NIH Public Access Author Manuscript Eur J Pharmacol. Author manuscript; available in PMC 2008 September 24.

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1 NIH Public Access Author Manuscript Published in final edited form as: Eur J Pharmacol September 24; 571(1): Time dependent decreases in central α 7 nicotinic acetylcholine receptors associated with haloperidol and risperidone treatment in rats Alvin V. Terry Jr. and Debra A. Gearhart Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia, 30912, United States Abstract α 7 nicotinic acetylcholine receptor deficits may contribute to cognitive dysfunction in schizophrenia; however, the contribution of antipsychotic drug exposure to these deficits is unknown. In this study, rats were treated orally with haloperidol (2.0 mg/kg/day) or risperidone (2.5 mg/kg/day) for 15 or 90 days. Subsequent immunoassays indicated that both antipsychotics were associated with α 7 nicotinic receptor decreases in the basal forebrain and prefrontal cortex when administered for 90 (but not 15) days, a result that was confirmed in autoradiographic experiments. These data suggest that haloperidol and risperidone may be associated with time dependent decreases in an important neurobiological substrate of memory. Keywords schizophrenia; memory; cognition; antipsychotic; acetylcholine; cholinergic 1. Introduction Low affinity α 7 nicotinic acetylcholine receptors comprise approximately 10% of the total number of nicotinic receptors in mammalian brain, yet they are believed to modulate a variety of calcium-dependent events in neurons including neurotransmitter release (McGehee et al., 1995; Gray et al., 1996), postsynaptic signaling (Chang & Berg, 1999; Hefft et al., 1999) and neuronal survival (Messi et al., 1997; Berger et al., 1998). There is also a significant body of evidence to support an important role of α 7 nicotinic receptors in the cognitive dysfunction associated with certain chronic psychiatric illnesses such as schizophrenia. This premise is supported by postmortem evidence of α 7 nicotinic receptor deficits in the hippocampus and frontal cortex of schizophrenic patients (Guan et al., 1999) and by linkage analysis implicating chromosome 15q14 (the region that includes the α 7 neuronal nicotinic acetylcholine receptor gene). Polymorphisms in the core promoter of the α 7 neuronal nicotinic acetylcholine receptor gene (CHRNA7; GeneBank accession no. Z23141) are associated with reduced inhibition of the P50 evoked response to repeated auditory stimuli (i.e., indicative of sensory gating abnormalities) in schizophrenia (reviewed, Freedman et al., 2003). α 7 nicotinic receptor deficits may also contribute to abnormalities of smooth pursuit eye movements, sustained attention, Corresponding Author: Alvin V. Terry Jr., Ph.D., Professor of Pharmacology and Toxicology, CJ-1020, Medical College of Georgia, 1120 Fifteenth Street, Augusta, Georgia , United States, Phone , Fax , Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimersthat apply to the journal pertain.

2 Terry and Gearhart Page 2 and cognitive performance often observed in schizophrenia (reviewed Martin et al., 2004). Collectively, these findings have lead to the suggestion that a reduction in α 7 nicotinic receptor expression could serve as a biomarker for schizophrenia (Perl et al., 2003; Freedman et al., 2005). It is important to note, however, that the extent to which antipsychotic drugs contribute to α 7 nicotinic receptor alterations in schizophrenia is unknown given that the brains of antipsychotic-naïve patients have rarely been analyzed. This is an important consideration since many schizophrenia patients are treated with antipsychotic drugs for decades. In previous work in our laboratory, we have detected time dependent alterations in several cholinergic marker proteins including choline acetyltransferase, the vesicular acetylcholine transporter, and the high affinity choline transporter in rats treated with either representative first or second generation antipsychotics (see Terry et al., 2007 and review, Terry and Mahadik, 2007). Typically, we have observed elevations in cholinergic markers early in treatment (e.g., 15 days) followed by decreases below control levels at longer time periods (e.g., 90 days and beyond). We have also observed persistent decreases in α 7 nicotinic receptors in association with risperidone administered for 180 days (Terry et al., 2005), but have not determined if such a biphasic effect (as observed with other cholinergic proteins) occurs with α 7 nicotinic receptors. Therefore, in the present study in rats, the effects chronic oral treatment with a representative first generation antipsychotic, haloperidol, and a representative second generation antipsychotic, risperidone, on the levels of α 7 nicotinic receptors in several (memory-related) brain regions were investigated. The antipsychotics were delivered in drinking water and dosed on a mg/kg/24 hour basis for 15 or 90 days. This dosing approach was shown previously to result in plasma levels in rats that are considered within the therapeutic range for humans (Terry et al., 2005; Terry et al., 2007). α 7 nicotinic receptors were subsequently measured by ELISA and autoradiographic methods. 2. Materials and Methods 2.1 Subjects and Treatment Three month old Wistar rats were treated with haloperidol (2.0 mg/kg/day) or risperidone (2.5 mg/kg/day) orally in drinking water as described previously (see Terry et al., 2007) for periods of 15 or 90 days. All procedures employed during this study were reviewed and approved by the Medical College of Georgia Institutional Animal Care and Use Committee and are consistent with AAALAC guidelines. Measures were taken to minimize pain or discomfort in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No ) revised Significant efforts were also made to minimize the total number of animals used while maintaining statistically valid group numbers. 2.2 ELISA for α 7 nicotinic acetylcholine receptors Euthanasia of rats, brain dissections, and preparation of brain lysates were done according to Gearhart et al. (2006). Briefly, basal forebrain, hippocampal formation, cortex, and prefrontal cortex were dissected and then homogenized in RIPA buffer containing protease inhibitors and glycerol. The Micro BCA Protein Assay Kit (Pierce Biotechnology; Rockford, IL) was used to determine the total protein concentration in each brain lysate. An indirect ELISA was devised to measure the relative levels α 7 nicotinic receptor protein in brain lysates. As an internal control for day-to-day variation in the ELISA methods, brain lysates from vehicle, haloperidol, and risperidone-treated rats were assayed on the same ELISA plate. Brain lysates (15-25 μg total protein/μl) were diluted in sodium carbonate buffer (ph 9.6), and then the diluted lysates (0.5 ug protein/50 μl/well) were adsorbed to Nunc Maxisorp TM ELISA plates (overnight at 5 C). Microwells were rinsed once with 300 μl of wash buffer (ph 7.4 phosphate-buffered saline containing 0.05% (v/v) Triton X-100), before blocking the wells for one hour with 300 μl/well

3 Terry and Gearhart Page 3 1% (w/v) nonfat dry milk in PBST. Wells were rinsed with wash buffer (300 μl/well), before adding 50 μl/well of α 7 nicotinic receptor antibody (Chemicon AB5637; rabbit polyclonal; diluted 1:100 in blocking buffer). Incubation with primary antibody was carried out at room temperature for two hours, and then the microwells were rinsed with wash buffer (five times, 300 μl/well). The primary antibody was detected using peroxidase (HRP)-conjugated goat antirabbit IgG (Jackson Immunoresearch # , diluted 1:10,000 in blocking buffer, 50 μl/well). After one hour at room temperature, the microplate was rinsed five times with wash buffer (300 μl/well). Tetramethylbenzidine (100 μl/well, KPL ) was the peroxidase substrate; after minutes at room temperature, 1M HCl (100 μl/well) was added to stop the peroxidase reaction (color changed from blue to yellow). The absorbance of the yellow reaction product was measured at 450 nm using a microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT). 2.3 Quantitative Receptor Autoradiography 2.4 Statistical Analyses 3. Results 3.1 ELISA Data In order to further assess the effects of chronic antipsychotic administration on α 7 nicotinic receptor densities at the 90 day treatment time point, autoradiographic analyses of brain tissues harvested from rats treated with haloperidol, risperidone, or vehicle were conducted with the subtype specific cholinergic radioligand [ 125 I]-α-bungarotoxin. Test subjects (N=7-8) were sacrificed by decapitation and the brains flash frozen. Using a Microm HM cryostat (-18 C), the left hemisphere of each brain was serially sectioned (16 μm) up to the midline, and sections were adhered onto chrome alum/gelatin-coated slides. The concentration of [ 125 I]-αbungarotoxin used was 1.0 nm, the incubation time was 120 minutes, and the duration of film exposure was 160 hours. Please refer to previous publications for more detailed descriptions of the autoradiographic procedures (Hernandez and Terry, 2005; Terry et al., 2005). Statistical analyses were made using a one or two-way analysis of variance (ANOVA) with repeated measures when indicated. Dunnett s procedure was used to examine post hoc differences (P value <0.05 considered significant). Figure 1 depicts the ELISA results from the four selected brain regions in rats treated with vehicle, haloperidol, or risperidone for 15 or 90 days. Results from drug-treated rats are presented as a percent of control (vehicle-treated rats). Statistically significant time x brain region interactions were observed for both haloperidol and risperidone, which had a P-value for the interaction of <0.05. The statistically significant interaction indicates that the effect of time on the normalized OD values was different in the different brain regions. As indicated in Figure 1, post hoc comparisons revealed that both haloperidol and risperidone were associated with significant decreases (P<0.05) in α 7 nicotinic receptor protein in the basal forebrain as well as prefrontal cortex. Significant decreases were also observed in the cortex at 90 days in animals administered risperidone. 3.2 Autoradiographic Data The results of autoradiographic experiments are depicted in Figure 2. Representative autoradiograms are illustrated in the top left of the figure. The pattern of [ 125 I]-α-bungarotoxin binding was similar to that observed in previous studies (Hernandez and Terry, 2005;Terry et al., 2005). Statistical analyses revealed that there were significant treatment-related differences in binding densities F 2,18 =4.2, P=0.03, a highly significant difference in binding in the different brain regions F 6,12 =253.7, P<0.001, without a significant treatment x brain region

4 Terry and Gearhart Page 4 4. Discussion Acknowledgements References interaction F 105,143 =1.3, P=0.31. Post hoc statistical comparisons revealed the following: 1) risperidone was associated with statistically significant decreases (P<0.05) in [ 125 I]-αbungarotoxin binding in the prefrontal cortex, and the CA1, CA2, and CA3 regions of the hippocampus, while haloperidol was associated with significant decreases (P<0.05) in binding in the CA1 and CA2 regions of the hippocampus. A perusal of the histograms in Figure 2 reveals however, that [ 125 I]-α-bungarotoxin binding was at least slightly decreased by both haloperidol and risperidone treatment in all of the brain regions that were analyzed (an effect that replicates an earlier study at 90 days, Terry et al., 2005). The results of this study can be summarized as follows: 1) neither haloperidol nor risperidone significantly affected the density of α 7 nicotinic receptors in memory-related brain regions in the rat when they were administered for 15 days; 2) both antipsychotics were, however, associated with significant α 7 nicotinic receptor decreases in the basal forebrain and prefrontal cortex when administered for 90 days (i.e., by as much as 22% in the case of haloperidol and 15% in the case of risperidone); 3) quantitative receptor autoradiography experiments with the radioligand [ 125 I]-α-bungarotoxin on tissues from animals that were treated with antipsychoitcs for 90 days revealed layer specific decreases in α 7 nicotinic receptors in the hippocampus and confirmed ELISA results in the prefrontal cortex. These data support an accruing body of evidence (see Terry and Mahadik, 2007) that representative first and second generation antipsychotics exert a time-dependent effect on cholinergic marker proteins in memory-related regions of mammalian brain. While we did detect a time dependent decrease in α 7 nicotinic receptors in the present study, we did not observe an increase above baseline at the early time point followed by a decease below baseline with longer treatment as we have found with other cholinergic proteins such as the vesicular acetylcholine transporter (Terry et al., 2007) in animals treated with haloperidol or risperidone. The mechanism for the time-dependent effect of these antipsychotics on cholinergic proteins is currently unknown, but it has also been observed in association with first generation antipsychotics in older studies which focused on elucidating the mechanism for their adverse motor effects (Miller and Chouinard, 2003). Given the important role of α 7 nicotinic receptors in neuronal function, cognition, and the pathophysiology of schizophrenia, it is important that the mechanism for this effect be elucidated. It is also important to determine if such receptor effects are associated with other antipsychotics given their widespread use in the schizophrenia population as well in a variety of other psychiatric and neurologic illnesses (e.g., autism, bipolar disease, Parkinson s disease, Alzheimer s disease). A better understanding of such effects might facilitate the development of new antipsychotics that do not affect α 7 nicotinic receptors (or other cholinergic proteins) and the development of adjunctive therapeutic agents designed to improve cognition. Specifically, a thorough knowledge of the chronic effects of different antipsychotic drugs on cholinergic marker proteins might facilitate more patient specific (i.e., tailored) therapeutic strategies that involve a cholinergic replacement strategy. The authors wish to acknowledge the technical support of Kristy Bouchard, Samantha Warner, Sameera Warsi, and Mary-Louise Middlemore. This work was supported by the National Institute of Mental Health (R01 MH to AVT). Berger F, Gage FH, Vijayaraghavan S. Nicotinic receptor-induced apoptotic cell death of hippocampal progenitor cells. J Neurosci 1998;18: [PubMed: ]

5 Terry and Gearhart Page 5 Chang KT, Berg DK. Nicotinic acetylcholine receptors containing alpha7 subunits are required for reliable synaptic transmission in situ. J Neurosci 1999;19: [PubMed: ] Freedman R, Olincy A, Ross RG, Waldo MC, Stevens KE, Adler LE, Leonard S. The genetics of sensory gating deficits in schizophrenia. Curr Psychiatry Rep 2003;5: [PubMed: ] Freedman R, Ross R, Leonard S, Myles-Worsley M, Adams CE, Waldo M, Tregellas J, Martin L, Olincy A, Tanabe J, Kisley MA, Hunter S, Stevens KE. Early biomarkers of psychosis. Dialogues Clin Neurosci 2005;7: [PubMed: ] Gearhart DA, Middlemore ML, Terry AV Jr. ELISA methods to measure cholinergic markers and nerve growth factor receptors in cortex, hippocampus, prefrontal cortex, and basal forebrain from rat brain. J Neurosci Meth 2006;150: Gray R, Rajan AS, Radcliffe KA, Yakehiro M, Dani JA. Hippocampal synaptic transmission enhanced by low concentrations of nicotine. Nature 1996;383: [PubMed: ] Guan ZZ, Zhang X, Blennow K, Nordberg A. Decreased protein level of nicotinic receptor alpha7 subunit in the frontal cortex from schizophrenic brain. Neuroreport 1999;10: [PubMed: ] Hefft S, Hulo S, Bertrand D, Muller D. Synaptic transmission at nicotinic acetylcholine receptors in rat hippocampal organotypic cultures and slices. J Physiol 1999;515: [PubMed: ] Hernandez CM, Terry AV Jr. Repeated nicotine exposure in rats: effects on memory function, cholinergic markers and nerve growth factor. Neuroscience 2005;130: [PubMed: ] Martin LF, Kem WR, Freedman R. Alpha-7 nicotinic receptor agonists: potential new candidates for the treatment of schizophrenia. Psychopharmacology (Berl) 2004;174: [PubMed: ] Messi ML, Renganathan M, Grigorenko E, Delbono O. Activation of alpha7 nicotinic acetylcholine receptor promotes survival of spinal cord motoneurons. FEBS Lett 1997;411: [PubMed: ] McGehee DS, Heath MJ, Gelber S, Devay P, Role LW. Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors. Science 1995;269: [PubMed: ] Miller R, Chouinard G. Loss of striatal cholinergic neurons as a basis for tardive and L-dopa-induced dyskinesias, neuroleptic-induced supersensitivity psychosis and refractory schizophrenia. Biol Psychiatry 1993;34: [PubMed: ] Perl O, Ilani T, Strous RD, Lapidus R, Fuchs S. The alpha7 nicotinic acetylcholine receptor in schizophrenia: decreased mrna levels in peripheral blood lymphocytes. FASEB J 2003;17: [PubMed: ] Terry AV Jr, Gearhart DA, Mahadik SP, Warsi S, Davis LW, Waller JL. Chronic exposure to typical or atypical antipsychotics in rodents: temporal effects on central alpha 7 nicotinic acetylcholine receptors. Neuroscience 2005;136: [PubMed: ] Terry AV Jr, Mahadik SP. Time Dependent Cognitive Deficits Associated with First and Second Generation Antipsychotics: Cholinergic Dysregulation as a Potential Mechanism. J Pharmacol Exper Ther 2007;320: [PubMed: ] Terry AV Jr, Gearhart DA, Warner SE, Zhang G, Bartlett MG, Middlemore ML, Beck WD, Mahadik SP, Waller JL. Oral haloperidol or risperidone treatment in rats: temporal effects on nerve growth factor receptors, cholinergic neurons, and memory performance. Neuroscience 2007;146: [PubMed: ]

6 Terry and Gearhart Page 6 Fig 1. ELISA results for α 7 nicotinic receptor protein expressed as percent of vehicle-treated group (mean ± S.E.M.) in the brain regions indicated from rats treated with vehicle, haloperidol (2.0 mg/kg/24 h), or risperidone (2.5 mg/kg/24 h) for 15 or 90 days. Rats were euthanized at each of the time points indicated and then the dissected brain regions were processed for ELISA. *P<0.05 = significantly different from vehicle control. N=5-6.

7 Terry and Gearhart Page 7 Fig 2. Top Left: Representative autoradiograms illustrating receptors labeled by [ 125 ]-α-bungarotoxin (ligand at the α 7 nicotinic receptor), in 16 μm sagittal sections of brains from Wistar rats treated with vehicle, haloperidol (2.0 mg/kg/24 h), or risperidone (2.5 mg/kg/24 h) for 90 days. PFC = prefrontal cortex; HIPP = hippocampus; CTX = cortex (layers I-VI); SI = substantia inominata. Histograms illustrate the effects of 90 days of antipsychotic treatment on the density of receptors labeled by [ 125 ]-α-bungarotoxin in different (memory-related) brain regions in the rat. *P<0.05 = significantly different from vehicle control. VEH = vehicle, HAL = haloperidol; RISP = risperidone. N=7-8 rats per group.