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Mapping Pavlovian Conditioning Effects on the Brain: Blocking, Contiguity, and Excitatory Effects DIRK JONES AND F. GONZALEZ-LIMA Department of Psychology and Institute for Neuroscience, University of Texas at Austin, Austin, Texas 78712 Received 26 December 2000; accepted in final form 17 April 2001 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 (GonzalezLima 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 GonzalezLima 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 Address for reprint requests: F. Gonzalez-Lima, Behavioral Neuroscience, Mezes Hall 330, University of Texas, Austin, TX 78712 (E-mail: [email protected]). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. INTRODUCTION www.jn.org 0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society 809 Downloaded from http://jn.physiology.org/ by 10.220.33.3 on May 15, 2017 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 toneblocked 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. 810 D. JONES AND F. GONZALEZ-LIMA 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; J Neurophysiol • VOL 86 • AUGUST 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on May 15, 2017 FIG. 1. Schematic depiction of the ideas of contiguity and the Kamin blocking effect. The lines indicate time of presentation of various stimuli. In Pavlovian conditioning to a single stimulus, the conditioned and unconditioned stimuli (CS) and (US) are presented in close temporal proximity (A) and a conditioned emotional response is elicited. B: the 2 phases of training in the blocking design. CSE is the light paired with shock US that is conditioned as excitor (elicits a conditioned response) in phase I. CSB is the tone CS that is paired with the shock US in compound with the light during phase II training. In the tone-blocked group, this tone CS is blocked (fails to elicit a conditioned response) due to phase I training. 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 NEUROIMAGING OF CONDITIONING AND BLOCKING EFFECTS 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 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 lowfrequency 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). 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 toneexcitor 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 toneexcitor 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 TABLE 1. Summary of behavioral training Description Day Behavioral protocol Drinking baseline in operant chamber Phase I trials with four paired or unpaired light & US presentations Phase II excitatory training with 4 reinforced compound trials Post train probe of behavior to context, no discrete stimuli presented Post train probe of conditioning, 3 presentations of each stimulus Reinstatement training with 4 reinforced compound trials Probe on day of FDG test, 3 presentations of tone alone FDG post injection session with target tone 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. Total number of stimulus presentations is equal in the three groups. Only reinforcement history with the light stimulus in phase I is different between blocked and excitor groups. The pseudorandom group had only one paired trial with the light and one with the tone. US, unconditioned stimulus; FDG, fluorodeoxyglucose. J Neurophysiol • VOL 86 • AUGUST 2001 • www.jn.org 1–4 5–8 9–10 11 12–13 14 15 15 Downloaded from http://jn.physiology.org/ by 10.220.33.3 on May 15, 2017 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. 811 812 D. JONES AND F. GONZALEZ-LIMA TABLE 2. Experimental design Groups Phase I Phase II Tone CER FDG Test Tone blocked Tone excitor Pseudorandom L3S L, S L, S TL 3 S TL 3 S T, L, S No Yes No T T T T, 1 to 2-kHz tone FM sweep; L, flashing light; TL, tone/light compound; S, footshock. Both the blocked and excitor groups have tone conditioned stimulus (CS)-US contiguity in phase II, but the blocked group does not show a conditioned emotional response (CER) to the tone while the excitor group does. The pseudorandom group has no reliable contiguity and does not show a CER. 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 Ci/100 g body wt of universally labeled [14C]2-deoxy-2fluoro-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 J Neurophysiol • VOL 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 m 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. 86 • AUGUST 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on May 15, 2017 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. 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 m 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 microscale 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). NEUROIMAGING OF CONDITIONING AND BLOCKING EFFECTS 813 Zij(group 2))/公(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. 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) ⫺ J Neurophysiol • VOL 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 toneexcitor 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 FIG. 3. Freezing data for the 3 groups to the different test stimuli in the probe sessions. Behavioral counts represent the mean of observed behavior during the 2nd 2 15-s CS presentations. Each 15-s CS period was broken down into 5 bins of 3 s each. The significantly lower freezing count (*) in the tone-blocked relative to the tone-excitor group is further support that the light was effective in blocking the behavioral expression of conditioned emotional response (CER) to the tone. As expected, both blocked and excitor groups showed significantly greater freezing to the light alone or in compound with the tone as compared with the pseudorandom group. 86 • AUGUST 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on May 15, 2017 FIG. 2. Suppression ratios for the tone alone probe session on day 15. A score near 0.0 indicates strong conditioned responding, while a score near 0.5 indicates no change in drinking with the presentation of the tone. Some suppression seen on the 1st presentation of the tone was expected as animals will suppress to any initial stimulus presentation when first in the training boxes. The significantly lower suppression ratios (*) in the tone-excitor group compared with the tone-blocked group are evidence of the blocking effect [F(1, 12) ⫽ 101.47, P ⬍ 0.0001, repeated-measures ANOVA]. 814 D. JONES AND F. GONZALEZ-LIMA 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 toneexcitor 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 J Neurophysiol • VOL 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. 86 • AUGUST 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on May 15, 2017 FIG. 4. Images taken from the autoradiographs representing the approximate levels at which readings were taken. Regions of interest are indicated, and areas that reached statistical significance (P ⬍ 0.01) following the ANCOVA analysis are labeled. Bregma levels are indicated below each level. NEUROIMAGING OF CONDITIONING AND BLOCKING EFFECTS TABLE 815 3. FDG uptake in the auditory structures Structure Dorsal cochlear nucleus (DCN) Ventral cochlear nucleus-posterior (VCP) Ventral cochlear nucleus-anterior (VCA) Lateral superior olivary nucleus (LSO) Medial superior olivary nucleus (MSO) Trapezoid body nucleus (TBN) Lateral lemniscus nucleus-intermediate (ILL) Lateral lemniscus nucleus-ventral (VLL) Inferior colliculus nucleus-dorsal (ICD) Inferior colliculus nucleus-central (ICC) Inferior colliculus nucleus-external (ICE) Medial geniculate nucleus-dorsal (MGD) Medial geniculate nucleus-medial (MGM) Medial geniculate nucleus-ventral (MGV) Auditory cortex, area 1 (TE1) Auditory cortex, area 2 (TE2) Auditory cortex, area 3 (TE3) Mean Lower 99% Upper 99% Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor1 Pseudorandom Blocked1 Excitor1 Pseudorandom Blocked1 Excitor1 Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked1 Excitor1 Pseudorandom Blocked1 Excitor1 Pseudorandom Blocked1 Excitor1 Pseudorandom 1051 ⫾ 56 1091 ⫾ 52 1004 ⫾ 67 792 ⫾ 39 830 ⫾ 36 762 ⫾ 46 803 ⫾ 34 813 ⫾ 31 730 ⫾ 40 1084 ⫾ 52 1122 ⫾ 48 946 ⫾ 62 977 ⫾ 46 1011 ⫾ 42 876 ⫾ 55 660 ⫾ 30 668 ⫾ 28 640 ⫾ 36 645 ⫾ 28 666 ⫾ 26 617 ⫾ 33 746 ⫾ 35 745 ⫾ 33 704 ⫾ 42 778 ⫾ 38 793 ⫾ 35 764 ⫾ 45 729 ⫾ 39 780 ⴞ 36 641 ⫾ 46 1605 ⴞ 74 1598 ⴞ 69 1175 ⫾ 89 916 ⴞ 41 944 ⴞ 38 773 ⫾ 49 765 ⫾ 49 733 ⫾ 46 598 ⫾ 59 747 ⫾ 43 796 ⫾ 40 718 ⫾ 51 820 ⫾ 46 922 ⫾ 42 927 ⫾ 55 1028 ⴞ 50 1079 ⴞ 47 835 ⫾ 60 724 ⴞ 34 750 ⴞ 31 565 ⫾ 41 767 ⴞ 37 799 ⴞ 35 591 ⫾ 45 888 940 809 680 726 628 705 722 612 932 981 765 844 887 716 573 587 536 563 589 518 644 650 582 667 690 631 616 675 506 1388 1397 916 797 834 631 622 600 427 622 680 568 686 798 768 881 943 660 625 659 447 658 697 461 1214 1243 1199 905 935 897 902 905 848 1236 1263 1127 1111 1135 1035 748 749 745 728 742 715 849 840 827 889 895 896 843 886 777 1821 1799 1434 1035 1054 915 908 866 769 872 913 867 953 1046 1087 1175 1216 1011 823 842 683 876 900 722 FDG units are in nCi/g tissue wet weight. Regional mean ⫾ SE and 99% confidence intervals are shown for each group. Values are expressed as estimated marginal means following ANCOVA analysis using white-matter readings as a covariate. Superscript number and boldface indicate group mean greater than pseudorandom (P ⬍ 0.01). There were no auditory structures with significant differences between the blocked and excitor groups. 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 toneblocked 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. J Neurophysiol • VOL This region showed the largest mean percent difference (25%) between tone-blocked and -excitor groups. However, the toneexcitor 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. 86 • AUGUST 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on May 15, 2017 Lateral lemniscus nucleus-dorsal (DLL) Group 816 TABLE D. JONES AND F. GONZALEZ-LIMA 4. FDG uptake in the extra-auditory structures Structure Medial frontal cortex (MFC) Lateral frontal cortex (LFC) Sulcal frontal cortex (SFC) Cingulate cortex, anterior (CgA) Insular cortex (Ins) Medial septal nucleus (MS) Accumbens (ACB) Vertical diagonal band nucleus (VDB) Parietal cortex, anterior (Par1a) Caudate putamen, rostral (rCPU) Medial preoptic area (MPO) Horizontal diagonal band (HDB) Anterior hypothalamic nucleus (AHy) Paraventricular hypothalamic nucleus (PVH) Cingulate cortex, posterior (CgP) Mediodorsal thalamic nucleus (MD) Ventroposterior thalamic nucleus (VPL) Central amygdaloid nucleus (CeA) Basolateral amygdaloid nucleus (BLA) Medial amygdaloid nucleus (MeA) Ventromedial hypothalamic nucleus (VMH) Posterior parietal cortex (Par1p) Mean Lower 99% Upper 99% Blocked Excitor1 Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor1,3 Pseudorandom Blocked Excitor1,3 Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor3 Pseudorandom Blocked Excitor1 Pseudorandom Blocked Excitor Pseudorandom Blocked3 Excitor3 Pseudorandom Blocked Excitor3 Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor1,3 Pseudorandom Blocked Excitor3 Pseudorandom Blocked Excitor3 Pseudorandom Blocked Excitor3 Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor3 Pseudorandom Blocked Excitor3 Pseudorandom Blocked Excitor3 Pseudorandom 870 ⫾ 57 1086 ⴞ 53 943 ⫾ 68 899 ⫾ 40 990 ⫾ 37 878 ⫾ 48 1090 ⫾ 54 1219 ⫾ 51 1102 ⫾ 65 904 ⫾ 42 1073 ⴞ 39 892 ⫾ 51 678 ⫾ 32 771 ⴞ 29 630 ⫾ 38 692 ⫾ 34 765 ⫾ 31 695 ⫾ 40 524 ⫾ 28 603 ⫾ 26 508 ⫾ 33 586 ⫾ 29 668 ⴞ 27 520 ⫾ 35 682 ⫾ 21 756 ⴞ 20 683 ⫾ 25 921 ⫾ 47 979 ⫾ 44 938 ⫾ 56 854 ⴞ 22 871 ⴞ 21 762 ⫾ 27 529 ⫾ 41 666 ⴞ 38 456 ⫾ 49 817 ⫾ 30 859 ⫾ 28 780 ⫾ 36 470 ⫾ 42 603 ⴞ 39 437 ⫾ 50 514 ⫾ 35 616 ⴞ 32 455 ⫾ 41 1128 ⫾ 69 1204 ⴞ 64 932 ⫾ 82 912 ⫾ 44 1015 ⴞ 41 792 ⫾ 53 907 ⫾ 49 938 ⫾ 46 835 ⫾ 59 432 ⫾ 19 470 ⫾ 18 409 ⫾ 23 762 ⫾ 58 777 ⫾ 54 742 ⫾ 69 452 ⫾ 25 516 ⴞ 24 428 ⫾ 30 452 ⫾ 27 503 ⴞ 25 402 ⫾ 32 940 ⫾ 39 1010 ⴞ 36 870 ⫾ 47 703 931 744 782 881 738 930 1072 912 781 958 743 585 685 520 593 674 579 443 528 411 502 590 419 620 699 609 783 851 773 789 810 683 409 555 314 730 778 676 349 490 292 413 522 335 926 1017 691 783 897 639 763 805 663 377 419 343 593 620 540 378 448 339 373 430 308 825 904 732 1036 1241 1142 1016 1098 1018 1249 1367 1292 1029 1188 1040 770 857 741 790 856 813 605 678 605 671 747 621 744 813 756 1060 1107 1103 920 932 840 648 777 599 904 939 883 591 715 582 616 710 576 1329 1391 1172 1040 1134 946 1051 1072 1006 488 522 475 930 933 943 526 585 516 531 576 496 1055 1117 1007 J Neurophysiol • VOL 86 • AUGUST 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on May 15, 2017 Lateral septal nucleus (LS) Group NEUROIMAGING OF CONDITIONING AND BLOCKING EFFECTS TABLE 817 4. (continued) Structure Lateral habenula (Hb) Hippocampus CA1, rostral (rCA1) Hippocampus CA3, rostral (rCA3) Dentate gyrus (DG) Perirhinal cortex (Per) Caudate putamen, caudal (cCPU) Retrosplenial cortex (RSpl) Presubiculum (Psub) Subiculum (Sub) Hippocampus CA1, caudal (cCA1) Hippocampus CA2, caudal (cCA2) Hippocampus CA3, caudal (cCA3) Cerebellar hemisphere, lateral (CHL) Cerebellar hemisphere, medial (CHM) Deep cerebellar nucleus (DpCb) Medial vestibular nucleus (MVe) Solitary tract nucleus (Sol) External cuneate nucleus (ECu) Spinal trigeminal nucleus (Sp5I) Lateral reticular nucleus (LRt) Magnocellular reticular nucleus (MCRt) Mean Lower 99% Upper 99% Blocked Excitor Pseudorandom Blocked3 Excitor3 Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked3 Excitor3 Pseudorandom Blocked Excitor1,3 Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked3 Excitor3 Pseudorandom Blocked Excitor3 Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked3 Excitor3 Pseudorandom Blocked2 Excitor Pseudorandom Blocked2 Excitor Pseudorandom Blocked Excitor Pseudorandom Blocked Excitor Pseudorandom 976 ⫾ 55 1023 ⫾ 51 997 ⫾ 66 710 ⴞ 33 707 ⴞ 30 546 ⫾ 39 693 ⫾ 30 693 ⫾ 28 606 ⫾ 36 620 ⫾ 25 662 ⫾ 24 587 ⫾ 30 792 ⴞ 41 830 ⴞ 38 631 ⫾ 49 708 ⫾ 41 840 ⴞ 38 569 ⫾ 49 718 ⫾ 32 689 ⫾ 29 653 ⫾ 38 1105 ⫾ 51 1072 ⫾ 47 1005 ⫾ 61 892 ⫾ 36 888 ⫾ 34 884 ⫾ 44 847 ⫾ 40 788 ⫾ 37 820 ⫾ 48 611 ⫾ 25 603 ⫾ 23 571 ⫾ 30 639 ⫾ 26 625 ⫾ 24 560 ⫾ 31 703 ⫾ 32 693 ⫾ 30 605 ⫾ 38 805 ⴞ 18 788 ⴞ 17 646 ⫾ 22 622 ⫾ 13 639 ⴞ 12 581 ⫾ 16 900 ⫾ 24 916 ⫾ 22 915 ⫾ 29 1031 ⫾ 31 1047 ⫾ 29 989 ⫾ 37 627 ⴞ 12 627 ⴞ 12 577 ⫾ 15 738 ⴞ 23 673 ⫾ 21 672 ⫾ 27 758 ⴞ 22 692 ⫾ 21 723 ⫾ 27 631 ⫾ 27 618 ⫾ 25 585 ⫾ 31 672 ⫾ 16 669 ⫾ 15 650 ⫾ 20 815 874 804 614 619 433 605 611 501 546 593 498 673 720 489 590 729 427 626 604 542 957 934 828 786 789 756 731 680 681 537 535 483 563 555 469 609 606 493 752 738 582 583 602 534 830 849 831 940 962 880 591 594 533 671 612 593 693 631 645 554 546 492 624 625 593 1138 1173 1190 805 795 660 781 775 712 694 730 675 911 940 773 827 950 711 810 775 763 1254 1210 1183 999 986 1011 963 896 959 685 671 659 715 696 651 797 780 717 859 838 711 661 675 627 971 980 1000 1122 1131 1098 663 661 620 804 735 751 823 752 801 709 689 677 720 714 707 FDG units are in nCi/g tissue wet weight. Regional mean ⫾ SE and 99% confidence intervals are shown for each group. Values are expressed as estimated marginal means following ANCOVA analysis using white matter readings as a covariate. Superscript numbers and boldface indicate: 1, excitor greater than blocked; 2, blocked greater than excitor; and 3, indicated group greater than pseudorandom (P ⬍ 0.01). J Neurophysiol • VOL 86 • AUGUST 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on May 15, 2017 Entorhinal cortex (Ent) Group 818 D. JONES AND F. GONZALEZ-LIMA 5. Summary of extra-auditory regions with effects of conditioning 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 toneblocked 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). TABLE I. Blocking effects (unique to blocking group) 1. Blocked lower than excitor Medial frontal cortex (MFC) 2. Blocked greater than excitor Spinal trigeminal nucleus (Sp5I) External cuneate nucleus (ECu) II. Contiguity effects (common to excitor and blocked groups) 1. Excitor and blocked both greater than pseudorandom Caudate putamen, rostral (rCPU) Perirhinal cortex (Per) Hippocampus CA1, rostral (rCA1) Cerebellar hemisphere, lateral (CHL) Solitary tract nucleus (Sol) III. Excitatory effects (unique to excitor group) 1. Excitor greater than both blocked and pseudorandom Insular cortex (Ins) Cingulate cortex, anterior (CgA) Vertical diagonal band nucleus (VDB) Anterior hypothalamic nucleus (AHy) Caudate putamen, caudal (cCPU) 2. Excitor greater than pseudorandom Accumbens (ACB) Medial preoptic area (MPO) Paraventricular hypothalamic nucleus (PVH) Mediodorsal thalamic nucleus (MD) Medial amygdaloid nucleus (MeA) Cingulate cortex, posterior (CgP) Ventromedial hypothalamic nucleus (VMH) Posterior parietal cortex (PPC) Cerebellar hemisphere, medial (CHM) DISCUSSION 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 J Neurophysiol • VOL FIG. 5. Examples of differences in fluorodeoxyglucose (FDG) incorporation in the medial (MFC) area of the prefrontal cortex (A–C, left) and corresponding profiles through a representative level of the autoradiograph (A–C, right). D: a schematic of the area (box) and level (dotted line) at which readings for the profile were taken. Although darker (more FDG) labeling may be seen in this region by the unaided eye, pictures of autoradiographs are examples only and as such some individual variation may be seen. The results are not merely based on visual inspection of autoradiographs but also on quantitative group data collected from the actual films with a calibrated densitometer as reported in Tables 3 and 4. 86 • AUGUST 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on May 15, 2017 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 admin- NEUROIMAGING OF CONDITIONING AND BLOCKING 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 J Neurophysiol • VOL 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 processingfailure 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 86 • AUGUST 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on May 15, 2017 istration. 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. 819 820 D. JONES AND F. GONZALEZ-LIMA J Neurophysiol • VOL 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 GonzalezLima 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 GonzalezLima 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 (ZolaMorgan et al. 1989). In the rat Per, lesion studies have also 86 • AUGUST 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on May 15, 2017 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 NEUROIMAGING OF CONDITIONING AND BLOCKING EFFECTS 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 J Neurophysiol • VOL 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 toneexcitor 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 86 • AUGUST 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on May 15, 2017 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 toneexcitor group may reflect Pavlovian excitatory effects sustaining CERs to tones paired with shock. 821 822 D. JONES AND F. 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