Download Reflex Facilitation During Eyeblink Conditioning and Subsequent

Behavioral Neuroscience
2002, Vol. 116, No. 6, 1052–1058
Copyright 2002 by the American Psychological Association, Inc.
0735-7044/02/$5.00 DOI: 10.1037//0735-7044.116.6.1052
Reflex Facilitation During Eyeblink Conditioning and Subsequent
Interpositus Nucleus Inactivation in the Rabbit (Oryctolagus cuniculus)
Jan Wikgren
Timo Ruusuvirta
University of Jyväskylä
University of Helsinki
Tapani Korhonen
University of Jyväskylä
In eyeblink conditioning in the rabbit (Oryctolagus cuniculus), not only is a conditioned response (CR)
acquired, but also the original reflex is modified as a function of training. In Experiment 1, by comparing
unconditioned responses in unpaired and paired groups, 3 types of reflex facilitation were distinguished.
One type was linked to exposure to the unconditioned stimuli (USs) and/or experimental setting. The 2nd
type was related to the formation of the memory trace for conditioned eyeblink. The 3rd type was linked
to the conditioned stimulus immediately preceding the US in the paired group. In Experiment 2,
reversible inactivation of the interpositus nucleus (IPN) abolished the CR and reduced the CR-related
reflex facilitation, indicating that the latter depends on the plasticity of the IPN.
by reflex modification, a change in the unconditioned responses
(URs; Harvey, Gormezano, & Cool-Hauser, 1985; Weisz & LoTurco, 1988; Weisz & McInerney, 1990; Weisz & Walts, 1990).
This is defined as a change in the latency and/or amplitude of the
UR. In the case of eyeblink conditioning with a relatively short
interstimulus interval (less than 1 s), modification in the form of
reflex facilitation occurs (Schreurs, Oh, Hirashima, & Alkon,
1995). The previous findings indicate the existence of at least three
types of reflex facilitation, the first type being linked simply to
mere exposure to the experimental setting (including presentations
of the US; Schreurs et al., 1995). This type could be termed
experience-related reflex facilitation, as it refers to reflex facilitation that is not related to the forward CS–US pairings. The second
type is linked to the temporal proximity of the CS, causing greater
URs in paired trials as compared with US-alone trials, even in the
phase of learning at which CRs have not yet emerged (e.g., Weisz
& LoTurco, 1988). We refer to this as CS-mediated reflex facilitation, which is most likely linked to emotive learning of the
association between the CS and the aversiveness of the US. The
third type occurs in the phase of learning at which CRs emerge. It
is linked to URs in US-alone trials, which are higher in their
amplitude after extensive training than before such training
(Schreurs et al., 1995). As this type of reflex facilitation is most
probably linked to sensory learning, it is termed CR-related reflex
facilitation.
The contribution of emotive learning in reflex facilitation is
further supported by the finding that whereas the interpositus
nucleus (IPN) is necessary for the conditioned eyeblink response
(e.g., Anderson & Steinmetz, 1994; Steinmetz, Lavond, Ivkovich,
Logan, & Thompson, 1992; Weisz & LoTurco, 1988), the amygdala is involved in both emotive learning and reflex facilitation
(Weisz, Harden, & Xiang, 1992).
The present study examines different types of reflex facilitation
within a single experiment with the aim of excluding those types
of reflex facilitation that are dependent on the sensorimotor learn-
Different dichotomies of associations during classical conditioning have been proposed, such as diffuse– discrete, preparatory–
consummatory, or autonomic–somatic (Brandon & Wagner, 1991;
Konorski, 1967). The rationale for these dichotomies is that in any
conditioning procedure that involves an emotionally significant
unconditioned stimulus (US), there are at least two types of learning taking place in parallel: emotive and sensorimotor (Brandon &
Wagner, 1991). These types, it has been suggested, are relatively
independent and differ in terms of the brain sites critical to their
emergence (cerebellum for sensorimotor learning, amygdala and
related areas for emotive learning). However, it has been demonstrated that both types of learning are involved in learning that
seems only sensorimotor in nature, such as classical conditioning
of the eyeblink response in rabbits (Gormezano, Schneiderman,
Deaux, & Fuentes, 1962). This involves the pairing of a conditioned stimulus (CS; e.g., a tone) with a US (e.g., an airpuff toward
the cornea). Repetitious temporal forward pairing of these stimuli
results in the acquisition of a conditioned response (CR), that is, a
movement of the nictitating membrane (NM) as a response to the
CS. For example, Brandon and Wagner (1991) demonstrated the
involvement of emotive learning in eyeblink conditioning by finding that the context CS (the emotive CS) potentiated the learning
of the eyeblink reflex to a new CS.
In addition to changes in CR amplitude, another means of
studying the emotive learning in eyeblink conditioning is provided
Jan Wikgren and Tapani Korhonen, Department of Psychology, University of Jyväskylä, Jyväskylä, Finland; Timo Ruusuvirta, Cognitive
Brain Research Unit, Department of Psychology, University of Helsinki,
Helsinki, Finland.
This study was supported by the Graduate School of Psychology, Turku,
Finland. We thank Lauri Viljanto, Asko Tolvanen, Piia Astikainen, Satu
Barman, and Michael Freeman for their help.
Correspondence concerning this article should be addressed to Jan
Wikgren, Department of Psychology, University of Jyväskylä, P.O.
Box 35, 40351 Jyväskylä, Finland. E-mail: [email protected]
1052
REFLEX FACILITATION IN EYEBLINK CONDITIONING
ing circuit (IPN). In Experiment 1, we compared the development
of reflex facilitation over the time course of the paired treatment
with its development over the time course of the explicitly unpaired treatment. In Experiment 2, we reversibly inactivated the
IPN by a cold probe in well-trained rabbits to reveal those types of
reflex facilitation that are linked to the normal functioning of this
nucleus.
We made four predictions. First, some reflex facilitation should
occur solely because of the experience of the experimental setting
during both treatments (experience-related reflex facilitation). Second, in the paired treatment, reflex facilitation should be more
vigorous when the CS immediately precedes the US than when the
US is presented alone (CS-mediated reflex facilitation). Third,
after a robust level of CR is achieved in the paired treatment, reflex
facilitation should be higher in the US-alone trials than in the
corresponding trials in the unpaired treatment (CR-related reflex
facilitation). Fourth, the reversible inactivation of the IPN in
well-trained rabbits should affect CR-related reflex facilitation but
not necessarily CS-mediated reflex facilitation.
Experiment 1
Method
Subjects. The subjects were 21 adult New Zealand albino rabbits
(Oryctolagus cuniculus) weighing 2.5–3.7 kg at the time of surgery. The
animals were individually housed in metal cages on a 12:12-hr light-dark
cycle, with free access to food and water. All the experiments were carried
out in accordance with the European Union Directive 86/609/EEC regarding the care and use of animals for experimental procedures (Netherlands
Centre Alternatives to Animal Use, 2002). The Ethics Committee for
Animal Research of the University of Jyväskylä, Jyväskylä, Finland gave
its consent to the study.
Surgery. The animals were anesthetized with intramuscular injections
of a ketamine–xylazine cocktail (Ketaminol, 50 mg/ml, 5.6 ml;
Rompun, 20 mg/ml, 2.2 ml; physiological saline, 2.2 ml). The initial
dosage was 3– 4 ml, and the anesthesia was maintained by additional
injections of 2 ml every 20 – 40 min. After a deep general anesthesia had
been achieved, we placed the animals in a stereotaxic instrument (David
Kopf Instruments, Tujunga, CA) with the bregma 1.5 mm above the
lambda. A longitudinal incision was made to reveal the skull, onto which
the headstage (designed to hold the minitorque potentiometer, an airpuff
delivery nozzle and tone tubing) was cemented using four stainless steel
anchoring screws.
Cold probes were implanted only for the unpaired group (they served as
subjects in Experiment 2). It is our experience that implantation of a cold
probe near the IPN does not affect subjects’ learning capabilities (in
Experiment 2, their learning curve was normal). As both groups otherwise
underwent the same surgical protocols, there should be no other differences
between the groups than those caused by their respective experimental
treatments. The construction of the cold probe was based on that presented
by Zhang, Ni, and Harper (1986). The shaft of the probe was not warmed,
as it has been shown that lesioning the cerebellar lobule HVI, which the
probe penetrated, does not interfere with the acquisition of the conditioned
eyeblink response (R. E. Clark, Zhang, & Lavond, 1992). In short, the cold
probe consists of two stainless steel tubes, one inside the other. The inner
tube delivers the coolant at a distance of 1 mm from the tip of the probe,
which is sealed by solder. The coolant exits through a plastic tube attached
to the outer cannula at a Y-shaped junction. The coolant used was freonlike 1,1,1,2-tetrafluoroethane (KLEA R-134-A). The implantation followed a method of recording-electrode implantation proposed by Korhonen (1991). The cold probe was implanted near the IPN using the
coordinates of 0.5 mm anterior and 5.0 mm lateral to the lambda. Anal-
1053
gesics (Temgesic, 0.3 mg/ml) were provided 2 hr after surgery and additionally if needed.
Experimental procedure. The subjects were given at least 1 week to
recover after surgery before the commencement of the experimental procedures. On the first day, adaptation to the experimental situation was done
by placing the animals in a Plexiglas restraining box in a soundproof
conditioning chamber. The rabbits were divided in two groups: the unpaired (UP) group (n ⫽ 9) and the classical conditioning (CC) group (n ⫽
11). A UP session consisted of 70 presentations of the tone (1000 Hz, 85
dB, 350 ms) and 70 presentations of the airpuff (2.1 N/cm2 source pressure,
100 ms) given in a pseudorandom order with an intertrial interval (ITI)
varying between 15 and 25 s (mean ITI ⫽ 20 s). A CC session consisted
of 60 CC trials, in which the tone was followed by the airpuff and 10
CS-alone and 10 US-alone test trials in a pseudorandom order. The paired
trials in the CC group were presented in a delayed fashion so that the
stimuli coterminated. The ITI varied between 30 and 50 s (mean ITI ⫽
40 s). The subjects were treated for five successive daily sessions. Peak
amplitude values for CRs and URs were defined as the maximum extension
of the NM during a period of 250 ms immediately following the CS or US.
Any movement of the NM exceeding 0.5 mm was counted as a response.
Trials in which NM movement exceeded 0.3 mm during the 100 ms
immediately prior to the trial were excluded from the analysis.
Statistical procedures. URs were analyzed with respect to the different
trial types in the three phases of the experiment. The trial types consisted
of the US presented alone in the UP group (US/UP) and in the CC group
(US/CC) and the US paired with the CS in the CC group (CS ⫹ US/CC).
The phases of the experiment consisted of the first trial in the first session
(Phase A), the last trial in the first session (Phase B), and the last trial in
the last session (Phase C). The last trial in the last session in the CC group
was not compared with the US-alone trials in the other conditions because
of the presence of conditioned motor responding in that group. Single-trial
samples were used to minimize the possibility of interaction between the
CS and the US at the beginning of the experiment and thereby to capture
the responses when they were most naive. Experience has shown that in our
setting, 60 paired trials are usually not enough for the development of a
CR. Therefore, by the last trial in the first session, the URs should not yet
be influenced by a learned sensorimotor response. For the statistical analyses, analysis of variance for repeated measures was used. When the CC
group was compared with the UP group, treatment (CC vs. UP) served as
a between-subjects factor in a mixed model. In addition, t tests were used
to further assess differences between groups (independent samples) or trial
types during a certain training phase (paired samples).
Histology. After the experiments, the animals were anesthetized with
an intramuscular injection of ketamine–xylazine cocktail and then overdosed with an intravenous injection of pentobarbital. The rabbits were then
perfused via the ascending aorta with saline followed by 10% Formalin.
The brains were removed and fixed in Formalin–sucrose solution for at
least 1 week. Frozen coronal sections of 100 ␮m were taken from the site
of the cold probe. Slices were mounted on gelatinized slides and stained
with cresyl violet. The locations of the cold probes were determined
according to the stereotaxic atlas by Shek, Wen, and Wisniewski (1986).
Results
Conditioned responding. No CRs exceeding the criterion were
observed in either of the groups during the first session. The
analyses of the URs, therefore, are not contaminated by learned
overt motor responses. By the fifth session, the mean (⫾SEM) CR
percentage in the CC group was 77.4% ⫾ 7.2. No CRs above the
baseline level were observed in the UP group.
Unconditioned responding. Figure 1 depicts the development
of the UR amplitude for the US period during the experiment in
both groups and trial types. As can be seen, common to both
groups and to both trial types in the CC group is the tendency of
1054
WIKGREN, RUUSUVIRTA, AND KORHONEN
Figure 1. Mean (⫾SEM) peak amplitudes of the unconditioned responses (in millimeters) in unconditioned
stimulus (US)-alone trials in the classical conditioning group (CC; squares), CS ⫹ US trials in the CC group
(triangles), and US trials in the unpaired group (UP; circles) plotted as a function of the training phase in
Experiment 1. The mean peak amplitudes are plotted pairwise, as they were analyzed. Asterisks indicate
significant differences between the NM amplitudes (* p ⬍ .05, ** p ⬍ .01). CS ⫽ conditioned stimulus; NM ⫽
nictitating membrane; A ⫽ the first trial in the first session; B ⫽ the last trial in the first session; C ⫽ the last
trial in the last session.
the UR to increase in amplitude as a function of exposure to the
experimental setting, as indicated by the significant main effect of
training phase (A, B, and C) in both groups: in the CC group, F(2,
20) ⫽ 25.25, p ⬍ .01; in the UP group, F(2, 16) ⫽ 13.06, p ⬍ .01.
In the CC group, the UR amplitude grew more rapidly in the
CS ⫹ US trials than in the US-alone trials, as indicated by the
significant Training Phase (A, B, and C) ⫻ Trial Type (US-alone
vs. CS ⫹ US) interaction, F(2, 20) ⫽ 6.38, p ⬍ .05. A t test for
paired samples further revealed that the differences between the
trial types was significant only for the last trial of the first training
session, t(10) ⫽ 3.83, p ⬍ .01 (left-hand side in Figure 1).
Furthermore, there was no significant difference in the last trial of
the first session between the US/CC and US/UP conditions, indicating that the presence of the CS was a necessary factor in this
phase of training to maintain reflex facilitation.
Comparison of the US-alone trials between the CC and UP
groups is shown in the center of Figure 1. The Training Phase (A,
B, and C) ⫻ Group (CC vs. UP) interaction was significant, F(2,
36) ⫽ 2.88, p ⬍ .05. A t test for independent samples further
revealed a significant difference between these groups in the last
trial of the last session, t(18) ⫽ 2.24, p ⬍ .05, by which time the
CC group had already acquired a robust level of conditioned
responding. Taken together, these results indicate the presence of
reflex facilitation, especially in relation to the US-alone trials in
the CC group. When the CS ⫹ US/CC and US/UP trials were
compared (right-hand side of Figure 1), the difference in UR
amplitude was significant for the last trial of the first session,
t(18) ⫽ 2.24, p ⬍ .05, but not for the first trial of the first session,
where the subject had not yet experienced paired presentation of
the stimuli in the CC group.
The finding that the later URs were higher in amplitude in all
conditions suggests the presence of experience-related reflex facilitation. The presence of CS-mediated reflex facilitation was
indicated by the finding that the UR in the CC group was facilitated more if the US was preceded by the CS, despite the same
initial level of responding in all conditions. Although no difference
was found in URs to the US-alone trials between paired and
unpaired treatments, an indication of the US–UR circuit having not
yet been modified, these responses were higher in amplitude in the
paired treatment by the end of training. This indicates the presence
of CR-related reflex facilitation and, presumably, a relatively permanent modification of the US–UR circuit.
These results are in accordance with those of Schreurs et al.
(1995), who found CR-related reflex facilitation in US-alone trials
when a robust level of CRs was achieved but not when the extent
of conditioning was low (17% CRs after the first day of conditioning in their data). The data suggest that CS-mediated reflex
facilitation, at this phase of learning, reflects a conditioned change
in the organism. Whatever this change may be, it affects the US
processing in a facilitative manner.
To investigate whether reflex facilitation is dependent on the
functioning of the IPN (the plausible core of the sensorimotor
learning circuit), we set up Experiment 2. The aim was to test
whether the amount of reflex facilitation is altered when the CR is
blocked by inactivation of the IPN. Because the IPN is essential for
forming and maintaining the association between the CS and the
US, Experiment 2 should show whether any type of reflex facilitation is influenced by sensorimotor learning.
Discussion
Method
These results indicate that three types of reflex facilitation were
present during conditioning and that it is possible to dissociate
them by taking samples from appropriate phases of conditioning.
Subjects and surgery. The subjects were the same 9 adult female New
Zealand albino rabbits (Oryctolagus cuniculus) that were used in the UP
group in Experiment 1.
Experiment 2
REFLEX FACILITATION IN EYEBLINK CONDITIONING
Procedure. After the unpaired treatment, the rabbits were trained in the
same manner as the CC group in Experiment 1. After reaching the learning
criterion of eight out of nine consecutive CRs, with the addition of one
overtraining session, the rabbits underwent a session that involved cooling
of the IPN. The cooling session was divided into three blocks: precooling,
cooling, and postcooling. Each block consisted of 30 trials (4 CS only, 4
US only, and 22 CS ⫹ US trials). CRs were recorded from CS period in
CS-only and CS ⫹ US trials, and UR peak amplitudes were recorded
separately from the US-alone and CS ⫹ US trials, as in Experiment 1.
After the first block, the cold probe was activated and the gas flow was
adjusted so that the temperature in the cold probe fell below 5 °C. This took
about 2 min. After the cooling block, the experiment was again interrupted
for about 2 min to allow the temperature to return to normal. Generally, the
subjects showed no CRs during cooling, but, in practice, the temperature
fluctuates to some extent, which causes release from blocking in some of
the trials. To ensure that the URs measured in the paired trials during
cooling were not influenced by sudden CRs, the trials with NM movement
exceeding 0.5 mm during the CS period were excluded from the analyses.
Statistical procedure. We used repeated measures. For CRs, the three
blocks of the cooling session formed the cooling variable. URs in both trial
types (URs in paired trials and in US-alone trials) were analyzed separately
in the same manner as the CRs. Afterward, we ran an additional analysis
in which trial type was used as an additional variable. To provide an
objective measure for the functioning of the cooling, we performed t tests
for paired samples on each subject’s CR values during the cooling session
(before vs. during cooling). Single trials were treated as cases. In all
statistics, an alpha level of .05 was applied.
Results
Histology. The cold probe was correctly located (distance less
than 2 mm from the IPN) in 6 out of 9 subjects (see Figure 2).
Consistently, statistically significant reduction of the CR because
of cooling was found only in these animals. Consequently, data
from only these 6 subjects were analyzed further.
Conditioned responding. Figure 3 illustrates the average peak
amplitudes of CRs before, during, and after cooling. A significant
main effect of cooling, F(2, 10) ⫽ 7.91, p ⬍ .01, was found.
Unconditioned responding. The peak amplitude of the URs in
both the US-alone and CS ⫹ US trials in the three phases of the
cooling session are shown in Figure 4. The interaction of cooling
and trial type was significant, F(2, 10) ⫽ 8.89, p ⬍ .01, as were the
1055
Figure 3. Mean (⫾SEM) peak amplitudes of the conditioned responses
(in millimeters) in well-trained animals with accurate cold probe implantation during the different blocks in the cooling session as measured in
conditioned stimulus ⫹ unconditioned stimulus trials (22 trials per block)
in Experiment 2.
main effects of both cooling, F(2, 10) ⫽ 11.13, p ⬍ .01, and trial
type, F(2, 10) ⫽ 11.33, p ⬍ .05. Analyses performed for both trial
types separately indicated that the effect of cooling was significant
for the US-alone trials, F(2, 10) ⫽ 23.04, p ⬍ .01, but not for the
CS ⫹ US trials, F(2, 10) ⫽ 0.79, p ⫽ .48.
Discussion
Cooling of the IPN in well-trained animals abolished the CR and
significantly reduced the amplitude of URs in the US-alone trials
but not in the CS ⫹ US trials. Thus, cooling of the IPN both
abolished the actual discrete conditioned skeletal muscle response
and reduced the CR-related reflex facilitation but had no effect on
CS-mediated reflex facilitation. This indicated that the CR-related
reflex facilitation, in particular, is dependent on a functional IPN.
Consistently, in Experiment 1, this type of reflex facilitation was
associated with the presence of a robust CR, as it was not observed
in the initial phase of training.
General Discussion
Figure 2. Locations of the cold probe tips in all 9 experimental subjects
in Experiment 2. Circles stand for the subjects in which conditioned
responding was abolished during probe activation. The cold probe tip
locations for the animals that did not show statistically significant abolition
of conditioned response during cooling are marked with rectangles; their
data were excluded from further analysis. A ⫽ millimeters anterior to
lambda; M ⫽ midline; IP ⫽ interpositus nucleus; DE ⫽ dentate nucleus;
FA ⫽ fastigial nucleus.
The present experiments indicated the presence of three types of
reflex facilitation. The first type, experience-related reflex facilitation, emerged after 60 US-presentations even in the UP group,
indicating that the animals’ experience of the US and/or of the
context in which the US was presented must have been sufficient
for this effect. A possibility that the context might also play a role
in this type of reflex facilitation is based on the finding that the CR
amplitude, at least, can be increased by the context (Brandon &
Wagner, 1991; Schreurs et al., 1995). Thus, not only selective
sensitization of the US–UR circuit but also an association between
1056
WIKGREN, RUUSUVIRTA, AND KORHONEN
Figure 4. Mean (⫾SEM) peak amplitudes of the unconditioned responses
(in millimeters) in unconditioned stimulus (US)-alone trials and CS ⫹ US
trials as a function of phase in the cooling session in Experiment 2. CS ⫽
conditioned stimulus.
the US and its context might contribute to experience-related
reflex facilitation.
The second type of reflex facilitation, CR-related reflex facilitation, was linked to the emergence of the CR. Pairing the CS and
the US resulted in more vigorous URs in the US-alone trials but,
in line with the previous studies (Schreurs et al., 1995), only after
a robust level of conditioning was reached. Consistently, the
emergence of CR-related reflex facilitation was linked to the same
neural circuit, that is, the IPN, that is necessary for the emergence
of the CR itself, as it was affected by the temporal inactivation of
this nucleus. This result is in contrast with the unaffected URs in
the US-alone trials after the IPN lesion found in most of the earlier
studies (e.g., G. A. Clark, McCormick, Lavond, & Thompson,
1984; R. E. Clark et al., 1992; Lavond, Hembree, & Thompson,
1985; Steinmetz et al., 1992; Yeo, Hardiman, & Glickstein, 1985).
This finding is, however, in line with the study by Ivkovich,
Lockard, and Thompson (1993), who reported such a declining
trend in UR amplitudes to US-alone presentations.
One might argue that the prior unpaired treatment in Experiment 1 led to this result; namely, given that reflex facilitation
might be context related to some extent, one might propose that the
IPN is involved not only in the storage of the CS–US association
but also in that of the context–US association. However, in our
recent experiment (Wikgren & Korhonen, 2001), the reversible
inactivation of the IPN after the unpaired treatment was not found
to affect the UR amplitude in US-alone trials but, in line with the
present study, did so after the paired treatment. This evidence
further suggests that the CS–US association formed by the IPN
was linked to the processing of the US presented alone as well.
Also, the third type of reflex facilitation (CS-mediated reflex
facilitation) seems to have an associative basis. It was found to
emerge at an early phase of the paired treatment, even before a
robust level of CR was achieved but only after several CS–US
pairings had been presented. A lack of its emergence during the
very first trial of the experiment excludes a possibility that merely
the presence of a tone led, in a nonspecific manner, to more
vigorous URs, making valid comparisons between unpaired and
paired trials impossible (Young, Cegavske, & Thompson, 1976).
In contrast, even earlier studies have indicated that reflex facilitation can be specific to the physical features of the CS immediately
preceding the US (Weisz & LoTurco, 1988; Weisz & McInerney,
1990), a key feature necessary for the CS acting as a signal. The
exact nature of the CS-mediated reflex facilitation remains to be
seen. One possibility, however, is that this type of reflex facilitation might be related to a conditioned emotive state, that is, to the
ability of the US not only to possess sensory and response-eliciting
value as such but also to induce aversiveness (Richardson &
Thompson, 1984; Thompson, Thompson, Kim, Krupa, & Shinkman, 1998). This dissociation, at least in one direction, between
the emotive and nonemotive value of a US has been further
indicated by the finding that stimulation of the dorsal accessory
olive as a US can be associated with a CS without signs of
aversiveness (Mauk, Steinmetz, & Thompson, 1986). Thus, in the
present study, it is possible that the remaining emotive aspects of
the US might have become associated with the CS, independent of
the behavioral response. This interpretation is further supported by
the relative independence of the CS-mediated reflex facilitation on
the cooling of the IPN (Wikgren & Korhonen, 2001), indicating
that the neural bases necessary for the related association must be
other than the IPN. In the present data, this interpretation is
supported, as the UR amplitude in CS ⫹ US trials did not alter
during cooling of the IPN.
What, then, might anatomically contribute to CS-mediated reflex facilitation? It seems that the related pathways diverge from
those necessary for CR acquisition and maintenance of the CR at
the lower levels of the CS pathway. Whereas reflex facilitation can
be induced by the electrical stimulation of the cochlear nucleus as
a CS, this cannot be done when the nuclei located later along this
pathway (the superior olive, inferior colliculus, or medial geniculate nucleus) are stimulated (Nowak, Kehoe, Macrae, & Gormezano, 1999). This finding further converges with the findings
from another line of research, fear-potentiated startle effect (Davis,
1998); it refers to the findings that the amplitude of a UR to an
intensive stimulus (the startle reflex), such as a loud tone, can be
modified by inducing a state of fear. More specifically, pairing a
neutral stimulus with the startle-eliciting noise, the startle reflex
becomes stronger in paired trials, as measured by freezing behavior in rats or facial electromyograph responses in humans. Similarly to reflex facilitation, the startle-eliciting stimulus elicits the
related behavior via the lower levels of the CS pathway. The
related neural circuit consists of the connection between the sensory neurons in the cochlear root nucleus and the neurons in the
nucleus reticular pontis caudalis (Rosen, Hitchcock, Sananes,
Miserendino, & Davis, 1991), which, in turn, has a connection
with the motor nuclei and the amygdala. The central nucleus of the
amygdala in particular seems to be a critical region for the acquisition and maintenance of the fear-potentiated startle effect, as
suggested by lesions to the amygdala or its connections with the
startle pathway (Hitchcock & Davis, 1991; Hitchcock, Sananes, &
Davis, 1989).
REFLEX FACILITATION IN EYEBLINK CONDITIONING
Given that both the emotive aspects of learning and the early
parts of the auditory pathway contribute to CS-mediated reflex
facilitation and the fear-potentiated startle effect, it is possible that
they are similar also in the respect of both being dependent on the
same critical region of the brain, the amygdala. Consistently,
animals with amygdalar lesion are compromised in CR acquisition
and in exhibiting reflex facilitation during aversive conditioning
(Weisz et al., 1992), which indicates that the amygdala might be
involved in learning the aversiveness of the US during eyeblink
conditioning. The role of the amygdala in reflex facilitation is
further supported by the finding that the electrical stimulation of
the amygdala prior to a US presentation facilitates the UR (Whalen
& Kapp, 1991). The neural substrate for this effect seems to
consist of the projections of the amygdalar central nucleus to the
lateral tegmental field in the thalamus, which, in turn, projects to
a variety of cranial motor nuclei (Hopkins & Holstege, 1978;
Takeuchi, Satoda, Tashiro, Matsushima, & Uemura-Sumi, 1988).
Thus, by way of these connections, the amygdala might modulate
various reflexes, such as the NM reflex (Kapp, Supple, & Whalen,
1994; Whalen & Kapp, 1991), independently of the IPN.
Three types of reflex facilitation were found. Experience-related
reflex facilitation developed as a function of time spent in the
experimental setting with aversive stimuli and/or the resulting
sensitization of the US–UR circuit. In contrast, CR-related reflex
facilitation, in particular, was linked to the emergence of the CR
and consistently was affected by the inactivation of the neural
circuit necessary (IPN) for the CR development and maintenance.
CS-mediated reflex facilitation, despite emerging at an early phase
of learning after a few repetitions of CS–US pairings, was not
affected by the IPN inactivation. By being dependent on forward
CS–US pairings but not on the IPN, CS-mediated reflex facilitation might reflect emotive aspects of learning and, thereby, be
based on the same neural substrates as other forms of emotive
learning, such as fear-potentiated startle reflex.
References
Anderson, B. J., & Steinmetz, J. E. (1994). Cerebellar and brainstem
circuits involved in classical eyeblink conditioning. Reviews in the
Neurosciences, 5, 251–273.
Brandon, S. E., & Wagner, A. R. (1991). Modulation of a discrete Pavlovian conditioned reflex by a putative emotive Pavlovian conditioned
stimulus. Journal of Experimental Psychology: Animal Behavior Processes, 17, 299 –311.
Clark, G. A., McCormick, D. A., Lavond, D. G., & Thompson, R. F.
(1984). Effects of lesions of cerebellar nuclei on conditioned behavioral
and hippocampal neuronal responses. Brain Research, 291, 125–136.
Clark, R. E., Zhang, A. A., & Lavond, D. G. (1992). Reversible lesions of
the cerebellar interpositus nucleus during acquisition and retention of a
classically conditioned behavior. Behavioral Neuroscience, 106, 879 –
888.
Davis, M. (1998). Anatomic and physiologic substrates of emotion in an
animal model. Journal of Clinical Neurophysiology, 15, 378 –387.
Gormezano, I., Schneiderman, N., Deaux, E., & Fuentes, I. (1962, October
12). Nictitating membrane: Classical conditioning and extinction in the
albino rabbit. Science, 138, 33–34.
Harvey, J. A., Gormezano, I., & Cool-Hauser, V. A. (1985). Relationship
between heterosynaptic reflex facilitation and acquisition of the nictitating membrane response in control and scopolamine-injected rabbits.
Journal of Neuroscience, 5, 596 – 602.
Hitchcock, J. M., & Davis, M. (1991). The efferent pathway of the
1057
amygdala involved in conditioned fear as measured with the fearpotentiated startle paradigm. Behavioral Neuroscience, 105, 826 – 842.
Hitchcock, J. M., Sananes, C. B., & Davis, M. (1989). Sensitization of the
startle reflex by footshock: Blockade by lesions of the central nucleus of
the amygdala or its efferent pathway to the brainstem. Behavioral
Neuroscience, 103, 509 –518.
Hopkins, D. A., & Holstege G. (1978). Amygdaloid projections to the
mesencephalon, pons and medulla oblongata in the cat. Experimental
Brain Research, 32, 529 –547.
Ivkovich, D., Lockard, J. M., & Thompson R. F. (1993). Interpositus lesion
abolition of the eyeblink conditioned response is not due to effects on
performance. Behavioral Neuroscience, 107, 530 –532.
Kapp, B. S., Supple, W. F., Jr., & Whalen, P. J. (1994). Effects of electrical
stimulation of the amygdaloid central nucleus on neocortical arousal in
the rabbit. Behavioral Neuroscience, 108, 81–93.
Konorski, J. (1967). Integrative activity of the brain. Chicago: University
of Chicago Press.
Korhonen, T. (1991). A method for rapid implantation of multielectrode
systems. Physiology & Behavior, 49, 401– 403.
Lavond, D. G., Hembree, T. L., & Thompson, R. F. (1985). Effect of kainic
acid lesions of the cerebellar interpositus nucleus on eyelid conditioning
in the rabbit. Brain Research, 326, 179 –182.
Mauk, M. D., Steinmetz, J. E., & Thompson, R. F. (1986). Classical
conditioning using stimulation of the inferior olive as the unconditioned
stimulus. Proceedings of the National Academy of Sciences, USA, 79,
2731–2742.
Netherlands Centre Alternatives to Animal Use. (2002). Council directive
of 24 November 1986 on the approximation of laws, regulations and
administrative provisions of the member states regarding the protection
of animals used for experimental and other scientific purposes (86/609/
EEC). Retrieved September 20, 2002, from http://www.nca-nl.org/
English/Docs/86-609-eec_en.pdf
Nowak, A. J., Kehoe, E. J., Macrae, M., & Gormezano, I. (1999). Conditioning and reflex modification of the rabbit nictitating membrane response using electrical stimulation in auditory nuclei. Behavioural Brain
Research, 105, 189 –198.
Richardson, R. T., & Thompson, R. F. (1984). Amygdaloid unit activity
during classical conditioning of the nictitating membrane response in
rabbit. Physiology & Behavior, 32, 527–539.
Rosen, J. B., Hitchcock, J. M., Sananes, C. B., Miserendino, M. J. D., &
Davis, M. A. (1991). A direct projection from the central nucleus of the
amygdala to the acoustic startle pathway: Anterograde and retrograde
tracing studies. Behavioral Neuroscience, 105, 817– 825.
Schreurs, B. G., Oh, M. M., Hirashima, C., & Alkon, D. L. (1995).
Conditioning-specific modification of the rabbit’s unconditioned nictitating membrane response. Behavioral Neuroscience, 109, 24 –33.
Shek, J. W., Wen, G. Y., & Wisniewski, H. M. (1986). Atlas of the rabbit
brain and spinal cord. Basel, Switzerland: Karger.
Steinmetz, J. E., Lavond, D. G., Ivkovich, D., Logan, C. G., & Thompson,
R. F. (1992). Disruption of classical eyelid conditioning after cerebellar
lesions: Damage to a memory trace system or a simple performance
deficit? Journal of Neuroscience, 12, 4403– 4426.
Takeuchi, Y., Satoda, T., Tashiro, T., Matsushima, R., & Uemura-Sumi,
M. (1988). Amygdaloid pathway to the trigeminal motor nucleus via the
pontine reticular formation in the rat. Brain Research Bulletin, 21,
829 – 833.
Thompson, R. F., Thompson, J. K., Kim, J. J., Krupa, D. J., & Shinkman,
P. G. (1998). The nature of reinforcement in classical conditioning.
Neurobiology of Learning and Memory, 70, 150 –176.
Weisz, D. J., Harden, D. G., & Xiang, Z. (1992). Effects of amygdala
lesions on reflex facilitation and conditioned response acquisition during
nictitating membrane response conditioning in rabbit. Behavioral Neuroscience, 106, 262–273.
Weisz, D. J., & LoTurco, J. J. (1988). Reflex facilitation of the nictitating
1058
WIKGREN, RUUSUVIRTA, AND KORHONEN
membrane response remains after cerebellar lesions. Behavioral Neuroscience, 102, 203–209.
Weisz, D. J., & McInerney, J. (1990). An associative process maintains
reflex facilitation of the unconditioned nictitating membrane response
during the early stages of training. Behavioral Neuroscience, 104, 21–
27.
Weisz, D. J., & Walts, C. (1990). Reflex facilitation of the rabbit nictitating
membrane response by an auditory stimulus as a function of interstimulus interval. Behavioral Neuroscience, 104, 11–20.
Whalen, P. J., & Kapp, B. S. (1991). Contributions of the amygdaloid
central nucleus to the modulation of the nictitating membrane reflex in
the rabbit. Behavioral Neuroscience, 105, 141–153.
Wikgren, J., & Korhonen, T. (2001). Interpositus nucleus inactivation
reduces unconditioned response amplitude after paired but not explicitly
unpaired treatment in rabbit eyeblink conditioning. Neuroscience Letters, 308, 181–184.
Yeo, C. H., Hardiman, M. J., & Glickstein, M. (1985). Classical conditioning of the nictitating membrane response of the rabbit: I. Lesions of
the cerebellar nuclei. Experimental Brain Research, 60, 87–98.
Young, R. A., Cegavske, C. F., & Thompson, R. F. (1976). Tone-induced
changes in excitability in abducens motoneurons and of the reflex path
of nictitating membrane in rabbit (Oryctolagus cuniculus). Journal of
Comparative and Physiological Psychology, 90, 424 – 434.
Zhang, J., Ni, H., & Harper, R. M. (1986). A miniaturized cryoprobe for
functional neuronal blockade in freely moving animals. Journal of
Neuroscience Methods, 16, 79 – 87.
Received April 27, 2000
Revision received March 26, 2002
Accepted May 13, 2002 䡲