Behavioral Responsiveness And Receptor Expressions Are Altered By Repeated Adolescent Atypical Antipsychotic Exposure: A Preclinical Test In The Phencyclidine Hyperlocomotion Model

Schizophrenia is a debilitating mental illness that affects 1% of the population. Despite intensive study, its molecular etiology remains enigmatic. Like many common diseases, schizophrenia is multifactorial in origin, with both genetic and environmental contributions likely playing an important role in the manifestation of symptoms.In recent years, there has been a significant increase in the number of children and adolescents who are being treated with antipsychotic drugs. One survey in the US showed a six-fold increase in the number of prescriptions of antipsychotics for patients ≤20 years from 1993-2002. The percentage of the user population accounted for by children and adolescents(i.e., by patients <18 years old) has doubled from 7% in 1996-1997 to 15% in 2004-2005. Clinical studies demonstrate that atypical antipsychotics are effective for pediatric patients with early-onset schizophrenia and schizoaffective disorder, although they also cause more severe antipsychotic-related 14 adverse side effects(e.g. extrapyramidal side effects, sedation, prolactin elevation, weight gain) in pediatric patients than in adult patients.Current research on children and adolescent antipsychotic treatment has been mostly focusing on the efficacy, tolerability, and side effect profiles of individual drugs. There is a general lack of research on the long-term consequences of antipsychotic treatment on the brain and behavioral development of patients. Preclinical studies suggest that synaptic connections, receptor densities-especially those among the dopamine and serotonin systems-in the prefrontal cortex, striatum, and hippocampus undergo dramatic maturational changes during adolescence that may have implications for understanding the unique clinical response and side effects associated with adolescent antipsychotic treatment. Animal work also shows that antipsychotic exposure during adolescence alters various neuroreceptors, including dopamine D1, D2 and D4 receptors, serotonin 5-HT1 A and 5-HT2 A receptors, and ionotropic glutamate NMDA and AMPA in unique ways not seen in adult animals. All these findings strongly suggest that antipsychotic exposure during adolescence could alter the trajectory of the brain and behavioral development of pediatric patients which in turn, may change their later response to drug treatment as adults. As pediatric treatment occurs during the period of rapid brain and behavioral development, there is a need to evaluate the possible short-term and long-term impacts of antipsychotic medications on psychological and brain functions in order to determine their risk/benefits.In recent years, we have demonstrated that in adult rats, repeated treatment of olanzapine or clozapine(two atypical antipsychotic drugs commonly used in the treatment of pediatric schizophrenic patients) induces a potentiated(sensitization) or a decreased(tolerance) inhibition of the phencyclidine(PCP)-induced hyperlocomotion, respectively, a preclinical test for antipsychotic activity. The present study examined the extent to which early antipsychotic exposure during adolescence affects ‘exposure-dependent’ alterations in responsiveness to its pharmacological actions later during adulthood. Such alterations can be manifested as either tolerance, which is characterized by progressive reductions in responsiveness to certain drug effects, or sensitization(also known as reverse-tolerance), which is characterized by progressive increases in responsiveness to certain drug effects.The present study extended this line of research and investigated the persistence of olanzapine and risperidone sensitization and clozapine tolerance from adolescence to adulthood in this model. To assess how adolescent antipsychotic treatment and PCP treatment affect the brain and behavioral functions and developments of adolescent rats, we also examined the prepulse inhibition(PPI) of acoustic startle response(ASR) periodically throughout their developmental period. PPI refers to the phenomenon of a reduction in the startle magnitude when the startling stimulus is preceded by a low-intensity prepulse. It measures the sensorimotor gating ability which is found to be deficient in patients with schizophrenia and in animals treated with dopamine agonists and NMDA antagonists.In addition, we attempted to identify the possible molecular mechanisms underlying such a short-term or long-term behavioral effect. Brain-derived neurotrophic factor(BDNF) is a member of the neurotrophin family that is widely distributed in the brain with the highest levels found in the hippocampus and other cortical areas. It is involved in many functional processes critical for the brain development and experience-dependent neuroplasticity. Accumulating clinical evidence indicates that abnormal BDNF expressions may contribute to pathophysiology of schizophrenia, as the levels of BDNF have been found decreased in peripheral(blood) and central(brain) systems of patients with schizophrenia. Antipsychotic drugs can also alter the brain levels of BDNF, and prevent the stress-induced decrease in the levels of BDNF, indicating that it may also regulate the action of antipsychotic drugs. Given that repeated antipsychotic treatment is shown to induce a robust long-term change in BDNF, D2 and 5-HT2 A, three important biomarkers involved in brain plasticity and the action of chronic antipsychotic treatment. We hypothesized that adolescent antipsychotic treatment would also cause long-lasting changes in the expression of D2, 5-HT2 A and BDNF levels parallel to the behavioral sensitization or tolerance.Therefore, we established the PCP hyperlocomotion model to address the behavioral and molecular changes induced by repeated antipsychotic treatment in adolescent rats. In the first part, we examined how repeated olanzapine, clozapine or risperidone treatment in adolescence alters sensitivity to the same drug in adolescence. In the second part, we assessed how a short term antipsychotic exposure in adolescence impacts drug response in adulthood in the PCP-induced hyperlocomotion model. In the third part, we first investigated a short term impact of clozapine exposure in adulthood on drug responsiveness and related mechanisms in the PCP-induced hyperlocomotion model. Then we assessed the role of serotonin 5-HT2A/2C or dopamine D2/3 receptors in the repeated effects of risperidone in adolescent rats. The last part, we investigated potential molecular basis of repeated antipsychotic treatment in adolescent rats, focusing on BDNF.PartⅠ Repeated treatment effect of antipsychotic drugs in adolescence in the PCP-induced hyperlocomotion modelAIM:This study examined how repeated olanzapine(OLZ), clozapine(CLZ) or risperidone(RIS)treatment in adolescence alters sensitivity to the same drug in adolescence in the PCP-induced hyperlocomotion model.METHODS:Male adolescent Sprague-Dawley rats(P 44) were first treated with OLZ(1.0 or 2.0 mg/kg, sc), CLZ(10.0 or 20.0 mg/kg, sc), RIS(0.5 or 1.0 mg/kg, sc) or vehicle and tested in the PCP(3.2 mg/kg, sc)-induced hyperlocomotion model for five consecutive days(n=6-7/group). Then a challenge test with OLZ(0.5 mg/kg), CLZ(5.0 mg/kg) or RIS(0.5 mg/kg), respectively, was administered during adolescence(P 51) to assess the antipsychotic effect to the same drug re-exposure.RESULTS: 1) During adolescence, repeated OLZ treatment produced a persistent inhibition of PCP-induced hyperlocomotion across the five test days. In the challenge test during adolescence, rats previously treated with OLZ did not show a significantly stronger inhibition of PCP-induced hyperlocomotion than those previously treated with vehicle. 2) CLZ treatment caused a persistent inhibition of PCP-induced hyperlocomotion during the induction phase in a dose-dependent fashion. Those previously treated with CLZ showed a weaker inhibition than the control groups in challenge test. 3) Repeated administration of RIS dose-dependently increased its inhibition of the PCP induced hyperlocomotion across the 5 drug test session. Rats previously treated with RIS 1.0 mg/kg showed a stronger inhibition of the PCP-induced hyperlocomotion than those previously treated with vehicle.CONCLUSIONS: 1. Prior treatment of RIS dose-dependently enhanced animals’ sensitivity to thisdrug in inhibiting PCP-induced hyperlocomotion, a clear sign of RIS sensitization. The expression of OLZ sensitization effect three days later(still in adolescent) was relatively weak. While repeated CLZ treatment caused a tolerance in the inhibition of PCP-induced hyperlocomotion later on.Part Ⅱ Adult responsiveness to an antipsychotic drug is altered by antipsychotic exposure in adolescence in the PCP-induced hyperlocomotion modelAIMS: 1) To assess how a short term antipsychotic exposure in adolescence impacts drug response in adulthood in the PCP-induced hyperlocomotion model. 2) To investigate how adolescent antipsychotic and PCP treatment affect acoustic startle response throughout their developmental period by using prepulse inhibition(PPI) test periodically.METHODS: Male adolescent Sprague-Dawley rats(P 44) were first treated with OLZ(1.0 or 2.0 mg/kg, sc), CLZ(10.0 or 20.0 mg/kg, sc), RIS(0.3 or 1.0 mg/kg, sc), fluoxetine(FLX 5.0 or 10.0 mg/kg, ip) or vehicle and tested in the PCP(3.2 mg/kg, sc)-induced hyperlocomotion model for 5 consecutive days. Then a challenge test with OLZ(0.5 mg/kg), CLZ(5.0 mg/kg), RIS(0.3 mg/kg) or FLX(5.0 mg/kg) was administered after they matured into adults(P 76 and 91), respectively. A total of three separate PPI tests were conducted throughout the development period. The first one was done during the late adolescent period(P 49), 1 day after the five drug test days) and the second one during the early adulthoodperiod(P 67). The third one(P 77) was conducted 1 day after the first sensitization test.RESULTS: 1) Repeated OLZ treatment progressively increased its inhibition of PCP-induced hyperlocomotion across the 5 test days. In the subsequent two challenge tests, rats previously treated with either 1.0 or 2.0 mg/kg all showed a stronger inhibition of the PCP-induced hyperlocomotion than those previously treated with vehicle, indicating a robust long-term olanzapine sensitization. 2) Repeated CLZ treatment progressively induced a sensitized inhibition of PCP-induced hyperlocomotion during the adolescent drug treatment period in a dose dependent fashion. However, in the first challenge test, rats previously treated with CLZ 10 mg/kg showed a significant weaker inhibition than those previously treated with vehicle. CLZ 20 mg/kg group also showed a similar trend like the lower dose group, indicating a tolerance effect in inhibition of PCP-induced hyperlocomotion later on. For the second challenge test, no clozapine tolerance effect was detected. 3) Repeated RIS treatment progressively increased its inhibition of PCP-induced hyperlocomotion across the 5 test days. In the subsequent two challenge tests, rats previously treated with 0.3 mg/kg showed a stronger inhibition of the PCP-induced hyperlocomotion than those previously treated with vehicle, indicating a robust risperidone sensitization. 4) Antidepressant drug FLX did not inhibit the PCP-induced hyperlocomotion during the adolescent period. In the subsequent two challenge test, two FLX group showed neither sensitization nor tolerance effect. 5) Repeated OLZ, CLZ, RIS, FLX and PCP treatment did not significantly impair the sensorimotor gating ability from the three points of testing(P 49, 67 and 77).1. Repeated OLZ treatment during adolescence induced a strong sensitization effect during the adolescent treatment period and that this sensitization effect persisted into adulthood. This effect was long-lasting and dose-dependent. Adolescent RIS exposure induces a long-term increase in behavioral sensitivity to RIS that persists into adulthood. 2. 5 consecutive days clozapine treatment progressively inhibited the PCP-induced hyperlocomotion in induction phase. CLZ tolerance effect persisted into early adulthood but disappeared with the passage of time. 3. Antidepressant drug FLX does not share the property of antipsychotics in the PCP-induced hyperlocomotion model and this model can possibly be used to distinguish antipsychotics from antidepressant. 4. Adolescent drug treatment did not affect the sensorimotor gating ability throughout the development period. CONCLUSIONS:Part Ⅲ The behavior response and receptor mechanisms of repeated clozapine and risperidone treatment AIMS: 1) The present study investigated a short term clozapine exposure in adulthood impacts drug response and receptor mechanisms in the PCP-induced hyperlocomotion model. 2) To assess the role of serotonin 5-HT2A/2C or dopamine D2/3 receptors on the repeated effects of risperidone in adolescent rats. METHODS: 1) Male adult Sprague-Dawley rats(P 74) were first treated with CLZ(10.0 or 20.0 mg/kg, sc) or vehicle and tested in the PCP(3.2 mg/kg, sc)-inducedhyperlocomotion model for 5 consecutive days. 28 and 43 days after the fifth drug treatment, all rats were retested with CLZ(5.0 mg/kg, sc) to assess the long-term impact of prior CLZ treatment. A total of three separate PPI tests were conducted throughout the development period(P 79, 97,107). 2) Male adult Sprague-Dawley rats(P 59) were first treated with CLZ(10.0 mg/kg, sc) or vehicle every other day for 5 times. Two challenge tests with QUI or DOI were administered on P 70 or P 73, respectively. 3) Male adolescent Sprague-Dawley rats(P 44) were first treated with RIS(1.0 mg/kg, sc) or vehicle and tested in the PCP(3.2 mg/kg, sc)-induced hyperlocomotion model for 5 consecutive days. Then a challenge test with QUI(1.0 mg/kg) was administered after they matured into adults(P 76). RESULTS: 1) Repeated CLZ treatment progressively induced a sensitized inhibition of PCP-induced hyperlocomotion during the adult drug treatment period across 5 days. However, no clozapine tolerance effect was detected in the two separate challenge tests. No group difference was found in three separate PPI tests. 2) Rats treated with CLZ 10 mg/kg made significantly lower locomotion activity than the vehicle group across 5 days. In the first QUI(0.2 mg/kg) challenge test, rats previously treated with CLZ showed a significantly higher locomotor activity than those treated with vehicle. Similar result was also found in the second QUI(0.5 mg/kg) challenge test. However, no group significance was found in two separate DOI challenge tests. 3) In the adolescent treatment phase, repeated RIS treatment(1.0 mg/kg) progressively enhanced its inhibition of PCP-induced hyperlocomotion. However, in the QUI-induced hyperlocomotion test in adulthood, rats previously treated with RIS did not make significantly more activity than the vehicle group.1. Like the adolescent repeated CLZ treatment, CLZ also induced a persistent inhibition of PCP-induced hyperlocomotion in adult rats during the drug treatment period. However, when assess the long-term consequence of clozapine in adult rats, the tolerance effect disappeared with the passage of time. Repeated CLZ treatment in adult rats did not impair the sensorimotor gating ability. 2. Dopamine D2 receptor may be related to the tolerance effect of repeated CLZ treatment in adult rats but not the serotonin 5-HT2A/2C receptor. Dopamine D2/3 receptor may not be responsible for the effect of RIS sensitization response to adolescent treatment in the PCP-induced hyperlocomotion model. CONCLUSIONS:Part Ⅳ Potential molecular basis of repeated antipsychotic treatment in the PCP-induced hyperlocomotion model AIMS: 1) To investigate the levels of brain-derived neurotrophic factor(BDNF) underlying repeated treatment effect of OLZ, CLZ and RIS in adolescent rats in the PCP induced hyperlocomotion model. 2) To assess whether the levels of mature and precursor BDNF were altered by repeated treatment of OLZ and CLZ from adolescence to adulthood. METHODS: One day after the last challenge test from PartⅠand PartⅡ, rats were sacrificed by live decapitation and their brains were quickly removed. The prefrontal cortex(PFC), striatum(ST) and hippocampus(HC) were dissected out over ice according to the brain atlas of Watson and Paxions(5th edition). Tissues from theseareas were homogenized in RIPA buffer. Equal amount of proteins(40 ug/lane) was run on a sodium dodecyl sulfate(SDS)-12% polyacrylamide gel. After being separated by the electrophoresis, proteins were electrophoretically transferred onto polyvinylidene fluoride(PVDF) membranes. The PVDF membranes were blocked with 5% nonfat milk and incubated with primary antibodies( BDNF and β-actin) overnight at 4℃. Then the membranes were washed and incubated with secondary antibodies for 1 hour at room temperature. Then the bands were visualized and quantified by using Odyssey Fc Imager. RESULTS: 1) In adolescent rats, repeated OLZ(1.0 and 2.0 mg/kg) and PCP(3.2 mg/kg) treatment in adolescent rats resulted in a slight but not signi?cant increase of BDNF compared to the other two OLZ na?ve groups in ST and HC but not in PFC brain area. 2) In CLZ treated adolescent rats, no significant group difference in BDNF expressions was observed in three brain areas. 3) For the repeated treatment of RIS in adolescent rats, there was not any group or regional differences in BDNF protein levels. 4) In adult rats, mature or precursor BDNF were not altered by any repeated treatment(OLZ, CLZ or PCP or their combinations). CONCLUSIONS: 1. In adolescent rats, although repeated adolescent antipsychotic drugs induced sensitization or tolerance effect in the PCP-induced hyperlocomotion model in a dose-dependent fashion, whether mature or precursor BDNF is critically involved in this effect is still unsettled. In summary, our present study has the following new concerns: 1. Adolescent antipsychotic exposure can induce alternations in laterantipsychotic response. Repeated OLZ, CLZ and RIS progressively inhibited the hyperlocomotion induced by PCP across 5 consecutive days. Repeated CLZ treatment caused a tolerance effect in the inhibition of PCP-induced hyperlocomotion later on. Prior treatment of RIS dose-dependently enhanced animals’ sensitivity to this drug in inhibiting PCP-induced hyperlocomotion, a clear sign of RIS sensitization. 2. Adult response to antipsychotic treatment is altered by adolescent drug exposure. Repeated OLZ treatment during adolescence induced a strong sensitization effect during the adolescent treatment period and that this sensitization effect persisted into adulthood. This effect was long-lasting and dose-dependent. 5 consecutive days clozapine treatment progressively inhibited the PCP-induced hyperlocomotion in induction phase. CLZ tolerance effect persisted into early adulthood but disappeared with the passage of time. Adolescent RIS exposure induces a long-term increase in behavioral sensitivity to RIS that persists into adulthood. Antidepressant drug FLX does not share the property of antipsychotics in the PCP-induced hyperlocomotion model and this model can possibly be used to distinguish antipsychotics from antidepressant. Adolescent drug treatment did not affect the sensorimotor gating ability throughout the development period. 3. Adolescent CLZ treatment changed the sensitivity of dopamine D2/3 receptor in adult rats. Like the adolescent repeated CLZ treatment, CLZ also induced a persistent inhibition of PCP-induced hyperlocomotion in adult rats during the drug treatment period. However, when assess the long-term consequence of clozapine in adult rats, the tolerance effect disappeared with the passage of time. Repeated CLZ treatment in adult rats did not impair the sensorimotor gating ability. Dopamine D2receptor may be related to the tolerance effect of repeated CLZ treatment in adult rats but not the serotonin 5-HT2A/2C receptor. Dopamine D2/3 receptor may not be responsible for the effect of RIS sensitization response to adolescent treatment in the PCP model. 4. BDNF may not be the molecular basis underlying adolescent repeated antipsychotic treatment. In adolescent rats, OLZ sensitization may be mediated by BDNF expressions in the PFC and possibly in ST but not HC. BDNF is less likely involved in the mechanism of CLZ tolerance. Although repeated adolescent RIS administration induced a sensitization effect in the PCP-induced hyperlocomotion model in a dose-dependent fashion, whether BDNF is critically involved in this effect is still unsettled. In adult rats, mature or precursor BDNF may not be the molecular basis underlying either OLZ sensitization or CLZ tolerance.