Chronic haloperidol administration downregulates select BDNF transcript and protein levels in the dorsolateral prefrontal cortex of rhesus monkeys

Abstract

Post-mortem studies in the prefrontal cortex and hippocampal formation from schizophrenia patients have revealed significant disruptions in the expression molecules associated with cytoarchitecture, synaptic structure, function, and plasticity, known to be regulated in part by brain derived neurotrophic factor (BDNF). Interestingly, several studies using postmortem brain tissue from individuals diagnosed with schizophrenia have revealed a significant reduction in BDNF mRNA and protein levels in the dorsolateral prefrontal cortex (DLPFC), hippocampus and related areas; however, differentiating the effects of illness from antipsychotic history has remained difficult. We hypothesized that chronic antipsychotic treatment may contribute to the altered BDNF mRNA and protein expression observed in post-mortem brains of individuals diagnosed with schizophrenia. To address the influence of antipsychotic administration on BDNF expression in the primate brain, rhesus monkeys orally administered haloperidol, clozapine, or vehicle twice daily for 180 days. We found BDNF splice variants 4 and 5 in the DLPFC and variant 2 in the EC were significantly down-regulated following chronic administration of haloperidol. In addition, proBDNF and mature BDNF expression in the DLPFC, but not the EC, were significantly reduced. Based on the known regulation of BDNF expression by BDNF-AS, we assessed the expression of this lncRNA and found expression was significantly upregulated in the DLPFC, but not EC. The results of the present study provide evidence of haloperidol-induced regulation of BDNF mRNA and protein expression in the DLFPC and suggest an important role for BDNF-AS in this regulation. Given the role of BDNF in synaptic plasticity, neuronal survival and maintenance, aberrant expression induced by haloperidol likely has significant ramifications for neuronal populations and circuits in primate cortex.

Keywords: antipsychotic action; long non-coding RNA; messenger RNA (mRNA); neurotrophin (NT); non-human primate (macaque); protein; schizophrenia.

FIGURE 1

panBDNF mRNA levels in the DLPFC and EC of rhesus monkeys following 6-month antipsychotic administration regimen. Chronic antipsychotic administration resulted in a significant change in pan BDNF mRNA expression in the DLPFC [F_(3,32) = 3.321, P = 0.0320] (A). Post hoc analysis revealed a significant difference between the groups was LHAL > HHAL (P = 0.0197); however, none of the groups were significantly different from CTRL. In the EC, no significant differences in panBDNF expression were detected following chronic antipsychotic administration [F_(3,33) = 1.163, P = 0.3385] in the EC (B). pan BDNF expression values are presented as bars which represent the mean (±S.E.M.) values following determination from relative standard curves. For each subject, expression values were calculated as quantitative mean value of panBDNF divided by the average of the quantitative mean values for the two reference genes for the respective regions. Bars represent the mean ± S.E.M. for the respective groups. Circles denote expression levels for individual subjects per group. *p < 0.05.

FIGURE 2

BDNF variant mRNA levels in the DLPFC and EC of rhesus monkeys following 6-month antipsychotic administration regimen. BDNF variants 1–5 were assessed in the DLPFC and EC using qPCR. In the DLPFC (A–E), significant differences between the groups were identified for BDNF variant 1 [F_(3,34) = 6.88, P = 0.0011], variant 4 [F_(3,37) = 6.635, P = 0.0012], and variant 5 [F_(3,35) = 5.5, P = 0.0037]. Tukey’s multiple comparison test revealed the followings differences: Variant 1: LHAL > CTRL (P = 0.0163), (P = 0.0094); Variant 4: CTRL > HHAL (P = 0.0011), CLZ > HHAL (P = 0.0052); Variant 5: CTRL > HHAL (P = 0.0355). No significant difference among groups were detected for BDNF variants 2 and 3 in the DLPFC. For the EC (F–J), a significant difference between the groups was identified for BDNF variant 2 [F_(3,34) = 5.805, P = 0.0026]. Tukey’s multiple comparison test revealed CTRL > HHAL (P = 0.0014). No significant difference among groups was observed for the other BDNF variants in the EC. BDNF variant values are presented as bars which represent the mean (±S.E.M.) values following determination from relative standard curves. For each subject, BDNF variant expression values were calculated as quantitative mean value of the variant divided by the average of the quantitative mean values for the two reference genes for the respective regions. Bars represent the mean ± S.E.M. for the respective groups. Circles denote expression levels for individual subjects per group. *p < 0.05, ^**p < 0.01.

FIGURE 3

proBDNF and mature BDNF levels are decreased following chronic administration of 4.0 mg/kg/day haloperidol. In the DLPFC, proBDNF (A) was significantly different among the groups [F_(3,34) = 3.631, P = 0.0235] and Tukey’s multiple comparison test revealed CTRL > HHAL (P = 0.0161). Mature BDNF (B) was also significantly different among the groups in this region [F_(3,33) = 6.889, P = 0.0012] where Tukey’s multiple comparison test revealed CTRL > HHAL (P = 0.0011) and LHAL > HHAL (P = 0.0271). In the EC, proBDNF (C) was significantly different among the groups [F_(3,29) = 3.684, P = 0.0231] and Tukey’s multiple comparison test revealed CLZ > HHAL (P = 0.0271). No significant difference in mature BDNF (D) between the groups was observed following chronic antipsychotic administration [F_(3,33) = 1.697, P = 0.1867]. For each subject, proBDNF and mature BDNF expression levels were calculated as pg/μg tissue protein. Bars represent the mean ± S.E.M. for the respective groups. Circles denote expression levels for individual subjects per group. *p < 0.05, ^**p < 0.01.

FIGURE 4

BDNF-AS levels are increased in the DLPFC but not the EC following chronic administration in the HHAL group. In the DLPFC (A), BDNF-AS lncRNA levels were significantly greater in the HHAL group compared to the CTRL group (t = 2.414, df = 15, P = 0.029); however, in the EC (B) no significant differences were observed between the HHAL and CTRL groups (t = 0.5113, df = 15, P = 0.6166). For each subject, BDNF-AS levels were calculated as quantitative mean value divided by the average of the quantitative mean values for the two reference genes for the respective regions. Bars represent the mean ± S.E.M. for the respective groups. Circles denote expression levels for individual subjects per group. *p < 0.05.