Lesions of the Perirhinal Cortex Interfere With Conditioned Excitation but Not With Conditioned Inhibition of Fear

Transcription

1 Behavioral Neuroscience Copyright 1997 by the American Psychological Association, Inc. 1997, Vol. 111, No. 3, /97/$3.00 Lesions of the Perirhinal Cortex Interfere With Conditioned Excitation but Not With Conditioned Inhibition of Fear William A. Falls, Karl T. Bakken, and Scott A. Heldt Northern Illinois University Posttraining lesions of the perirhinal cortex (Prh) have been shown to interfere with the expression of fear. This study assessed whether Prh lesions would also disrupt the inhibition of fear as measured with conditioned inhibition of fear-potentiated startle. Following light + shock, noise ~ light-no shock conditioned-inhibition training, rats were given Prh lesions. The lesions interfered with the expression of fear-potentiated startle to the light. To assess whether conditioned inhibition was affected, the rats were given light + retraining without additional noise --* light - training. The noise-conditioned inhibitor retained its ability to inhibit fear-potentiated startle to the retrained light. These results suggest that the areas of the Prh that are essential for the initial expression of conditioned fear are not important for the expression of conditioned inhibition of fear. Considerable progress has been made in identifying the neural systems that are involved in conditioned fear (Davis, Campeau, Kim, & Falls, 1995; LeDoux, 1987; Mishkin & Appenzeller, 1987). With very few exceptions, these studies have focused on investigating the neural systems involved in the acquisition and expression of conditioned fear. However, fear is not only acquired and expressed, but under some circumstances fear can be reduced or inhibited. Very little is known about the neural systems that are involved in the inhibition of fear. Clearly, to more fully understand the neural systems involved in conditioned fear, it is necessary to also evaluate the neural systems that are involved in the inhibition of fear. Falls and Davis (1997) have detailed a procedure that may be particularly well suited for evaluating the neural systems that are involved in the inhibition of fear: conditioned inhibition of fear-potentiated startle. In the basic fearpotentiated startle paradigm, conditioned fear is operationally defined as elevated startle amplitude in the presence of a light that has been previously paired with shock (Davis & Astrachan, 1978). Conditioned inhibition of fear-potentiated startle was produced by giving rats training in which a light was paired with shock (i.e., light+), and a serial noise and light compound was presented without shock (i.e., noise---* light-). Following this training the rats showed fear-potentiated startle to the light, and significantly less fear-potentiated startle to the light when it was preceded by the noise-conditioned inhibitor, suggesting that the noise had acquired the ability to inhibit fear. Control experiments revealed that neither a novel noise nor a noise nonreinforced William A. Falls, Karl T. Bakken, and Scott A. Heldt, Department of Psychology, Northern Illinois University. We are grateful to Gary Coover and James Corwin for their comments on a draft of this article. Correspondence concerning this article should be addressed to William A. Falls, Department of Psychology, Northern Illinois University, De Kalb, Illinois Electronic mail may be sent via Internet to niu.edu. separately from the light (i.e., following light+, noise-, light- training) inhibited fear-potentiated startle to the light. These results suggest that the inhibitory effect of the noise was acquired through nonreinforcement of the serial noise --* light compound and cannot be explained solely in terms of an unconditioned effect of the noise on fearpotentiated startle to the light (cf. Papini & Bitterman, 1993; Swartzentruber, 1995). Additional experiments revealed that the noise-conditioned inhibitor passed both the transfer and retardation tests for conditioned inhibition (Rescorla, 1969). Specifically, following light+, noise ---* light- training, fear-potentiated startle to a separately trained conditioned stimulus (CS; e.g., a tactile CS) was also inhibited by the noise (transfer) and subsequent acquisition of conditioned fear to the noise was retarded (retardation). Similar results were obtained when the stimulus modalities were reversed. Together these results suggest that the inhibitory effect of the noise cannot be understood solely in terms of an increase in attention to the noise, generalization decrement, external inhibition, or a modality-specific effect of a noise stimulus on fear produced by a light (cf. Papini & Bitterman, 1993). Rather, these results strongly suggest that the noise acquired the ability to inhibit fear produced by the light (Falls, 1993; Falls & Davis, 1997). Hence, this procedure may be well suited for evaluating the neural systems that are involved in the inhibition of fear. Much is known about the neural systems involved in the expression of fear-potentiated startle (Davis et al., 1995). Although the details have not been worked out, it is believed that the conditioned stimulus is processed through various subcortical sensory structures (Campeau & Davis, 1995a) to either the perirhinal cortex or primary sensory cortex and on to the lateral and basolateral nuclei of the amygdala (Campeau & Davis, 1995a; Rosen et al., 1991; Sananes & Davis, 1992). These amygdala nuclei project to the central nucleus of the amygdala, which in turn projects to the sensorimotor interface of the startle reflex pathway (Hitchcock & Davis, 1991; Koch & Ebert, 1993; Yeomans & Polard, 1993). Falls and Davis (1995b) suggest that one way to approach 476

2 PERIRHINAL CORTEX LESIONS 477 identifying the neural systems involved in conditioned inhibition of fear-potentiated startle is to first outline the neural circuitry involved in the expression of fear and to then evaluate where along this circuitry a conditioned inhibitor affects fear-potentiated startle. Once this site is identified, it should be possible to determine those areas activated by a conditioned inhibitor by identifying the brain areas that project to this site in the fear-potentiated startle circuit. Following this logic, Fails and Davis (1995b) examined whether lesions of the central nucleus of the amygdala would affect the performance of conditioned inhibition. If a conditioned inhibitor affects fear-potentiated startle at the central nucleus of the amygdala, lesioning it should produce a deficit in the expression of previously acquired conditioned inhibition. However, an obstacle in this study was that lesions of the central nucleus would produce a deficit in the expression of fear-potentiated startle (Campeau & Davis, 1995b; Hitchcock & Davis, 1986; Kim & Davis, 1993). This deficit would preclude the assessment of conditioned inhibition because fear would have to be present in order to assess the inhibition of fear (Falls & Davis, 1995b; LoLordo & Fairless, 1985). To overcome this problem, Fails and Davis (1995b) capitalized on a previous observation showing that fear-potentiated startle could be reacquired following lesions of the central nucleus of the amygdala (Kim & Davis, 1993). Therefore, to evaluate the effect of central nucleus lesions on the expression of conditioned inhibition, rats were given conditioned inhibition training followed by lesions of the central nucleus of the amygdala. The rats were then given light + shock retraining with no further conditioned inhibition training (i.e., no further noise ~ light- training). Falls and Davis found that conditioned inhibition was retained following lesions of the central nucleus of the amygdala. This suggested that areas of the central nucleus important for the expression of fear-potentiated startle were not critical for the expression of conditioned inhibition. Furthermore, these results suggested that the conditioned inhibitor affected fear-potentiated startle elsewhere in the circuit, either afferent or efferent to the central nucleus of the amygdala. The perirhinal cortex (Prh) is also an important component of the neural systems involved in fear-potentiated startle. Lesions of the Prh produce deficits in the expression of fear-potentiated startle to both visual (Campeau & Davis, 1995a; Rosen et al., 1991) and auditory (Campeau & Davis, 1995a) conditioned stimuli. However, the role of the Prh in the inhibition of fear-potentiated startle is not known. It is possible that the conditioned inhibitor reduces fear by some action in the Prh, perhaps by altering the processing of fear-related (e.g., CS) information. Alternatively, the Prh could be a structure through which the conditioned inhibitor is processed en route to its insertion at lower levels of the fear-potentiated startle circuit. The purpose of the present study was to evaluate whether the Prh plays a role in the expression of conditioned inhibition of fear-potentiated startle. As with the central nucleus of the amygdala, evaluating the involvement of the Prh in the expression of conditioned inhibition is complicated by the fact that lesions of the Prh interfere with the expression of fear-potentiated startle. However, we have found that rats sustaining posttraining lesions of the Prh can be made to show fear-potentiated startle if they are given light+ retraining after the lesion. This allows us to evaluate whether lesions of the Prh interfere with expression of conditioned inhibition acquired before the lesion. Experiment 1 Experiment 1 evaluated whether the Prh is important for the expression of previously acquired conditioned inhibition. Two groups of rats were given light+, noise ---, lightconditioned inhibition training (Fails & Davis, 1997), Next, one group was given lesions of the Prh, and the other was given sham lesions. Both groups were then given light+ retraining with no further conditioned inhibition training. If the Prh is important for the expression of conditioned inhibition, the noise should not retain its ability to inhibit fear-potentiated startle to the retrained light. If, on the other hand, the Prh is not important for the expression of conditioned inhibition, the noise should retain its ability to inhibit fear-potentiated startle to the retrained light. Subjects Me~od A total of 20 experimentally naive, male, albino Sprague- Dawley rats were obtained from the Northern Ilfinois University Psychology Department rat colony. The rats weighed between 350 and 400 g at the start of the experiment. All rats were housed singly in hanging wire mesh cages (18 25 x 20 cm) and maintained on a 12-hr light-dark cycle (lights on at 7 a.m.) with food and water continuously available. Apparatus Conditioning and fear-potentiated startle testing were conducted in four identical stabilimeter devices. Each stabilimeter consisted of an 8 X cm Plexiglas and wire mesh cage suspended between compression springs within a steel frame. The floor of each stabilimeter consists of four 4-mm diameter stainless steel bars spaced 20 mm apart, through which shock could be administered. Cage movement results in displacement of an accelerometer with the resulting voltage being proportional to the velocity of displacement. The analog output of the accelerometer was amplified (Fintronics Accelerometer Amplifier, Model FA-560, Orange, CT) and digitized on a scale of 0 to 4,096 units by a MacADIOS II board (GW Instruments, Somerville, MA) interfaced to a Macintosh Power PC 7100/66. Startle amplitude was defined as the peak to peak accelerometer voltage that occurred during the first 200 ms after the onset of the startle stimulus. Each stabilimeter was located within a separate ventilated, sound attenuating chamber (58 x 41 x 36 cm; Industrial Acoustics, Bronx, NY). Ventilation fans produced background noise of 55 db sound pressure level (SPL). The startle stimulus was a 50-ms burst of white noise having a rise-decay time of 2.5 ms. The startle stimulus was provided through a high-frequency speaker (Radio Shack Super Tweeter, Tandy, Fort Worth, TX) located 2 cm from the rear of each stabilimeter. Three startle stimulus intensities were used, 95, 105, and 115 db SPL. The light CS was produced by an

3 478 FALLS, BAKKEN, AND HELDT 8-W fluorescent bulb located 2 cm from the rear of each stabilimeter. The light was controlled by a specially designed light control unit (Fintronics, Orange, CT) that allowed a near instantaneous rise time (100 ~ts) and control of light intensity. The light luminance was 630 footlamberts (6,781 ix). The noise CS was a 70-dB SPL white noise that was band-pass filtered with a Krohn-Hite bandpass filter (Model 3100A, Avon, MA) with high and low passes both set at 4 khz. The noise CS was delivered through an 8-in. full-range speaker (Radio Shack, Model C) located 15 cm to the left of each stabilimeter. The unconditioned stimulus was scrambled shock generated by two constant-current shock generators (Lafayette, Model 82404/5-SS, Lafayette, IN) located outside of the isolation chambers. Shock intensity was 0.6 ma. The timing and presentation of stimuli were controlled by SuperScope II data acquisition and control software (GW Instruments, Somerville, MA) running on a Macintosh Power PC 7100/66. Procedure Initial startle test. Before conditioned inhibition training, a brief test session was given to familiarize the rats to handling, the apparatus, and the startle stimulus. Rats were placed in the stabilimeter and 5 min later presented with 10 startle stimuli at each of three intensities, 95, 105, and 115 db, for a total of 30 startle stimuli. The three noise-burst intensities were presented in balanced irregular order with a 30-s interval between successive stimuli. These pretest data were not used. Training. The basic experimental procedure is outlined in Table 1. Training was conducted in two phases. In Phase 1 the rats were given pairings of a light and shock. This training was meant to establish the light as a conditioned excitor of fear. This was followed by Phase 2 training that included nonreinforcement of a serial noise --~ light compound meant to establish the noise as a conditioned inhibitor of fear. Phase 1 began 1 day after the pretest. On two consecutive days, the rats were placed in the stabilimeters and 5 min later given 15 light+ trials consisting of a 4-s light coterminating with a 500-ms, 0.6-mA shock. The mean intertrial interval (ITI; defined for training as the interval between successive lights) was 2 min (range min). Phase 2 training began 1 day after the completion of Phase 1. The rats were placed in the stabilimeters and 5 min later given the first training trial. Training consisted of 5 light+ trials identical to those given in Phase l, intermixed with 15 nonreinforced, serial noise --~ light compound trials. The noise stimulus was also 4 s long. The stimuli were presented serially such that the offset of the noise was coincident with the onset of the light. The two trial types were presented in a pseudorandom sequence with a mean ITI of 2 min (range 1,5-2.5 min). Presurgery testing. Conditioned inhibition of fear-potentiated startle was assessed 1 day after the last training day. The rats were placed in the stabilimeter and, after a 5-min period during which no stimuli were administered, given 10 startle stimuli at each of three intensities, 95, 105, and 115 db, for a total of 30 startle stimuli. This was followed immediately by five startle-stimulus-alone test trials, five light test trials, and five noise --, light test trials at each of three startle stimulus intensities. For light test trials, the startle stimulus occurred 3.5 s after the onset of the light (i.e., the time at which the shock unconditioned stimulus would have occurred). On noise --* light test trials the startle stimulus occurred 3.5 s after the onset of the light (i.e., 3.5 s after the offset of the noise). All trial types were presented in a pseudorandom sequence with the constraint that each trial type occur only once in each consecutive three-trial block. The interval between successive startle stimuli was 30 s. Surgery. One to 2 days after the presurgery test, rats were given bilateral lesions of the perirhinal cortex (n = 10) or sham operations (n = 10). The rats were anesthetized with chloral hydrate (400 mg/kg ip) and placed in a Kopf stereotaxic instrument. The skin was retracted, and the muscles attached to either side of the skull were cut and retracted. Holes were drilled in the skull, and the lateral surface of the skull was chipped away. Kopf Model NE-300 electrodes (0.25 mm diameter) insulated to within 0.5 mm of the tip were used to make DC anodal lesions. Campeau and Davis (1995a) reported that Prh lesions interfered with fear-potentiated startle only when the lesion included most of the rostrocaudal extent of the Prh. To accomplish this, we made three lesions along the rhinal sulcus on each side of the brain. The anterior-posterior axis was referenced from bregma, the lateral coordinates from the midline, and the dorsoventral coordinates from the surface of the skull measured at bregma. The coordinates were (1) AP - 1.6, ML _+ 6.2, DV -7.8; (2)AP -4, ML _+ 6.5, DV -7.7; and (3)AP -5, ML , DV Each lesion was made by passing 1.0 ma of current for 12 s. For sham-operated rats the electrode was lowered to the same coordinates, but no current was passed. Postsurgery testing. Conditioned inhibition of fear-potentiated startle was assessed 7 to 10 days after surgery. The test was identical to the presurgery test. Retraining. Light+ retraining began 1 day after the postsurgery test. On five consecutive days, the rats were placed in the stabilimeters and 5 min later given the first of 15 light+ trials identical to the trials given in Phase 1. Post-retraining testing. Conditioned inhibition of fear-potentiated startle was assessed 1 day after retraining. The test was identical to the previous two tests. Table 1 Experimental Designs Training phase Post light+ 1 2 Presurgery test Lesion Postsurgery test Retraining retraining test Experiment 1 Perirhinal cortex light+ light+, noise --~ light- light, noise --* light ~ ~ light, noise ~ light light+ Sham light, noise ---, light Experiment 2 Perirhinal cortex light+ light+ light, noise --* light ~ ~ / light, noise ~ light light+ light, noise --~ light Sham Note. Noise ---* light denotes serial presentation of stimuli; plus sign denotes shock; minus sign denotes the omission of shock. In testing, fear-potentiated startle was assessed in the presence and absence of the light when it was and was not preceded by the noise.

4 Histology. At the end of the experiment, animals were overdosed with chloral hydrate and perfused intracardially with 0.9% saline followed by 10% buffered formalin phosphate. The brains were removed and stored for at least 48 hr in 20% (wt/vol) sucrose. Frozen sections 60 Ima thick were taken through the lesion site and stained with cresyl violet. The lesions were transcribed onto atlas plates (Paxinos & Watson, 1986). Data reduction and statistical analyses. The mean startle amplitudes from the three startle stimulus intensities for the no-cs (i.e., startle-stimulus-alone test trials), light, noise ~ light, and noise test trials were pooled to form a single mean for each rat for each of these trial types. Difference scores were then calculated for each rat by subtracting the mean startle amplitude obtained on no-cs test trials from the mean startle amplitude obtained on trials in which a CS was presented (i.e., light, noise ~ light, and noise test trials). The resulting difference scores reflect the magnitude of fear-potentiated startle in the presence of each CS. Because statistical differences observed in the difference scores might be the result of differences in startle amplitude on no-cs test trials, mean startle amplitudes on no-cs test trials were analyzed separately with an analysis of variance (ANOVA). The mean startle amplitudes for no-cs test trials are presented in the captions for Figures 1 and 2. No significant differences were found in no-cs test trials. Consequently, difference scores for each of the three tests were computed and analyzed with an ANOVA with trial type (light or noise ~ light) as a within-subjects factor and group (i.e., lesion or sham) as a between-subjects factor. Subsequent comparisons were made with t tests. The difference scores calculated on noise test trials are not required for the assessment of conditioned inhibition and were analyzed separately to determine whether the lesion produced any unexpected changes in responding to the noiseconditioned inhibitor alone. PERIRHINAL CORTEX LESIONS 479 Histology Results and Discussion The data from I rat with unilateral sparing of the Prh were excluded from the data analysis. The remaining 9 lesioned rats had bilateral destruction of the Prh that extended along the rostrocaudal extent of the rhinal sulcus from bregma -1.8 to bregma -5.3 (Paxinos & Watson, 1986). The lesions typically encroached upon parietal cortex dorsal to the Prh and piriform cortex and entorhinal cortex ventral to the Prh. Lesions did not extend beyond the external capsule, and in no case was there identifiable damage to the lateral or basolateral nuclei of the amygdala. Figure 1 shows a serial reconstruction of the smallest and largest lesion. Behavioral Data One sham-operated rat died during surgery, so its presurgery data were excluded from the statistical analysis. Figure 2 shows the results of the presurgery, postsurgery, and postretraining tests for conditioned inhibition. Prior to surgery the noise inhibited fear-potentiated startle to the light in both groups, F(I, 16) = 30.32, p <.001. Following surgery, rats sustaining lesions of the Prh no longer showed fear-potentiated startle to the light. The ANOVA conducted on the postsurgery data revealed a significant effect of group, F(1, 16) = 11.79, p <.01, and trial type, F(1, 16) = 4.86, p <.05, but no group by trial type interaction, F(1, 16) = Figure 1. A serial reconstruction showing the extent of the largest (hatched) and smallest (solid) perirhinal cortex lesion. Plates follow Paxinos and Watson (1986). Coordinates are relative to bregma. 1.23, p >.05. Sham-lesioned rats showed fear-potentiated startle, t(8) = 2.87, p <.05, whereas Prh-lesioned rats did not, t(8) = 1.67, p <.05, leading to a significant difference between the two groups on light test trials, t(16) = 4.54, p <.00 I. Closer inspection of Figure 2 shows that the magnitude of fear-potentiated startle to the light was reduced from the presurgery to the postsurgery test in sham-operated rats. A similar reduction in fear-potentiated startle has been reported under conditions of repeated testing (Falls & Davis, 1997) and is most likely a consequence of extinction that occurs over the course of repeated testing (Falls, 1993; Falls & Davis, 1997). Despite this, the noise inhibited this

5 480 FALLS, BAKKEN, AND HELDT Presurgery Post,surgery [] Light Noise~Light [] Difference Post-retraining r~ 100 o e~ -100 ~ ~ Perirhinal Cortex Sham Lesions Lesions Perirhinal Cortex Sham Lesions Lesions Perirhinal Cortex Sham Lesions Lesions Figure 2. Conditioned inhibition of fear-potentiated startle measured before surgery, after surgery, and after light + shock retraining in rats sustaining perirhinal cortex (Prh) lesions or sham lesions. The data shown are the mean startle difference scores calculated by subtracting the amplitude of startle obtained on no-cs (conditioned stimulus) test trials from the amplitude of startle obtained on trials in which a CS was presented (i.e., light and serial noise --, fight test trials). Also shown are the differences between the two trial types. A negative difference represents conditioned inhibition. Mean startle amplitudes on no-cs test trials were as follows: Prh lesions: 383, 395, and 313 for presurgery, postsurgery, and post-retraining, respectively. Sham lesions: 471, 363, and 367 for presurgery, postsurgery, and post-retraining, respectively. smaller magnitude of fear-potentiated startle in shamlesioned rats, t(8) = 2.34, p <.05. The postretraining data show that following light + retraining, Prh-lesioned rats could still show fear-potentiated startle to the light. These data are consistent with those reported by Campeau and Davis (1995a) showing that posttraining lesions of the Prh did not interfere with the acquisition of fear-potentiated startle. An ANOVA on the postretraining data revealed a significant effect of trial type, F(1, 16) = 44.18, p <.001, and no effect of group, F(1, 16) = 1.46, p >.05, or group by trial type interaction, F < 1. Most importantly, the noise still significantly inhibited fear-potentiated startle to the retrained light in lesioned rats, t(8) = 4.44, p <.01, even though there was no further noise ---* light- retraining. The retention of conditioned inhibition in Prh lesions cannot be attributed to rapid reacquisition of conditioned inhibition during testing. Analysis of the first three trials of the postretraining test revealed a significant effect of trial type, F(1, 16) = 17.28,p <.001, and no effect of group, F(1, 16) = 2.78, p >.05, or group by trial type interaction, F < 1. This is strong evidence that conditioned inhibition was retained following Prh lesions. There were no differences between the groups in responding to the noise-conditioned inhibitor alone, F(1, 16) = 1.24, p >.05 (data not shown). Hence, the lesion did not produce any unexpected responding to the noise-conditioned inhibitor. The results of the present experiment suggest that Prh lesions interfere with the expression of conditioned fear but do not interfere with the expression of conditioned inhibition of fear. The results of several behavioral experiments suggest that following light+, noise --, light- training the noise acquires the ability to inhibit fear. Neither a novel noise nor a noise nonreinforced separately from the light (i.e., light+, noise-) significantly inhibits fear-potentiated startle to the light (Falls, 1993; Falls & Davis, 1997). However, it is possible that in animals sustaining lesions of the Prh, the nonassociative inhibitory effect of the noise is somehow enhanced. If true, this nonassociative inhibition could mask any effect the lesion might have had on conditioned inhibition. The purpose of Experiment 2 was to examine the nonassociative inhibitory effect of the noise in rats sustaining lesions of the Prh. Experiment 2 Experiment 2 was similar to Experiment 1 in all respects except that the rats were given light+ training only. Hence, the noise was neutral at the time of testing. Subjects Method A total of 19 experimentally naive, male, albino Sprague- Dawley rats were obtained from the Northern Illinois University Psychology Department rat colony. The rats weighed between 350 and 400 g at the start of the experiment. The rats were housed and maintained as described in Experiment 1.

6 PERIRHINAL CORTEX LESIONS ' ,2., 100 Presurgery Postsurgery Post-retraining I [] Light ] II Noise---~Light [] Difference V~ ] ~ i r~ -200 t Perirhinal Cortex Sham Lesions Perirhinal Cortex Sham Lesions Perirhinal Cortex Lesions Lesions Lesions Sham Lesions Figure 3. Nonassociative inhibition of fear-potentiated startle measured before surgery, after surgery, and after light + shock retraining in rats sustaining perirhinal cortex (Prh) lesions or sham lesions. Prior to surgery these rats were given light + shock training only. Hence, the noise was novel at the time of testing. The data shown are the mean startle difference scores calculated by subtracting the amplitude of startle obtained on no-cs (conditioned stimulus) test trials from the amplitude of startle obtained on trials in which a CS was presented (i.e., light and serial noise ----, light test trials). Also shown are the differences between the two trial types. A negative difference represents nonassociative inhibition. Mean startle amplitudes on no-cs test trials were as follows: Prh lesions: 444, 487, and 613 for presurgery, postsurgery, and post-retraining, respectively. Sham lesions: 454, 395, and 410 for presurgery, postsurgery, and post-retraining, respectively. Apparatus The apparatus was the same as that used in Experiment 1. Procedure Training. The basic experimental procedure is outlined in Table 1. Following the initial startle test, rats were given pairings of a light+ in two phases. In Phase 1 the rats were given 2 days of light+ training consisting of 15 light+ trials. In Phase 2 the rats were given 5 additional days of light+ training consisting of 15 light+ trials each day. Testing, surgery, and retraining. Nonassociative inhibition of fear-potentiated startle was assessed 1 day after the last training day. The test was identical to the tests described in Experiment 1. One to two days after the presurgery test, rats were given bilateral lesions of the Prh (n = 10) or sham operations (n = 9) as described for Experiment 1. Nonassociative inhibition of fear-potentiated startle was assessed 7 to 10 days after surgery and was followed by light+ retraining consisting of 15 light+ trials on each of 5 days. Finally, postretraining nonassociative inhibition was assessed 1 day after the last retraining day. Histology Results The 10 lesioned rats had complete bilateral lesions of the Prh similar to those described in Experiment 1. The lesions typically encroached upon parietal cortex, but in no case did the lesions extend medially beyond the external capsule. Behavioral Data Figure 3 shows the results of the presurgery, postsurgery and postretraining tests for nonassociative inhibition. The presurgery data show that the neutral noise did not inhibit fear-potentiated startle, F(1, 17) = 2.27, p >.05. As in Experiment 1, lesions of the Prh interfered with the expression of fear-potentiated startle to the light, t(17) = 4.30,p <.001. Following light+ retraining, Prh-lesioned rats again showed fear-potentiated startle to the light. Most importantly, in the postretraining test the neutral noise did not inhibit fear-potentiated startle in either Prh-lesioned, t(9) < 1, or sham-lesioned rats, t(8) = 1.99,p >.05. There were no differences between the groups in responding on noise-alone trials, F(1, 17) = 2.20, p >.05 (data not shown). Consistent with previous studies (Falls & Davis, 1995b; Falls & Davis, 1997), the neutral noise produced little nonassociative inhibition of fear-potentiated startle to the light CS. Hence, the retention of inhibition in Prh-lesioned animals of Experiment 1 cannot be attributed to a lesionproduced increase in nonassociative inhibition. Recall that behavioral experiments have indicated that light+, noise ----, light- training results in the noise acquiring the ability to inhibit conditioned fear (Falls, 1993; Falls & Davis, 1997). Because rats sustaining lesions of the Prh in Experiment 1 were given conditioned-inhibition training prior to the lesion, the noise must have acquired the ability to inhibit fear in these animals as well. The results of Experiment 2 suggest

7 482 FALLS, BAKKEN, AND HELDT that nonassociative inhibition is not enhanced following Prh lesions. Therefore, Prh-lesioned animals in Experiment 1 must have retained conditioned inhibition, suggesting that areas of the Prh that are important for the initial expression of fear-potentiated startle are not important for the expression of conditioned inhibition. General Discussion The results of this study confirm the importance of the Prh in the initial expression of conditioned fear (cf. Campeau & Davis, 1995a; Corodimas & LeDoux, 1995; Rosen et al., 1991). More importantly, they indicate that the Prh is not critical for the expression of previously acquired conditioned inhibition of fear. This strongly suggests that a conditioned inhibitor does not reduce fear by some action in the Prh, nor is the Prh a structure through which the conditioned inhibitor is processed en route to affecting conditioned fear elsewhere in the conditioned fear circuit. The role that the Prh plays in the expression of conditioned fear is still unclear. One view is that the Prh is included in the normal pathway through which auditory and visual information reaches the amygdala (Campeau & Davis, 1995a; Rosen et al., 1991). Posttraining lesions of the Prh interfere with the expression of conditioned fear because they disrupt this pathway. Reacquisition is thought to take place through alternative CS pathways that do not normally participate in the expression of conditioned fear (Campeau & Davis, 1995a). Under this hypothesis, the present results suggest that a conditioned inhibitor does not reduce fearpotentiated startle by altering the processing of the CS at this sensory convergent zone. In addition, the present results suggest that a conditioned inhibitor, unlike the CS, is not processed through the perirhinal cortex. Therefore, a conditioned inhibitor must act elsewhere in the fear-potentiated startle circuit, and it must use sensory pathways different from those used by the CS en route to its insertion point in the fear-potentiated startle circuit. However, it is possible that as a result of conditionedinhibition training, inhibition develops at the alternative CS pathway as well as at the Prh. For an auditory CS these alternative CS pathways may include direct projections from the auditory thalamus and auditory association areas to the amygdala (LeDoux, Cicchetti, Xagoraris, & Romanski, 1990). Analogous visual pathways to the amygdala have not been worked out. Nevertheless, this could explain how conditioned inhibition is retained following Prh lesions. However, it seems unlikely that inhibition would develop at structures that do not normally participate in the expression of fear-potentiated startle. Therefore, under the hypothesis that the Prh is included in the normal CS pathway to the amygdala, the present results suggest that a conditioned inhibitor affects fear-potentiated startle either afferent or efferent to the Prh. Furthermore, because the inhibition was retained, the present results suggest that the alternative CS pathway shares some of the same afferent or efferent circuitry with the Prh. This shared circuitry may include thalamic sensory structures, the amygdala, or structures efferent to the amygdala. Corodimas and LeDoux (1995) have suggested an alternative view of the role of the Prh in the expression of conditioned fear. In their view the Prh is important for a postacquisitional memory process. Lesions of the Prh interfere with the expression of conditioned fear because they disrupt either memory storage or retrieval (Coover & Mertes, 1995; Corodimas & LeDoux, 1995). Consistent with this, studies conducted in both primates and rats suggest that the Prh plays an important role in memory processes in non-fear-related tasks (Meunier, Bachevalier, Mishkin, & Murray, 1993; Nagahara, Otto, & Gallagher, 1995; Wiig & Bilkey, 1994, 1995; Zola-Morgan, Squire, Amaral, & Suzuki, 1989; Zola-Morgan, Squire, & Ramus, 1994). Under this hypothesis, the present results suggest that the conditioned inhibitor does not affect storage or retrieval by some action at the Prh. In addition, the memory-storage or retrieval function of the Prh necessary for the expression of conditioned fear is not necessary for the expression of conditioned inhibition. Corodimas and LeDoux (1995) suggested that the memory deficit induced by Prh lesions may be specifically related to an inability of animals to process contextual stimuli or use these stimuli to retrieve CS information. The present results suggest that this contextual information is not essential for conditioned inhibition of fear. This issue deserves further study, especially in light of behavioral data that indicate that inhibition can be context dependent (Bouton, 1991; Bouton & Bolles, 1979; Bouton & Nelson, 1994). For example, if conditioned inhibition of fear-potentiated startle is context dependent, the present results suggest that other areas of the brain control the contextual aspects of conditioned inhibition of fear. As previously mentioned, little is known about the neural systems responsible for conditioned inhibition of fear. There are several strategies one could use to identify candidate neural systems. One strategy would be to propose a conceptual definition of the inhibition of fear and then investigate the role of brain systems that might fit with this definition. For example, theorists have long thought that conditioned inhibition is the consequence of conditioning of some process that is antagonistic to the conditioned response of fear. Konorski (1948) argued that conditioned inhibition was the result of the accrual of inhibitory associations between nodal representations of the CS and US. These inhibitory associations would exist side by side with the excitatory, conditioned response-producing associations. Using this hypothesis, one might attempt to determine whether conditioned inhibition is intrinsic to the fear-potentiated startle pathway. Alternatively, its been argued that conditioned inhibition of fear is a consequence of incompatible affect elicited by the conditioned inhibitor (i.e., conditioning of a pleasure or "relief" response; Denny, 1971; McAllister & McAllister, 1992, 1995). Using this hypothesis, one might investigate the role of structures implicated in reward or in appetitive conditioning in conditioned inhibition of fear. Finally, there are behavioral data to suggest that conditioned inhibition might involve an interaction between the sensory representations of the CS and conditioned inhibitor (cf. Bouton & Nelson, 1994; Holland, 1985). Using this hypothesis, one might consider the possibility that conditioned

8 PERIRHINAL CORTEX LESIONS 483 inhibition results from an interaction between thalamic or subthalamic visual and auditory structures. An alternative strategy would be to consider the fact that a conditioned inhibitor of fear must inhibit fear by interacting with the known neural pathways responsible for the production of fear. Hence, it should be possible to evaluate where along these neural pathways a conditioned inhibitor affects fear-potentiated startle (Falls & Davis, 1995b). Once this site is identified, it should then be possible to determine those areas activated by a conditioned inhibitor by identifying the brain areas that project to this site in the fearpotentiated startle circuit. The advantage of this approach is that it does not require commitment to a specific conceptual definition of conditioned inhibition. Instead, it leaves open to empirical identification the nature of conditioned inhibition. Following the latter strategy, Falls and Davis (1995b) showed that a conditioned inhibitor does not affect fearpotentiated startle at the central nucleus of the amygdala. Other components of the fear-potentiated startle circuit where a conditioned inhibitor could act include (a) sensory structures afferent to the amygdala, (b) the lateral nucleus or basolateral nucleus of the amygdala, and (c) points along the pathway from the central nucleus of the amygdala to the startle-reflex pathway. It is possible that a conditioned inhibitor acts somewhere in the sensory system to affect processing of the CS. Falls and Davis (1995b) argued that insertion of inhibition in the CS pathway was unlikely given the demonstration of stimulus transfer. They argued that to account for stimulus transfer, a noise-conditioned inhibitor would have to act simultaneously at every sensory system. As a consequence, Falls and Davis favored the idea that the conditioned inhibitor acts at a more central site where stimuli from different modalities converge (e.g., the lateral or basolateral nuclei of the amygdala). However, as mentioned, behavioral data suggest that conditioned inhibition might involve an interaction between the sensory representations of the CS and conditioned inhibitor (cf. Bouton & Nelson, 1994; Holland, 1985). Recent neuroanatomical data suggest that conditioned inhibition may involve functional interactions between sensory systems. Mclntosh and Gonzalez-Lima (1995) measured fluorodeoxyglucose incorporation in rats given tone+, light/noise- conditioned inhibition training and found lower incorporation in the ventral division of medial geniculate in animals for which the light was inhibitory. This raises the possibility that the light inhibitor affects the processing of the tone CS by reducing activity in the lemniscal auditory pathways. The lateral and basolateral nuclei are critical for the acquisition and expression of conditioned fear: Lesions of these nuclei interfere with conditioned fear to auditory (Campeau & Davis, 1995b; LeDoux et al., 1990; Maren, Aharonov, & Fanselow, 1996), visual (Campeau & Davis, 1995b; Lee, Walker, & Davis, 1996; Sananes & Davis, 1992), and contextual CSs (Maren et al., 1996). It is possible that a conditioned inhibitor acts at the level of the lateral or basolateral nuclei. However, evaluating the role of these nuclei in the expression of conditioned inhibition is compli- cated by the fact that both pretraining and posttraining lesions of the lateral and basolateral nuclei eliminate fearpotentiated startle (Campeau & Davis, 1995b; Lee et al., 1996). And it is unlikely that conditioned fear can be reacquired following posttraining lesions (Maren et al., 1996). Therefore, alternatives to the lesion technique must be used. For example, GABA receptors are abundant in the basolateral nucleus of the amygdala (Carlsen, 1988; Nitecka & Ben-Ari, 1987; Rainnie, Asprodini, & Shinnick-Gallagher, 1991). It is possible that a conditioned inhibitor reduces fear-potentiated startle by causing the release of GABA in the basolateral nucleus. If so, direct infusion of a GABA antagonist into the basolateral nucleus might reduce conditioned inhibition. A conditioned inhibitor could act efferent to the central nucleus of the amygdala. The central nucleus projects to the caudal pontine reticular nucleus (Campeau & Davis, 1995b; Hitchcock & Davis, 1991), an essential sensorimotor interface of the acoustic startle reflex pathway (Davis, Gendelman, Tischler, & Gendelman, 1982; Koch & Ebert, 1993; Koch, Lingenhohl, & Pilz, 1992; Lingenhohl & Friauf, 1992, 1994). This projection may be both direct (Campeau & Davis, 1995b; Hitchcock & Davis, 1991) and indirect via the midbrain (Fendt, Koch, & Schnitler, 1994; Frankland & Yeomans, 1995; Yeomans & Polard, 1993). Although it does not appear that the conditioned inhibitor directly reduces the startle reflex (Falls & Davis, 1993), a conditioned inhibitor might affect the ability of the central nucleus to modulate the startle reflex either in the midbrain or at the caudal pontine reticular nucleus. If so, a conditioned inhibitor should reduce startle facilitated by either electrical (Rosen & Davis, 1988) or chemical (Koch & Ebert, 1993) stimulation of the central nucleus of the amygdala. Once the site where a conditioned inhibitor acts to inhibit fear-potentiated startle is identified, it will be possible to identify the structures that project to that site that may potentially inhibit it. At the present time there is no direct information concerning the identity of the brain areas that might be activated by a conditioned inhibitor. However, a few brain areas are noteworthy (see Falls & Davis, 1995a). Work by Yadin and Thomas has consistently implicated the lateral septal nucleus in the reduction of fear-related behavior (Thomas, 1988; Yadin & Thomas, 1981, 1992). There have also been suggestions that the inhibition of fear may involve primary sensory cortex (LeDoux, Romanski, & Xagoraris, 1989; Teich et al., 1989) and infralimbiccingulate cortex (Holson, 1986; Jaskiw & Weinberger, 1992; Morgan & LeDoux, 1995; Morgan, Romanski, & LeDoux, 1993). However, there are data to suggest that these areas may not be involved in the reduction of fear in all situations (Falls & Davis, 1993; Gewirtz, Falls, & Davis, in press). For example, Gewirtz et al. found that rats sustaining lesions of the infralimbic-cingulate cortex showed normal conditioned inhibition of fear-potentiated startle suggesting that the infralimbic-cingulate cortex may not be activated by a conditioned inhibitor. In an attempt to begin to identify the brain areas activated by a conditioned inhibitor, Campeau et al. (in press) measured the induction of the mrna for the early immedi-

9 484 FALLS, BAKKEN, AND HELDT ate gene c-fos in rats that were trained for conditioned inhibition. In rats that were presented with the conditioned inhibitor immediately before being killed, specific induction was observed in the ventral nucleus of the bed nucleus of the stria terminalis and associated septohypothalamic nucleus, the locus coeruleus, and the pedunculopontine nucleus. The bed nucleus of the stria terminalis projects to many of the same brainstem areas as the central nucleus of the amygdala (Holstedge, Meiners, & Tan, 1985), but in several instances it seems to have a functional effect on fear and stress opposite to that of the central nucleus of the amygdala (Henke, 1984; Morrow, Grijalca, Geiselman, & Novin, 1993). The pedunculopontine nucleus, like the central nucleus of the amygdala, projects to the caudal pontine reticular nucleus and has been implicated in the modulation of the startle reflex (Koch et al., 1993). The fact that a conditioned inhibitor specifically activates these structures suggests that the conditioned inhibitor might affect the ability of the central nucleus to modulate the startle reflex. Studies are underway to evaluate this possibility as well as to evaluate whether lesions of these areas will interfere with the expression of conditioned inhibition. In summary, the present data show that lesions of the Prh interfere with the expression of conditioned fear but do not affect the expression of conditioned inhibition of fear. Although the present data do not directly address the debate over whether the perirhinal cortex is involved in the transmission of stimulus information to the amygdala or in some postacquisitional process such as memory storage or retrieval, they do suggest that not all fear-related stimulus information or memory processes involve the perirhinal cortex. 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