Department of Psychology and Institute for Neuroscience, University of Texas at Austin, Austin, Texas 78712
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Jones, Dirk and F. Gonzalez-Lima. Mapping Pavlovian Conditioning Effects on the Brain: Blocking, Contiguity, and Excitatory Effects. J. Neurophysiol. 86: 809-823, 2001. Pavlovian conditioning effects on the brain were investigated by mapping rat brain activity with fluorodeoxyglucose (FDG) autoradiography. The goal was to map the effects of the same tone after blocking or eliciting a conditioned emotional response (CER). In the tone-blocked group, previous learning about a light blocked a CER to the tone. In the tone-excitor group, the same pairings of tone with shock US resulted in a CER to the tone in the absence of previous learning about the light. A third group showed no CER after pseudorandom presentations of these stimuli. Brain systems involved in the various associative effects of Pavlovian conditioning were identified, and their functional significance was interpreted in light of previous FDG studies. Three conditioning effects were mapped: 1) blocking effects: FDG uptake was lower in medial prefrontal cortex and higher in spinal trigeminal and cuneate nuclei in the tone-blocked group relative to the tone-excitor group. 2) Contiguity effects: relative to pseudorandom controls, similar FDG uptake increases in the tone-blocked and -excitor groups were found in auditory regions (inferior colliculus and cortex), hippocampus (CA1), cerebellum, caudate putamen, and solitary nucleus. Contiguity effects may be due to tone-shock pairings common to the tone-blocked and -excitor groups rather than their different CER. And 3) excitatory effects: FDG uptake increases limited to the tone-excitor group occurred in a circuit linked to the CER, including insular and anterior cingulate cortex, vertical diagonal band nucleus, anterior hypothalamus, and caudoventral caudate putamen. This study provided the first large-scale map of brain regions underlying the Kamin blocking effect on conditioning. In particular, the results suggest that suppression of prefrontal activity and activation of unconditioned stimulus pathways are important neural substrates of the Kamin blocking effect.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In modern learning theory,
Pavlovian conditioning has come to be understood as more than simple
associations; evidence shows that organisms learn new responses based
on their current knowledge (Domjan et al. 2000). The
blocking effect on Pavlovian conditioning (Kamin 1969
)
provided an early demonstration that learning about a stimulus depended
not simply on contiguity but on previous learning. In the Kamin
blocking effect, the presence of a previously conditioned stimulus
blocks the conditioned response to a newly introduced stimulus. For
example, subjects exposed to a compound stimulus (tone plus light)
paired with shock (Fig. 1A)
exhibit conditioned responses to both the tone and the light. However,
subjects first exposed to a number of light and shock pairings (phase
I, Fig. 1B) followed later by the tone/light compound paired
with the shock (phase II, Fig. 1B), show conditioned
responses to the light but not to the tone. Of importance is that all
subjects received the same tone-shock contiguity. In other words, in
both groups the tone occurred in close temporal proximity with the
shock. However, conditioned responses to the tone are "blocked" in
subjects with a history of light-shock pairings. This blocking
phenomenon has been regarded as one of the most significant
observations in Pavlovian conditioning (Fanselow 1998
).
In addition, recent findings (Jones et al. 1997
) showing
that schizophrenics lack the development of blocking render this
phenomenon of interest to a wider audience.
|
Neuroimaging techniques using glucose analogues (Sokoloff
1992) such as fluorodeoxyglucose (FDG) autoradiography have
increased the ability to study the functioning nervous system,
including behavioral and learning effects (Gonzalez-Lima
1992
). This approach has made possible the simultaneous
examination of functional maps of learning-related activity
throughout the rat brain. For example, researchers using this approach
were the first to report functional brain maps produced by habituation
and arousal (Gonzalez-Lima and Scheich 1984
,
1985
; Gonzalez-Lima et al. 1989
; Toga
and Collins 1981
), conditioned excitation
(Gonzalez-Lima and Scheich 1984
, 1986
), conditioned
inhibition (McIntosh and Gonzalez-Lima 1994
, 1998
), and
extinction (Nair and Gonzalez-Lima 1999
). In addition, differential conditioning of tones has been shown to evoke specific metabolic differences in a thalamostriate circuit as well as in the
auditory system (Gonzalez-Lima 1992
;
Gonzalez-Lima and Agudo 1990
; Scheich et al.
1997
). McIntosh and Gonzalez-Lima (1993
-1995
) have shown FDG increases in the auditory system, basal forebrain, and
cerebellum evoked by a tone conditioned as an excitor relative to the
same tone conditioned as inhibitor. However, little work has been done
on the neural mechanisms of the Kamin blocking effect.
Most of the available neurobiological studies of blocking have assessed
the effects of lesions in the hippocampus or the amygdala (Gallo
and Candido 1995; Holland and Gallagher 1993
;
Rickert et al. 1978
; Solomon 1977
).
Lesions of the amygdala did not prevent blocking (Holland and
Gallagher 1993
), and hippocampal lesions have had mixed results
(Garrud et al. 1984
; Rickert et al. 1978
; Solomon 1977
). Hippocampal unit responses to a blocked
light appear at the first compound trial but do not occur at the end of
conditioning (Neuenschwander-El Massioui et al. 1991
).
Identification of the neural mechanisms involved in the blocking effect
may help to differentiate among various theories of blocking of
Pavlovian conditioning based on behavioral studies because there is
conflicting behavioral evidence for each theory.
For example, some theories limit attention to a redundant conditioned
stimulus (CS) as an explanation of why a blocked CS fails to elicit a
conditioned response (Kamin 1969; Mackintosh 1983
; Pearce and Hall 1980
). These theories
emphasize changes in processing of the blocked CS. In contrast, the
influential Rescorla-Wagner model (1972)
limits the
amount of learning that an unconditioned stimulus (US) can support.
This theory emphasizes changes in US processing as an explanation of
the blocking effect. A third group of theories views blocking as an
instance of conditioned response (CR) inhibition independent of changes
in CS or US processing (Balaz et al. 1982
; Miller
et al. 1993
; Schachtman et al. 1983
). Although
these three types of theories emphasize either CS, US or CR behavioral
mechanisms, their implications can be considered in light of functional
neural data.
For example, differences in neural modification of CS pathways by
excitor and blocked CSs would provide support for CS inattention interpretations of blocking (e.g., Mackintosh 1983;
Pearce and Hall 1980
). Neuronal recordings in the visual
system have been carried out during administration of a modified
blocking procedure (Kinkaide and Walley 1974
), but a
clear interpretation of the resulting data regarding the blocking
effect is confounded by the presence of inhibitory training. Recent
work using the presence of fos protein has implicated the auditory and
visual parts of the thalamic reticular nucleus as mediating attentional
properties involved in blocking (McAlonan et al.
2000
). Although interesting, this fos study showed no
convincing evidence of blocking because the compound stimuli were as
effective as the blocked stimuli. Hence the results can be explained
not by a blocking effect but by differential excitatory effects of CSs
as we have shown previously in the reticular thalamic nucleus using FDG
uptake after differential conditioning (Gonzalez-Lima
1992
).
Auditory CS learning-dependent modification of neuronal activity in the
primary auditory system and its secondary projection fields is well
documented (Gonzalez-Lima and Agudo 1990;
Gonzalez-Lima and Scheich 1984
, 1986
; Scheich et
al. 1997
; Weinberger 1995
; Weinberger and
Diamond 1987
). Some have interpreted these increases in
neuronal activity as underlying increases in attention to a particular
CS (Mesulam 1990
; Posner et al. 1987
).
This laboratory published a report of changes in the auditory system
during blocking of a tone CS using a marker of metabolic capacity,
cytochrome oxidase (Poremba et al. 1997
). The regions
with cytochrome oxidase differences were limited to areas of the
auditory system receiving US somatosensory inputs, such as the dorsal
cochlear nucleus, the inferior colliculus, and the secondary auditory
cortex. These regions receive direct anatomical convergence of the CS
(auditory) and US (somatosensory) pathways, suggesting that the
influence of US pathways may be relevant for the blocking effect. This
provided support for a US-based explanation of blocking. Additional
evidence suggests that blocking of eyeblink conditioning depends on a
negative feedback circuitry related to the US (Kim et al.
1998
); this is also consistent with the US-based interpretation
of blocking afforded by the Rescorla-Wagner model (Rescorla and
Wagner 1972
).
The current paper investigates the Kamin blocking effect and contiguity
effects in a FDG mapping of rat auditory and extra-auditory regions
implicated in learning-dependent, evoked activity differences in our
previous FDG studies of conditioning. The results suggest that the
blocking of conditioned emotional responses (CER) to a tone is not
simply dependent on reductions in CS auditory processing pathways as
would be anticipated by CS inattention theories or reductions in
extra-auditory circuits implicated in CS-US contiguity as would be
anticipated by an attention or CS-US associative deficit. Rather,
blocking of an excitatory tone CER may result from metabolic suppression of the prefrontal cortex as would be anticipated by theoretical accounts of blocking based on CR inhibition (Balaz et al. 1982; Miller et al. 1993
;
Schachtman et al. 1983
) and activation of lower US
somatosensory pathways as would be anticipated by theoretical accounts
of blocking based on US saturation (Rescorla and Wagner
1972
).
D. Jones submitted this work in partial fulfillment of the requirements for a Ph.D. degree at the University of Texas at Austin.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Subjects and apparatus
Subjects were 32 male Long-Evans black-hooded rats weighing an
average of 100 g when received from the supplier (Harlan,
Indianapolis, IN). Subjects were housed in Association for Assessment
and Accreditation of Laboratory Animal Care-approved facilities
under standard laboratory conditions, two to three to a cage with free
access to food and water. Following 1 wk of acclimation and handling,
all subjects were placed on restricted water for the duration of
training. Training was conducted in a standard operant conditioning
chamber as described elsewhere (Poremba et al. 1997,
1998
). Two operant chambers (28 × 22 × 22 cm, model
No. 80000, MED Associates, St. Albans, VT) were used for training and
testing. These chambers were enclosed in sound-attenuating boxes to
eliminate extraneous acoustic stimuli. The front and back walls of the
operant chambers were aluminum and the sides were clear Plexiglass. A
5-cm hole in the front wall of each chamber allowed access to a
drinking tube. An infrared beam from a photo source module was focused directly in front of the drinking tube through clear glass rods (MED
Associates). This was used to monitor drink time and ensure that the
beam was broken only by drinking rather than some other activity (i.e.,
exploration of recess, freezing with head in the recess). Time spent
drinking was recorded on a AT&T PC 6300. Water was provided for 15 min
in the training context during baseline training trials and for a
45-min period in the home cage daily. Protocols for these experiments
were approved by the University of Texas at Austin Institutional Animal
Care and Use Committee, and conform to all applicable federal and
National Institutes of Health guidelines for the care and use of
animals in research.
Experimental stimuli
Two Wavetek sweep/modulation generators produced the acoustic stimuli. Tones were presented through two speakers, one mounted on top and one mounted in the front of the operant chamber, to produce whole-field stimulation. Two frequency-modulated (FM) acoustic stimuli were used: a low-frequency FM tone (1-2 kHz, 65 dB SPL) and a high-frequency FM tone (10-20 kHz, 65 dB SPL). The low-frequency tone was used as the CS, while the high-frequency tone was used as a comparator stimulus. A visual stimulus was provided by two flashing white lights (intensity of 200 foot-candles measured 2 cm from the source). The light source was outside the clear Plexiglas side of the chamber approximately 5 cm from the front. Background illumination was provided by a red light mounted outside the rear of the chamber (intensity of 100 ft.-cd measured 2 cm from the source). The grid floor of the chamber was wired for US delivery to a Lafayette Instruments master shocker (Lafayette IN). The US was a 0.75-s, 0.5-mA footshock. In CS-US paired trials, the US co-terminated with the 15-s CS. Intertrial intervals ranged randomly between 60 and 180 s. Presentation of stimuli and collection of drink data were controlled by computer programs created using MED-PC behavioral programming language (MED Associates).
Behavioral protocol
Training involved the CER or conditioned fear paradigm. In this protocol, training establishes a stimulus as predictive of an aversive event and the degree of conditioning is assessed by measuring disruption of ongoing behavior such as drinking. Total drink time during a 15-min period in the training context was collected on days 1-4. This average level of baseline drinking was used to order subjects and alternatively assign them to the three groups. This was done to obviate any group differences in individual water consumption prior to formal training (Table 1). During the course of the experiments, several subjects were lost to difficulties in tissue processing and data acquisition. This resulted in n = 6 for the tone-blocked group, n = 7 for the tone-excitor group, and n = 7 for the pseudorandom group. The previous groups were equated on stimulus history, therefore the use of additional CS alone or US alone groups were avoided as they would be confounded by different stimulus histories.
|
Training followed three basic phases; phase I consisted of light training, phase II consisted of compound training, and phase III was FDG testing. In the tone-blocked group, phase I excitatory training consisted of conditioning the flashing light as an excitatory CS (i.e., light will evoke CER) through four reinforced trials a day on days 5-8. In the tone-excitor group, the light and shock were presented four trials a day for days 5-8. Unlike the tone-blocked group, all but one of the light and US presentations were unpaired in the tone-excitor group. The light presentations were given in one session, and US presentations were in a different session on the same day. These sessions were separated by at least 1 h. The remaining light and shock stimulus in the tone-excitor group was given as a paired presentation that randomly occurred on training day 6 or 7. This resulted in a 100% probability of light-shock pairing in the tone-blocked group and a probability of 0.0625 for light-shock pairing in the tone-excitor group. This was done to minimize any potential for the light in the tone-excitor group to acquire properties consistent with conditioned excitation or inhibition. Phase II proceeded identically for both groups. The tone/light compound was paired with footshock four times a day on days 9, 10, and 14 for a total number of 12 reinforced presentations.
The pseudorandom group received pseudorandom presentations of the same
tones, lights, and foot shocks. These were not explicitly unpaired
stimuli presentations because there was less than a 0.04 probability of
tone or light being paired with footshock (1 light/footshock and 1 tone/footshock pairing over 7 days). This served to prevent the stimuli
from acquiring a reliable predictive value of footshock delivery
(Poremba et al. 1998). Therefore the number of days and stimuli presentations was the same in all groups. For a summary of the
experimental design, see Table 2.
|
In all subjects, 3 days of probe trials measured the degree of conditioning to the context, light, tone, and a comparator stimulus on days 11-13 and 15. Day 11 consisted of exposure to the training context and observation of any contextual fear conditioning. The probe sessions on days 12 and 13 consisted of tests of responding to three presentations of the discrete stimuli as well as summation tests of compound stimuli (Table 1). On day 15, 1 h prior to the FDG session, a final probe of three presentations of the CS tone alone was used to confirm behavioral effects before the FDG administration. Measures of drink time and behavioral counts of freezing were taken during the probe sessions.
As an index of excitatory conditioning, suppression of drinking was
measured and reported in the form of a suppression ratio. Time spent
drinking was collected during a 15-s period prior to the onset of the
CS and during the 15-s CS presentation period. Drink time prior to CS
presentation (pre-CSdrink-time) was used as an
indication of baseline drinking, while drink time during the CS
presentation (CSdrink-time) indicated any
disruption caused by the stimulus presentation. A ratio that expressed
this behavioral suppression was calculated as follows:
CSdrink-time/(pre-CSdrink-time + CSdrink-time) (Kamin 1969).
Suppression ratios range from 0 to 0.5. A value of zero indicates
strong conditioned suppression to the CS, and 0.5 indicates no change
in drinking behavior as a result of the CS presentation.
In addition to suppression measures, direct measures of freezing behavior were taken. Researchers blind to experimental condition monitored each subject's behavior. Behavior was monitored in 3-s intervals during the 15-s CS presentations. In each period, a freezing response was recorded if the rat was standing motionless with short rapid breathing as indicated by slight body movement. A total of five counts were possible for each 15-s CS period. For example, if a subject exhibited freezing behavior continuously during the CS period, a count of five for freezing was recorded.
At the time of the FDG uptake test, all subjects were presented with
the same low-frequency tone unreinforced in a context changed from that
of training. Adding a smooth metal plate on the floor, placing
horizontal black lines over the Plexiglas front and swabbing an iodine
surgical scrub solution (Betadine) on the walls and floor of the
chamber changed the context. Since rats show learned responses to the
training context (Miller et al. 1993), it was important
to change the context to control for context generated cues. While the
effect of associations involving contextual cues on brain metabolism is
interesting, the present study was aimed at revealing the effects of a
discrete CS. Presentation of the CS outside of the training context
leads to robust CERs in this paradigm (McIntosh and
Gonazalez-Lima 1993
-1995
, 1998
). Furthermore, changes in
context following phase II training does not attenuate the blocking
effect (Giftakis et al. 1998
). During the FDG uptake
period, the brain effects were evoked by the tone CS rather than other
learned cues.
Tissue processing and autoradiography
On the day of FDG administration, all animals were allowed
15-min free access to water prior to the FDG uptake period. This was
done to equate groups on water access and avoid dehydration during the
FDG uptake period. FDG administration [an intraperitoneal injection of
18 µCi/100 g body wt of universally labeled
[14C]2-deoxy-2-fluoro-glucose (2-DG; 360 mCi/mM
specific activity; American Radiolabeled Chemicals, St. Louis,
MO)] in 0.36 ml sterile saline followed. FDG was used, as opposed to
2-DG, because rates of transport of FDG across the blood-brain barrier
and phosphorylation in the cell are significantly better than those of
2-DG and because FDG universal labeling allows for a greater signal
(Gonzalez-Lima 1992). Immediately following injection,
the subject was placed in the test chamber, and stimulus presentation
was initiated. The test period was 60 min, during which 5-s tone (1-2
kHz sweep) presentations were separated by 1-s intervals as this
stimulation has resulted in optimal evoked FDG uptake and no evidence
of CER extinction under our testing conditions (McIntosh and
Gonzalez-Lima 1993
-1995
, 1998
).
Following completion of the testing period, animals were removed from
the testing room and immediately decapitated. Brains were removed
rapidly and frozen in isopentane at 40°C. The brain was then
mounted and sectioned at 40 µm at
20°C in a cryostat (Reichert-Jung 2800 Frigocut E). Sections were picked up on slides at
room temperature and dried on a hot plate at 60°C for later processing with X-ray film.
Dried slides were mounted on black paper, opposed to Kodak EB-1 X-ray film and placed in Kodak X-O-Matic cassettes. Acrylic micro-scale standards of known 14C concentrations (Amersham, Arlington Heights, IL) were placed with each piece of film. Following a 2-wk exposure period, film was developed in Kodak D-19 developer at 25°C for 2 min, rinsed in Kodak indicator stop, fixed for 10 min, and rinsed in water for 10 min. Variability in exposure time and development was obviated by the use of standards, and further readings were automatically expressed as 14C isotope incorporation (nCi/g tissue).
Imaging analysis
Using the same slides previously exposed to X-ray film, sections
were stained for Nissl substance with cresyl violet and used to
corroborate selection of regions of interest for analysis. A
stereotaxic atlas (Paxinos and Watson 1986) and our
Nissl/cytochrome oxidase atlas (Gonzalez-Lima and Cada
1998
) of the rat brain were used to identify brain sections
that approximately matched the levels of sections between Bregma +3.7
and
13.3 mm. At each Bregma level, three adjacent sections up to 120 µm of anteroposterior distance were sampled (Fig. 4). Both gray and
white matter landmarks were used to locate each region analyzed at the
same level in all subjects. The regions of interest (ROI) were selected
based on our previous FDG studies of conditioning that showed tone
conditioning effects on auditory and extra-auditory integrative regions
(Gonzalez-Lima 1989
, 1992
; Gonzalez-Lima and
Agudo 1990
; Gonzalez-Lima and Scheich 1984
-1986
; Gonzalez-Lima et al. 1989
). In
addition, control regions were analyzed including white matter (optic
tract and spinal trigeminal tract) and regions to control for
nonspecific motor and arousal effects such as primary motor cortex
(lateral frontal cortex) and reticular formation (lateral and
magnocellular reticular nuclei).
The autoradiographs were analyzed using an image-processing system consisting of a high-gain video camera, a Targa-M8 image capture board, a 486 computer, Sony color monitor, DC-powered illuminator, and JAVA software (Jandel Scientific, San Rafael, CA). The system was calibrated using the plastic 14C standards from Amersham.
Multiple readings were taken from left and right sides of each section to avoid artifacts. For each region measured, the size of the densitometer window was set to approximate the size of the whole region. In cases of odd-shaped ROIs, a smaller size window was used, and adjacent measurements were taken and averaged to sample the entire region. The averaged measures resulted in one mean value per region for each subject. The size of the window was held constant across subjects as was the number of readings for each ROI. Thus the area measured was constant across all animals and was limited to the anatomical region of interest.
Statistical analysis
In addition to the individual regions of interest, a measure of
global brain activity was taken by averaging the isotope incorporation from the entire set of sections in the series of a given subject. This
global measure of activity was used as a reference for group differences in overall metabolic activity. These global measures did
not differ in the experimental groups (P < 0.98, 2-tailed t-test), but there was a significantly lower level
of global FDG labeling in the pseudorandom group. Due to this overall
difference, regional FDG incorporation was adjusted based on
white-matter readings, and the estimated marginal means were compared.
Following this correction, measures of whole brain activity did not
differ across groups. An ANOVA was performed following the statistical removal of nonspecific differences in overall FDG incorporation. Data
are presented in the form of mean FDG uptake for each ROI adjusted by
the covariate white-matter readings. The statistical significance of
the results at P < 0.01 was tested with confidence intervals produced through the ANCOVA analysis. Each ROI comparison is
treated as independent of the others (a premise of regional brain mapping) and one simply accepts that 1 of 100 comparisons (for
P = 0.01) may be type 1 errors. This is a standard
procedure in neuroimaging studies that cannot apply any correction for
multiple comparisons due to the large number of ROIs analyzed
(Nobrega 1992). All structures were analyzed at
P < 0.01 level, and systematic bias within group error
variance were minimized through the use of the ANCOVA procedure.
Behavioral data were analyzed by repeated measures ANOVA with tests for
simple effects where appropriate. The covariance structure of the ROIs
was also examined. Pairwise correlations were calculated between pairs
of normalized values of regional glucose metabolic activity within each
group using SPSS 8.0 software. To control for inflation of family-wise
error rates due to the large number of correlations relative to the
sample size, a "jackknife" procedure was used (Shao and
Dongsheng 1995). In this procedure, the calculation of pairwise
correlations is repeated for each possible n
1 subset of the sample. Only those correlations that are significant
across all subsets are used for the final analysis. Thus spuriously
high correlations that are dependent on a single subject are screened out.
Areas that survived the jackknife procedure were tested to see if they
had a correlation coefficient that differed significantly between
groups. The Fisher Z transformation was used to convert each
correlation to a Z score. A test statistic of the form
Z = (Zij(group 1) Zij(group
2))/
(1/ng1) + (1/ng2) was used to compare across
group differences evaluated at
= 0.01 level. The use of this
data set of correlations for structural equation modeling revealed
evidence of multicollinearity: i.e., there were several correlations
above 0.8 in each group. This presents problems in drawing inferences
about the nature of the path coefficients in structural modeling
(Asher 1983
). The total coefficient of determination (an
index of the amount of variation in the observed variables explained by
the model) should have a value between zero and one. This ratio of the
determinants of the residual to fitted covariance matrices
(Jöreskog and Sörbom 1989
) was below 0.5 for
all groups, suggesting the model was accounting for little of the
observed variance. In some cases, this value was negative
(pseudorandom =
1.16; excitor =
0.84), indicating that
the observed variance was being accounted for by residual values rather
than the path coefficients. For these reasons, it was concluded that
covariance structural equation modeling was not informative as applied
to this particular data set.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Behavioral results
Conditioned suppression was confirmed in probe trials prior to FDG administration. The presentation of the tone CS was expected to produce distinct behaviors in the three groups. In the tone-blocked group, little or no interruption or suppression of drinking was predicted for the tone, whereas in the tone-excitor group, significant suppression of drinking was expected for the tone. This constitutes the traditional comparison used to demonstrate the Kamin blocking effect. The light was expected to produce CERs in both groups, as it was paired with shock in phase I of the tone-blocked group and phase II of both groups (Table 2). The pseudorandom group was expected to show no CERs to the tone or the light.
The conditioned suppression ratio for the tone CS in the tone-blocked group was always significantly higher than that of the tone-excitor group [F(1, 12) = 101.47, P < 0.0001, repeated-measures ANOVA], indicating the previous training with the light effectively blocked conditioned suppression to the tone in the tone-blocked group (Fig. 2).
|
However, the novel high-frequency comparator tone failed to produce significantly greater suppression of drinking in the tone-blocked group (0.28 ± 0.05; mean ± SE). A nonsignificant suppression ratio to the comparator tone in the tone-excitor group (0.18 ± 0.06) is most probably indicative of some generalization from the CS tone. As expected, both conditioned groups showed a strong suppression to the light alone (tone excitor, 0.14 ± 0.04; tone blocked, 0.03 ± 0.02) and to the compound (tone excitor, 0.06 ± 0.02; tone blocked, 0.03 ± 0.02).
Furthermore there was significantly less freezing to the CS tone in the tone-blocked group than in the tone-excitor group [F(1, 11) = 9.18, P < 0.03; Fig. 3]. As anticipated, the pseudorandom group showed no evidence of CERs, with mean behavioral counts of freezing of 0.93 ± 0.13 (Fs < 1.0).
|
The possibility that group differences in responding to the tone result from tone-excitor group enhanced responding to the tone due to putative inhibitory conditioning to the light is unlikely. The possibility of an inhibitory association with the light was lessened by the lack of an explicitly unpaired light-US relationship (probability of pairing = 0.0625). Additionally, the similar level of freezing to the light in both the tone-excitor and tone-blocked group is inconsistent with inhibitory conditioning of the light in the tone-excitor group (Fig. 3).
FDG uptake in the auditory system
Figure 4 illustrates the location of
ROIs and highlights significant differences across groups. There were
no differences in mean FDG uptake evoked by the tone in the auditory
system between tone-blocked and -excitor groups (Table
3). But both conditioned groups showed
regional means significantly greater than the pseudorandom group at
P < 0.01. These conditioning effects were localized to the inferior colliculus and the auditory cortex. The largest effect was
in the central nucleus of the inferior colliculus, as in the first FDG
studies of Pavlovian conditioning (Gonzalez-Lima and Scheich
1984, 1986
). The central nucleus in tone-blocked and -excitor groups showed mean increases of 36% as compared with the effect of the
same tone in the pseudorandom group.
|
|
FDG uptake in structures with tone-blocked versus -excitor differences
The comparison of mean FDG uptake in extra-auditory areas showed
significant differences at P < 0.01 between
tone-blocked and -excitor groups in three regions (Table
4 and Table
5, I). FDG uptake in the medial
prefrontal cortex [MFC, also known as prelimbic (Zilles and
Wree 1985)] was largest in the tone-excitor group and smallest
in the tone-blocked group. This region showed the largest mean
percent difference (25%) between tone-blocked and -excitor groups.
However, the tone-excitor mean was not significantly greater than the
pseudorandom mean, suggesting that the MFC effect involved not just FDG
increase in the tone-excitor group but also some decrease in the
tone-blocked group (Fig. 5).
|
|
|
The spinal trigeminal nucleus (Sp5I) and the external cuneate nucleus (ECu) were the only regions found with greater means in the tone-blocked group than the tone-excitor group. However, the tone-excitor and pseudorandom groups showed similar means in Sp5I and ECu. Therefore these two structures are somatosensory medullary regions with FDG increases unique to the tone-blocked group, indicating an active role of US somatosensory pathways in the blocking effect.
FDG uptake in structures with group means greater than pseudorandom
These structures can be subdivided into two categories: regions with common effects in the tone-blocked and -excitor groups (i.e., both groups greater than pseudorandom; Table 5, II) and regions with effects unique to the tone-excitor group (Table 5, III). The first category included five regions: rostral caudate putamen (rCPU), perirhinal cortex (Per), CA1 of dorsal hippocampus (rCA1), lateral cerebellar hemisphere (CHL), and solitary tract nucleus (Sol). These effects were similar to those seen in auditory regions (Table 3) and suggest common blocked-excitor conditioning effects based on their CS-US contiguity not present in the pseudorandom group. Other regions showing greater means in the tone-excitor group as compared with both the tone-blocked and pseudorandom groups were the insular (Ins) and anterior cingulate cortex (CgA), vertical diagonal band nucleus (VDB, with P = 0.01 in tone-excitor vs. pseudorandom), anterior hypothalamic nucleus (AHy), and caudoventral caudate putamen (cCPU). Other regions listed in Table 5 showed FDG uptake increases unique to the tone-excitor group, which may be based on the CERs evoked by the tone in this group only.
Covariance analysis
Although there were many group differences in mean activational
effects, the inter-regional correlations of FDG uptake appeared similar
in the three groups with one exception. The correlation between the
anterior parietal cortex (US somatosensory cortex) and the lateral
septum was 0.98 in the tone-blocked group. This was significantly
different from the corresponding values in the tone-excitor and
pseudorandom groups (0.60 and
0.24, respectively). Other
nonsignificant results are summarized here by presenting the mean and
range of correlation coefficients (r) in the three groups:
tone-blocked mean = 0.38 (range 0.95-0.01), tone-excitor mean = 0.46 (range 0.91-0.04), and pseudorandom mean = 0.39 (range 0.91-0.03).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study served in localizing brain regions displaying Pavlovian conditioning effects on FDG uptake in response to the same physical tone. Brain activity differences are proposed to result from group differences in previous training because all subjects received the same tone stimulation after FDG administration. The main focus of this study is to describe FDG differences between the two conditioning groups (tone-blocked vs. tone-excitor), while the pseudorandom group provides a control for unconditioned tone effects. In both the tone-blocked and -excitor groups, the subjects' experience with the tone was identical. At the time of FDG testing, the resulting differences in behavior and concomitant changes in FDG incorporation are most likely due to each groups history with the light presentations. While the tone was contiguous with the US during phase II training in both conditioning groups, the tone did not elicit a conditioned response in the tone-blocked group as the light was predictive of the US following phase I training. In the tone-excitor group, this was not the case; during phase I, the light did not predict the occurrence of the US, and both the light and tone came to elicit a conditioned response following phase II training. The pseudorandom control group received unconditioned stimuli identical to both experimental groups but lacked contiguity and CER effects. Subjects in this control group were not exposed to paired light-US presentations (similar to the tone-excitor group) and pseudorandom subjects did not display a CER to the tone. This absence of a CER was also seen in the tone-blocked group.
The brains of tone-blocked and -excitor rats differed in their average
metabolic responses in certain regions that may be implicated in the
Kamin blocking effect on Pavlovian conditioning. Hence this study
provides a first large-scale map of brain regions underlying the
blocking effect. However, all brain regions identified as having group
differences are not necessarily components of the blocking effect per
se. Conditioning effects that result from a blocking training protocol
may include various associative and nonassociative mechanisms.
Inferring other functional aspects of FDG uptake differences remains an
interpretive challenge (Gonzalez-Lima 1992), but one
that offers an opportunity to integrate relevant research into a
systems-level model. In light of previous studies, we attempt to do
just this, proposing neural pathways that may underlie three major
conditioning effects within the constraints of our experimental design:
blocking effects unique to the tone-blocked group; contiguity effects
common to the tone-blocked and -excitor groups; and excitatory effects
unique to the tone-excitor group. Table 5 provides a summary of these effects.
Blocking effects
There were two types of blocking effects on the brain, involving
higher (frontal) and lower (medullary) regions that may be engaged in
complementary behavioral mechanisms related to the blocking phenomenon.
One type of brain effect was localized to the medial prefrontal cortex.
This region showed a 25% mean difference between the tone-blocked and
-excitor groups. While the tone-excitor mean was greater than the
tone-blocked mean, the tone-excitor was not significantly greater than
the pseudorandom. Therefore the data suggest that the blocked-excitor
difference in the medial prefrontal cortex was not only due to
increased FDG uptake in the tone-excitor group, but also it involved
reduced activity in the tone-blocked group. Anatomically, the
identified medial prefrontal cortex region sends a projection to the
anterior cingulate (Beckstead 1979) and thereby can
influence Pavlovian excitatory effects found in the anterior cingulate.
This effect in the medial prefrontal cortex is consistent with previous
studies of this region, which has well-documented roles in behavioral
inhibition and selective attention that may be relevant for blocking.
This effect may be also of clinical significance because schizophrenics characterized with abnormal FDG uptake in the prefrontal cortex are
unable to develop the blocking effect on Pavlovian conditioning (Jones et al. 1997
).
The second type of brain effect of blocking was the activation of two subcortical somatosensory nuclei, the spinal trigeminal and the external cuneate nuclei in the medulla. These are major relay nuclei for the two main somatosensory pathways that transmit the ascending effects of the aversive US (electric shock) from the periphery to the brain: the trigeminal pain pathway (spinal trigeminal nucleus) and the dorsal column-medial lemniscus tactile pathway (external cuneate nucleus). Interestingly, our analysis of inter-regional correlations of FDG uptake revealed only the correlation between the primary somatosensory cortex (anterior parietal cortex) and lateral septal nucleus to be different in the tone-blocked group as compared with the other two groups. Therefore it may be concluded that a blocked tone activates somatosensory US pathways in a way different from the same tone as an excitor.
How these blocking effects on the brain relate to theoretical mechanisms derived from behavioral studies is discussed in relation to three classes of theories that emphasize either CS, US, or CR mechanisms.
Mackintosh (1983) proposed a CS-based theoretical
mechanism that explains blocking through decreased or selective
attention shifted away from a stimulus that has no added predictive
value during phase II of compound training (the tone in this
experiment). An attractive contribution for the frontal and anterior
cingulate areas in blocking is one of selective attention
(Mesulam 1990
; Posner et al. 1987
). The
medial prefrontal and CgA cortices have been implicated in attentional
processing by a wide array of imaging and neurophysiological studies.
We argue that the absence of blocking effects in the auditory
structures rules out a CS processing failure account, but this is true
only if it is believed that associate effects in primary sensory
structures must be attentional in character. The data suggest the
intriguing alternative that the acoustic associative changes are
preattentive and that attentional modulation is only exhibited in
association cortical areas such as the prefrontal cortex and anterior
cingulate. In other words, if the association cortical component is
compromised while the acoustic processing component is intact, CS
processing may still fail.
However, the similar excitatory effects of the tone CS on the auditory
system in both tone-blocked and -excitor groups argue against an
attention shift away from the blocked tone as suggested by popular
views of blocking. The enhanced effect of the blocked tone in the
auditory system provides neural evidence against CS/attentional explanations of blocking. Some of the inattention accounts of blocking,
including that of Kamin (1969), imply that the CS is ignored or somehow rejected so that no proper CS-US association may be
formed during blocking. These accounts have been referred to as CS
processing-failure explanations of blocking (Mackintosh 1983
). However, our findings of similar FDG increases to the
tone in the auditory system of tone-blocked and -excitor groups do not
support this explanation of blocking. It does appear that the blocked
tone is as effective at producing conditioned activation of auditory
regions as the tone conditioned as an excitor. Therefore blocking is
not likely the result of simple reductions in CS processing pathways,
and it would be hard to reconcile a role of decreased CS processing as
part of the blocking effect.
Our previous study of blocking effects on the auditory system using
cytochrome oxidase histochemistry (Poremba et al. 1997) may shed some light on how blocking develops during training. But there
has to be a clear understanding of the different kind of information
provided by our cytochrome oxidase study. The main difference is that
the cytochrome oxidase study reflects effects of all stimuli
through out training (i.e., tone, shock, and light). In contrast,
the present FDG study reflects responses to the tone after
training (i.e., during a posttraining FDG uptake period with tone
CS alone). There is no analogous posttraining uptake period in a
cytochrome oxidase study, so any differences in cytochrome oxidase at
the time of brain histochemistry reflect cumulative CS and US effects
building-up during the entire training period. Interestingly,
cytochrome oxidase suggests the auditory system is activated
during blocking only in regions receiving anatomical connections from US somatosensory pathways: dorsal cochlear nucleus, inferior colliculus, and secondary auditory cortex (Poremba et al. 1997
). So it is unlikely that blocking develops due to a
lack of tone-shock CS-US association in the brain as implied by some CS
inattention accounts of this phenomenon (Mackintosh
1983
). Together these studies point to the US influence in the
brain as more relevant than the CS influence during blocking.
The neural activational effect on US pathways can be interpreted as
support of the most well-known behavioral theory that deals with the
blocking effect (Rescorla and Wagner 1972). The Rescorla-Wagner model of learning suggests that blocking involves the
modification of US pathways. That is, there may be a limit in the
amount of learning that a US can support, and the blocking effect may
result from saturating activity in US relay pathways. The specific
neuroimaging finding of differences in subcortical somatosensory nuclei
seems to support this US-based theoretical mechanism, at least in part.
Thus we derive support for the Rescorla-Wagner model from the enhanced
activation of the somatosensory areas, suggesting that this model
predicts that blocking is due to modification of the US pathway. Yet
the Rescorla-Wagner model suggests that a given US supports a fixed
amount of associative strength and blocking occurs because some or all
of the available strength was preinvested in the light-shock
association. These dynamics seem to be a matter of interference or
associative competition rather than US efficacy per se.
The suppressive effect found in the medial frontal cortex suggests that
blocking may also involve CR mechanisms. A third theoretical mechanism
based on behavioral studies suggests that blocking involves a
conditioned response inhibition (Miller et al. 1993).
That is, tone-shock contiguity may support a CS-US association, but the CER is inhibited after phase II training in blocking. To the extent that the medial prefrontal cortex is involved in CR inhibition, our
neural findings support CR mechanisms that emphasize response failure
in blocking (Balaz et al. 1982
; Miller et al.
1993
; Schachtman et al. 1983
). Therefore the
brain regions modified in this study suggest a combined strategy with
higher (frontal) and lower (medullary) regions engaged in two
complementary mechanisms of CR inhibition and US saturation. Blocking
effects may then result from the interplay of these mechanisms in the
brain as they both contribute to the same behavioral outcome. This
general conclusion is consistent with the contiguity and excitatory
effects discussed in the next sections as well as our previous FDG
mapping studies of learning that indicate that the rat brain uses a
combined strategy to modify behavior as a result of experience
(Gonzalez-Lima 1992
).
Contiguity effects
The tone-blocked and -excitor groups showed a number of similar FDG effects relative to the pseudorandom group. These similar effects were interpreted as the result of tone-shock (CS-US) contiguity common to the conditioned groups but absent in the pseudorandom group. These common effects are unlikely due to the elicitation of a CER because, while the tone-excitor group showed a CER, the tone-blocked and pseudorandom groups did not. Similarly the common effects cannot be accounted for by the number of stimuli because they were the same in all groups. Thus the most likely explanation is that tone-shock pairings changed the way certain brain regions responded to the tone as compared with pseudorandom tone presentations.
This interpretation is reinforced by the finding of strong FDG uptake
increases in auditory regions such as the inferior colliculus and
auditory cortex in response to the tone in the tone-blocked and
-excitor groups. It has been a consistent finding in our FDG studies
since 1984 (reviewed in Gonzalez-Lima and McIntosh 1996) that pairing of a tone with shock or another aversive US leads to a
subsequent greater metabolic response to the tone in auditory structures. This is the case in the rat with tone-shock pairings as
well as in humans during eyeblink conditioning to a tone
(Molchan et al. 1994
).
Similar CS-US contiguity effects are also present in the current FDG
study between experimental groups in certain extra-auditory regions
(Table 5): rCPU, Per, rCA1, CHL, and Sol. These regions showed FDG
increases similar but lower than those seen in auditory regions. These
effects may indicate conditioning effects based on the CS-US contiguity
not present in the pseudorandom group. We have previously reported that
pairing of a tone with US reticular stimulation leads to FDG uptake
increases in caudate putamen and CA1, but the other regions were not
examined in that study (Gonzalez-Lima and Scheich 1986).
However, in a subsequent FDG study using tone-shock pairings, a tone
conditioned as an excitor compared with a tone conditioned as a
Pavlovian inhibitor revealed differences in Per and cerebellum
(McIntosh and Gonzalez-Lima 1994
). In that study, the
excitor effects on the brain could be accounted for by either CS-US
pairing or CER expression. The present results aid in the interpretation of our previous studies because CS-US pairing and CER
expression is separated across groups. The conditioning effects in
these regions are likely due to the common factor of CS-US contiguity
in the blocked and excitor groups rather than CER expression, which is
absent in blocked and inhibitor conditions.
Other studies involving learned associations have consistently
implicated these regions we attributed to contiguity effects, like the
rabbit CA1 and cerebellum in eyeblink conditioning (Lavond et
al. 1993) or the monkey Per in delayed nonmatching to sample and object retention memory (Zola-Morgan et al. 1989
).
In the rat Per, lesion studies have also shown that it is a key
structure in the circuit for conditioned fear-potentiated acoustic
startle (Rosen et al. 1992
). Finally, the solitary
nucleus has been implicated in conditioning effects by both neuronal
recording (Giza et al. 1997
) and lesion studies
(Grigson et al. 1997
). In particular, aversive
conditioning effects are greater than preference conditioning effects
in the rat solitary nucleus (Giza et al. 1997
), and
these activation effects do not disappear after conditioned response extinction (McCaughey et al. 1997
). Together with our
data, previous studies support the conclusion that these regions likely
mediate associative learning effects rather than effects due to
conditioned response expression. Results from the current experiment
are uniquely suited to support such a conclusion as CS-US pairings are
similar across experimental groups while the presence of a CER is
separated by groups.
Excitatory effects
Effects unique to the tone-excitor group (tone elicits CER) are referred to as excitatory effects in Pavlovian terminology. That is, they refer to conditioned response excitation in a behavioral sense rather than excitation in a neuronal electrophysiological sense. The tone is a CS excitor when it elicits a conditioned response as in the tone-excitor group but not in the tone-blocked or pseudorandom groups. There were five regions with FDG uptake in the tone-excitor group greater than the other two groups (i.e., tone-excitor > tone-blocked and pseudorandom in Table 5): the Ins, CgA, VDB, AHy, and cCPU.
The simplest explanation for these excitatory effects is that they are
due to CER components rather than any CS-US contiguity effects common
to tone-excitor and -blocked groups. However, these excitatory effects
are not simply the result of nonspecific motor or arousal effects
because relevant regions of the primary motor system (lateral frontal
cortex) and reticular system (lateral and magnocellular reticular
nuclei) showed no such effects. Each of the affected regions appeared
more specifically related to the CER. First, there is mounting evidence
that the Ins mediates visceral reactions and avoidance responses to
aversive stimuli. For example, lesions or reversible inactivation of
the rat Ins produce amnesia for inhibitory avoidance
(Bermudez-Rattoni et al. 1991). The Ins receives taste
and visceral information from the thalamus and sends direct projections
to the Sol along with the anterior hypothalamus (van der Kooy et
al. 1984
). Thus the insular and anterior hypothalamic
projections to the Sol may mediate visceral components of the CER in
the tone-excitor group.
Similarly, the role of the CgA in avoidance responses to learned
aversive stimuli has been well documented in a series of elegant
studies by Gabriel and collaborators. For example, Gabriel (1990) has shown that a locomotive avoidance response to a tone that signals a shock is retarded after lesions of the cingulate cortex
in rabbits. Interestingly, early-developing training-induced unit
activity in the posterior cingulate (area 29) was absent in rabbits
with anterior cingulate (area 24) lesions, indicating that the anterior
cingulate is a source of early-developing plasticity in avoidance
conditioning (Gabriel et al. 1991
). Presumably Pavlovian CERs develop early in avoidance conditioning before an instrumental response is observed. Therefore the anterior cingulate change in the
tone-excitor group may reflect Pavlovian excitatory effects sustaining
CERs to tones paired with shock.
A region showing consistent tone-evoked excitatory effects in our FDG
conditioning studies is the cCPU. These effects occur whether an
aversive (Gonzalez-Lima and Scheich 1986) or an
appetitive US (Gonzalez-Lima 1992
) is used, as long as a
tone is the CS. We have proposed that this region links the auditory
system with a striatal motor response system (Gonzalez-Lima
1989
). The rat cCPU receives direct projections from both
auditory cortex (Roger and Arnault 1989
) and medial
geniculate (LeDoux et al. 1990
). Thus the cCPU contains
a secondary auditory projection field that is an anatomical
continuation of the primary auditory pathways into the neostriatum. The
contribution of this auditory striatal field to auditory Pavlovian
conditioning has been examined with lesion studies that showed that its
damage eliminates tone-evoked conditioned responses in rats
(Iwata et al. 1986
). Thus the cCPU may contribute a
conditioned striatal motor component to the CER elicited by the tone in
the tone-excitor group.
In addition, there were multiple distributed effects on regional
metabolism in other regions that were similar in tone-excitor and
-blocked groups but that showed FDG uptake increases in the tone-excitor group as compared with the pseudorandom group (listed in
Table 5). These other regions showing excitatory effects limited to the
tone-excitor group have been observed in our previous FDG studies. For
example, we identified these regions in a study of CER elicited by a
tone conditioned to reticular stimulation (Gonzalez-Lima and
Scheich 1986) as well as in a study using tone-shock stimuli (McIntosh and Gonzalez-Lima 1994
) comparing a tone as
excitor (elicits CER) versus the same tone as inhibitor (inhibits CER). Pavlovian conditioned excitation and inhibition affected the same circuits, but this effect was through a shift in sign in the
correlative activity between the regions from positive in the
tone-excitor condition to negative in the tone-inhibitor condition
(McIntosh and Gonzalez-Lima 1994
). Thus it appears that
Pavlovian conditioned inhibition and the Kamin blocking effect are
mediated by different brain mechanisms.
It also appears that lesion studies aimed at impairing a conditioned
response provide an incomplete picture of the effects of Pavlovian
conditioning in the intact brain. For example, damaging a relatively
small number of brain stem regions may impair a simple conditioned
response involving eyeblink or nictitating membrane closure
(Lavond et al. 1986). While lesions of the amygdala and related regions may impair the expression of a CER (Iwata et al. 1986
), it appears that large-scale networks are affected by
tone-shock conditioning in the intact brain.
For example, the medial frontal cortex may have important functional
contributions to tone blocking effects on the identified excitatory
brain circuits. The cortical limbic circuit receives auditory CS
information, and can influence the basal forebrain through medial
frontal cortex projections to the accumbens (Beckstead 1979; Phillipson and Griffths 1985
) and through
its reciprocal projection with the VDB (Saper 1984
). The
efferent projection from the medial frontal cortex to the cCPU
(Beckstead 1979
) may serve to compare different levels
of auditory CS processing. Auditory CS information can enter this
system at various levels. The strongest of these are the projections
from the medial geniculate and auditory cortex to the caudal caudate
putamen (LeDoux et al. 1985
), but the medial geniculate
also has efferents to the medial amygdaloid nucleus (Ottersen
and Ben-Ari 1979
). Auditory CS information can also reach the
medial preoptic area indirectly through the medial amygdaloid nucleus
(Conrad and Pfaff 1976
; McDonald 1987
).
The medial amygdaloid nucleus may play a role in stimulus aversive emotional value. C-fos immunoreactivity is reported to increase in the
medial amygdala of rats subjected to tail pinch (Smith et al.
1996
), and this nucleus has been shown to be important in
regulation of antinociceptive processes (Werka 1997
).
This would suggest that the role of the medial amygdaloid nucleus be related to US emotional properties evoked by the CS. It is clear from
this and previous neuroimaging studies that large-scale networks are
involved in the behavioral effects of tone-shock Pavlovian conditioning
in intact brains (McIntosh and Gonzalez-Lima 1998
). Therefore Pavlovian conditioning effects are of various kinds and are
pervasive throughout the brain of intact organisms, including humans
(Molchan et al. 1994
).
![]() |
ACKNOWLEDGMENTS |
---|
We thank M. Domjan for advice concerning the behavioral studies and A. Crane for advice on the manuscript.
This work was supported by National Institute of Neurological Disorders and Stroke Grant RO1 NS-37755 to F. Gonzalez-Lima.
![]() |
FOOTNOTES |
---|
Address for reprint requests: F. Gonzalez-Lima, Behavioral Neuroscience, Mezes Hall 330, University of Texas, Austin, TX 78712 (E-mail: gonzalez-lima{at}psy.utexas.edu).
Received 26 December 2000; accepted in final form 17 April 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|