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Temporary inactivation of the nucleus accumbens disrupts acquisition and expression of fear-potentiated startle in rats
Brain Research 1027 (2004) 87 – 93 www.elsevier.com/locate/brainres
Research report
Temporary inactivation of the nucleus accumbens disrupts acquisition and expression of fear-potentiated startle in rats Isabel Schwienbachera,*, Markus Fendta, Rick Richardsonb, Hans-Ulrich Schnitzlera a
Animal Physiology, University of Tuebingen, Auf der Morgenstelle 28, D-72076 Tuebingen, Germany b School of Psychology, University of New South Wales, Sydney, Australia Accepted 19 August 2004 Available online 18 September 2004
Abstract Recent research suggests that in addition to its prominent role in appetitive learning, the nucleus accumbens (NAC) may also be involved in fear conditioning. In the present study, we investigated whether temporary inactivation of the NAC, by injection of tetrodotoxin (TTX), affects acquisition and expression of conditioned fear, as measured by fear-potentiated startle (FPS). TTX injection into the NAC totally blocked acquisition and markedly decreased expression of conditioned fear to a discrete visual conditioned stimulus (CS). Interestingly, temporary inactivation of the NAC did not affect shock sensitization of startle, indicating that both the perception of the shock and short-term contextual conditioning was not affected by intra-accumbal TTX injection. Taken together, these results show that the NAC is crucial for acquisition and expression of long-term conditioned fear, as measured by fear-potentiated startle, to discrete CSs, but not short-term conditioned fear to a context. D 2004 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Learning and memory: systems and functions—animals Keywords: Nucleus accumbens; Learned fear; Acoustic startle response; Fear potentiation of startle; Short-term memory; Long-term memory; Tetrodotoxin; Sensitization
1. Introduction The nucleus accumbens (NAC) receives converging afferents from the amygdala, hippocampus, and prefrontal cortex—areas that form a complex network which has been shown to be involved in emotional learning [2,4]. There is also evidence suggesting that the NAC itself is involved in emotional learning, particularly learning about aversive events (i.e., fear conditioning). For example, Campeau et al. [1] reported that presentation of a conditioned stimulus (CS) previously paired with shock enhanced expression of c-fos within the NAC. The results of a number of lesion studies have also suggested that the NAC is involved in the * Corresponding author. Tel.: +49 7071 2976134; fax: +49 7071 292618. E-mail address: [email protected] (I. Schwienbacher). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.08.037
acquisition and expression of conditioned fear [5,6,12,15, 17,18]. However, the exact pattern of results reported in these studies has been inconsistent. For example, Haralambous and Westbrook [5] reported that temporary inactivation of the NAC, with local infusion of the anaesthetic bupivacaine, disrupted context conditioning but had no effect on either acquisition or expression of learned fear to a discrete CS. Similarly, Riedel et al. [17] reported that electrolytic lesions of the NAC shell disrupted learning about a context but had no effect on learning about a discrete CS. Jongen-Relo et al. [6] also found reduced context conditioning following excitotoxic lesions of the NAC shell, but in contrast to the two studies described above, reported that these lesions disrupted conditioning to a discrete CS as well. Kubos et al. [7] reported that lesions of the NAC core had no effect on either type of learning. In stark contrast, Parkinson et al. [12] reported that excitotoxic
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lesions of the NAC shell had no effect on learning about either a context or a discrete CS, but lesions of the NAC core blocked fear conditioning to a discrete CS and enhanced fear conditioning to a context. In other words, the relatively small literature on the effects of lesions of the NAC on learned fear has yielded nearly every possible outcome, and, in some cases, the results have been completely contradictory. Thus, although it seems that the NAC plays a role in fear conditioning, the exact nature of this role is unclear because of the inconsistent findings in the literature. Some of these inconsistencies could be due to procedural differences (e.g., number of training trials, intensity of shock, etc.) between the various studies, or to the use of different lesioning techniques (i.e., temporary vs. permanent; excitotoxic vs. electrolytic). It should also be noted that most of the studies described above assessed acquisition/expression of fear by measuring freezing. Although this is a very common measure of learned fear in rats, it may be problematic to completely rely on freezing as a measure when examining the role of the NAC in learned fear. Specifically, lesions of the NAC have been shown to affect motor activity in some situations [7,11,19], and this effect could have influenced the results reported in some of the studies described above. One solution to this potential difficulty would be to use a measure of fear that is based on the enhancement of an involuntary reflex, rather than the cessation of movement. Such a measure is the fear potentiation of the acoustic startle response (ASR). The aim of the present study was to investigate whether the NAC is required for acquisition and expression of conditioned fear, as measured by fear-potentiated startle (FPS). In this study, the NAC was temporarily inactivated by local injections of tetrodotoxin (TTX), a selective and reversible voltage-gated sodium channel blocker. The effects of local TTX injections into the NAC on acquisition and expression of FPS to a discrete cue was examined first. Then, the effects of intra-accumbal TTX injection on shock sensitization of the ASR were assessed, in order to determine whether inactivation of the NAC (1) reduced perception of the shock stimulus, and (2) affected short-term contextual fear memory.
2. Materials and methods 2.1. Subjects Male Sprague–Dawley rats (Charles River, Sulzfeld, Germany) weighing 200 to 320 g at the time of surgery were housed in groups of 5–6 rats/cage in a temperaturecontrolled colony room under a 12:12-h light/dark cycle (lights on at 07:00 h). They were fed 12 g standard rat chow/ animal/day, and tap water was available ad libitum. Each rat was handled daily before and after surgery. The experiments were performed in accordance with international ethical
guidelines for the care and use of experimental animals and were approved by the local council of animal care (Regierungspr7sidium Tqbingen, ZP 4/02). 2.2. Surgery Rats were anesthetized with ketamine/xylazine (9:1, 100 mg/kg i.p.) and placed into a stereotaxic frame. The skull was exposed and stainless steel guide cannulas (0.7 mm diameter) were implanted bilaterally aiming at the NAC (toothbar +5 mm above the interaural plane; distance from Bregma: rostrocaudal +3.4 mm, mediolateral F1.5 mm, dorsoventral 7.2 mm; coordinates from Ref. [14]). The guide cannulas were fixed to the skull with acrylic cement (Kulzer, Wehrheim, Germany) and three anchoring screws. After surgery, the cannulas were fitted with stylets in order to maintain patency. Experiments began 3 to 4 days after surgery, when all rats had fully recovered. 2.3. Fear conditioning The rats were trained in two identical boxes (386028 cm). The floor of each box consisted of stainless steel rods, 6 mm in diameter, spaced 22 mm apart, center to center. The CS was produced by a 15-W white light bulb located at the top of the box. The boxes were unilluminated at all times except for when the CS was presented. The unconditioned stimulus (US), a 0.6-mA shock, was produced by a shock generator (custom-made, University of Tqbingen) and delivered through the grid floor. Presentations of the CS and the US were controlled by a PC. Animals were placed into the training boxes, allowed to adapt for 5 min, and then given either 5 or 10 CS–US pairings (see Behavioral procedures), with a mean interstimulus interval of 2 min (range: 1–3 min). The US was presented during the last 0.5 s of the 3.7 s light CS. 2.4. Fear-potentiated startle test The magnitude of the ASR was measured in two identical sound-attenuating chambers (1008060 cm). The rats were placed in wire mesh test cages (201012 cm) with solid steel floors that were placed onto piezoelectric accelerometers (custom-made, University of Tqbingen). Movements of the rats caused changes of the accelerometer voltage output. These changes were amplified, digitized, and analyzed by a PC. The ASR magnitude was computed as the difference of the peak-to-peak voltage output of the accelerometer 80 ms before and 80 ms after the onset of the acoustic startle stimulus (100 dB SPL, 10 kHz tone pulse, 20 ms duration including 0.4 ms rise and fall times). Acoustic stimuli were generated by a functionsynthesizer (Hortmann, Neckartenzlingen, Germany) and delivered through loudspeakers mounted 40 cm away from the test cages. A continuous background white noise (55 dB SPL) was present throughout the test session. All sound
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intensity measurements were done with a 0.5-in. condenser microphone and a measuring amplifier (Brqel and Kjaer, Copenhagen, Denmark) after bandpass (0.25–80 kHz) filtering. In addition to the startle data, spontaneous motor activity was also measured in the test cages. Spontaneous motor activity was calculated as the root mean square value of the accelerometer output during a 28-s period between startle stimulus presentations. After a 5-min acclimatization period during which the rats received no stimuli except the background noise, 10 initial startle stimuli (30 s interstimulus intervals) were presented in order to induce a stable baseline of ASR magnitude. Thereafter, each rat was exposed to 20 startle stimuli, of which 10 were presented 3.2 s after onset of the light CS (light-tone trials) and 10 were presented in the absence of the light CS (tone-alone trials) in a pseudorandomized order (30 s interstimulus interval). The tone-alone trials provided a measure of baseline startle. The difference between the tone-alone and the light-tone trials serves as an operational measure of fear (reviewed in Ref. [4]). 2.5. Shock sensitization The same equipment was used here as for the fearpotentiated startle test. The only difference was that the floor of the wire mesh test cages consisted of steel bars, 2 mm in diameter, spaced 15 mm apart, center to center. For this test, rats were first given a 5-min adaptation period, and then received 10 startle stimuli. Those trials served to produce a stable baseline of ASR and were not considered for further analysis. The mean amplitude of the next 30 trials was taken as the mean preshock ASR amplitude. Then, 10 shocks (0.6 mA, 0.5 s duration, 1 Hz) were given. Following this, an additional 40 trials occurred. The mean ASR amplitude for stimuli 6–35 after the shock was taken as the postshock ASR-amplitude. The responses to the first 5 startle stimuli following the shocks were not included in the analysis because the startle sensitization effect is often not detected until 2–4 min after presentation of the shocks [3]. The difference between the pre- and the postshock ASR amplitudes provided a measure of the sensitization effect. Motor activity during shock presentation was also measured and taken as an indication of shock reactivity.
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TTX (n=13) immediately before fear conditioning (Experiment 1). Two days later, FPS was tested; neuronal activity has been shown to return to normal levels within 48 h of TTX infusion [10]. No drug was given prior to testing these rats. To assess whether NAC inactivation affected expression of conditioned fear, rats (n=15) were first fear-conditioned, without any injections (Experiment 2). These rats were then tested twice, 48 h apart. The rats received either saline or TTX immediately before testing (in a counterbalanced order). To reduce extinction of the conditioned fear during the two test days, these rats were retrained (only 5 CS–US pairings) on each test day, 4 h before testing. For the shock sensitization of startle experiment, rats (n=12) were tested twice, 48 h apart. Each rat was tested following saline and TTX infusions, in a counterbalanced order (Experiment 3). 2.8. Histology After behavioral testing had been completed, the rats were sacrificed with an overdose of pentobarbital (Nembutal). Their brains were removed and immersion fixed in 8% paraformaldehyde and 20% sucrose. Frontal sections (50 Am) were cut on a freezing microtome and Nissl stained with thionine. The injection sites were localized and the extent of tissue lesions due to cannulation was examined under a light microscope. The injection sites were drawn onto plates taken from a rat brain atlas [13]. 2.9. Statistical analysis For the acquisition and expression experiments, the startle amplitudes on each trial type (i.e., tone-alone and light-tone) were compared with ANOVA, and group differences in FPS were tested by comparing (with a ttest) difference scores (i.e., ASR amplitude on light-tone trials—ASR amplitude on tone-alone trials). For the shock sensitization experiment, the startle amplitudes on the pre- and postshock trials were compared with ANOVA, and group differences in sensitization were tested by comparing (with a t-test) difference scores (i.e., ASR amplitude on postshock trials —ASR amplitude on preshock trials).
2.6. Drugs and treatment TTX (Tocris, Kfln, Germany) was dissolved in saline and administered bilaterally (10 ng/1 Al per side, according to Ref. [10]) into the NAC with a velocity of 0.5 Al/min. The injection cannulas were left in place for an additional 2 min to allow diffusion of the solution away from the cannulas. 2.7. Behavioral procedures To assess the effects of NAC inactivation on acquisition of conditioned fear, rats received either saline (n=11) or
3. Results Fig. 1A and B shows the cannula placements for the rats that were included in the statistical analysis of the three experiments; the injection sites for all of these rats were located in the core region of the NAC. Some rats in this study (6 rats in Experiment 1 and 4 rats in Experiment 2) had misplaced cannulas (e.g., located in the ventricle or dorsal striatum); the data from these rats were excluded from the statistical analysis.
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Fig. 1. Serial drawings of frontal sections of the NAC depicting the injection sites for rats given TTX either before CS–US training (panel A, black dots) or CS testing (panel A, black squares), and for rats tested in the shock sensitization experiment (panel B; AcbC—nucleus accumbens core, AcbSh—nucleus accumbens shell).
3.1. Experiment 1 Pretraining injections of TTX into the NAC blocked acquisition of fear-potentiated startle (saline: n=8, TTX: n=10; Fig. 2A). ANOVA of startle amplitudes on the tonealone and light-tone trials revealed a significant effect of treatment [ F(1,16)=6.24, p=0.024] and a nonsignificant effect of trial type [ F(1,16)=3.24, p=0.091]. The interaction of trial typetreatment was also significant [ F(1,16)=17.06; p=0.001]. Pairwise comparisons of FPS (i.e., difference scores) revealed that this interaction was due to the TTXinfused rats exhibiting much less FPS than did the salineinfused rats [independent samples t(16)=4.13; p=0.001]. TTX injections 2 days prior to test did not affect baseline (i.e., tone-alone) ASR [t(16)=1.57; p=0.14] or motor activity during the interstimulus intervals [t(16)=1.2; p=0.25]. 3.2. Experiment 2 Expression of fear-potentiated startle was decreased after pretest TTX injections into the NAC (n=11; Fig. 2B). ANOVA of startle amplitudes on the tone-alone and lighttone trials revealed a significant effect of treatment [ F(1,10)=83.62, pb0.001] and trial type [ F(1,10)=21.19, p=0.001]. The interaction of trial typetreatment was also significant [ F(1,10)=16.09, p=0.002]. This interaction was due to the rats exhibiting greater FPS following saline
infusions than after TTX infusions [dependent samples t(10)=4.036, p=0.002]. Pretest TTX injections into the NAC did not affect either baseline startle amplitude [t(10)=1.59; p=0.14; Fig. 2B] or levels of general motor activity during the interstimulus intervals [t(10)=1.75; p=0.11; saline: 262F57; TTX: 162F28]. 3.3. Experiment 3 TTX injections into the NAC did not affect shock sensitization of the ASR (n=12; Fig. 3). Rats exhibited as much shock sensitization of startle following TTX infusions as they did following saline infusions. Statistical analysis of the data supported this description of the results. Specifically, an initial analysis indicated that test order (i.e., TTX or saline first) did not impact on the results ( Fb1.0), so the data were collapsed across this variable for all subsequent analyses. A repeated-measures ANOVA, with both block (pre- vs. postshock) and drug (TTX vs. saline) as repeated measures, yielded a significant effect of block [ F(1,11)=11.36, p=0.006], an effect of drug that approached traditional levels of statistical significance [ F(1,11)=4.48, p=0.058], and a nonsignificant interaction [ F(1,11)=2.50, p=0.14]. The significant effect of block was due to both groups exhibiting a similar increase in startle response amplitude after the shock [dependent samples t(11)=1.58; pN0.14; Fig. 3]. The near-significant effect of drug was due to a slightly larger shock sensitization effect on TTX trials compared to saline
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Fig. 2. Effects of TTX infusions into the NAC immediately before fear conditioning (A) or immediately before testing of FPS (B). Histograms depict the mean ASR magnitude in the absence (black bars) or presence (white bars) of the light CS. Hatched bars represent the difference between tone-alone and light-tone trials, which was taken as an index of fear. TTX infusions into the NAC disrupted both acquisition and expression of conditioned fear (**pb0.01).
trials (see Fig. 3). Preshock ASR amplitudes were not affected by TTX injections [t(11)=0.78; p=0.45]. The rats did exhibit slightly more motor activity during shock when given TTX than when given saline (saline: 62.96F19.73, TTX: 111.1F23.740), but this difference was not statistically significant [t(11)=1.98; p=0.07]. Post hoc analysis of the data on just the first test day (ns=6) yielded a similar pattern of results as reported above, except the magnitude of the sensitization effect was nearly identical for the two groups (for saline rats, preshock ASR=525 and postshock ASR=872; for TTX rats, preshock ASR=613, postshock ASR=938).
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al. were permanent, and were performed before conditioning, it is unclear whether their results were due to the lesions affecting learning or affecting performance. The present study, where the NAC was temporarily inactivated by TTX either just prior to training or just prior to testing, clearly shows that this structure is involved in both the acquisition and the expression of fear to a discrete CS. TTX, a selective and reversible voltage-gated sodium channel blocker, is a particularly effective agent for temporarily inactivating specific areas in the brain. Infusion of this drug completely blocks generation of action potentials in the target area, an effect that has completely dissipated 48 h after infusion [10]. It has to be noted that fibers of passage are also affected by TTX. The main fibers of passage through the NAC are the anterior commissure. Because this fiber bundle is mainly involved in transferring olfactory information between the two hemispheres [8], we are confident that the effects reported in the present study are based on a temporary lesion of the NAC and not due to a blockade of activity in the anterior commissure. The observed impairments in learning to fear a discrete CS are not a consequence of intra-NAC infusions of TTX diminishing the rat’s perception of the shock US. Specifically, the results of the shock sensitization experiment show that intra-accumbal TTX infusions do not reduce either (1) the intense burst of motor activity elicited by the shock, or (2) the shock sensitization of startle effect (see Fig. 3). Indeed, rats given intra-NAC TTX exhibited slightly but not statistically significant greater (1) motor activity following the shock, and (2) sensitization of startle effect. These results clearly show that the TTX rats perceived the shock to be at least as intense as the saline-treated rats did. However, the finding that temporarily inactivating the NAC with TTX had no effect on the shock sensitization effect raises another issue. Specifically, it has been shown that the shock sensitization of startle effect is due to
4. Discussion The present study implicates the NAC, particularly its core region, in both the acquisition and the expression of conditioned fear to a discrete CS, as measured by fear potentiation of startle. Parkinson et al. [12] also found that excitotoxic lesions of the core region of the NAC caused a significant impairment in rats acquiring conditioned fear to a discrete CS. However, because the lesions by Parkinson et
Fig. 3. Effects of TTX infusions into the NAC immediately before shock sensitization of the ASR. Histograms depict the difference between the preand postshock ASR amplitudes. TTX infusions into the NAC did not affect the shock sensitization of startle effect.
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rapid contextual fear conditioning [16]. Therefore, given that NAC inactivation impairs both acquisition and expression of learned fear to a discrete CS, why doesn’t NAC inactivation also reduce the shock sensitization of startle effect? One possibility is that the NAC is involved in learning to fear discrete CSs but is not involved in learning to fear a context. There are numerous examples in the literature of a manipulation affecting CS and context conditioning differentially. For example, lesions of the hippocampus have been reported to disrupt context conditioning but not to affect conditioning to a discrete CS (for review, see Ref. [4]). The present study would be the first demonstration, to our knowledge, of a manipulation impairing conditioning to a discrete CS but not affecting conditioning to a context. Another possibility is that the NAC is not involved in acquisition and expression of short-term fear, but is involved in the transfer of that information into some long-term store. This latter possibility can be tested in two ways: (1) test for fear of a discrete CS shortly after (e.g., 5–10 min) the training trials, and (2) test for fear of a context after a delay (i.e., after 24–48 h). If this idea is correct, then temporarily inactivating the NAC would be found to impair context conditioning but not affect learning about a discrete CS in these cases. It should be noted that Haralambous and Westbrook [5] have reported that temporary inactivation of the NAC disrupts context conditioning. Critically, the test occurred after a delay in that study. Further support, albeit indirect, for the possibility that the NAC is involved in long-term learned fear of a context is provided by the results of the present study. Because the rats in the shock sensitization experiment of the present study were tested twice (i.e., once after saline infusions and once after TTX infusions; 48 h between the two tests; test order counterbalanced), one might have expected to see an elevated startle response during the preshock period on the second test (i.e., because of the context fear). Such a result was not observed however (mean startle amplitude on Day 1=568, on Day 2=529), although rats in both conditions exhibited a pronounced sensitization effect on Day 1 (see Experiment 3 data regarding this). This failure to observe long-term context fear could have been due to the TTX infusions. That is, those rats given TTX prior to the first test could have had impaired long-term acquisition of the context-shock association, and those rats given saline prior to the first test could have had their expression of this learning impaired (because of the TTX infusions prior to the second test). After 10 shocks, one would expect for there to be some context conditioning, but it must be acknowledged that we do not have any explicit evidence that the procedures used here actually result in long-term context fear (i.e., the experiment did not include a group of rats given saline prior to both tests). Future studies will need to explore these ideas more systematically, but whatever the outcome of these future studies, the results of the present study clearly shows that
the fear potentiation of startle procedure is an effective way of examining the role of the NAC in learned fear. All injection sites in the present study were located within the core region of the NAC. However, considering that 1 Al of injectate was infused, we cannot exclude the possibility that some of the TTX diffused into the NAC shell. Empirical support for this possibility is provided by Lorenzini et al. [10], who reported that infusion of a similar concentration and volume of TTX (10 ng/Al) into the NAC inactivated neurons in an area up to 1 mm around the injection site. However, given the location of the cannula tips, it must be the case that the primary site of action of the infused TTX in the present study was in the core region of the NAC. Based on our results, we hypothesize that the NAC core modulates Pavlovian fear conditioning, at least to discrete CSs. At present, it is unclear exactly how the NAC is involved in acquisition/expression of learned fear. More specifically, the present results do not show whether the NAC is directly involved in learned fear or whether the NAC is involved only because of its interconnections with other relevant neural structures (i.e., the amygdala). Furthermore, the present study does not show what neurotransmitters within the NAC might be involved in acquisition/expression of learned fear. There is some suggestion that intra-NAC dopamine could be involved, but the evidence here is very inconsistent (for review, see Ref. [9]). Before exploring these more specific questions, however, it was first necessary to determine if a more consistent pattern of results would be obtained if some measure other than freezing was used to assess learned fear. As described earlier, there are a number of inconsistencies in the literature on the role of the NAC in learned fear. Because that research had largely relied on freezing as a measure of learned fear, the primary goal of the present study was to determine if the fear potentiation of startle procedure could also be used in this area of research. The results clearly show that this procedure can be effectively used to examine the role of the NAC in learned fear. Future studies can now determine if a more consistent pattern of results are obtained with this measure than with freezing, and if so, then begin to explore these more detailed questions.
Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (SFB 550 C8 and Graduiertenkolleg Kognitive Neurobiologie Tqbingen). We thank Mrs. Helga Zillus for her excellent technical assistance.
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Research report
Temporary inactivation of the nucleus accumbens disrupts acquisition and expression of fear-potentiated startle in rats Isabel Schwienbachera,*, Markus Fendta, Rick Richardsonb, Hans-Ulrich Schnitzlera a
Animal Physiology, University of Tuebingen, Auf der Morgenstelle 28, D-72076 Tuebingen, Germany b School of Psychology, University of New South Wales, Sydney, Australia Accepted 19 August 2004 Available online 18 September 2004
Abstract Recent research suggests that in addition to its prominent role in appetitive learning, the nucleus accumbens (NAC) may also be involved in fear conditioning. In the present study, we investigated whether temporary inactivation of the NAC, by injection of tetrodotoxin (TTX), affects acquisition and expression of conditioned fear, as measured by fear-potentiated startle (FPS). TTX injection into the NAC totally blocked acquisition and markedly decreased expression of conditioned fear to a discrete visual conditioned stimulus (CS). Interestingly, temporary inactivation of the NAC did not affect shock sensitization of startle, indicating that both the perception of the shock and short-term contextual conditioning was not affected by intra-accumbal TTX injection. Taken together, these results show that the NAC is crucial for acquisition and expression of long-term conditioned fear, as measured by fear-potentiated startle, to discrete CSs, but not short-term conditioned fear to a context. D 2004 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Learning and memory: systems and functions—animals Keywords: Nucleus accumbens; Learned fear; Acoustic startle response; Fear potentiation of startle; Short-term memory; Long-term memory; Tetrodotoxin; Sensitization
1. Introduction The nucleus accumbens (NAC) receives converging afferents from the amygdala, hippocampus, and prefrontal cortex—areas that form a complex network which has been shown to be involved in emotional learning [2,4]. There is also evidence suggesting that the NAC itself is involved in emotional learning, particularly learning about aversive events (i.e., fear conditioning). For example, Campeau et al. [1] reported that presentation of a conditioned stimulus (CS) previously paired with shock enhanced expression of c-fos within the NAC. The results of a number of lesion studies have also suggested that the NAC is involved in the * Corresponding author. Tel.: +49 7071 2976134; fax: +49 7071 292618. E-mail address: [email protected] (I. Schwienbacher). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.08.037
acquisition and expression of conditioned fear [5,6,12,15, 17,18]. However, the exact pattern of results reported in these studies has been inconsistent. For example, Haralambous and Westbrook [5] reported that temporary inactivation of the NAC, with local infusion of the anaesthetic bupivacaine, disrupted context conditioning but had no effect on either acquisition or expression of learned fear to a discrete CS. Similarly, Riedel et al. [17] reported that electrolytic lesions of the NAC shell disrupted learning about a context but had no effect on learning about a discrete CS. Jongen-Relo et al. [6] also found reduced context conditioning following excitotoxic lesions of the NAC shell, but in contrast to the two studies described above, reported that these lesions disrupted conditioning to a discrete CS as well. Kubos et al. [7] reported that lesions of the NAC core had no effect on either type of learning. In stark contrast, Parkinson et al. [12] reported that excitotoxic
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lesions of the NAC shell had no effect on learning about either a context or a discrete CS, but lesions of the NAC core blocked fear conditioning to a discrete CS and enhanced fear conditioning to a context. In other words, the relatively small literature on the effects of lesions of the NAC on learned fear has yielded nearly every possible outcome, and, in some cases, the results have been completely contradictory. Thus, although it seems that the NAC plays a role in fear conditioning, the exact nature of this role is unclear because of the inconsistent findings in the literature. Some of these inconsistencies could be due to procedural differences (e.g., number of training trials, intensity of shock, etc.) between the various studies, or to the use of different lesioning techniques (i.e., temporary vs. permanent; excitotoxic vs. electrolytic). It should also be noted that most of the studies described above assessed acquisition/expression of fear by measuring freezing. Although this is a very common measure of learned fear in rats, it may be problematic to completely rely on freezing as a measure when examining the role of the NAC in learned fear. Specifically, lesions of the NAC have been shown to affect motor activity in some situations [7,11,19], and this effect could have influenced the results reported in some of the studies described above. One solution to this potential difficulty would be to use a measure of fear that is based on the enhancement of an involuntary reflex, rather than the cessation of movement. Such a measure is the fear potentiation of the acoustic startle response (ASR). The aim of the present study was to investigate whether the NAC is required for acquisition and expression of conditioned fear, as measured by fear-potentiated startle (FPS). In this study, the NAC was temporarily inactivated by local injections of tetrodotoxin (TTX), a selective and reversible voltage-gated sodium channel blocker. The effects of local TTX injections into the NAC on acquisition and expression of FPS to a discrete cue was examined first. Then, the effects of intra-accumbal TTX injection on shock sensitization of the ASR were assessed, in order to determine whether inactivation of the NAC (1) reduced perception of the shock stimulus, and (2) affected short-term contextual fear memory.
2. Materials and methods 2.1. Subjects Male Sprague–Dawley rats (Charles River, Sulzfeld, Germany) weighing 200 to 320 g at the time of surgery were housed in groups of 5–6 rats/cage in a temperaturecontrolled colony room under a 12:12-h light/dark cycle (lights on at 07:00 h). They were fed 12 g standard rat chow/ animal/day, and tap water was available ad libitum. Each rat was handled daily before and after surgery. The experiments were performed in accordance with international ethical
guidelines for the care and use of experimental animals and were approved by the local council of animal care (Regierungspr7sidium Tqbingen, ZP 4/02). 2.2. Surgery Rats were anesthetized with ketamine/xylazine (9:1, 100 mg/kg i.p.) and placed into a stereotaxic frame. The skull was exposed and stainless steel guide cannulas (0.7 mm diameter) were implanted bilaterally aiming at the NAC (toothbar +5 mm above the interaural plane; distance from Bregma: rostrocaudal +3.4 mm, mediolateral F1.5 mm, dorsoventral 7.2 mm; coordinates from Ref. [14]). The guide cannulas were fixed to the skull with acrylic cement (Kulzer, Wehrheim, Germany) and three anchoring screws. After surgery, the cannulas were fitted with stylets in order to maintain patency. Experiments began 3 to 4 days after surgery, when all rats had fully recovered. 2.3. Fear conditioning The rats were trained in two identical boxes (386028 cm). The floor of each box consisted of stainless steel rods, 6 mm in diameter, spaced 22 mm apart, center to center. The CS was produced by a 15-W white light bulb located at the top of the box. The boxes were unilluminated at all times except for when the CS was presented. The unconditioned stimulus (US), a 0.6-mA shock, was produced by a shock generator (custom-made, University of Tqbingen) and delivered through the grid floor. Presentations of the CS and the US were controlled by a PC. Animals were placed into the training boxes, allowed to adapt for 5 min, and then given either 5 or 10 CS–US pairings (see Behavioral procedures), with a mean interstimulus interval of 2 min (range: 1–3 min). The US was presented during the last 0.5 s of the 3.7 s light CS. 2.4. Fear-potentiated startle test The magnitude of the ASR was measured in two identical sound-attenuating chambers (1008060 cm). The rats were placed in wire mesh test cages (201012 cm) with solid steel floors that were placed onto piezoelectric accelerometers (custom-made, University of Tqbingen). Movements of the rats caused changes of the accelerometer voltage output. These changes were amplified, digitized, and analyzed by a PC. The ASR magnitude was computed as the difference of the peak-to-peak voltage output of the accelerometer 80 ms before and 80 ms after the onset of the acoustic startle stimulus (100 dB SPL, 10 kHz tone pulse, 20 ms duration including 0.4 ms rise and fall times). Acoustic stimuli were generated by a functionsynthesizer (Hortmann, Neckartenzlingen, Germany) and delivered through loudspeakers mounted 40 cm away from the test cages. A continuous background white noise (55 dB SPL) was present throughout the test session. All sound
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intensity measurements were done with a 0.5-in. condenser microphone and a measuring amplifier (Brqel and Kjaer, Copenhagen, Denmark) after bandpass (0.25–80 kHz) filtering. In addition to the startle data, spontaneous motor activity was also measured in the test cages. Spontaneous motor activity was calculated as the root mean square value of the accelerometer output during a 28-s period between startle stimulus presentations. After a 5-min acclimatization period during which the rats received no stimuli except the background noise, 10 initial startle stimuli (30 s interstimulus intervals) were presented in order to induce a stable baseline of ASR magnitude. Thereafter, each rat was exposed to 20 startle stimuli, of which 10 were presented 3.2 s after onset of the light CS (light-tone trials) and 10 were presented in the absence of the light CS (tone-alone trials) in a pseudorandomized order (30 s interstimulus interval). The tone-alone trials provided a measure of baseline startle. The difference between the tone-alone and the light-tone trials serves as an operational measure of fear (reviewed in Ref. [4]). 2.5. Shock sensitization The same equipment was used here as for the fearpotentiated startle test. The only difference was that the floor of the wire mesh test cages consisted of steel bars, 2 mm in diameter, spaced 15 mm apart, center to center. For this test, rats were first given a 5-min adaptation period, and then received 10 startle stimuli. Those trials served to produce a stable baseline of ASR and were not considered for further analysis. The mean amplitude of the next 30 trials was taken as the mean preshock ASR amplitude. Then, 10 shocks (0.6 mA, 0.5 s duration, 1 Hz) were given. Following this, an additional 40 trials occurred. The mean ASR amplitude for stimuli 6–35 after the shock was taken as the postshock ASR-amplitude. The responses to the first 5 startle stimuli following the shocks were not included in the analysis because the startle sensitization effect is often not detected until 2–4 min after presentation of the shocks [3]. The difference between the pre- and the postshock ASR amplitudes provided a measure of the sensitization effect. Motor activity during shock presentation was also measured and taken as an indication of shock reactivity.
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TTX (n=13) immediately before fear conditioning (Experiment 1). Two days later, FPS was tested; neuronal activity has been shown to return to normal levels within 48 h of TTX infusion [10]. No drug was given prior to testing these rats. To assess whether NAC inactivation affected expression of conditioned fear, rats (n=15) were first fear-conditioned, without any injections (Experiment 2). These rats were then tested twice, 48 h apart. The rats received either saline or TTX immediately before testing (in a counterbalanced order). To reduce extinction of the conditioned fear during the two test days, these rats were retrained (only 5 CS–US pairings) on each test day, 4 h before testing. For the shock sensitization of startle experiment, rats (n=12) were tested twice, 48 h apart. Each rat was tested following saline and TTX infusions, in a counterbalanced order (Experiment 3). 2.8. Histology After behavioral testing had been completed, the rats were sacrificed with an overdose of pentobarbital (Nembutal). Their brains were removed and immersion fixed in 8% paraformaldehyde and 20% sucrose. Frontal sections (50 Am) were cut on a freezing microtome and Nissl stained with thionine. The injection sites were localized and the extent of tissue lesions due to cannulation was examined under a light microscope. The injection sites were drawn onto plates taken from a rat brain atlas [13]. 2.9. Statistical analysis For the acquisition and expression experiments, the startle amplitudes on each trial type (i.e., tone-alone and light-tone) were compared with ANOVA, and group differences in FPS were tested by comparing (with a ttest) difference scores (i.e., ASR amplitude on light-tone trials—ASR amplitude on tone-alone trials). For the shock sensitization experiment, the startle amplitudes on the pre- and postshock trials were compared with ANOVA, and group differences in sensitization were tested by comparing (with a t-test) difference scores (i.e., ASR amplitude on postshock trials —ASR amplitude on preshock trials).
2.6. Drugs and treatment TTX (Tocris, Kfln, Germany) was dissolved in saline and administered bilaterally (10 ng/1 Al per side, according to Ref. [10]) into the NAC with a velocity of 0.5 Al/min. The injection cannulas were left in place for an additional 2 min to allow diffusion of the solution away from the cannulas. 2.7. Behavioral procedures To assess the effects of NAC inactivation on acquisition of conditioned fear, rats received either saline (n=11) or
3. Results Fig. 1A and B shows the cannula placements for the rats that were included in the statistical analysis of the three experiments; the injection sites for all of these rats were located in the core region of the NAC. Some rats in this study (6 rats in Experiment 1 and 4 rats in Experiment 2) had misplaced cannulas (e.g., located in the ventricle or dorsal striatum); the data from these rats were excluded from the statistical analysis.
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Fig. 1. Serial drawings of frontal sections of the NAC depicting the injection sites for rats given TTX either before CS–US training (panel A, black dots) or CS testing (panel A, black squares), and for rats tested in the shock sensitization experiment (panel B; AcbC—nucleus accumbens core, AcbSh—nucleus accumbens shell).
3.1. Experiment 1 Pretraining injections of TTX into the NAC blocked acquisition of fear-potentiated startle (saline: n=8, TTX: n=10; Fig. 2A). ANOVA of startle amplitudes on the tonealone and light-tone trials revealed a significant effect of treatment [ F(1,16)=6.24, p=0.024] and a nonsignificant effect of trial type [ F(1,16)=3.24, p=0.091]. The interaction of trial typetreatment was also significant [ F(1,16)=17.06; p=0.001]. Pairwise comparisons of FPS (i.e., difference scores) revealed that this interaction was due to the TTXinfused rats exhibiting much less FPS than did the salineinfused rats [independent samples t(16)=4.13; p=0.001]. TTX injections 2 days prior to test did not affect baseline (i.e., tone-alone) ASR [t(16)=1.57; p=0.14] or motor activity during the interstimulus intervals [t(16)=1.2; p=0.25]. 3.2. Experiment 2 Expression of fear-potentiated startle was decreased after pretest TTX injections into the NAC (n=11; Fig. 2B). ANOVA of startle amplitudes on the tone-alone and lighttone trials revealed a significant effect of treatment [ F(1,10)=83.62, pb0.001] and trial type [ F(1,10)=21.19, p=0.001]. The interaction of trial typetreatment was also significant [ F(1,10)=16.09, p=0.002]. This interaction was due to the rats exhibiting greater FPS following saline
infusions than after TTX infusions [dependent samples t(10)=4.036, p=0.002]. Pretest TTX injections into the NAC did not affect either baseline startle amplitude [t(10)=1.59; p=0.14; Fig. 2B] or levels of general motor activity during the interstimulus intervals [t(10)=1.75; p=0.11; saline: 262F57; TTX: 162F28]. 3.3. Experiment 3 TTX injections into the NAC did not affect shock sensitization of the ASR (n=12; Fig. 3). Rats exhibited as much shock sensitization of startle following TTX infusions as they did following saline infusions. Statistical analysis of the data supported this description of the results. Specifically, an initial analysis indicated that test order (i.e., TTX or saline first) did not impact on the results ( Fb1.0), so the data were collapsed across this variable for all subsequent analyses. A repeated-measures ANOVA, with both block (pre- vs. postshock) and drug (TTX vs. saline) as repeated measures, yielded a significant effect of block [ F(1,11)=11.36, p=0.006], an effect of drug that approached traditional levels of statistical significance [ F(1,11)=4.48, p=0.058], and a nonsignificant interaction [ F(1,11)=2.50, p=0.14]. The significant effect of block was due to both groups exhibiting a similar increase in startle response amplitude after the shock [dependent samples t(11)=1.58; pN0.14; Fig. 3]. The near-significant effect of drug was due to a slightly larger shock sensitization effect on TTX trials compared to saline
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Fig. 2. Effects of TTX infusions into the NAC immediately before fear conditioning (A) or immediately before testing of FPS (B). Histograms depict the mean ASR magnitude in the absence (black bars) or presence (white bars) of the light CS. Hatched bars represent the difference between tone-alone and light-tone trials, which was taken as an index of fear. TTX infusions into the NAC disrupted both acquisition and expression of conditioned fear (**pb0.01).
trials (see Fig. 3). Preshock ASR amplitudes were not affected by TTX injections [t(11)=0.78; p=0.45]. The rats did exhibit slightly more motor activity during shock when given TTX than when given saline (saline: 62.96F19.73, TTX: 111.1F23.740), but this difference was not statistically significant [t(11)=1.98; p=0.07]. Post hoc analysis of the data on just the first test day (ns=6) yielded a similar pattern of results as reported above, except the magnitude of the sensitization effect was nearly identical for the two groups (for saline rats, preshock ASR=525 and postshock ASR=872; for TTX rats, preshock ASR=613, postshock ASR=938).
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al. were permanent, and were performed before conditioning, it is unclear whether their results were due to the lesions affecting learning or affecting performance. The present study, where the NAC was temporarily inactivated by TTX either just prior to training or just prior to testing, clearly shows that this structure is involved in both the acquisition and the expression of fear to a discrete CS. TTX, a selective and reversible voltage-gated sodium channel blocker, is a particularly effective agent for temporarily inactivating specific areas in the brain. Infusion of this drug completely blocks generation of action potentials in the target area, an effect that has completely dissipated 48 h after infusion [10]. It has to be noted that fibers of passage are also affected by TTX. The main fibers of passage through the NAC are the anterior commissure. Because this fiber bundle is mainly involved in transferring olfactory information between the two hemispheres [8], we are confident that the effects reported in the present study are based on a temporary lesion of the NAC and not due to a blockade of activity in the anterior commissure. The observed impairments in learning to fear a discrete CS are not a consequence of intra-NAC infusions of TTX diminishing the rat’s perception of the shock US. Specifically, the results of the shock sensitization experiment show that intra-accumbal TTX infusions do not reduce either (1) the intense burst of motor activity elicited by the shock, or (2) the shock sensitization of startle effect (see Fig. 3). Indeed, rats given intra-NAC TTX exhibited slightly but not statistically significant greater (1) motor activity following the shock, and (2) sensitization of startle effect. These results clearly show that the TTX rats perceived the shock to be at least as intense as the saline-treated rats did. However, the finding that temporarily inactivating the NAC with TTX had no effect on the shock sensitization effect raises another issue. Specifically, it has been shown that the shock sensitization of startle effect is due to
4. Discussion The present study implicates the NAC, particularly its core region, in both the acquisition and the expression of conditioned fear to a discrete CS, as measured by fear potentiation of startle. Parkinson et al. [12] also found that excitotoxic lesions of the core region of the NAC caused a significant impairment in rats acquiring conditioned fear to a discrete CS. However, because the lesions by Parkinson et
Fig. 3. Effects of TTX infusions into the NAC immediately before shock sensitization of the ASR. Histograms depict the difference between the preand postshock ASR amplitudes. TTX infusions into the NAC did not affect the shock sensitization of startle effect.
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rapid contextual fear conditioning [16]. Therefore, given that NAC inactivation impairs both acquisition and expression of learned fear to a discrete CS, why doesn’t NAC inactivation also reduce the shock sensitization of startle effect? One possibility is that the NAC is involved in learning to fear discrete CSs but is not involved in learning to fear a context. There are numerous examples in the literature of a manipulation affecting CS and context conditioning differentially. For example, lesions of the hippocampus have been reported to disrupt context conditioning but not to affect conditioning to a discrete CS (for review, see Ref. [4]). The present study would be the first demonstration, to our knowledge, of a manipulation impairing conditioning to a discrete CS but not affecting conditioning to a context. Another possibility is that the NAC is not involved in acquisition and expression of short-term fear, but is involved in the transfer of that information into some long-term store. This latter possibility can be tested in two ways: (1) test for fear of a discrete CS shortly after (e.g., 5–10 min) the training trials, and (2) test for fear of a context after a delay (i.e., after 24–48 h). If this idea is correct, then temporarily inactivating the NAC would be found to impair context conditioning but not affect learning about a discrete CS in these cases. It should be noted that Haralambous and Westbrook [5] have reported that temporary inactivation of the NAC disrupts context conditioning. Critically, the test occurred after a delay in that study. Further support, albeit indirect, for the possibility that the NAC is involved in long-term learned fear of a context is provided by the results of the present study. Because the rats in the shock sensitization experiment of the present study were tested twice (i.e., once after saline infusions and once after TTX infusions; 48 h between the two tests; test order counterbalanced), one might have expected to see an elevated startle response during the preshock period on the second test (i.e., because of the context fear). Such a result was not observed however (mean startle amplitude on Day 1=568, on Day 2=529), although rats in both conditions exhibited a pronounced sensitization effect on Day 1 (see Experiment 3 data regarding this). This failure to observe long-term context fear could have been due to the TTX infusions. That is, those rats given TTX prior to the first test could have had impaired long-term acquisition of the context-shock association, and those rats given saline prior to the first test could have had their expression of this learning impaired (because of the TTX infusions prior to the second test). After 10 shocks, one would expect for there to be some context conditioning, but it must be acknowledged that we do not have any explicit evidence that the procedures used here actually result in long-term context fear (i.e., the experiment did not include a group of rats given saline prior to both tests). Future studies will need to explore these ideas more systematically, but whatever the outcome of these future studies, the results of the present study clearly shows that
the fear potentiation of startle procedure is an effective way of examining the role of the NAC in learned fear. All injection sites in the present study were located within the core region of the NAC. However, considering that 1 Al of injectate was infused, we cannot exclude the possibility that some of the TTX diffused into the NAC shell. Empirical support for this possibility is provided by Lorenzini et al. [10], who reported that infusion of a similar concentration and volume of TTX (10 ng/Al) into the NAC inactivated neurons in an area up to 1 mm around the injection site. However, given the location of the cannula tips, it must be the case that the primary site of action of the infused TTX in the present study was in the core region of the NAC. Based on our results, we hypothesize that the NAC core modulates Pavlovian fear conditioning, at least to discrete CSs. At present, it is unclear exactly how the NAC is involved in acquisition/expression of learned fear. More specifically, the present results do not show whether the NAC is directly involved in learned fear or whether the NAC is involved only because of its interconnections with other relevant neural structures (i.e., the amygdala). Furthermore, the present study does not show what neurotransmitters within the NAC might be involved in acquisition/expression of learned fear. There is some suggestion that intra-NAC dopamine could be involved, but the evidence here is very inconsistent (for review, see Ref. [9]). Before exploring these more specific questions, however, it was first necessary to determine if a more consistent pattern of results would be obtained if some measure other than freezing was used to assess learned fear. As described earlier, there are a number of inconsistencies in the literature on the role of the NAC in learned fear. Because that research had largely relied on freezing as a measure of learned fear, the primary goal of the present study was to determine if the fear potentiation of startle procedure could also be used in this area of research. The results clearly show that this procedure can be effectively used to examine the role of the NAC in learned fear. Future studies can now determine if a more consistent pattern of results are obtained with this measure than with freezing, and if so, then begin to explore these more detailed questions.
Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (SFB 550 C8 and Graduiertenkolleg Kognitive Neurobiologie Tqbingen). We thank Mrs. Helga Zillus for her excellent technical assistance.
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