Different effects of unexpected changes in environmental conditions on prepulse inhibition in rats and humans

Different effects of unexpected changes in environmental conditions on prepulse inhibition in rats and humans L.G. De la Casa1, A. Fernandez1, J. Larrauri2, A. Mena1, A. Puentes1, E. Quintero1, and N. Schmajuk2 1 Department of Experimental Psychology, University of Seville, 41018 Seville, Spain 2 Department of Psychology and Neuroscience, Duke University, Durham, NC 27708, United States Address correspondence to: L.G. De la Casa Dpt. Psicologia Experimental Facultad de Psicología C/ Camilo José Cela, s/n 41018 Sevilla (Spain) Tel.: (34) 954557682 Fax: (34) 954551784 E-mail: [email protected] Abstract The reduction of the startle response to an auditory stimulus caused by the presentation of another stimulus of lower intensity closely preceding it, a phenomenon known as prepulse inhibition (PPI), can be modulated by changes in dopaminergic activity. Schmajuk, Larrauri, De la Casa, and Levin (2009) demonstrated that this dopaminergic modulation of PPI in rats can be influenced by manipulating the experimental context, specifically by introducing changes in the ambient lighting condition that include novel elements. In this paper we analyse the effects of introducing changes in context illumination on PPI in male rats (Experiment 1) and humans (Experiment 2). The results with rats showed a reduction of PPI when the illumination condition switched from dark to light, but not from light to dark. In the experiment with human participants the reduction of PPI occurred for both changes in illumination conditions. The animal experiment results are interpreted in terms of competing exploratory behavior that appear when the context is illuminated after the darklight transition; while in the case of human participants a perceptual and/or attentional mechanism after both illumination transitions is proposed, which may result in a reduced processing of the prepulse and subsequent lower PPI. Keywords: Prepulse inhibition; Novelty; Dopamine; Rats; Humans. 1. Introduction. The startle response includes, among other behaviors, the involuntary contraction of the skeletal muscles and occurs after the presentation of a stimulus of some intensity [1,2]. Typically, the startle response is assessed by quantifying the intensity of muscle contraction after the stimulus presentation. The startle response is susceptible to modulation through various manipulations such as the repeated presentation of the stimulus, which can lead to habituation or sensitization of the response [3,4], the induction of a particular emotional state prior to stimulus presentation [5,6] or to changes in environmental conditions in which the stimulus is presented [7]. Another form of modulation of the startle response that has received much attention from researchers in the field of psychophysiology in the last decades is prepulse inhibition (PPI), a phenomenon that occurs when a stimulus of lower intensity precedes the presentation of a startling stimulus, resulting in the reduction of the response to the latter [8]. The occurrence and intensity of PPI depends on a number of variables such as the time interval between the prepulse and pulse stimuli [9], their intensity [10] or the background noise level in which the stimuli are presented [11]. PPI is believed to reflect sensorimotor gating abilities, i.e., the ability to respond to potentially relevant stimuli, simultaneously inhibiting the processing of other stimuli and/or responses that might hinder the in-depth processing of a stimulus under analysis [12]. According to this point of view, PPI would reflect an effective inhibition of the motor response to the stimulus of greater intensity (pulse), ensuring an in-depth analysis of the prepulse stimulus (which preceded the pulse presentation), and thus representing a simple example of sensorimotor modulation [13]. The neural circuitry that regulates both the startle response to auditory stimuli as well as PPI has been characterized in detail [8]. The neural systems of both the startle response and PPI are modulated by a number of neurotransmitters such as dopamine, GABA, glutamate and acetylcholine, which regulate the magnitude of the startle response and its inhibition [8]. Zhang et al. [18] studied the effect of indirect dopamine (DA) agonists on PPI in rats, and showed that amphetamine administration resulted in a decrease in the magnitude of PPI. Studies with schizophrenic patients, a disorder characterized among other things by a hyperactivity of the dopaminergic system, support the hypothesis relating elevated dopamine release with PPI attenuation [21]. In addition to pharmacological manipulations, there are environmental changes that can cause changes in dopaminergic activity, such as the exposure to a novel stimulus or context [22,23]. An increase in cortical dopaminergic activity in rats indicates that a novel stimulus is presented to the animal, an effect that is not found with neutral or habituated stimuli [24]. With regards to the subcortical dopamine system, the release of dopamine in the nucleus accumbens is related to the perception of novelty [25]. According to the results presented above, external changes involving the introduction of new elements in the experimental situation, such as a manipulation of the illumination condition in the area where the subject is located, would impact the dopaminergic activity and produce a modulating effect on PPI [26]. Our aim in this paper is to analyze the effect that environmental changes –namely, a variation in the illumination conditionshave on the startle response and PPI in both rats and humans. Based on the results reported by Schmajuk et al [26] we expect that exposure to novel environmental situations would produce a transient increase in dopamine release in both cortical and subcortical regions [24] and therefore cause a reduction in PPI in both rats and humans. However, it is possible that the effects on PPI may depend on the type of novel stimulus that appears in the experimental situation, since previous results with rats indicate that PPI is reduced after a dark to light transition, but not when the change is from light to dark [26]. It is possible that this lack of symmetry in animals is restricted to the transition from dark to light due to the increase in surrounding visual stimuli that occurs with a sudden illumination increase. 2. Experiment 1 The first experiment is designed to reproduce the results obtained by Schmajuk et al. [26], namely the reduction of the startle response and PPI after a change of illumination in the experimental condition. Some changes to the Schmajuk et al. [26] design were introduced in this experiment: first, a 90 dB SPL prepulse was used instead of a 70 dB SPL stimulus, since in pilot studies in our laboratory no consistent PPI was found with the latter prepulse intensity. A second major change was the use of male rats in our experiment, thus eliminating the source of variability related to the hormonal changes that occur periodically in female rats in estrus function [27], that have been shown to influence PPI [28]. Third, in our experiments Wistar rats were used, instead of the Sprague-Dawley strain tested in Schmajuk et al. [26]. Fourth, in our experiment the animals were maintained under a regular light-dark cycle, instead under a reversed cycle (thus, in our experiment the animals were tested in their light phase). A final important change in the design was that this experiment used a between-subjects design in which each group of animals received only one of the environmental changes (light to dark [L/D] or dark to light [D/L]) versus the within-subject design used in Schmajuk et al. [26]. Based on the previous results obtained by Schmajuk et al. [26], we expect that the illumination change would produce an attenuation of PPI, but only when the change involves a transition from dark to light. 2.1. Method 2.1.1. Subjects Sixteen male Wistar rats, experimentally naïve, participated in this experiment. The mean weight at the start of the experiment was 342 gr. (range 297-410). Food and water were available ad libitum throughout the experiment. Rats were individually housed in the colony with a regular light-dark cycle of 12:12 hours. All testing occurred during the 12-h light period (starting at 10:00 AM). Four days before the start of the experimental sessions, each of the animals was handled 5 minutes daily. 2.1.2. Apparatus and stimuli Four Panlab chambers (model LE 111) designed to detect and record the startle response in rats were used. Each chamber was enclosed in a soundproof module (model LE 116), and inside each chamber a perspex cylinder of 8 cm in diameter was attached to the floor of the experimental chamber, resting on a platform that registered and recorded each animal’s movement. A loudspeaker was present at the top of each chamber, which produced a constant background white noise of 65 dB SPL. The pulse stimulus was a 20 ms, 120 dB SPL white noise, and the pre-pulse was a 20 ms, 90 dB SPL white noise. The lead interval in the prepulse-pulse trials was 100 ms, and the intertrial interval was 30 sec (+/- 5). A 24V, 2W key light (light intensity of approximately 180 lx) was located on the left side of the chamber. Vibrations of the Plexiglas enclosure caused by the whole-body startle response of the animal were converted into analog signals by a piezoelectric unit attached to the platform. These signals were digitized and stored by a computer as a linear parameter. The average startle activity was measured in a 100-ms time window starting at the onset of the sound stimulus, whereas the average baseline activity was measured by selecting the highest response in the interval between trials. 2.1.3. Procedure For the L/D group (n = 8) the key light inside the experimental chamber was on from the beginning of the experiment, while in the case of the D/L group (n = 8) the key light was off. Once the rats were introduced in the experimental chambers, they went through a 5-minute acclimation period in which the only auditory stimulation presented was the constant 65-dB SPL background noise, which remained throughout the experiment. Following the acclimation period, 4 pulse-alone stimuli were presented with a mean inter-trial interval ITI of 30 sec. After 6.5 additional minutes, 6 pulse-alone and 6 prepulse-pulse trials were randomly presented, with a mean ITI of 30 sec (+/- 5). In prepulse-pulse trials the interval between the prepulse and pulse was 100 ms. Following this sequence of trials, the change in lighting condition (light to dark or vice versa, depending on the group) was introduced, and the same 6 pulse-alone and 6 prepulse-pulse trial sequence was presented. 2.1.4. Results A preliminary 4 x 2 ANOVA (Trials x Condition: D/L vs. L/D) conducted on mean startle responses to the 4 pulse-alone trials presented at the beginning of the session revealed no significant main effects or interactions (all ps>.19) Figure 1 shows startle responses (expressed in arbitrary units) to pulsealone and prepulse-pulse trials during the experimental phase. It also shows mean response during inter trial intervals (no stimulus trials) computed by collapsing the maximum spontaneous response by contiguous Pulse and Prepulse-pulse ITIs. Panel A presents the startle responses for rats in group L/D, in which the first block of trials took place with the key light on, and the second block with the key light off. Panel B shows the results of rats in group D/L, in which the illumination conditions were reversed. As seen in both panels of Figure 1, during the first block the difference between responses to pulsealone and prepulse-pulse trials (i.e., PPI) remained constant. However, the introduction of changes in illumination conditions had a differential effect on the startle response in pulse-alone trials: while no change was observed in group L/D (Panel A), PPI disappeared transiently in group D/L during the first trials in the presence of light (immediately after the transition) to gradually recover over the remaining trials (panel B). The responses during the no-stimulus periods remains low and stable across the entire duration of the experiment, thus discarding any possible floor effect of the startle response on the experimental trials, but showing a general increase after the illumination transition. ----------------------------------------- Figure 1 about here ----------------------------------------- These impressions were confirmed by a subsequent statistical analysis. Specifically, a 6 x 2 x 2 x 2 ANOVA test (Trials x Trial type: pulse-alone vs. prepulse-pulse x Position: first block of six trials vs. second block x Group: L/D vs. D/L, with the first three factors being within-subject) on mean startle responses revealed a significant main effect of Trial Type, F(1,14) = 35.54; p < .001, η2 = .72, reflecting the overall PPI effect. A significant Trials x Position interaction was also found, F(5,70) = 6.77; p < .001, η2 = .33, due to a general trend of startle amplitude to decrease across trials in the first block of six trials, and to increase in the second block. Finally, the 3-way Trial type x Position x Group interaction was also significant, F(1,14) = 7.61; p < .05, η2 = .35. No additional significant main effects or interactions between factors were found (all ps>.09). In order to identify the source of the 3-way interaction, we conducted separate 2 x 2 ANOVAs (Trial type x Position) for L/D and for D/L groups. The ANOVA for the L/D group revealed only a significant main effect of Trial type, F(1,7) = 14.05; p < .01, η2 = .66, due to the general effect of PPI. The ANOVA test on mean startle responses for the D/L group revealed a significant main effect of Trial type, F(1,7) = 21.95; p < .01, η2 = .77, due to the PPI effect, and a trend toward a significant Trial type x Block interaction was found, F(1,7) = 4.84; p = .064, η2 = .35. The interaction reflects a trend towards a lower startle responses in pulse-alone trials in the first block of trials (mean = 42.24, SEM = interaction was also significant, F(3,66) = 3.26; p < .05, η2 = .13, reflecting the overall decrease of the startle response across trials due to habituation in the pre-transition block that did not appear in the post-transition trials, where the startle response was already habituated. No additional main effects or interactions were found to be significant (all ps>.11). Figure 2 shows mean startle responses to pulse-alone and prepulsepulse trials, expressed in arbitrary units. Panel A presents the startle response for group D/L (the first trials in darkness and the last tests in the presence of light), while Panel B shows these responses for group L/D (with the first six trials in the presence of light and the last six in the dark). In both cases, the response pattern was similar, with more intense responses in the first six pulsealone trials compared to prepulse-pulse trials (reflecting the PPI) and an increase in response to prepulse-pulse trials in the second block of trials, after the introduction of the illumination change (from light to dark or from dark to light). -------------------------------------- Figure 2 about here -------------------------------------- A mixed 6 x 2 x 2 x 2 ANOVA (Trials x Trial type: pulse-alone vs. prepulse-pulse x Position: first block of six trials vs. second block x Group: L/D vs. D/L, the first three factors being within-subject) revealed significant main effects of Trials and Trial type, F(5,110) = 8.67; p < .001, η2 = .29, and F(1,22) = 32.44; p < .001, η2 = .60, respectively. The main effect of Trials reflects a general decrease of the startle response across trials due to a habituation process; the main effect of Trial type was due to an overall PPI effect, with higher startle response to pulse-alone than prepulse-pulse trials (mean = 0.56, SD = 0.38, and mean = 0.42, SD = 0.30, respectively). The Trials x Trial type interaction was also significant, F(5,110) = 2.36; p < .05, η2 = .10, reflecting a decrease of startle intensity across trials for the Pulse-alone, but not for the Prepulse-Pulse trials. The Trial type x Position was also significant, F(1,22) = 14.39; p = .001, η2 = .40, due to the PPI effect obtained in the first block of trials that vanished on the second block. Finally, the Trials x Trial type x Position interaction was significant, F(5,110) = 4.06; p < .01, η2 = .16. An exploration of this 3-way interaction revealed it was due to a decrease across trials of the PPI effect on the first block of trials, and an absence of such PPI effect on the second block of trials. No additional main effects or interactions were found to be significant (all ps>.16) 4. Discussion The results of both experiments show that the presentation of a novel stimulus, specifically the introduction of a change in ambient illumination conditions, has an important effect on the startle response and PPI. In the first experiment we found that the startle response of rats in pulse-alone trials decreases as a result of novelty –but only after the change was from dark to light-, causing a decrease PPI. In Experiment 2, changes in illumination conditions –regardless of their directioncaused an increase in the startle response on prepulse-pulse trials in humans, and a consequent decrease in PPI. Although yielding a similar outcome in terms of PPI reduction, the results of both experiments are clearly different attending to the way in which changes in environmental conditions affect the startle response in pulse-alone and prepulse-pulse trials. The results of animal experiments reproduce exactly those obtained by Schmajuk et al. [26], namely the decrease in startle response in pulse-alone trials. Although our Experiment 1 did not offer any direct neurobiological data, the behavioral data are consistent with the hypothesis that considers the observed startle reduction to the pulse mediated by the release of dopamine in the NAC induced by the introduction of a novel stimulus (the change in ambient illumination). According to this view, the activation of the dopaminergic system would lead to the development of exploratory behaviors that would compete with the generation of startle responses in pulse-alone trials, thus decreasing their amplitude [26]. Several findings support this hypothesis: First, there is experimental evidence showing that increased exploratory behavior is negatively correlated with the intensity of the startle response to intense stimuli [32]; in addition, several studies have revealed that contextual changes involving novelty are positively correlated with increased dopamine release in the NAC [33,34]. Thus, when Sprague-Dawley rats were exposed to an unfamiliar novel environment, an increase of dopamine located in the NAC that persists for about 25 seconds was observed [35] Rebec et al. (1997). Using microdialysis studies, dopamine release in the NAC increased when LongEvans rats were exposed to an environment containing novel objects [36]. Such phasic dopamine increase seems to be linked to the activity of neurons in Ventral Subiculum (VS), since neurons in this region activate glutamate receptors in the VTA (Ventral Tegmental Area) that results in an increase of dopamine in the NAC to novel stimuli. There is also the possibility that VS direct glutamatergic projections of neurons to the NAC induce an increase in dopamine to novelty, or that it is related to a circuit involving VS projections to the prefrontal cortex, which in turn involves glutamatergic projections to VTA [36]. In the case of change in the ambient illumination condition from light to dark, it is possible that the exploratory behaviors that compete with the startle response in pulse-alone trials do not appear since the number of visual stimuli available for exploration in this condition decreases. This possibility was confirmed by the analysis of exploratory behavior conducted on two trials before and after the illumination change: an increase in exploratory behavior was observed only in the Dark-to-Light group. The results of Experiment 2 with human participants exhibit a different pattern from those obtained with animals. First, the PPI reduction effect after the introduction of the illumination change was symmetrical, i.e., it was observed in changes from dark to light as well as from light to dark. A second important difference with the results obtained in the experiment with rats is related to the origin of the observed reduction in PPI. While for rodents PPI decreased as a consequence of a reduction of the startle response in the first pulse-alone trials after the introduction of the environmental illumination change, in humans the reduction was due to an increase in startle responses in the first prepulse-pulse trials following the illumination change. An additional difference between rats and humans results is related to the length of the effect that was restricted to the first two trials in the experiment with rats, but extended across the six postchange trials in the experiment with humans. However, as can be seen in Figure 2, the startle reduction to the Pulse, probably due to habituation, had contributed to the apparent attenuation of PPI. Therefore, according to the experimental results, the cause of the observed PPI reduction in humans is necessarily different from that observed in rodents, since all changes (not only that induced by the emergence of novel stimuli in the environment introduced in the light condition, but also the change caused by the disappearance of cues when the light was turned off) reduced PPI. An important factor to consider when analyzing these differences is related to the magnitude of the light source used in the experiments with rodents and humans. While in Experiment 1 the stimulus used was a 2 W light, in the case of Experiment 2 a 36 W LED bulb located 100 cm above the head of the participants was employed. It is possible that the illumination provided in the rodent’s case could have facilitated the appearance of an orienting response to the novel stimuli, causing the observed reduction in PPI [26], whereas in the human experiment, the more salient light could have launched perceptual and/or attentional processes in addition to the ones exhibited by rodents. Thus, a possible explanation –albeit speculativefor our results, is that the introduction of severe environmental changes in the human experiment may have temporarily changed the detection threshold of auditory stimuli. Using different preparations to the ones employed in the present experiments (e.g., cross-modal oddball tasks), evidence of the effects of presenting distracting stimuli on the processing of stimuli of different sensory modality with human participants has been reported. Thus, using mainly auditory [37] and tactile [38] stimuli, these researchers showed the existence of what they call “post-novel distraction”, a process that reflects the need to redirect attentional resources from a novel to a target stimulus in the experimental situation [39]. Although admittedly speculative, in addition to this distracting effect, the introduction of changes in ambient illumination conditions in Experiment 2 may trigger a second effect related to the attentional shift caused by the presentation of an unexpected visual stimulus, which would slow the processing of an auditory stimulus appearing immediately afterwards [4041]. This slowing effect, which results in increased response times in reaction time tasks, appears to be more significant when the stimuli presented are unexpected [42]. In this case, the observed PPI would not indicate a sensorimotor gating effect, but reflect a failure to detect the prepulse. 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