhibitory neurons in the rabbit, (d) electrical stimulation of the central nucleus in rabbit produces profound, short-latency bradycardia and depressor responses, with maximum bradycardia elicited from the site of origin of the central nucleus-dorsal medulla projection, and (e) during the course of the conditioning procedure increases in central nucleus neuronal activity develop to the conditioned stimulus (CS) at the time when the conditioned bradycardia response develops. In some cases the magnitude of the increase in neuronal activity to the CS was significantly correlated with the magnitude of the bradycardia response to the CS over the course of the conditioning session. The results are consistent with the hypothesis that at least one function of the amygdala central nucleus in the acquisition of conditioned bradycardia may be in the motoric expression of the conditioned response to the CS by modulation of vagal preganglionic cardioinhibitory neurons within the dorsal medulla.