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Stress Facilitates Acquisition of the Classically Conditioned Eyeblink Response at Both Long and Short Interstimulus Intervals
Learning and Motivation 32, 178–192 (2001) doi:10.1006/lmot.2000.1071, available online at http://www.idealibrary.com on
Stress Facilitates Acquisition of the Classically Conditioned Eyeblink Response at Both Long and Short Interstimulus Intervals Richard J. Servatius,*,† Francis X. Brennan,*,† Kevin D. Beck,† Dawn Beldowicz,† and Kelly Coyle-DiNorcia† *Department of Neuroscience, New Jersey Medical School; and †Department of Veterans Affairs, New Jersey Health Care System, East Orange, New Jersey Exposure to inescapable stress facilitates acquisition of the classically conditioned eyeblink response in freely moving rats. Here, we determined whether facilitated acquisition of the eyeblink conditioned response (CR) depended on the interstimulus interval (ISI). Rats were either stressed (40 2-mA tailshocks delivered over a single 1-h session) or served as nonstressed controls. Paired training was accomplished with a 300-, 500-, 700-, or 1200-ms ISI. Separate groups of stressed and nonstressed rats were exposed to explicitly unpaired stimuli. Consistent with eyeblink conditioning using humans or rabbits as subjects, acquisition depended on the ISI; a ‘‘Ushaped’’ curve was described for nonstressed rats. Acquisition was fastest at the 700-ms ISI; the order was 700 ⬎ 500 ⬎ 300 ⬎ 1200 ms. The unconditioned response decreased with training. Except for the 700-ms ISI, exposure to stress facilitated acquisition. Exposure to stress did not affect the amplitude of the unconditioned response. Facilitated acquisition of the eyeblink CR after exposure to inescapable stressors is independent of nonassociative changes in reactivity to the conditioned stimulus or unconditioned stimulus. 2001 Academic Press
Classical conditioning of the eyeblink response has proven to be a valuable model system for understanding the neural basis of associative learning. This paradigm affords a high degree of specificity; nonassociative and performance factors can be independently assessed (Gormezano, 1966; Gormezano & Kehoe, 1975). For another, extensive research in the rabbit has established distinct neural dependencies for the conditioned stimulus (CS) (Tracy, Thompson, Krupa, & Thompson, 1998; Steinmetz & Sengelaub, 1992), the unconditioned stimulus (US) (Mauk, Steinmetz, & Thompson, 1986; Sears & Steinmetz, 1991), and the associative nexus residing in the deep cerebellar The authors thank the engineering cadre of the Neurobehavioral Unit for their expert support. Funds provided by the Cooperative VA/DOD Research Program and DVA Medical Research Funds to R.J.S. Address correspondence and reprint requests to Richard J. Servatius, Neurobehavioral Unit, 127A, DVA Medical Center, 88 Ross St., East Orange, NJ 07019. 178 0023-9690/01 $35.00 Copyright 2001 by Academic Press All rights of reproduction in any form reserved.
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nuclei (Yeo, Hardiman, & Glickstein, 1985; Kim & Thompson, 1997; McCormick & Thompson, 1984; Steinmetz et al., 1987). Although not necessary, activity in the prefrontal cortex (Powell, 1992; Powell, Buchanan, & Gibbs, 1990), amygdala (Shors & Mathew, 1998), and nigrostriatal pathway (Kao & Powell, 1986) affect acquisition of the eyeblink response. Acquisition of the eyeblink response will occur in the absence of these structures, although at a slower pace. The utility of the eyeblink conditioning paradigm will ultimately be judged on the concordance of animal research to the human condition. Accordingly, research in humans with various pathologies (Bracha, Zhao, Wunderlich, Morrissy, & Bloedel, 1997; Woodruff-Pak & Papka, 1996; Woodruff-Pak, 1993) has largely supported the neural contributions identified in the lower mammalian models. To expand the scope of mammalian model systems there has been a recent movement to extend the eyeblink conditioning paradigm to rats and mice. The topography of the rodent conditioned response (CR) resembles that of rabbits and humans. Moreover, initial work suggests that there is a concordance in the neural dependencies for rabbit and rats (Schmajuk, Lam, & Christiansen, 1994; Christiansen & Schmajuk, 1992; Freeman, Barone, & Stanton, 1995; Freeman, Carter, & Stanton, 1995; Castro-Alamancos & Torres-Aleman, 1994; Weiss et al., 1999; Weiss & Thompson, 1992; Weiss, Bouwmeester, Power, & Disterhoft, 1999). However, many of these studies are based on the tacit assumption that acquisition of the eyeblink CR in rats is parametrically similar to that in rabbits. As the rat model gains wider acceptance, the need for parametric demonstrations will be greater (Weiss et al., 1999). One factor determining acquisition of the eyeblink CR is the timing between CS onset and US onset, the interstimulus interval (ISI). Acquisition of the eyeblink CR does not occur with simultaneous and backward conditioning paradigms. For humans, the concave function (‘‘inverted U’’) for acquisition peaks at an ISI of 400–600 ms (Prescott, Durkin, Furchtgott, & Powell, 1992). Learning is less efficient at 100 and 800 ms, but robust conditioning will occur with a 1000- to 1200-ms ISI (Kimble, Leonard, & Perlmuter, 1968; Prescott et al., 1992; Little, Lipsitt, & Rovee, 1984). For rabbits, the curve describing acquisition as a function of ISI is shifted slightly to the left. Acquisition is optimal at shorter ISIs relative to humans, ranging from 200 to 500 ms (Smith, 1968; Smith, Coleman, & Gormezano, 1969; Port, Mikhail, & Patterson, 1985a; Steinmetz, 1990; Sears, Baker, & Frey, 1979; Coleman & Gormezano, 1971). An important next step in the development of the rat model of eyeblink conditioning is a parametric manipulation of interstimulus interval. The interest of our laboratory and others is the proactive effects of exposure to inescapable stressors on the processes of learning and coping with subsequent aversive events. Exposure to intense inescapable stressors facilitates acquisition of the classically conditioned eyeblink response (CCER) in
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rats (Shors, Weiss, & Thompson, 1992); an effect that persists for days after stressor cessation (Servatius & Shors, 1994). Appearance of facilitated acquisition depends on the intensity of the stressor (Shors & Servatius, 1997) and may be extended through reexposure to contextual cues associated with the stressful experience (Shors & Servatius, 1997). While much is known about the conditions by which facilitation occurs, the nature of the stress-induced enhancement remains controversial. Exposure to stress may affect processing of the CS or the US individually or affect responding to the CS after exposure to the US (pseudoconditioning). Prior work demonstrated that stressed rats had increased reactivity to the CS prior to training (Servatius et al., 1994; Servatius & Shors, 1996) and pseudoconditioned to a greater degree (Servatius et al., 1994) than nonstressed rats. These data suggested that facilitated acquisition was secondary to enhanced processing of the CS. However, stressed rats whose reactivity to the CS normalized after extensive unpaired training still exhibited facilitated acquisition. This manipulation did not account for the possibility that the change in contingency between the CS and US reinstated enhanced processing of the CS. Thus, the question remains as to whether stress proactively facilitates the associative process or whether facilitated acquisition is secondary to enhanced processing of the CS. A manipulation of the ISI should illuminate this issue. If the primary disturbance after exposure to stress were an enhancement of sensory reactivity, one would expect that facilitation would be evident at shorter ISIs. In contrast, the advantage of quicker processing and responding would be a disadvantage as the ISI lengthened. However, if stress affects the associative process linking the CS and US, then facilitated acquisition should be apparent regardless of the ISI (ceiling effects notwithstanding). Therefore, we manipulated stress and ISI to (1) establish parametric data for the freely moving rat preparation and (2) evaluate the effects of stress on acquisition of the eyeblink CR as a function of ISI. Last, exposure to an inescapable stressor may increase sensitivity to the US. It is well known that acquisition of the eyeblink CR is positively related to US intensity (Smith, 1968). However, previous work did not allow for an analysis of the unconditioned response (UR) in our freely moving rat preparation. Recently, we described a method using a square-wave stimulus as the US that allowed the measure of eyelid opening after stimulation in EMG activity (Servatius, 2000). Using this preparation, we demonstrated that the UR reliably tracks US intensity and learning of the eyeblink CR. Therefore, the present study evaluated the UR as a function of stress and ISI. METHOD
Subjects Subjects were 105 male Sprague–Dawley rats obtained from Charles River (MA). All rats were housed individually in shoebox cages with free
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access to food and water. The individual cages were kept in chambers designed to control light cycles, filter air, control temperature, and attenuate noise; up to 16 rat cages may be housed within a single chamber. Rats were maintained on a 12 :12 light : dark cycle with lights on at 07:00. Surgery All rats were surgically implanted with electrodes for obtaining eyelid electromyograph (EMG) signals and the delivery of the unconditioned stimulus (US) as described previously (Servatius & Shors, 1994). Briefly, rats were anesthetized with sodium pentobarbital and fitted with head stages. Electrodes (two for EMG and two for US delivery) were implanted subcutaneously and emerged through the eyelid. Postoperatively, rats were given 24 h access to Children’s Non-aspirin Pain Elixir diluted 1:100 in the drinking water. Rats were allowed at least 4 days to recover from surgery prior to training. Stress As previously described (Servatius, Ottenweller, Bergen, Soldan, & Natelson, 1994), stressed rats were exposed to a single session of loose restraint in hardware cloth and exposed to 40 3-s tailshocks (2 mA) over a 1-h period. For the delivery of shock, a PC-compatible computer running Viewdac software (Keithly-Metrabyte, Tauton, MA) controlled a Coulbourne Instruments (Allentown, PA) shocker. Shock amplitude was monitored and adjusted online to account for sessionwide changes in resistance. Apparatus Conditioning was accomplished in a modular test cage within an isolation chamber (Coulbourne Instruments). The EMG recording electrodes were connected to a differential AC amplifier with a 300- to 500-Hz bandpass filter (A-M Systems Model 1700, Everett, WA) and were amplified by 10K. A PC computer equipped with an A/D board (Keithley-MetraByte DAS 1602) collected the EMG signals. The timing of stimulus presentation and EMG data collection was controlled using a program written in LabView (National Instruments). The CS was produced by shaping the output of a white noise generator (82 dB) with a rise/fall gate (10-ms rise/fall) (Coulbourne Instruments). A Bioelectric Stimulus Isolator (Coulbourne Instruments) produced the US. The US was a 10- ms square-wave stimulus of 10 V. Procedure All rats completed the surgical procedure outlined above. Rats were weighted and then randomly assigned to groups in an incomplete 4 ⫻ 2 ⫻ 2 (ISI ⫻ Stress ⫻ Training) between-subjects design. We have the capability to examine learning in four rats at one time. Therefore, we only examined
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a single ISI during a particular experimental run. Each experimental run had an equal number of stressed and nonstressed rats. Animals were habituated to the experimental chamber for 1 h during which spontaneous eyeblinks were recorded. After this acclimation session, rats were either transferred to the stress room or returned to their home cages. Training commenced on the next day during the same time period in which the initial acclimation period occurred. Rats participated in two daily training sessions. The ISI for training was 300, 500, 700, or 1200 ms. A delay-type paradigm was employed; that is, the CS and US coterminated. Each session consisted of 100 trials divided equally into 10 blocks of 10 trials. Each block began with a CS-alone trial, was followed by 4 paired trials, then a US-alone trial, and ended with 4 more paired trials. These trials were separated by 20–40 s. Explicitly unpaired training consisted of exposing rats to the 300-, 500-, or 700-ms CS and the US. Rats were exposed to an equal number of CS and US presentations; therefore, a session of unpaired training consisted of 180 alternating CS-alone and US-alone trials. The intertrial interval for unpaired training was 10–20 s. A typical experimental run had two nonstressed and two stressed rats, half of whom received paired training. This balanced the factors of stress and training over a given experimental run. Thus, ISI was not manipulated in any experimental run. Last, we did not perform unpaired training at the 1200ms ISI. Preliminary data suggested that unpaired training yielded very little pseudoconditioning at the 700-ms ISI. We therefore did not expect unpaired training at the 1200-ms ISI to yield quantitatively different results from that of the 300-, 500-, and 700-ms ISIs; hence, the incomplete 4 ⫻ 2 ⫻ 2 design. Data Reduction and Statistics Eyelid EMG recordings were sampled at 1 kHz. The EMG signal was low pass filtered using a locally weighted regression filter (Lowess, Stat-Sci, Tacoma WA) with a time constant of 0.025 and a smoothing interval of 5. Using this algorithm an eyeblink corresponds to activity greater than 0.4 (unitless). For the acclimation period, the entire EMG record was processed for the appearance of eyeblinks. For the recordings in which a stimulus was delivered, each EMG record was subdivided into a 200-ms prestimulus baseline. If an eyeblink occurred during the 200-ms prestimulus baseline, that trial was thrown out and an NA was recorded. During conditioning, an eyeblink response was scored as an α-response (orienting response) if the onset occurred within 40 ms of the CS onset. An eyeblink was scored as a conditioned response (CR) if it occurred at least 60 ms after the CS onset and before the onset of the US. On CS-alone trials the CR interval was extended 100 ms. In addition to collecting the CR data, we also collected data on the UR (Servatius, 2000). The square-wave stimulation caused a significant stimulus artifact in the EMG record that dissipated by 40 ms after US offset. The
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post-US record was low pass filtered to remove the ‘‘ringing’’ of the amplifier after US offset. Videographic evidence demonstrated that filtered EMG activity 45–245 ms after US offset corresponded to the eyelid opening after the cessation of US-induced closure. Further, the amplitude of this event was sensitive to US intensity and within session changes in amplitude associated with learning. Therefore, peak EMG activity in the post-US period was considered a surrogate of the UR (Servatius, 2000). The CR and UR data were analyzed with analysis of variance models. The data for each ISI were analyzed separately; these models included data from rats given unpaired training. An overall analysis was also performed that directly evaluated acquisition as a function of ISI; this model did not include unpaired controls. In addition, acquisition was evaluated as the number of trials to a criterion of greater than 16 eyeblink CRs in 20 consecutive trials. If a rat failed to reach the criterion by the second training session, 250 was scored (midpoint of the next session) as a placeholder. Post hoc analyses were accomplished with F tests for simple effects and Dunnett’s tests for a priori comparisons with a control mean. RESULTS
300-ms ISI Acquisition of the eyeblink CR for rats previously exposed to inescapable stress was facilitated compared to nonstressed rats. Moreover, pseudoconditioning was not apparent in stressed rats given unpaired training. These impressions were supported by the 2 ⫻ 2 ⫻ 2 ⫻ 9 (Stress ⫻ Training ⫻ Session ⫻ Trial Block) mixed ANOVA. The significant main effects and two-way interactions were subordinate to the Stress ⫻ Training ⫻ Session interaction, F(1, 391) ⫽ 10.8, P ⬍.001 (see Fig. 1A). For nonstressed rats, acquisition of the eye-blink CR at the 300 ms ISI was generally accomplished during the second session of training (104 ⫾ 35 trials). In contrast, stressed rats acquired the eyeblink CR during the first training session (53 ⫾ 18 trials). The amplitude of URs differed as a function of stress and training. For nonstressed rats, the URs of paired trained rats were greater than those emitted by unpaired trained rats. The URs of stressed rats given paired training did not differ from nonstressed rats during the first session of paired training; nonstressed rats exhibited greater URs during the initial stages of the second training session. Stressed rats given unpaired training exhibited greater URs compared to nonstressed rats given unpaired training over both training sessions (data not shown). In support, the analysis yielded a significant Stress ⫻ Training ⫻ Session interaction, F(1, 390) ⫽ 7.3, P ⬍.001 (see Fig. 1B). 500-ms ISI A similar pattern of results was obtained for the rats trained at the 500ms ISI, that is, facilitated acquisition of the eyeblink CR in previously
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FIG. 1. (Left) Percentage of CRs across the two sessions, using a 300-ms ISI delay paradigm. (Right) UR amplitude across the two sessions during the US-alone trials.
stressed rats. Again pseudoconditioning was not apparent in stressed rats given unpaired training (see Fig. 2A). Consistent with these impressions, the analysis indicated a significant Stress ⫻ Training ⫻ Block interaction, F(1, 408) ⫽ 4.1, P ⫽ .04. Nonstressed rats generally acquired the eyeblink CR between the first and second training sessions (99 ⫾ 11 trials). Stressed rats exhibited faster acquisition of the eyeblink CR (72 ⫾ 29 trials). For paired trained rats URs generally decreased with training in both stressed and nonstressed rats. Stress rats given unpaired training, however, exhibited greater URs during the initial trial blocks (data not shown). The analysis yielded a significant Stress ⫻ Training ⫻ Trial Block interaction, F(1, 388) ⫽ 2.0, P ⫽ .05 (see Fig. 2B). 700-ms ISI Acquisition of the eyeblink CR did not differ between the stressed and nonstressed rats. The only significant factors in the mixed ANOVA were main effects of Training, Session, and Trial Block and the two interactions of Training ⫻ Session and Training ⫻ Trial Block, all Ps ⬍.01 (see Fig. 3A). Here, acquisition was generally accomplished in the first session of training for both stressed (80 ⫾ 34 trials) and nonstressed (69 ⫾ 30 trials) rats.
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FIG. 2. (Left) Percentage of CRs across the two sessions, using a 500-ms ISI delay paradigm. (Right) UR amplitude across the two sessions during the US-alone trials.
For paired trained rats URs generally decreased with training in both stressed and nonstressed rats. There were no stress-related differences in UR amplitudes. The main effects of Training, Session, and Trial Block as well as the Training ⫻ Session and Training ⫻ Trial Block interactions were significant, all Ps ⬍ .05 (see Fig. 3B). 1200-ms ISI Facilitated acquisition of the eyeblink CR was again evident in previously stressed rats. In support, the analysis yielded a significant Stress ⫻ Session interaction, F(1, 357) ⫽ 10.0, P ⬍.001 (see Fig. 4A). For stressed rats, acquisition of the eyeblink CR generally occurred in the second training session (109 ⫾ 37 trials). In contrast, few nonstressed rats acquired the eyeblink CR by the second training session (187 ⫾ 35 trials). The UR amplitudes decreased with training. There were no differential effects attributable to stressor exposure. The analysis yielded only significant main effects of Session and Trial Block, Ps ⬍.001 (see Fig. 4B). Overall Acquisition of the eyeblink CR depended on ISI during paired training (see Fig. 5). For nonstressed rats, efficient acquisition was apparent with the
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FIG. 3. (Left) Percentage of CRs across the two sessions, using a 700-ms ISI delay paradigm. (Right) UR amplitude across the two sessions during the US-alone trials.
intermediate levels of ISI, i.e., 500 and 700 ms; all nonstressed rats reached criterion by the end of the second session. Acquisition of the eyeblink CR was slower in rats trained with the 300-ms ISI. Rats trained with the 300ms ISI acquired the eyeblink response by the end of the second session of training; only 1 rat of 9 failed to reached criterion by the end of the second session. Acquisition was slowest in rats trained with the 1200-ms ISI; only 5 of 11 rats acquired the eyeblink CR by the end of the second training session. As for stress, facilitated acquisition was evident at the 300-, 500-, and 1200-ms ISI, but not the 700-ms ISI. Although stressed rats trained at the 1200-ms ISI acquired the eyeblink CR faster than nonstressed rats, learning was poorer at the 1200-ms ISI than at the other ISIs. Analysis of the acquisition data as a function of stress and ISI demonstrated a significant Stress ⫻ ISI ⫻ Session interaction, F(1, 1156) ⫽ 12.8, P ⬍ .001. DISCUSSION
The primary goal of the present study was to assess acquisition of the eyeblink CR as a function of ISI. Acquisition of the eyeblink CR in a delaytype task differed as a function of the ISI. Similar to humans and rabbits, acquisition in freely moving rats could be described by an inverted ‘‘U’’shaped function. The fastest acquisition of the eyeblink CR was apparent in
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FIG. 4. (Left) Percentage of CRs across the two sessions, using a 1200-ms ISI delay paradigm. (Right) UR amplitude across the two sessions during the US-alone trials.
rats trained with a 700-ms ISI. Slower but still efficient acquisition was apparent with a 500-ms ISI. For both the 500- and 700-ms ISIs, acquisition was generally accomplished within the first session of training. Learning was less efficient with a 300-ms ISI. Nonstressed rats required more trials to acquire the eyeblink CR; acquisition was generally apparent during the second training session. Although the 300-ms ISI was not optimal for acquisition, only 11% of nonstressed rats failed to acquire the eyeblink CR by the second session of training. Moreover, these data at the 300 ms ISI are consistent with the acquisition curves described by others (Weiss et al., 1999; Servatius et al., 1994; Shors et al., 1998). Acquisition degraded substantially at the 1200-ms ISI; only 45% of nonstressed rats acquired the eyeblink CR by the end of the second session of training. We did not continue training to determine the proportion of rats that would fail to acquire the eyeblink response with further training. However, the data clearly demonstrate that acquisition at 1200 ms was more difficult for freely moving rats. The acquisition function as it relates to ISI differed from those described for rabbits and humans. For humans, acquisition peaks at 400- to 600-ms ISIs (Prescott et al., 1992). For rabbits, acquisition is optimal between 200and 500-ms ISIs (Smith, 1968; Smith et al., 1969; Port et al., 1985a; Steinmetz, 1990; Sears et al., 1979; Coleman et al., 1971). Thus, the optimal
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FIG. 5. (Left) Mean percentage of CRs across the two sessions at all ISIs. (Right) UR amplitude across the two sessions during the US-alone trials at all ISIs.
ISIs for freely moving rats appear to be similar to those for humans, with both being longer than for rabbits. Although not specifically manipulated here, we expect that rats would be similar to humans and rabbits in the inability to acquire the eyeblink CR with simultaneous or backward conditioning. A second goal of the current study was to examine the impact of stress on acquisition of the eyeblink CR. Exposure to inescapable stressors facilitated acquisition of the eyeblink CR at less than optimal ISIs. Thus, facilitation was evident at the 300-, 500-, and 1200-ms ISIs. The facilitation was most prevalent at the 1200-ms ISI. Here, nonstressed rats generally did not acquire the eyeblink CR over the 200 training trials, whereas stressed rats did. Facilitation was not evident at the 700-ms ISI. However, acquisition was fastest in nonstressed rats at this interval. Therefore, the lack of facilitation may be due to a ‘‘ceiling’’ effect. Similarly, in a series of studies by Port and colleagues, hippocampal lesions facilitated acquisition at short and long ISIs, but not at the optimal ISI (Port et al., 1985a; Port, Mikhail, & Patterson, 1986b). How then does exposure to inescapable stressors facilitate acquisition? One possible explanation centers on enhanced processing of the CS. Exposure to inescapable stressors affects reactivity to acoustic stimuli for some time after stressor cessation (Servatius et al., 1994; Davis, 1989; Servatius, Ottenweller, & Natelson, 1995). At shorter ISIs, such a response may resem-
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ble an associative response. Rabbit research demonstrated that the timing of the eyeblink CR is relative to US onset. As the ISI lengthens, the time between CS onset and the initiation of the CR should also lengthen. In contrast, a reactive response would be evident as a quick, long-lasting response. Inspection of the EMG activity did not reveal such response topography in the stressed rats trained at the 1200-ms ISI. Thus, facilitation is not merely a function of enhanced reactivity or responsivity to the CS. Enhanced reactivity to the US could also account for faster acquisition of the eye-blink CR. Reactivity to the US was assessed as the magnitude of the UR, as previously defined (Servatius, 2000). Consistent with our earlier work and that of others (Canli et al., 1992; Donegan, 1981), the UR in USalone trials decreased with acquisition of the eye-blink CR. Facilitation of the UR was also apparent; the UR was larger in rats given paired CS–US exposures than in rats given explicitly unpaired CS and US exposures (Schreurs et al., 1995). Exposure to inescapable stressors did not affect UR magnitudes in rats given paired training. Therefore, enhanced reactivity to the US does not appear to account for facilitated acquisition of the eyeblink CR. However, larger URs were apparent in stressed rats given unpaired training compared to those of nonstressed rats. These data suggest that exposure to stress enhanced nonassociative processes modulating UR magnitude. In summary, the learning curves for rats trained in a delay-conditioning task resemble those generated for rabbits and humans, although the optimal ISI appears to be slightly longer for rats. Although exposure to inescapable stressors persistently facilitated acquisition of the eyeblink CR at long and short ISIs, facilitation was not apparent at the optimal ISI. Facilitated acquisition was not the result of enhanced sensitivity to the US. REFERENCES Bracha, V., Zhao, L., Wunderlich, D. A., Morrissy, S. J., & Bloedel, J. R. (1997). Patients with cerebellar lesions cannot acquire but are able to retain conditioned eyeblink reflexes. Brain, 120, 1401–1413. Canli, T., Detmer, W. M., & Donegan, N. H. (1992). Potentiation or dimunition of discrete motor unconditioned responses (rabbit eyeblink) to an aversive Pavlovian unconditioned stimulus by two associative processes: Conditioned fear and a conditioned dimunition of unconditioned stimulus processing. Behavioral Neuroscience, 106, 498–508. Castro-Alamancos, M. A., & Torres-Aleman, I. (1994). Learning of the conditioned eye-blink response is impaired by an antisense insulin-like growth factor I oligonucleotide. Proceedings of the National Academy of Sciences USA, 91, 10203–10207. Christiansen, B. A., & Schmajuk, N. A. (1992). Hippocampectomy disrupts the topography of the rat eyeblink response during acquisition and extinction of classical conditioning. Brain Research, 595, 206–214. Coleman, S. R., & Gormezano, I. (1971). Classical conditioning of the rabbit’s (Oryctolagus cuniculus) nictitating membrane response under symmetrical CS–US interval shifts. Journal of Comparative and Physiological Psychology, 77, 447–455. Davis, M. (1989). Sensitization of the acoustic startle reflex by footshock. Behavioral Neuroscience, 103, 495–503.
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during acquisition of the rabbit classically conditioned eyelid response. Brain Research, 545, 114–122. Servatius, R. J. (2000). Eyeblink conditioning in the freely-moving rat: Square-wave stimulation as the unconditioned stimulus. Journal of Neuroscience Methods, 102, 35–42. Servatius, R. J., Ottenweller, J. E., Bergen, M. T., Soldan, S., & Natelson, B. H. (1994). Persistent stress-induced sensitization of adrenocortical and startle responses. Physiology & Behavior, 56, 945–954. Servatius, R. J., Ottenweller, J. E., & Natelson, B. H. (1995). Delayed startle sensitization distinguishes rats exposed to one or three stress sessions: Further evidence toward an animal model of PTSD. Biological Psychiatry, 38, 539–546. Servatius, R. J., & Shors, T. J. (1994). Exposure to inescapable stress persistently facilitates associative and nonassociative learning in rats. Behavioral Neuroscience, 108, 1101– 1106. Servatius, R. J., & Shors, T. J. (1996). Early acquisition, but not retention, of the classically conditioned eyeblink response is N-methyl-d-aspartate (NMDA) receptor dependent. Behavioral Neuroscience, 110, 1040–1048. Shors, T. J., & Mathew, P. R. (1998). NMDA receptor antagonism in the lateral/basolateral but not central nucleus of the amygdala prevents the induction of facilitated learning in response to stress. Learning and Memory, 5, 220–230. Shors, T. J., & Servatius, R. J. (1997). The contribution of stressor intensity, duration, and context to the stress-induced facilitation of associative learning. Neurobiology of Learning and Memory, 67, 92–96. Shors, T. J., Weiss, C., & Thompson, R. F. (1992). Stress-induced facilitation of classical conditioning. Science, 257, 537–539. Smith, M. C. (1968). CS–US interval and US intensity in classical conditioning of the rabbit’s nictitating membrane response. Journal of Comparative and Physiological Psychology, 66, 679–687. Smith, M. C., Coleman, S. R., & Gormezano, I. (1969). Classical conditioning of the rabbit’s nictitating membrane response at backward, simultaneous, and forward CS–US intervals. Journal of Comparative and Physiological Psychology, 69, 226–231. Steinmetz, J. E. (1990). Classical nictitating membrane conditioning in rabbits with varying interstimulus intervals and direct activation of cerebellar mossy fibers as the CS. Behavioral Brain Research, 38, 97–108. Steinmetz, J. E., Logan, C. G., Rosen, D. J., Thompson, J. K., Lavond, D. G., & Thompson, R. F. (1987). Initial localization of the acoustic conditioned stimulus projection system to the cerebellum essential for classical eyelid conditioning. Proceedings of the National Academy of Sciences USA, 84, 3531–3535. Steinmetz, J. E., & Sengelaub, D. R. (1992). Possible conditioned stimulus pathway for classical eyelid conditioning in rabbits. I. Anatomical evidence for direct projections from the pontine nuclei to the cerebellar interpositus nucleus. Behavioral and Neural Biology, 57, 103–115. Tracy, J. A., Thompson, J. K., Krupa, D. J., & Thompson, R. F. (1998). Evidence of plasticity in the pontocerebellar conditioned stimulus pathway during classical conditioning of the eyeblink response in the rabbit. Behavioral Neuroscience, 112, 267–285. Weiss, C., Bouwmeester, H., Power, J. M., & Disterhoft, J. F. (1999). Hippocampal lesions prevent trace eyeblink conditioning in the freely moving rat. Behavioral Brain Research, 99, 123–132. Weiss, C., Knuttinen, M. G., Power, J. M., Patel, R. I., O’Connor, M. S., & Disterhoft, J. F.
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(1999). Trace eyeblink conditioning in the freely moving rat: Optimizing the conditioning parameters. Behavioral Neuroscience, 113, 1100–1105. Weiss, C., & Thompson, R. F. (1992). Delayed acquisition of eyeblink conditioning in aged F1 hybrid (Fischer-344 ⫻ Brown Norway) rats. Neurobiology of Aging, 13, 319–323. Woodruff-Pak, D. S. (1993). Eyeblink classical conditioning in H. M.: Delay and trace paradigms. Behavioral Neuroscience, 107, 911–925. Woodruff-Pak, D. S., & Papka, M. (1996). Huntington’s disease and eyeblink classical conditioning: Normal learning but abnormal timing. Journal of the International Neuropsychological Society, 2, 323–334. Yeo, C. H., Hardiman, M. J., & Glickstein, M. (1985). Classical conditioning of the nicitating membrane response of the rabbit. I. Lesions of the cerebellar nuclei. Experimental Brain Research, 60, 87–98. Received June 21, 2000, revised September 8, 2000 Published online February 16, 2001
Stress Facilitates Acquisition of the Classically Conditioned Eyeblink Response at Both Long and Short Interstimulus Intervals Richard J. Servatius,*,† Francis X. Brennan,*,† Kevin D. Beck,† Dawn Beldowicz,† and Kelly Coyle-DiNorcia† *Department of Neuroscience, New Jersey Medical School; and †Department of Veterans Affairs, New Jersey Health Care System, East Orange, New Jersey Exposure to inescapable stress facilitates acquisition of the classically conditioned eyeblink response in freely moving rats. Here, we determined whether facilitated acquisition of the eyeblink conditioned response (CR) depended on the interstimulus interval (ISI). Rats were either stressed (40 2-mA tailshocks delivered over a single 1-h session) or served as nonstressed controls. Paired training was accomplished with a 300-, 500-, 700-, or 1200-ms ISI. Separate groups of stressed and nonstressed rats were exposed to explicitly unpaired stimuli. Consistent with eyeblink conditioning using humans or rabbits as subjects, acquisition depended on the ISI; a ‘‘Ushaped’’ curve was described for nonstressed rats. Acquisition was fastest at the 700-ms ISI; the order was 700 ⬎ 500 ⬎ 300 ⬎ 1200 ms. The unconditioned response decreased with training. Except for the 700-ms ISI, exposure to stress facilitated acquisition. Exposure to stress did not affect the amplitude of the unconditioned response. Facilitated acquisition of the eyeblink CR after exposure to inescapable stressors is independent of nonassociative changes in reactivity to the conditioned stimulus or unconditioned stimulus. 2001 Academic Press
Classical conditioning of the eyeblink response has proven to be a valuable model system for understanding the neural basis of associative learning. This paradigm affords a high degree of specificity; nonassociative and performance factors can be independently assessed (Gormezano, 1966; Gormezano & Kehoe, 1975). For another, extensive research in the rabbit has established distinct neural dependencies for the conditioned stimulus (CS) (Tracy, Thompson, Krupa, & Thompson, 1998; Steinmetz & Sengelaub, 1992), the unconditioned stimulus (US) (Mauk, Steinmetz, & Thompson, 1986; Sears & Steinmetz, 1991), and the associative nexus residing in the deep cerebellar The authors thank the engineering cadre of the Neurobehavioral Unit for their expert support. Funds provided by the Cooperative VA/DOD Research Program and DVA Medical Research Funds to R.J.S. Address correspondence and reprint requests to Richard J. Servatius, Neurobehavioral Unit, 127A, DVA Medical Center, 88 Ross St., East Orange, NJ 07019. 178 0023-9690/01 $35.00 Copyright 2001 by Academic Press All rights of reproduction in any form reserved.
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nuclei (Yeo, Hardiman, & Glickstein, 1985; Kim & Thompson, 1997; McCormick & Thompson, 1984; Steinmetz et al., 1987). Although not necessary, activity in the prefrontal cortex (Powell, 1992; Powell, Buchanan, & Gibbs, 1990), amygdala (Shors & Mathew, 1998), and nigrostriatal pathway (Kao & Powell, 1986) affect acquisition of the eyeblink response. Acquisition of the eyeblink response will occur in the absence of these structures, although at a slower pace. The utility of the eyeblink conditioning paradigm will ultimately be judged on the concordance of animal research to the human condition. Accordingly, research in humans with various pathologies (Bracha, Zhao, Wunderlich, Morrissy, & Bloedel, 1997; Woodruff-Pak & Papka, 1996; Woodruff-Pak, 1993) has largely supported the neural contributions identified in the lower mammalian models. To expand the scope of mammalian model systems there has been a recent movement to extend the eyeblink conditioning paradigm to rats and mice. The topography of the rodent conditioned response (CR) resembles that of rabbits and humans. Moreover, initial work suggests that there is a concordance in the neural dependencies for rabbit and rats (Schmajuk, Lam, & Christiansen, 1994; Christiansen & Schmajuk, 1992; Freeman, Barone, & Stanton, 1995; Freeman, Carter, & Stanton, 1995; Castro-Alamancos & Torres-Aleman, 1994; Weiss et al., 1999; Weiss & Thompson, 1992; Weiss, Bouwmeester, Power, & Disterhoft, 1999). However, many of these studies are based on the tacit assumption that acquisition of the eyeblink CR in rats is parametrically similar to that in rabbits. As the rat model gains wider acceptance, the need for parametric demonstrations will be greater (Weiss et al., 1999). One factor determining acquisition of the eyeblink CR is the timing between CS onset and US onset, the interstimulus interval (ISI). Acquisition of the eyeblink CR does not occur with simultaneous and backward conditioning paradigms. For humans, the concave function (‘‘inverted U’’) for acquisition peaks at an ISI of 400–600 ms (Prescott, Durkin, Furchtgott, & Powell, 1992). Learning is less efficient at 100 and 800 ms, but robust conditioning will occur with a 1000- to 1200-ms ISI (Kimble, Leonard, & Perlmuter, 1968; Prescott et al., 1992; Little, Lipsitt, & Rovee, 1984). For rabbits, the curve describing acquisition as a function of ISI is shifted slightly to the left. Acquisition is optimal at shorter ISIs relative to humans, ranging from 200 to 500 ms (Smith, 1968; Smith, Coleman, & Gormezano, 1969; Port, Mikhail, & Patterson, 1985a; Steinmetz, 1990; Sears, Baker, & Frey, 1979; Coleman & Gormezano, 1971). An important next step in the development of the rat model of eyeblink conditioning is a parametric manipulation of interstimulus interval. The interest of our laboratory and others is the proactive effects of exposure to inescapable stressors on the processes of learning and coping with subsequent aversive events. Exposure to intense inescapable stressors facilitates acquisition of the classically conditioned eyeblink response (CCER) in
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rats (Shors, Weiss, & Thompson, 1992); an effect that persists for days after stressor cessation (Servatius & Shors, 1994). Appearance of facilitated acquisition depends on the intensity of the stressor (Shors & Servatius, 1997) and may be extended through reexposure to contextual cues associated with the stressful experience (Shors & Servatius, 1997). While much is known about the conditions by which facilitation occurs, the nature of the stress-induced enhancement remains controversial. Exposure to stress may affect processing of the CS or the US individually or affect responding to the CS after exposure to the US (pseudoconditioning). Prior work demonstrated that stressed rats had increased reactivity to the CS prior to training (Servatius et al., 1994; Servatius & Shors, 1996) and pseudoconditioned to a greater degree (Servatius et al., 1994) than nonstressed rats. These data suggested that facilitated acquisition was secondary to enhanced processing of the CS. However, stressed rats whose reactivity to the CS normalized after extensive unpaired training still exhibited facilitated acquisition. This manipulation did not account for the possibility that the change in contingency between the CS and US reinstated enhanced processing of the CS. Thus, the question remains as to whether stress proactively facilitates the associative process or whether facilitated acquisition is secondary to enhanced processing of the CS. A manipulation of the ISI should illuminate this issue. If the primary disturbance after exposure to stress were an enhancement of sensory reactivity, one would expect that facilitation would be evident at shorter ISIs. In contrast, the advantage of quicker processing and responding would be a disadvantage as the ISI lengthened. However, if stress affects the associative process linking the CS and US, then facilitated acquisition should be apparent regardless of the ISI (ceiling effects notwithstanding). Therefore, we manipulated stress and ISI to (1) establish parametric data for the freely moving rat preparation and (2) evaluate the effects of stress on acquisition of the eyeblink CR as a function of ISI. Last, exposure to an inescapable stressor may increase sensitivity to the US. It is well known that acquisition of the eyeblink CR is positively related to US intensity (Smith, 1968). However, previous work did not allow for an analysis of the unconditioned response (UR) in our freely moving rat preparation. Recently, we described a method using a square-wave stimulus as the US that allowed the measure of eyelid opening after stimulation in EMG activity (Servatius, 2000). Using this preparation, we demonstrated that the UR reliably tracks US intensity and learning of the eyeblink CR. Therefore, the present study evaluated the UR as a function of stress and ISI. METHOD
Subjects Subjects were 105 male Sprague–Dawley rats obtained from Charles River (MA). All rats were housed individually in shoebox cages with free
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access to food and water. The individual cages were kept in chambers designed to control light cycles, filter air, control temperature, and attenuate noise; up to 16 rat cages may be housed within a single chamber. Rats were maintained on a 12 :12 light : dark cycle with lights on at 07:00. Surgery All rats were surgically implanted with electrodes for obtaining eyelid electromyograph (EMG) signals and the delivery of the unconditioned stimulus (US) as described previously (Servatius & Shors, 1994). Briefly, rats were anesthetized with sodium pentobarbital and fitted with head stages. Electrodes (two for EMG and two for US delivery) were implanted subcutaneously and emerged through the eyelid. Postoperatively, rats were given 24 h access to Children’s Non-aspirin Pain Elixir diluted 1:100 in the drinking water. Rats were allowed at least 4 days to recover from surgery prior to training. Stress As previously described (Servatius, Ottenweller, Bergen, Soldan, & Natelson, 1994), stressed rats were exposed to a single session of loose restraint in hardware cloth and exposed to 40 3-s tailshocks (2 mA) over a 1-h period. For the delivery of shock, a PC-compatible computer running Viewdac software (Keithly-Metrabyte, Tauton, MA) controlled a Coulbourne Instruments (Allentown, PA) shocker. Shock amplitude was monitored and adjusted online to account for sessionwide changes in resistance. Apparatus Conditioning was accomplished in a modular test cage within an isolation chamber (Coulbourne Instruments). The EMG recording electrodes were connected to a differential AC amplifier with a 300- to 500-Hz bandpass filter (A-M Systems Model 1700, Everett, WA) and were amplified by 10K. A PC computer equipped with an A/D board (Keithley-MetraByte DAS 1602) collected the EMG signals. The timing of stimulus presentation and EMG data collection was controlled using a program written in LabView (National Instruments). The CS was produced by shaping the output of a white noise generator (82 dB) with a rise/fall gate (10-ms rise/fall) (Coulbourne Instruments). A Bioelectric Stimulus Isolator (Coulbourne Instruments) produced the US. The US was a 10- ms square-wave stimulus of 10 V. Procedure All rats completed the surgical procedure outlined above. Rats were weighted and then randomly assigned to groups in an incomplete 4 ⫻ 2 ⫻ 2 (ISI ⫻ Stress ⫻ Training) between-subjects design. We have the capability to examine learning in four rats at one time. Therefore, we only examined
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a single ISI during a particular experimental run. Each experimental run had an equal number of stressed and nonstressed rats. Animals were habituated to the experimental chamber for 1 h during which spontaneous eyeblinks were recorded. After this acclimation session, rats were either transferred to the stress room or returned to their home cages. Training commenced on the next day during the same time period in which the initial acclimation period occurred. Rats participated in two daily training sessions. The ISI for training was 300, 500, 700, or 1200 ms. A delay-type paradigm was employed; that is, the CS and US coterminated. Each session consisted of 100 trials divided equally into 10 blocks of 10 trials. Each block began with a CS-alone trial, was followed by 4 paired trials, then a US-alone trial, and ended with 4 more paired trials. These trials were separated by 20–40 s. Explicitly unpaired training consisted of exposing rats to the 300-, 500-, or 700-ms CS and the US. Rats were exposed to an equal number of CS and US presentations; therefore, a session of unpaired training consisted of 180 alternating CS-alone and US-alone trials. The intertrial interval for unpaired training was 10–20 s. A typical experimental run had two nonstressed and two stressed rats, half of whom received paired training. This balanced the factors of stress and training over a given experimental run. Thus, ISI was not manipulated in any experimental run. Last, we did not perform unpaired training at the 1200ms ISI. Preliminary data suggested that unpaired training yielded very little pseudoconditioning at the 700-ms ISI. We therefore did not expect unpaired training at the 1200-ms ISI to yield quantitatively different results from that of the 300-, 500-, and 700-ms ISIs; hence, the incomplete 4 ⫻ 2 ⫻ 2 design. Data Reduction and Statistics Eyelid EMG recordings were sampled at 1 kHz. The EMG signal was low pass filtered using a locally weighted regression filter (Lowess, Stat-Sci, Tacoma WA) with a time constant of 0.025 and a smoothing interval of 5. Using this algorithm an eyeblink corresponds to activity greater than 0.4 (unitless). For the acclimation period, the entire EMG record was processed for the appearance of eyeblinks. For the recordings in which a stimulus was delivered, each EMG record was subdivided into a 200-ms prestimulus baseline. If an eyeblink occurred during the 200-ms prestimulus baseline, that trial was thrown out and an NA was recorded. During conditioning, an eyeblink response was scored as an α-response (orienting response) if the onset occurred within 40 ms of the CS onset. An eyeblink was scored as a conditioned response (CR) if it occurred at least 60 ms after the CS onset and before the onset of the US. On CS-alone trials the CR interval was extended 100 ms. In addition to collecting the CR data, we also collected data on the UR (Servatius, 2000). The square-wave stimulation caused a significant stimulus artifact in the EMG record that dissipated by 40 ms after US offset. The
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post-US record was low pass filtered to remove the ‘‘ringing’’ of the amplifier after US offset. Videographic evidence demonstrated that filtered EMG activity 45–245 ms after US offset corresponded to the eyelid opening after the cessation of US-induced closure. Further, the amplitude of this event was sensitive to US intensity and within session changes in amplitude associated with learning. Therefore, peak EMG activity in the post-US period was considered a surrogate of the UR (Servatius, 2000). The CR and UR data were analyzed with analysis of variance models. The data for each ISI were analyzed separately; these models included data from rats given unpaired training. An overall analysis was also performed that directly evaluated acquisition as a function of ISI; this model did not include unpaired controls. In addition, acquisition was evaluated as the number of trials to a criterion of greater than 16 eyeblink CRs in 20 consecutive trials. If a rat failed to reach the criterion by the second training session, 250 was scored (midpoint of the next session) as a placeholder. Post hoc analyses were accomplished with F tests for simple effects and Dunnett’s tests for a priori comparisons with a control mean. RESULTS
300-ms ISI Acquisition of the eyeblink CR for rats previously exposed to inescapable stress was facilitated compared to nonstressed rats. Moreover, pseudoconditioning was not apparent in stressed rats given unpaired training. These impressions were supported by the 2 ⫻ 2 ⫻ 2 ⫻ 9 (Stress ⫻ Training ⫻ Session ⫻ Trial Block) mixed ANOVA. The significant main effects and two-way interactions were subordinate to the Stress ⫻ Training ⫻ Session interaction, F(1, 391) ⫽ 10.8, P ⬍.001 (see Fig. 1A). For nonstressed rats, acquisition of the eye-blink CR at the 300 ms ISI was generally accomplished during the second session of training (104 ⫾ 35 trials). In contrast, stressed rats acquired the eyeblink CR during the first training session (53 ⫾ 18 trials). The amplitude of URs differed as a function of stress and training. For nonstressed rats, the URs of paired trained rats were greater than those emitted by unpaired trained rats. The URs of stressed rats given paired training did not differ from nonstressed rats during the first session of paired training; nonstressed rats exhibited greater URs during the initial stages of the second training session. Stressed rats given unpaired training exhibited greater URs compared to nonstressed rats given unpaired training over both training sessions (data not shown). In support, the analysis yielded a significant Stress ⫻ Training ⫻ Session interaction, F(1, 390) ⫽ 7.3, P ⬍.001 (see Fig. 1B). 500-ms ISI A similar pattern of results was obtained for the rats trained at the 500ms ISI, that is, facilitated acquisition of the eyeblink CR in previously
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FIG. 1. (Left) Percentage of CRs across the two sessions, using a 300-ms ISI delay paradigm. (Right) UR amplitude across the two sessions during the US-alone trials.
stressed rats. Again pseudoconditioning was not apparent in stressed rats given unpaired training (see Fig. 2A). Consistent with these impressions, the analysis indicated a significant Stress ⫻ Training ⫻ Block interaction, F(1, 408) ⫽ 4.1, P ⫽ .04. Nonstressed rats generally acquired the eyeblink CR between the first and second training sessions (99 ⫾ 11 trials). Stressed rats exhibited faster acquisition of the eyeblink CR (72 ⫾ 29 trials). For paired trained rats URs generally decreased with training in both stressed and nonstressed rats. Stress rats given unpaired training, however, exhibited greater URs during the initial trial blocks (data not shown). The analysis yielded a significant Stress ⫻ Training ⫻ Trial Block interaction, F(1, 388) ⫽ 2.0, P ⫽ .05 (see Fig. 2B). 700-ms ISI Acquisition of the eyeblink CR did not differ between the stressed and nonstressed rats. The only significant factors in the mixed ANOVA were main effects of Training, Session, and Trial Block and the two interactions of Training ⫻ Session and Training ⫻ Trial Block, all Ps ⬍.01 (see Fig. 3A). Here, acquisition was generally accomplished in the first session of training for both stressed (80 ⫾ 34 trials) and nonstressed (69 ⫾ 30 trials) rats.
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FIG. 2. (Left) Percentage of CRs across the two sessions, using a 500-ms ISI delay paradigm. (Right) UR amplitude across the two sessions during the US-alone trials.
For paired trained rats URs generally decreased with training in both stressed and nonstressed rats. There were no stress-related differences in UR amplitudes. The main effects of Training, Session, and Trial Block as well as the Training ⫻ Session and Training ⫻ Trial Block interactions were significant, all Ps ⬍ .05 (see Fig. 3B). 1200-ms ISI Facilitated acquisition of the eyeblink CR was again evident in previously stressed rats. In support, the analysis yielded a significant Stress ⫻ Session interaction, F(1, 357) ⫽ 10.0, P ⬍.001 (see Fig. 4A). For stressed rats, acquisition of the eyeblink CR generally occurred in the second training session (109 ⫾ 37 trials). In contrast, few nonstressed rats acquired the eyeblink CR by the second training session (187 ⫾ 35 trials). The UR amplitudes decreased with training. There were no differential effects attributable to stressor exposure. The analysis yielded only significant main effects of Session and Trial Block, Ps ⬍.001 (see Fig. 4B). Overall Acquisition of the eyeblink CR depended on ISI during paired training (see Fig. 5). For nonstressed rats, efficient acquisition was apparent with the
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FIG. 3. (Left) Percentage of CRs across the two sessions, using a 700-ms ISI delay paradigm. (Right) UR amplitude across the two sessions during the US-alone trials.
intermediate levels of ISI, i.e., 500 and 700 ms; all nonstressed rats reached criterion by the end of the second session. Acquisition of the eyeblink CR was slower in rats trained with the 300-ms ISI. Rats trained with the 300ms ISI acquired the eyeblink response by the end of the second session of training; only 1 rat of 9 failed to reached criterion by the end of the second session. Acquisition was slowest in rats trained with the 1200-ms ISI; only 5 of 11 rats acquired the eyeblink CR by the end of the second training session. As for stress, facilitated acquisition was evident at the 300-, 500-, and 1200-ms ISI, but not the 700-ms ISI. Although stressed rats trained at the 1200-ms ISI acquired the eyeblink CR faster than nonstressed rats, learning was poorer at the 1200-ms ISI than at the other ISIs. Analysis of the acquisition data as a function of stress and ISI demonstrated a significant Stress ⫻ ISI ⫻ Session interaction, F(1, 1156) ⫽ 12.8, P ⬍ .001. DISCUSSION
The primary goal of the present study was to assess acquisition of the eyeblink CR as a function of ISI. Acquisition of the eyeblink CR in a delaytype task differed as a function of the ISI. Similar to humans and rabbits, acquisition in freely moving rats could be described by an inverted ‘‘U’’shaped function. The fastest acquisition of the eyeblink CR was apparent in
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FIG. 4. (Left) Percentage of CRs across the two sessions, using a 1200-ms ISI delay paradigm. (Right) UR amplitude across the two sessions during the US-alone trials.
rats trained with a 700-ms ISI. Slower but still efficient acquisition was apparent with a 500-ms ISI. For both the 500- and 700-ms ISIs, acquisition was generally accomplished within the first session of training. Learning was less efficient with a 300-ms ISI. Nonstressed rats required more trials to acquire the eyeblink CR; acquisition was generally apparent during the second training session. Although the 300-ms ISI was not optimal for acquisition, only 11% of nonstressed rats failed to acquire the eyeblink CR by the second session of training. Moreover, these data at the 300 ms ISI are consistent with the acquisition curves described by others (Weiss et al., 1999; Servatius et al., 1994; Shors et al., 1998). Acquisition degraded substantially at the 1200-ms ISI; only 45% of nonstressed rats acquired the eyeblink CR by the end of the second session of training. We did not continue training to determine the proportion of rats that would fail to acquire the eyeblink response with further training. However, the data clearly demonstrate that acquisition at 1200 ms was more difficult for freely moving rats. The acquisition function as it relates to ISI differed from those described for rabbits and humans. For humans, acquisition peaks at 400- to 600-ms ISIs (Prescott et al., 1992). For rabbits, acquisition is optimal between 200and 500-ms ISIs (Smith, 1968; Smith et al., 1969; Port et al., 1985a; Steinmetz, 1990; Sears et al., 1979; Coleman et al., 1971). Thus, the optimal
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FIG. 5. (Left) Mean percentage of CRs across the two sessions at all ISIs. (Right) UR amplitude across the two sessions during the US-alone trials at all ISIs.
ISIs for freely moving rats appear to be similar to those for humans, with both being longer than for rabbits. Although not specifically manipulated here, we expect that rats would be similar to humans and rabbits in the inability to acquire the eyeblink CR with simultaneous or backward conditioning. A second goal of the current study was to examine the impact of stress on acquisition of the eyeblink CR. Exposure to inescapable stressors facilitated acquisition of the eyeblink CR at less than optimal ISIs. Thus, facilitation was evident at the 300-, 500-, and 1200-ms ISIs. The facilitation was most prevalent at the 1200-ms ISI. Here, nonstressed rats generally did not acquire the eyeblink CR over the 200 training trials, whereas stressed rats did. Facilitation was not evident at the 700-ms ISI. However, acquisition was fastest in nonstressed rats at this interval. Therefore, the lack of facilitation may be due to a ‘‘ceiling’’ effect. Similarly, in a series of studies by Port and colleagues, hippocampal lesions facilitated acquisition at short and long ISIs, but not at the optimal ISI (Port et al., 1985a; Port, Mikhail, & Patterson, 1986b). How then does exposure to inescapable stressors facilitate acquisition? One possible explanation centers on enhanced processing of the CS. Exposure to inescapable stressors affects reactivity to acoustic stimuli for some time after stressor cessation (Servatius et al., 1994; Davis, 1989; Servatius, Ottenweller, & Natelson, 1995). At shorter ISIs, such a response may resem-
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ble an associative response. Rabbit research demonstrated that the timing of the eyeblink CR is relative to US onset. As the ISI lengthens, the time between CS onset and the initiation of the CR should also lengthen. In contrast, a reactive response would be evident as a quick, long-lasting response. Inspection of the EMG activity did not reveal such response topography in the stressed rats trained at the 1200-ms ISI. Thus, facilitation is not merely a function of enhanced reactivity or responsivity to the CS. Enhanced reactivity to the US could also account for faster acquisition of the eye-blink CR. Reactivity to the US was assessed as the magnitude of the UR, as previously defined (Servatius, 2000). Consistent with our earlier work and that of others (Canli et al., 1992; Donegan, 1981), the UR in USalone trials decreased with acquisition of the eye-blink CR. Facilitation of the UR was also apparent; the UR was larger in rats given paired CS–US exposures than in rats given explicitly unpaired CS and US exposures (Schreurs et al., 1995). Exposure to inescapable stressors did not affect UR magnitudes in rats given paired training. Therefore, enhanced reactivity to the US does not appear to account for facilitated acquisition of the eyeblink CR. However, larger URs were apparent in stressed rats given unpaired training compared to those of nonstressed rats. These data suggest that exposure to stress enhanced nonassociative processes modulating UR magnitude. In summary, the learning curves for rats trained in a delay-conditioning task resemble those generated for rabbits and humans, although the optimal ISI appears to be slightly longer for rats. Although exposure to inescapable stressors persistently facilitated acquisition of the eyeblink CR at long and short ISIs, facilitation was not apparent at the optimal ISI. Facilitated acquisition was not the result of enhanced sensitivity to the US. REFERENCES Bracha, V., Zhao, L., Wunderlich, D. A., Morrissy, S. J., & Bloedel, J. R. (1997). Patients with cerebellar lesions cannot acquire but are able to retain conditioned eyeblink reflexes. Brain, 120, 1401–1413. Canli, T., Detmer, W. M., & Donegan, N. H. (1992). Potentiation or dimunition of discrete motor unconditioned responses (rabbit eyeblink) to an aversive Pavlovian unconditioned stimulus by two associative processes: Conditioned fear and a conditioned dimunition of unconditioned stimulus processing. Behavioral Neuroscience, 106, 498–508. Castro-Alamancos, M. A., & Torres-Aleman, I. (1994). Learning of the conditioned eye-blink response is impaired by an antisense insulin-like growth factor I oligonucleotide. Proceedings of the National Academy of Sciences USA, 91, 10203–10207. Christiansen, B. A., & Schmajuk, N. A. (1992). Hippocampectomy disrupts the topography of the rat eyeblink response during acquisition and extinction of classical conditioning. Brain Research, 595, 206–214. Coleman, S. R., & Gormezano, I. (1971). Classical conditioning of the rabbit’s (Oryctolagus cuniculus) nictitating membrane response under symmetrical CS–US interval shifts. Journal of Comparative and Physiological Psychology, 77, 447–455. Davis, M. (1989). Sensitization of the acoustic startle reflex by footshock. Behavioral Neuroscience, 103, 495–503.
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