Effects of acute and chronic methylphenidate on prepulse inhibition: A sex difference study in Wistar rats

Physiology & Behavior 278 (2024) 114526 Available online 24 March 2024 0031-9384/© 2024 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Effects of acute and chronic methylphenidate on prepulse inhibition: A sex difference study in Wistar rats F. Montiel-Herrera , A. Batanero-Geraldo, J.C. L´ opez , J.P. Vargas , E. Quintero , E. Díaz * Laboratory of Animal Behavior and Neuroscience, Department of Experimental Psychology, University of Seville, Seville, Spain ARTICLE INFO Keywords: Methylphenidate ADHD Sex differences Chronic administration Sensorimotor gating ABSTRACT Background: The utilization of methylphenidate (MPH) is experiencing a notable surge within the adult population. This growth can be attributed to two key factors: its recreational and cognitive enhancement purposes, as well as the rising prevalence of ADHD diagnoses within this population. This study examined acute and chronic oral MPH effects on attention in male and female Wistar rats. To this end, we used a prepulse inhibition (PPI) task, which is widely used to assess psychoactive drug effects in both humans and rodents. This task allowed us to evaluate changes in attention by analyzing sensorimotor gating associated with stimulus selection process. Methods: Animals were administered a clinically relevant dose of MPH (5 mg/kg) daily for seven days. The estrous cycle phases of the female rats were measured during behavioral sessions. The PPI task was conducted 20 min after drug administration on day 1 (acute), day 7 (chronic), and 48 h post-treatment. Results: Results indicated that both acute and chronic MPH treatment impaired PPI expression in male rats, but not in female rats, regardless of their estrous cycle phase. Furthermore, a differential effect of chronic MPH treatment on the PPI task was found in male rats. Specifically, on the seventh treatment day, the PPI effect was observed when animals undertook the PPI task for the first time but was impaired in those animals in which the initial PPI session occurred under the acute influence of the drug (day 1). Conclusions: These findings suggest that the impact of MPH on sensorimotor gating responses may vary based on sex and task experience, possibly leading to state-dependent effects in healthy individuals. 1. Introduction The utilization of methylphenidate (MPH) is experiencing a notable surge within the adult population. This growth can be attributed to two key factors: its recreational and cognitive enhancement purposes, as well as the rising prevalence of ADHD diagnoses within this population [1]. Acute and chronic administration of MPH can lead to various physiological and behavioral effects, which may be modulated by factors such as sex, age, and dosage. MPH blocks dopamine (DA) and norepinephrine (NE) transporters, resulting in increased concentrations of these neurotransmitters in the synaptic cleft [2–5]. Dopamine (DA) is released in several brain regions, including the amygdala, nucleus accumbens, prefrontal cortex and ventral striatum. It plays a central role in mediating attention, reinforcement, and the stimulant effects of drugs [6–8]. While MPH shows improvements in ADHD-related behavioral symptoms, its effects on cognition are not fully understood. Human and preclinical studies have shown inconsistent and mixed results regarding its effects on learning, working memory, executive function and attentional processes [9–18]. Importantly, most of these studies have analyzed the chronic effects of early exposure to MPH, and little is known about the long-term effects of MPH treatment during adulthood. Adolescent and adult rats respond differently to psychostimulants [19]. For example, adults, but not adolescents, show sensitization of locomotor activity during and after treatment with catecholaminergic agonists and adolescent rats show increased responsiveness to dopaminergic antagonists [20]. In addition, several studies have shown that treatment with psychostimulants during adolescence influences the adult response to these drugs [21]. The response of adult rats to substances such as MPH varies depending on their history of adolescent use. For example, adults exposed to methylphenidate during adolescence show a sensitization effect when reintroduced to it in adulthood, unlike those who were only exposed to MPH during adulthood [22,23]. Furthermore, and surprisingly, despite evidence for sex differences in dopaminergic and behavioral responses to psychostimulants [24,25], * Corresponding author. E-mail address: [email protected] (E. Díaz). Contents lists available at ScienceDirect Physiology & Behavior journal homepage: www.elsevier.com/locate/physbeh https://doi.org/10.1016/j.physbeh.2024.114526 Received 25 January 2024; Received in revised form 28 February 2024; Accepted 4 March 2024 Physiology & Behavior 278 (2024) 114526 2 many of the above studies have used only male rats or have not examined differential effects of sex. Some studies have shown that estrogens increase the acute neurochemical and behavioral response to dopamine, amphetamine and cocaine in female rats [26], and in both humans and rodents, females have been shown to be more sensitive to psychostimulants [22,27–30]. Most rodent studies examining sex differences in the effects of MPH have focused primarily on locomotor responses [31–34]. These studies suggest that females are more sensitive to the behavioral activating effects of MPH, particularly in the induction of locomotor sensitization [31]. In addition, female rats treated with MPH show increased susceptibility to cross-sensitization to other drugs such as amphetamine [29]. Overall, previous research has found that the effects of psychostimulants vary according to both age and gender. However, research into the effects of MPH has focused predominantly on childhood and adolescence, with a particular emphasis on males. Given the increasing diagnosis of ADHD in adults and the widespread use and misuse of methylphenidate for recreational or cognitive enhancement purposes, further research is needed to understand how MPH affects cognition in adults and whether these effects are influenced by gender. The aim of the present study was to investigate the effects of acute and chronic oral MPH administration in adult male and female rats using a prepulse inhibition task (PPI). The PPI test was selected because it is a neurophysiological measure of the ability to filter out irrelevant sensory information and has been implicated in the selection of relevant stimuli, a process that appears to be abnormal in ADHD [35]. Furthermore, sensorimotor gating is a widely used behavioral test to evaluate the effects of psychoactive drugs in both rodents and humans and is modulated by central dopaminergic substrates [36–40]. In the current study, we treated male and female Wistar rats daily for 7 days with a clinically relevant dose of MPH (5 mg/kg) administered orally [41]. We administered a 7-day treatment because previous research has shown that a regimen of six single daily injections of MPD can be considered a chronic treatment for rats [33,42]. Behavioral experiments began 20 min after drug administration. In a typical PPI paradigm, trials with a loud and intense stimulus are interspersed with trials where the intense stimulus is preceded by a less intense pre-stimulus, known as the "prepulse" [43]. In healthy subjects, the loud stimulus typically triggers a startle reflex, which is normally attenuated when preceded by a prepulse [44]. PPI was measured on three occasions: on day 1 (acute effect), on day 7 (chronic effect), and 48 h after drug treatment ended to determine whether MPH produces lasting effects after drug. Since ovarian steroids affect the functionality of the dopaminergic system [45], we measured the phase of the estrous cycle in female rats after PPI sessions to analyze whether sex hormones modulate the behavioral effect of MPH. In addition, and because previous studies have shown that drug injections combined with repeated PPI assessments could alter PPI effects [46], a new group was added in which the first PPI session was administered on day 7 of treatment (during chronic treatment), with no PPI sessions during the acute phase of treatment. 2. Material and methods 2.1. Animals Twenty eigth Wistar male (300–500 g) and twenty nine Wistar female (200–260 g) rats aged 12 weeks (bred at the University of Seville) were used. The animals were housed in pairs in a 12 h light/dark regime with temperatures ranging from 22 to 24 ◦C and free access to a standard diet and water. Experimental manipulations were performed during the light period. At the arrival at the laboratory, the animals were housed in groups of 2 in type IIIH cages (820 cm 2 ), with wood shavings as bedding and other materials available in the cages (pieces of fabric, cardboard and wood, stones, etc.). The animals were allowed 7 days of acclimation to this environment before starting any experimental manipulations. A week before the start of the experimental sessions, each of the animals was handled daily for 5 min. Given the need for oral administration of drug, rats were trained to drink a saccharin solution (0.04 %) with the syringe-feeding technique. The Ethics Committee for Animal Research of the University of Seville approved all experimental procedures (code number CEEA-US2020-11) that were carried out in accordance with the guidelines established by the EU Directive 2010/63/EU for animal experiments and the Spanish R.D. 53/2013. 2.2. Apparatus For the PPI procedure two startle chambers were used within a box soundproofed cover with foam and a fan on the left wall for air renewal (Panlab, model LE 111). These chambers consisted of a black methacrylate wall with a transparent front door. The floor was prepared with a platform and a load cell that recorded the animal activity/immobility or startle response. There was a Plexiglas cylinder attached to the floor inside each chamber. The movement of the animals inside the Plexiglas retractor was recorded by transforming it into piezoelectric analog signals installed on the platform. In each box, a 17 cm loudspeaker was placed above the retractors to reproduce the sounds of pulses and prepulses. The boxes were programmed with PackWin software, which recorded the amplitude of the startle responses (ASR), measured for 20 ms, in the different trials. For drug administration non-sterile 3 ml Pasteur pipettes were used. 2.3. Drug A dose of 5 mg/kg MPH and 0.04 % saccharin were dissolved in 5 ml of tap water and administered to rats 30 min before the PPI test in a volume of 0.5 ml/kg. The dose dissolution was based on the Arnsten and Dudley [47] study. For the control group, only a dose of 0.04 % saccharin was used. 2.4. Procedure All experimental sessions were conducted between 16:00 and 19:00. The schedule of the groups is shown in Table 1. Animals were divided into four groups according to sex and drug: 11 females/ 11 males received saccharin and 13 females/ 13 males received MPH. We gave a total of 3 PPI sessions (PPI-1, PPI-2 and PPI-3), 2 within 7 days of treatment: day 1 (acute) and day 7 (chronic) and the last session was given 48 h after treatment ended, day 9 (drug withdrawal). The effect of estrous phases was also analyzed. Vaginal smears were taken after each PPI test. Each estrous phase was determined according to the method used in the study by Cora et al., (2015). A proestrus smear consists of a predominance of nucleated epithelial cells, an estrus smear consists primarily of anucleated cornified cells, a metaestrus smear consists of the same proportion of leukocytes, cornified, and nucleated epithelial cells, and a diestrus smear consists of a predominance of leukocytes. To analyze whether repeated PPI assessments (experience) could modulate the MPH effects, we added a new group that received 7 days of treatment before the first PPI test and a second session 48 h after the end Table 1 Schedule of drug treatment and prepulse inhibition sessions. Groups Day 1 Days 2–6 Day 7 Day 8 Day 9 MPH Acute and chronic effects MPH groups (F vs. M) MPHPPI-1 MPH MPHPPI-2 Washout PPI- 3 CON groups (F vs. M) CONPPI-1 Sac CONPPI-2 – PPI- 3 MPH chronic effect Nonexperience groups (F vs. M) MPH MPH MPHPPI-1 Washout PPI- 2 Note: MPH: Methylphenidate, CON: Control, Sac: Saccharin, F: Female, M: Male. F. Montiel-Herrera et al. Physiology & Behavior 278 (2024) 114526 3 of treatment (N: 4 male/5 female). 2.4.1. PPI test The PPI procedure used in the current study was similar to that previously validated in our laboratory by De la Casa et al. (2012). The PPI test included a startle stimulus (120 dB) and three different prepulses (75 dB, 85 dB, 90 dB). The protocol was as follows: with the rat inside the Plexiglas cylinder, a 5 min acclimation period with a 65 dB background noise was started, which will remain constant throughout the session. This was followed by the basal pulse phase, where the startle response to the pulse was measured for 5 trials. These trials were used to stabilize the startle response; thus they were not included in the analyzes. After this session, the prepulsepulse session begins which consists of 48 trials: 15 trials measurement in the absence of stimuli, 15 pulsealone trials, 1 trial for each prepulse-alone, and 5 prepulse-pulse trials for each prepulse (75 dB, 85 dB, 90 dB). The mean interval of both pulse and prepulse was 20 ms, while the prepulsepulse interval was 100 ms. The mean duration of the interval trials was randomly between 10 and 20 ms. PPI was measured as the difference between the presentation of pulsealone trials and the prepulse-pulse trials and expressed as percent [100 x (mean of startle response to the auditory stimulus – ASR- amplitude in pulse alone trials –mean ASR amplitude in prepulse-pulse trials) / mean ASR amplitude in pulse alone trials] [48–52]. 2.5. Statistical analysis In this study, mixed-design analysis of variance (ANOVA) models were used to test the effects of groups on the amplitude of the ASR. Drug and Sex were used as between-subject variables and Trial blocks (5) and PPI session (as within-subject variables). For the analysis of the percentage of PPI, we used mixed ANCOVAS with the ASRs as covariable. Prepulse intensities (75, 85 and 90 dB) and PPI session (PPI-1, PPI-2 and PPI-3) were used as within-subjects variables. Drug (CONT and MPD) and Sex (Female and Male rats) were used as between-subject variables. In order to evaluate the estrous cycle effect, we used three levels for the Sex variable: males (M), metaestrous–diestrous females (MD), Proestrous–estrous females (PE). Because the rats were in a different estrous cycle on days 1, 7 and 9, we conducted a separate analysis for each of these days. Prepulse intensities and Drug and Sex were used as variables with Prepulse intensities as a within-subjects factor. Finally, we added a new group to analyze the effect of experience with PPI task and ANCOVAs with regimen treatment (acute and chronic), sex and prepulse intensities were conducted. To correct for violation of the sphericity assumption in repeatedmeasures ANOVA, we used Greenhouse-Geisser adjustments for the within-session variables. Significant main and interaction effects were followed up by Bonferroni post-hoc test. For all analyzes, we used a α of 0.05. The effect size of the individual effects is reported using partial η 2. All analyzes were conducted using IBM SPSS statistic 21 (SPSS, Inc.). 3. Results 3.1. Acute and chronic effects 3.1.1. Pulse Fig. 1 shows the startle amplitude mean over 15 pulse trials (5 blocks of three trials each) on day 1 (acute effect), day 7(chronic effect) of MPH treatment, and on day 9 (drug withdrawal). As can be seen, the mean of startle amplitude was lower in female rats (A) than in male rats (B) in both the control and MPH groups. For PPI-1 session, the data of two male rats in the control group were lost due to a computer problem. The mixed ANOVA showed a significant effect of Sex, F(1, 42) = 58.58, p <0.001 ( η p 2 =0.58), Sessions, F(2, 84) =18.97, p <0.001( η p 2 = 0.31) and Sessions x Sex, F(2, 84) =3.62 ( η p 2 =0.08). No other effect was Fig. 1. Mean startle amplitudes (+SEM) to 120 dB pulse over blocks of three trials for female (A) and male rats (B) in the three PPI sessions (PPI-1, PPI-2, PPI-3) on day 1 (acute effect) and day 7 (chronic effect) of treatment (MPH vs. CONT) and day 9 (48 h after the end of treatment). For all sessions, ASR was higher in male rats than in female rats (p <0.01). MPH had no effect on ASR amplitudes (MPH vs. CONT groups, p >0.05). F. Montiel-Herrera et al. Physiology & Behavior 278 (2024) 114526 4 significant. ANOVAs revealed a significant effect of Sex on PPI-1 F (1, 44) = 10.79, p =0.002 ( η p 2 =0.20), PPI-2 F (1,44) =71.15, p <0.001 ( η p 2 = 0.62) and PPI-3 F (1, 44) =14.03, p <0.01 ( η p 2 =0.242) due to a lower magnitude of the startle response in female rats. Interestingly, the comparison between the three PPI sessions showed that in the male groups, the startle response to the pulse remained constant in sessions 1 and 2, but decreased in session 3, (mean (SEM) ASR for PPI-1 79.95 (2.03), PPI-2 73.68 (2.17) and PPI-3 68.27 (2.78)). However, in both groups of female rats (control and MPH groups), the startle response of PPI decreased significantly from the first session to 2 and 3 (both ps < 0.05), (mean (SEM) ASR for S1 63.52 (2.15), S2 49.44 (2.05) and S3 54.01 (2.63)). These sex differences in startle reactivity to pulse-alone trials could pose a problem for the interpretation of PPI results. Several human and rodent studies have shown that the percentage of PPI varies with the amplitude of the baseline startle response, with this percentage being higher the lower the intensity of the startle response [44,53–57]. To overcome this problem in the interpretation of the data, the use of the baseline startle response as a covariate has been proposed. This would allow a second screening to verify the differences obtained in the ANOVAs. 3.1.2. PPI Data were analyzed using a mixed ANCOVA with Drug and Sex (2: Male vs Female) as between-subject factors and PPI session (2: acute vs. chronic) and prepulse intensity (3: 75, 85 and 90) as within-subject repeated measures and Pulses of these sessions as covariables. There was a main effect of Drug, F (1, 42) =5.35, p <0.03( η p 2 =0.11), prepulse intensity, F (2,84) =10.30, p <0.01 ( η p 2 =0.20) and PPI sessions, F (1,42) =4.86, p <0.04 ( η p 2 =0.10). Importantly, there was a significant interaction Prepulse intensity x Drug x Sex, F (2, 84) =4.06, p <0.03 ( η p 2 =0.09) and Prepulse intensity x PPI sessions x Sex, F (2,84) =5,02, p < 0.01 ( η p 2 =0,11). Further analysis of these significant results showed that for all prepulses, males treated with MPH had significantly lower percentages of PPI than CONT males (all ps <0.05, Fig. 2). However, in female rats, MPH only reduced the percentages of PPI for the lower prepulse (PPI75, p <0.02, Fig. 2). Analysis per session showed that the differences between the MPH and CONT groups of male rats were mainly due to the acute effect of the drug. Thus, at session 1, PPI90 and PPI85 were lower in the MPH group than in the CONT group (both ps <0.05), but after 7 days of treatment, we only found a near significant reduction in PPI90 (p =0.06). However, comparison between sessions showed that this lack of differences on day 7 was mainly due to the fact that the control group significantly reduced the percentage of PPI75 and PPI85 from day 1 to day 7 (both ps <0.05). In MPH male groups, this reduction occurred only for the PPI75 (p <0.01). Finally, on day 9 after 48 h without drug, we found no differences between the control and MDP groups for males or females. This indicates that the effect of MPH is reversed after cessation of administration. 3.2. Estrous cycle effect Previous studies have suggested that the estrous cycle may interact with the effects of the several drugs to enhance or diminish this effect [49,58,59]. Thus, we divided the female rats into two groups according to their estrus cycle stages: metaestrus–diestrus stages (MD groups) and proestrus-estrus stages (PE groups), and we analyzed whether the Pulse and PPI of these groups differed in the effect of acute and treatment chronic with MPH. On day 1(PPI-1), the number of subjects for this new distribution was males: 24, MD females: 10 and PE females: 14. On day 7 (PPI-2) only two out of the 24 female rats were classified as MD and on day 9 (PPI-3) only two rats in MPH group. Therefore, in these sessions, we conducted the statistical analysis with males and PE females. 3.2.1. Pulse In PPI-1 session, mixed ANOVA revealed a significant effect of Cycle, F (2, 40) =14.12, p <0.001( η p 2 =0.41). This effect was due to ASRs of both groups of female rats were lower than those of male rats (both ps < 0.01). On day 7 (PPI-2), PE groups of female rats also differed from the group of male rats, F (1, 42) =61.84, p <0.001( η p 2 =0.60) (mean (SEM) ASR for PE rats 49.68 (2.48), and for male rats 73.05 (1.65). On day 9, we also found a significant effect of Cycle, F (1, 37) =12.43, p <0.01( η p 2 =0.25) with the ASRs of the male groups being higher than those of the Fig. 2. Mean percentage of PPI (+SEM) of female and male rats over the three PPI sessions (PPI-1, PPI-2, PPI-3) on day 1 (acute effect) and day 7 (chronic effect) of treatment (MPH vs. CONT) and on day 9 (48 h after the end of treatment) for experienced groups. * indicates a significant pairwise difference between MPH and CONT groups (p <0.05) and # indicates an almost significant difference between these groups (p =0.06). F. Montiel-Herrera et al. Physiology & Behavior 278 (2024) 114526 5 females PE groups (mean (SEM) ASR for PE rats 54.49(3.10), and for male rats 67.70 (2.37)). 3.2.2. PPI Table 2 shows mean PPI percentages after acute (PPI-1) and chronic (PPI-2) MPH administration for MD females and PE females. In PPI-1 session, ANCOVA showed a significant interaction drug x prepulse intensity x cycle, F (4, 82) =3.80, p <0.01 ( η p 2 =0.16). Males and females had similar PPI effects for all prepulse intensities in the control groups. However, in the drug groups, we obtained differences in PPI percentages when a prepulse of 90 dB was used. MD female rats showed a greater reduction in the startle response (more PPI) than male rats (p <0.01). Comparisons between control and drug groups showed that MPH significantly reduced PPI in the male group for PPI90 and PPI85 (both ps <0.05). Although drug reduced the PPI75 percentages in both female groups compared to the control groups, the effect was only significant for MD female group (p <0.05). In PPI-2 session, the percentages of PPI75 in the male control group were lower than PE females (p <0.01). Although overall, in the MPH groups the percentages of PPI were lower in males than in females, we only found significant differences for PPI75 (p <0.01). In PPI-3 session, ANCOVA showed that only the intensities of the prepulse had a significant effect, F (2, 72) =7.34, p <0.01( η p 2 =0.17). 3.3. PPI test experience vs. nonexperience effect In PPI-1 session, MPH treatment resulted in a significant decrease in PPI percentages in the male rat group. However, in PPI-2 session, differences between the MPH and control male rat groups were found only for PPI90. This lack of difference may be due to the control group rather than an effect of tolerance to MPH. Thus, in the male control group, the percentage of PPI decreased from PPI-1 to PPI-2. In order to dissociate the effects of experience from the chronic effects of the drug on the percentages of PPI, a new group was added (MPH non-experience) in which the first session of PPI was administered on the seventh day of treatment. 3.3.1. Pulse The ASRs of the first PPI session were analyzed using an ANOVA 3 (Groups: CONT, MPH-acute, MPH-chronic) x 2 (Sex) x 5 (blocks). Only the main effect of Sex was significant, F (1, 49) =21.02, p <0.001 ( η p 2 = 0.30) (mean (SEM) ASR for female rats 64.22 (1.72), and for male rats 79.01 (2.01)). On day 9, only the Sex effect was significant, F (1, 51) =16.44, p < 0.001( η p 2 =0.24) (mean (SEM) ASR for female rats 53.77 (2.30), and for male rats 67.90 (2.26)). 3.3.2. PPI Comparisons between PPI percentages in the first PPI session (see Table 3) showed a significant Sex x prepulse intensity x Groups (CON- acute, MPH-acute, MPH-chronic non-experience) interaction, F (4, 100) =2.30, p <0.03 ( η p 2 =0.11). Post-hoc tests revealed significantly reduced PPI in male rats with acute treatment (MPH-acute) compared with chronic treatment (MPH-chronic non-experience) (p <0.05, η p 2 = 0.14 for PPI75, and p =0.05, η p 2 =0.12 for PPI90). There were no differences between the control and chronic groups (CON-acute vs. MPH- chronic non-experience). Comparison between chronic male rat groups suggested that the PPI-disruptive effects of MPH depend on experience. We obtained significantly lower PPI percentages in MPH- acute versus MPH-chronic non-experience male rats for the lowest prepulse intensity (<0.03) and a trend toward this effect for the middle and highest prepulse intensities (p =0.08 and 0.076 respectively). In the female rat groups, animals treated with acute MPH (MPH- acute) showed a significant decrease in PPI percentage at a prepulse intensity of 75 dB. However, in animals receiving the first session of PPI after seven days of MPH treatment (MPH-chronic non-experience), the percentage of PPI75 was not reduced compared to the control group (CON-acute). We also obtained a significant decrease in the percentages of PPI75 in the MPH-chronic group compared to the MPH-chronic nonexperience group (p <0.04). Interestingly, when the animals were tested in a drug-free context (day 9) we found no effect of either sex or drug. 4. Discussion Our main results suggested that acute and chronic MPH treatment had differential effects on auditory sensorimotor gating depending on the sex. Neither acute nor chronic MPH treatment affected female rats, regardless of their estrous cycle. However, acute MPH treatment impaired the PPI effect in male rats. Additionally, chronic MPH treatment had different effects depending on whether the male rats had received a PPI session during the acute treatment. Male groups that received a PPI session on the first day of drug treatment and a second PPI session after 7 days of treatment showed a disruption of the PPI effect in both sessions (acute and chronic). However, male rats that received the first MPH session after 7 days of treatment exhibited a like-tolerance effect to the drug. Furthermore, 48 h after treatment was stopped, the PPI effect was similar to that in the control groups. 4.1. Pulse The results consistently showed that the ASR was higher in male rats compared to female rats in all sessions. This effect remained unaffected by the drug, regardless of acute or chronic exposure. Therefore, in both Table 2 Mean (SEM) PPI percentages of females rats by cycle and drug. PPI-1 PPI-2 PPI-3 Cycle Prepulse (dB) CONT MPH CONT MPH CONT MPH 75 12.82 (7.24) 5.87 (8.14) 16.14 (5.74) 6.648 (5.03) 5.96 (9.39) 15.92 (6.67) PE 85 11.36 (8.46) 16.97 (9.51) 16.44 (7.01) 21.19 (6.14) 14.62 (9.14) 14.43 (6.49) 90 34.68 (7.42) 20.61 (8.345) 22.99 (6.92) 25.14 (6.07) 36.10 (10.62) 30.57 (7.54) 75 25.08 (11.57) −7.30 (7.55) MD 85 28.03 (13.53) 24.24 (8.82) 90 43.19 (11.86) 38.40 (7.73) Note: MPH: Methylphenidate, CONT: control, S: PPI session, PE: Proestrus-Estrus,. MD: Metaestrus–Diestrus. F. Montiel-Herrera et al. Physiology & Behavior 278 (2024) 114526 6 the control and MPH groups, males exhibited greater response amplitude than females. However, this finding contradicts evidence from human studies, as previous research has not identified any ASR differences between men and women [60–62]. Methodological concerns might explain these divergent results. Lehmann et al. [63] proposed that the body weight of male and female rats could influence the intensity of the startle reflex when measured as movement acceleration. In contrast, human studies measure the amplitude of startle using eye electrodes, making weight irrelevant. Regarding ASR sex differences in rats, there have been limited investigations, yielding mixed findings. Variability in rat strains may explain the observed discrepancies. Studies using Sprague–Dawley or Long Evans strains have reported no sex differences in ASR [49,62,64, 65]. However, in line with our results, Lehmann et al. [63] found higher startle response amplitudes in male Wistar rats compared to females. However, contrary findings have also been obtained in other studies [58, 66]. Another plausible explanation for our findings may be related to sex differences in stress sensitivity, as males, but not females, have been shown to experience stress following initial exposure to the startle stimulus [66]. Thus, our findings indicate a habituation effect in females, with a decrease in startle responses from the first session to subsequent sessions (days 7 and 9). On the contrary, male responses remained consistently high across all sessions, indicating sustained arousal. 4.2. Effect of sex on PPI Despite the differences observed in ASR, the effects of PPI were similar in the male and female control groups. Studies examining the effects of sex on PPI have produced inconsistent results in both human and preclinical research. While several studies in humans have not found differences between men and women [67,68], most investigations have reported a reduced PPI effect in women compared to men [62,69–71]. In rats, some studies have found no differences between male and female rats [62,65,72], while others have observed a decrease in PPI in female rats [63,66]. These apparent discrepancies have been attributed to fluctuations in the estrous cycle. Thus, some studies examining the effects of phases of the menstrual cycle on PPI have shown a lower effect of PPI in luteal women, when estrogen and progesterone levels are higher [60]. Koch [49] found similar overall levels of PPI in male and female rats. However, when considering the estrous cycle, the analysis revealed significantly lower PPI scores in proestrous female rats compared to males. Furthermore, PPI levels in males were comparable to those in diestrous or estrous females. In our study, we did not find any differences between male and female rats regardless of the estrous cycle. Although on day 1, MD female rats exhibited higher PPI percentages than PE female and male rats for all prepulses, these findings were not supported by a significant main effect of the cycle. On day 7, we only had sufficient statistical power to compare the PE female and male groups, revealing a higher PPI effect in the PE female group for PPI75. Discrepancies with the limited studies examining the effect of the estrous cycle could be related to the amplitude of ASR and the lack of its control on the PPI effect. Thus, in studies in which the ASR amplitude tends to be greater in male rats compared to female rats, there is a higher likelihood of detecting sex differences in PPI. However, in our study, baseline startle responses were used as a covariate, allowing us to control for differences in PPI percentages attributable to ASR scores. Another possible explanation for the differences in results could be related to intra- and inter-litter variability. According to Lehmann et al. [63], using animals from different litters may facilitate the detection of statistically independent differences compared to using animals from the same litter, which could be problematic when treating males and females as independent samples. In our study, we used some animals from the same litter, which may have hindered the detection of differences. The timing of testing is another variable that could explain our results. Previous research has demonstrated that inhibition of the startle response in proestrous female rats is not affected by the time of day, while in males and diestrous female rats, it is reduced in the afternoon and evening compared to the morning [73]. Our experiments were carried out in the afternoon, which might have led to lower PPI percentages in the male and MD female groups. This could have masked the typical differences observed between the EP female group and the other two groups. 4.3. Effects of MPH and sex on PPI The effects of MPH on PPI were influenced by sex and task experience. Acute treatment with MPH resulted in a significant decrease in PPI percentages in the male rat group compared to the control group. As there were no changes in ASR amplitude due to MPH, these differences in PPI can be attributed to impairments in inhibiting the motor response in the presence of a prepulse. These results are consistent with the limited number of studies that have analyzed the effect of MPH on PPI [48,74], as well as investigations exploring the acute effects of other direct or indirect dopamine agonists [49,75–77]. Similar to observations in rodents, direct and indirect DA agonists disrupt sensory gating in humans [78–80]. Perhaps more noteworthy is the lack of the MPH effect in the female groups. PPI levels in MPH treated female rats were not different from those in female the control group. Specifically, this group had percentages of PPI similar to those of the female control group for both 85 and 90 dB prepulses. The effects of DA agonists on PPI have been broadly analyzed, and it has been shown that these effects depend on several parameters (see Sherdlow et al. 2000, for a revision). For example, certain strains of rats are more sensible to a direct DA agonist like apomorphine [75]. Thus, in several studies, Swerdlow et al. [15,77] showed that Wistar rats were less sensitive to the disruptive effects of dopaminergic agonists on PPI than the Sprague Dawley (SD) rat strain. This reduced sensitivity of Wistar rats may explain the results obtained in the MPH group of female rats. However, the studies by Swerdlow et al. [15,77] were conducted in male rats, so MPH should not have affected our group of male rats either. In addition, several studies have shown that both direct DA agonists, such as apomorphine, and indirect agonists, as amphetamine, have more potent PPI disrupting effects in male Wistar rats than in other strains of rats, including SD rats [75,81, 82]. It has also been proposed that heritable phenotypic differences due to supplier substrain could explain differences in sensitivity to these DA drugs within the same rat strain [77]. However, this variable or parameter could explain different pharmacological effects within the same rat strain between different laboratories, but not, as in our case, within laboratories. To our knowledge, there are no studies in female rodents that have investigated the effect of MPH on PPI and the few Table 3 Mean (SEM) PPI percentages of control, acute and non-experience groups. PPI-1 CONT MPH-acute MPH-chronic non-experience PPI75 PPI85 PPI90 PPI75 PPI85 PPI90 PPI75 PPI85 PPI90 Males 18.13 (6.05) 30.08 (7.01) 23.84 (6.24) 4.58 (5.82) 9.67 (6.73) 6.31 (6.01) 19.14 (9.76) 23.60 (11.33) 27.14 (10.04) Females 16,15 (6,27) 15,90 (7,26) 37,00 (6,47) −1,22 (5,60) 20,88 (6,48) 30,19 (5,78) 12,20 (8,69) 21,81 (10,08) 27,34 (9,26) Note: 75, 85, 90: prepulses intensities. PPI-1: first session of PPI. F. Montiel-Herrera et al. Physiology & Behavior 278 (2024) 114526 7 studies that have investigated the effects of dopamine agonists on PPI in both sexes have shown different results. In line with our results, Talledo et al. [83], in a study conducted exclusively in women and female rats, showed that the indirect dopamine agonist amphetamine did not affect PPI in the women sample. In rats, amphetamine caused a decrease in PPI levels in female rats of the SD strain, but the percentage of PPI in female rats of the Long Evans strain was not affected by drug administration. In addition, under certain parameters, the drug caused an increase in the percentage of PPI in this strain of rats. In contrast to our results, Swerdlow et al. [77] found no sex differences in the PPI-disrupting effect of the dopamine agonist apomorphine. However, these results were obtained in pup rats and the sex variable was not analyzed in the adult rat groups. This is important because in the study by Kosh [49], when low doses of apomorphine were used; different effects were observed depending on the phase of the estrous cycle. Rats in estrus and oestrus, as well as males, showed a reduction in the percentage of PPI, although this reduction was not significant due to the low dose of apomorphine used. However, females in the proestrus, did not have this trend of decrease. Similar discrepancies between male and female in the disruptive effect of PPI have been observed in studies examining the effects of other drugs such as LSD or NDMA and serotonin agonists [58, 59,84]. This confluence of results allows us to raise the possibility that the lack of reduction in the percentage of PPI in female rats could be related to the long-term effect of female sex steroids on these receptors. Previous studies have shown that estrogen increases the synthesis of DA and serotonin and decreases their degradation, reuptake, and recapture. It also upregulates dopaminergic receptors [45]. Therefore, a possible explanation for our results could be that exposure to steroids during their natural cycle could increase the pool of dopaminergic receptors, especially during the EP phase of the cycle. This may provide some protection to female rats from the disruptive effects of dopaminergic stimulants. This could have affected either the potency or efficacy of MPH. However, without conducting a dose-response curve, it is not possible to draw conclusions about how sexes differ. 4.4. PPI experience and chronic treatment with MPH Chronic treatment with MPH also produced a different effect on PPI depending on the sex of the animals. Chronic use of the drug did not affect the female groups. It’s important to clarify that we were unable to assess whether female rats developed tolerance to MPH. The reduction in MPH efficacy with repeated treatment may not have been detectable due to the lack of an acute effect of the drug. However, in the male groups, there was a differential effect of MPH as a function of the number of PPI sessions received (test experience effect). In the chronic group without experience with PPI, there was a tolerance-like effect to MPH, as the PPI effects in this group were greater than those in the acute and chronic experienced groups but similar to those in the control group without experience with PPI. However, there was no liketolerance effect in the chronic experienced group because the reduction of PPI effects were similar to those in the acute group and greater than those in the control group and the chronic inexperienced group. Since the startle response to the pulse in this second session did not differ from that of the first session (PPI-1-PPI-2 comparison) or from that of the control group (CON-chronic), we can rule out an explanation of this result in terms of pulse habituation or the chronic effect of MPH on ASR. To the best of our knowledge, there are no studies that have investigated the effects of repeated exposure to MPH on PPI. However, studies that have examined the effects of repeated exposure to DA agonists have produced conflicting results. Thus, both tolerance and sensitization effects or lack of differences with respect to the acute effect of the drug have been found [46,85,86]. Consistent with our findings, studies that have examined the chronic effect of administration of dopaminergic stimulants such as amphetamine, apomorphine, or cocaine [85,87] prior to the PPI test have shown that the interruption of the PPI effect produced by acute administration of the drug is not observed after chronic administration of the drug (tolerance effect). However, studies in which the PPI test was repeated during chronic administration of drugs such as quimpirole [86], apomorphine [88], or amphetamine [46] found a similar or greater disruption of the PPI effect than that obtained with acute administration of the drug. Factors related to test apparatus, dose, treatment regimen, stimulus parameters, or environmental factors have been used to explain these differences in results. Regarding to the latter type of factor, it has been suggested that the effects of the drug could interact with the effects of repeated presentation of the PPI test [46]. In particular, repeated exposure to a moderately stressful stimulus such as pulse may elicit an emotional response that alters the drug’s impact on PPI over time. In this context, the association between drug effects and the startle test has been proposed to play a crucial role in the sensitization to dopaminergic stimulants in PPI [81,88,89]. Additionally, it has been suggested that pre-exposure to these drugs without the presence of the startling test might attenuate a specific aspect of the sensitization context. Therefore, we can state that the decrease in the PPI effect resulting from acute application determined the establishment of an association between this drug effect and the PPI context. Consequently, exposure to the PPI context following chronic administration of the drug might have contributed to the restoration of the effect associated with the PPI context (i.e., decrease in the PPI effect). In the absence of previous experience of PPI impairment under the influence of the drug, such an association will not be formed, and as a result, a tolerance effect may be observed after chronic administration of the drug. In summary, the findings of this study suggest that MPH has differential effects on sensorimotor gating response (PPI) based on sex and task experience in rats. Acute MPH treatment significantly decreased PPI levels in male rats but had no effects on female rats. Furthermore, chronic MPH treatment showed a differential effect in male rats depending on the presence of a drug-associated context. Finally, these findings have important implications for the treatment of ADHD, as MPH is one of the most commonly prescribed medications for this condition. The results suggest that the effect of MPH on the sensorimotor gating response may vary based on sex, indicating that treatment strategies may need to be tailored differentially for males and females with ADHD. Further research is required to better understand these findings and improve treatment approaches for ADHD, taking into account sex differences and task experience [90]. This could lead to more effective and personalized treatment for individuals with ADHD, maximizing therapeutic benefits while minimizing potential adverse effects. Funding This work was supported by PID2019-110739GB-I00 grants (Ministerio de Ciencia, Innovaci´ on y Universidades, FEDER, UE). Significance statement Methylphenidate (MPH) is a first-line treatment for ADHD. Its use on adult population is increased due to the increasing rates of ADHD diagnosis in this age group, and the widespread employment and occasional misuse of MPH as a cognitive enhancer. This work analyzes the effects of acute and chronic oral administration of MPH in adult male and female Wistar rats on an attentional process of filtering and selection of stimuli (prepulse inhibition, PPI). The results indicated that acute and chronic MPH treatment impaired PPI expression in male rats, but not in female rats, regardless of their estrous cycle phase. Furthermore, among male rats, the development of tolerance or the attenuation of PPI effects was contingent upon whether the subjects had undergone a PPI session during the acute drug influence. Understanding these differential effects is crucial for optimizing ADHD treatment and preventing potential adverse outcomes associated with psychostimulant use. F. Montiel-Herrera et al. Physiology & Behavior 278 (2024) 114526 8 Data availability The data underlying this article will be shared on reasonable request to the corresponding author. It has been deposited in the institutional repository of the University of Seville, idUS (https://doi.org/10.12795/ 11441/155734). CRediT authorship contribution statement F. Montiel-Herrera: Writing – original draft, Visualization, Investigation, Formal analysis, Conceptualization. A. Batanero-Geraldo: Writing – review & editing, Visualization, Investigation, Formal analysis. J.C. L´ opez: Writing – review & editing, Writing – original draft, Visualization, Supervision, Resources, Formal analysis, Conceptualization. J.P. Vargas: Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition, Formal analysis, Conceptualization. E. Quintero: Writing – review & editing, Supervision, Investigation, Formal analysis, Conceptualization. E. Díaz: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization. Declaration of competing interest None. Acknowledgments None. References [1] R.B. Millstein, T.E. Wilens, J. Biederman, T.J. Spencer, Presenting ADHD symptoms and subtypes in clinically referred adults with ADHD, J. Atten. Disord. 2 (1997) 159–166, https://doi.org/10.1177/108705479700200302. [2] S.J. Gatley, D. 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