Effects of periadolescent fluoxetine and paroxetine on elevated plus-maze, acoustic startle, and swimming immobility in rats while on and off-drug

Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressants that inhibit the reuptake of serotonin (5-HT) into the presynaptic terminal by binding to the serotonin transporter (SERT). SSRIs have been used with success in adults for treatment of depression, premenstrual syndrome, obsessive compulsive behavior, panic disorder, generalized anxiety disorder, social anxiety disorder, and post-traumatic stress disorder [1–3]. A meta-analysis of adult trials indicates that suicidality may be increased shortly after antidepressant treatment is begun regardless of whether the drug is an SSRI or tricyclic antidepressant [4].

SSRIs have also been used in children and adolescents, primarily for depression [5]. It has been suggested that children and adolescents may be at higher risk of adverse effects perhaps because SSRIs are contraindicated for some types of depression (such as bipolar disorder) that are often unrecognized in younger patients [5]. A recent meta-analysis of randomized controlled trials of SSRI treatment for depression in children and adolescents found benefit from SSRI treatment, but not all SSRIs were equally effective. Fluoxetine, sertraline, and citalopram were most effective in this age group compared with other SSRIs [6].

Both efficacy and adverse effects of SSRIs in younger patients are ongoing areas of investigation. Modeling the effects of juvenile SSRI treatment in rats [7, 8] and mice [9] demonstrated that between postnatal (P) day 21 and 35, there are differences in drug response [10]. However, data on SSRI exposure during adolescence in rodents has received less attention. There are two issues concerning age-related effects of SSRI exposure: (1) efficacy and (2) long-term or adverse effects.

In one experiment, male Wistar rats were treated with 15 mg/kg of paroxetine or 30 mg/kg of fluvoxamine by gavage from postnatal days (P) 33-62 and tested 20-21 days later for sexual behavior, 23-24 days later in the elevated plus-maze (EPM), 31 days later for acoustic startle response (ASR) with prepulse inhibition (PPI), 35-36 days later in the Porsolt forced swim test (FST), and 56 days later in an elevated T-maze [11]. Both drugs decreased time in open arms in the EPM, but in an elevated T-maze in which 5 trials were given from a closed stem to open arms, only on trial-2 did fluvoxamine significantly increase time to enter an open arm. On the converse arrangement (open to closed entry), no change was seen. No change in ASR/PPI, immobility in the FST, or in 8-OH-DPAT-induced 5-HT_1A-related behaviors was observed (lower lip retraction or sexual facilitation). A reduction in ejaculatory frequency was found in the drug groups during the third week of testing, but not during weeks 1 and 2; no differences were found for ejaculatory latency, mount frequency, or intromissions.

More recently the effects of fluoxetine were tested in adolescent mice [12]. C57BL/6J and BALB/cJ male mice were treated with fluoxetine in drinking water at concentrations of 80 or 160 mg/L on P21-42 or P56-84 with testing 30 days later on open-field, EPM, cued conditioned fear and extinction, the FST, plasma corticosterone, and brain 5-HT and 5-HIAA. The lower dose was calculated to be 9-10 mg/kg/day and the higher dose 17-18 mg/kg/day. In neither strain, at neither dose did significant effects emerge [12]. No changes in 5-HT, 5-HIAA, or corticosterone after the FST were seen.

Hence, the evidence is divided as to whether SSRI exposure during adolescent brain development has immediate and/or untoward lasting effects. The purpose of the present experiment was to test whether adolescent exposure to two widely used SSRIs (fluoxetine and paroxetine) has immediate or long-term effects. The previous experiments tested animals 21-56 or 30 days after drug exposure. To determine if effects occur while on-drug and persist after drug cessation, we treated two cohorts of rats identically and tested one cohort while on-drug and the other after drug discontinuation.

We used the Porsolt FST because it is related to depression, the therapeutic target of these drugs. We assessed sensorimotor gating using ASR/PPI because this is a widely used marker for abnormalities in neuropsychiatric disorders [13–16]. We used the EPM to determine whether SSRIs cause changes in anxiety, and we assessed plasma corticosterone after FST to determine if stress reactivity was altered by SSRI exposure.

There are many SSRIs. We focused on paroxetine because it showed residual effects previously [11] and fluoxetine because it is FDA approved for use in juvenile and adolescent patients (6-18 years of age). We used the same treatment days as de Jong et al. [11], i.e., P33-62, an age range from puberty to young adulthood in rats. One cohort was tested while on-drug after 25 days of prior treatment (tested on P57-62) and the other cohort off-drug (P122-127; 60 days post-treatment). We tested doses from the literature showing effects after adolescent exposure [11, 12]. A third dose of paroxetine was included to match the dose used by de Jong et al. [11].

Fluoxetine, paroxetine, and many other SSRIs have proven to be efficacious for the treatment of adults with depression, premenstrual syndrome, obsessive compulsive behavior, panic disorder, generalized anxiety disorder, social anxiety disorder, and post-traumatic stress disorder [1–3]. These drugs have been found to be safer and have fewer side-effects than tricyclic antidepressants. SSRIs have also been used in children and adolescents, and fluoxetine is approved for use in those 6-18 years of age. In children whose nervous system is still developing, there are concerns about the long-term effects of these drugs.

In a recent preclinical experiment examining paroxetine and fluvoxamine, it was reported that these drugs reduced time-in-open in an EPM [11]. Decreased time in open in the EPM is the most widely accepted index for this test of increased anxiety-like behavior [23]. In the experiment by deJong et al. [11] this change occurred for both drugs 20-30 days after the end of treatment. No change was seen in the number of zone crossings. These authors also tested the animals in an elevated T-maze (the EPM was used with one arm blocked). Out of 5 trials for time to enter an open arm after being placed in a closed arm, both SSRI-treated groups entered an open arm faster on trial-2 compared with controls but not on trial-1 or 3-5. When the situation was reversed and they were given an additional trial by being placed in an open arm and timed for entry into a closed arm, no differences were found. No differences in ASR/PPI were obtained nor were changes obtained on immobility time in the FST.

We sought to further test the effects of adolescent exposure to SSRIs and followed many of the design features used previously [11], except using more dose levels of each drug and including separate groups of animals, half tested while on-drug (Group A) and half tested off-drug (Group B; 60 days post-treatment). If long-term effects of SSRI treatment were obtained, it would potentially represent a concern for the safety of the drugs.

Both drugs produced body weight reductions at the highest doses tested by P44, i.e., after 12 days of treatment, and these reductions remained through the end of treatment (P62). But the effects showed recovery to non-significant differences one week post-treatment. Behaviorally, no treatment-related effects on the EPM were obtained on the principal measure of anxiety, i.e., time-in-open. In addition, no differences were found for number of zone crossings or latency to first open arm entry. The Group A Par17 animals showed a significant reduction in head-dips, an effect seen in no other group, however this effect was not seen in Group B when tested 60 days after drug cessation. deJong et al. [11] did not report head-dips, therefore, it is not possible to make a direct comparison on this variable, however, a reduction in head-dips is generally interpreted as an increase in anxiety [24], although it is an index of anxiety that is secondary to time-in-open and therefore provides less persuasive evidence of a significant change in anxiety. However, this effect would be consistent with the finding of deJong et al. [11]. A recent study showed that rats treated from P25-49 with 12 mg/kg of fluoxetine had reduced time-in-open in the EPM [25] 7 days post-treatment, a finding consistent with reduced head-dips that we found while animals were on drug. It is unclear why adolescent exposed rats and mice show greater inconsistency in EPM responses to SSRIs than adult rodents. Some of it may be related to the different exposure ages as there is no consistent definition of adolescence in rodents. Doses and routes of drug administration as well as duration of treatment and end of treat to test interval vary widely across studies, increasing the difficulty of discerning patterns.

Neither we nor deJong et al. (2006) found any effect on the FST test of swimming despair [17, 18] and this finding is different than that reported by Homberg et al. [25] but they treated rats earlier (P25-46); moreover, they found no differences in rats treated at a later age. Other studies have found opposite or paradoxical effects of adolescent exposure to fluoxetine in mice, but only in one of the two strains tested [26]. To the extent that the FST is a valid preclinical test of depression, the data do not suggest that adolescent exposure to fluoxetine or paroxetine result in long-term changes in immobility. This is consistent with a newer report of chronic fluoxetine treatment in mice from P14-42 showing no FST effects [26] and the data cited above [12]. We scored the FST for immobility time as originally described by Porsolt et al. [17, 18]. We did not score the test using the recently suggested indices of active swimming and wall climbing [20]. While we cannot rule-out the possibility that these additional indices might have uncovered other effects, we did not include them because our interest was not differentiating among different classes of SSRIs, which was the reason these measures were introduced, but rather whether immobility was a long-term consequence of drug exposure after adolescent treatment.

Others have administered SSRIs by osmotic pump in order to maintain plasma concentrations in animals within the same range as human therapeutic concentrations [27, 28]. The intent of these studies was different than ours. These studies were designed to assess the molecular targets of SSRIs. For this purpose maintaining a constant drug concentration is desirable but this goal was not necessary for our purposes of determining if these drugs have persistent long-term effects long after cessation.

deJong et al. [11] reported no changes in ASR/PPI. We replicated their parameters and in agreement, observed no changes in ASR or PPI from either drug 60 days post-treatment. However, while on-drug, we saw a trend toward ASR facilitation that did not interact with PPI. To further explore the data, we conducted follow-up analyses by combining the two fluoxetine groups into one pooled group by averaging the data for the fluoxetine animals in each litter together to create a single, merged group. We did the same for the paroxetine, merging the data from all three dose levels together among littermates to create a single paroxetine group. Two follow-up analyses were performed. These were: (1) with all prepulse trials included and (2) with no-prepulse trials included, i.e., only the unmodified, basic ASR trials. Both analyses resulted in the same finding: the pooled fluoxetine and pooled paroxetine groups exhibited increased ASR amplitude compared with Controls. By contrast, when Group B ASR-PPI data were analyzed the same way, no residual effect or trend toward an effect was observed. This is in contrast to a study that found that P24-46 fluoxetine reduced ASR at a higher dose than we used (12 mg/kg), but similar to our data, found no effects at later ages [25].

The present data should be viewed within the limitations of the study. As in the deJong et al. [11] and Norcross et al. [12] studies, we tested only males. It may be worthwhile to test females as many drugs exhibit sexually dimorphic responses. It is also worth considering that future experiments might test a broader range of doses in order to more thoroughly test for possible adverse effects. Finally, other behavioral tests might be worth considering; Norcross et al. [12] also included open-field and cued fear conditioning with extinction, but even more tests might be considered such as fear-induced acoustic startle facilitation. There are many tests of anxiety, conditioned fear, sensorimotor gating, and depression, of which only a subset were used here or in the other studies cited that tested for residual effects. A more extensive battery of tests might reveal effects not detected herein.

Overall, the data show that for the doses of fluoxetine and paroxetine tested by an oral route during adolescent to early adult brain development (operationally defined as P33-62), caused minor and only transient reductions in body weight gain, a small but significant ASR facilitation during treatment that did not remain 60 days post-treatment, and a high-dose only (Par17) reduction in EPM head-dips while on-drug but not off-drug. The findings, when considered in terms of the power to detect differences in the present experimental design (≥ 20 animals per group), multiple dose levels, use of a within-litter design that improves subject matching, and inclusion of both on- and off-drug cohorts, suggest that there is no signal of adverse effects present in the data that might raise concern over the long-term safety of the drugs when treatment is during an interval spanning adolescent brain development in rats, in agreement with previous findings in mice [12]. It was recently reported that fluoxetine given by continuous infusion via minipumps from P14-42 results in anxiogenic effects in mice while on-drug on tests of novelty-induced hypophagia in Swiss-Webster (SW) and C57BL/6 Charles River (B6) mice, however there were no effects in the EPM in the SW strain and reduced time-in-open in the B6 strain but no effects on open-field center time or FST in either strain. These effects disappeared off-drug [26] which is entirely consistent with the present findings. Thus, while SSRIs induce relatively reliable effects on-drug in adult rodents, their effects from adolescent exposure are less consistent. Our data are consistent in that we saw only small effects while on drug and no long-term effects long after drug discontinuation. The latter provides some evidence that these drugs are not neurotoxic when given during adolescent stages of brain development at least on the behavioral indices used here.