Pharmacology, Biochemistry and Behavior 172 (2018) 50–58 Contents lists available at ScienceDirect Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh Neurochemical changes underlying schizophrenia-related behavior in a modified forced swim test in mice T Monika Woźniaka, Paulina Cieślika, Marcin Marciniaka, Tomasz Lendaa, Andrzej Pilca,b, ⁎ Joanna M. Wieronskaa, a Institute of Pharmacology, Polish Academy of Sciences, 12 Smętna St, 31-343 Kraków, Poland b Health Sciences Faculty, Institute of Public Health, Jagiellonian University Medical College, Kraków, Poland ARTICLE INFO ABSTRACT Keywords: The modified forced swim test (MFST) has excellent predictive validity for investigating the antipsychotic ac- Schizophrenia tivity of drugs, with particular emphasis on their activity toward negative symptoms of schizophrenia. However, GABAB its face and construct validity are less understood. Therefore, in the present study, some biochemical changes Negative symptoms within GABAergic and serotonergic neurotransmission that could be related to observed MK-801-induced dis- Animal models turbances and the activity of compounds active at those neurotransmitters were investigated. Antipsychotic effect In biochemical experiments, mice were treated acutely or chronically with MK-801 (13 days, 0.4 mg/kg). Modified forced swim test Western blotting Their brains were dissected and frontal cortices and hippocampi were taken for further analysis. The levels of RT-PCR neurotransmitters were investigated with HPLC, and the expression of surrogate markers of schizophrenia (5- HT1A receptors, GAD65, and GAD67, at both protein and mRNA levels) was measured via western blotting and qRT-PCR. The modified forced swim test and locomotor activity were used to assess the activity of GABAB and 5- HT1A-related compounds. Repeated MK-801 treatment (13 days, 0.4 mg/kg dose) led to decreases in the DOPAC/DA, 3MT/DA and HVA/DA metabolic ratios. Increased 5-HT1A protein expression and decreased GAD65 and GAD67 protein ex- pression was observed in both the cortex and hippocampus. mRNA levels for all proteins were decreased. The increased immobility in the forced swim test was reversed both by a GABAB agonist (SKF97541, 0.025 or 0.05 mg/kg), a positive allosteric modulator of GABAB receptor (racBHFF, 5 or 10 mg/kg) and by a 5-HT1A agonist ((R)-(+)-8-OH-DPAT 0.01 or 0.025 mg/kg). Our research supports the hypothesis that changes in the levels of GABA and/or 5-HT1A receptors may contribute to the schizophrenia-like phenotype, and GABAergic and serotonergic agents may be good candidates for treating negative symptoms of schizophrenia. 1. Introduction schizophrenia development. Based on pharmacological observations, several theories have been proposed. One of the most common of these Schizophrenia is a mental disorder considered to be one of the most theories, which has dominated over the decades, is the dopaminergic severe disabilities that affects 1% of the human population. The clinical theory of schizophrenia (Haracz, 1982; Heinz and Schlagenhauf, 2010; picture of schizophrenia is complicated and involves many different Munch-Petersen, 1955). The theory is based on the observations that symptoms that are divided into three groups: positive, negative and dopaminomimetics, such as amphetamine, induce positive symptoms of cognitive. schizophrenia in humans and, to a certain extent, in animal studies The background of schizophrenia inducement is not fully under- (hyperactivity, apomorphine-induced climbing). Following these ob- stood. Different trials, both in animal studies and in humans, have been servations, it was hypothesized that the increased dopamine outflow is undertaken to establish the neurochemical changes that contribute to responsible for at least certain schizophrenia symptoms, and Abbreviations: HVA, homovanilic acid; DOPAC, 3,4-dihydroxyphenylacetic acid 3-MT-3-methoxytyramine; 5-HIAA, 5-hydroxyindoleacetic acid; PCP, phencycli- dine; NMDA, N-methyl-D-aspartate; GAD, Glutamate decarboxylase; qRT-PCR, real time polymerase chain reaction; HPLC, high performance liquid chromatography; SDS, sodium dodecyl sulphate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase ⁎ Corresponding author. E-mail address: [email protected] (J.M. Wieronska). https://doi.org/10.1016/j.pbb.2018.06.003 Received 3 March 2018; Received in revised form 12 June 2018; Accepted 14 June 2018 Available online 18 June 2018 0091-3057/ © 2018 Published by Elsevier Inc. ź M. Wo niak et al. Pharmacology, Biochemistry and Behavior 172 (2018) 50–58 − consequently, the blockade of dopaminergic receptors could exert an- testing. The compounds were given in a volume of 10 ml·kg 1. The tipsychotic action. behavioral measurements were made by an observer unaware of the The observations that NMDA receptor antagonists, such as PCP or treatment. All procedures have been approved by the II Local Ethics MK-801, induce the full spectrum of schizophrenia symptoms in hu- Committee by the Institute of Pharmacology, Polish Academy of mans, including positive, negative and cognitive, stayed at the grounds Sciences in Krakow. Both the animal facility conditions and the pro- of the glutamatergic theory of schizophrenia (Javitt, 1987). The theory cedures are in accordance with EU Directive 2010/63/EU and sub- is based on the assumption that the dysfunctional NMDA receptors are sequent ordinances of Polish Ministry of Agriculture and Rural preferentially expressed on GABAergic interneurons in subcortical re- Development. gions that innervate thalamocortical pyramidal neurons which, due to the loss of inhibitory control from interneurons, overactivate the other 2.2. Drugs pyramidal neurons in the cortex (Conn et al., 2009; Moghaddam and Jackson, 2003). GABAB agonist SKF97541 (3-Aminopropyl(methyl)phosphinic Postmortem studies of schizophrenic patients' brains revealed that acid), GABAB positive allosteric modulator rac-BHFF (5,7-Bis(1,1-di- the changes in selected genes or proteins are typical for schizophrenia methylethyl)-3-hydroxy-3(trifluoromethyl)-2(3H)-benzofuranone), and are observed in the majority of schizophrenia cases, which are GABAB antagonist CGP55845 ((2S)-3-[[(1S)-1-(3,4-Dichlorophenyl) called the surrogate biomarkers of schizophrenia. The largest percen- ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid hydro- tage of changes observed are for developmental/synaptic processes and chloride), 5-HT1A agonist (RS)-8-OH-DPAT (2R)-(+)-8-Hydroxy-2-(di- the gamma aminobutyric acid system. The decreased levels of reelin, n-propylamino)tetralin hydrobromide, 5-HT1A antagonist WAY100635 parvalbumin, GAD65 and GAD67 proteins and their mRNAs were con- and NMDA antagonist MK-801 were purchased from Tocris, Bioscience, sistently observed in the majority of postmortem schizophrenic brains United Kingdom. SKF97541 and rac-BHFF were dissolved in EtOH (in the cortices and hippocampi) (Dracheva et al., 2004; Torrey et al., (200 μl) and then adjusted with 1% Tween-80 to 10 ml. (RS)-8-OH- 2005; Thompson et al., 2011; Guidotti et al., 2000; Fatemi et al., 2005). DPAT, WAY100635 (N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]- On the basis of those observations, the theory was raised that the GA- N-2-pyridinylcyclohexanecarboxamide maleate), MK-801 ((5S,10R)- BAergic dysfunction in schizophrenia is supposed to be a root of sy- (+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine naptic plasticity deficits and alteration of pyramidal neuron functioning maleate) was dissolved in 0.9% saline. SKF97541 and racBHFF were (Conn et al., 2009; Moghaddam and Jackson, 2003). Concomitantly, administered intraperitoneally (i.p) 30 min before the test, and the dysfunction of GABA synthesizing enzymes, GAD65 and GAD67, may CGP55845 was administered 10 min before those drugs. WAY100635 be considered to be the liability factors underlying schizophrenia. In the (0.1 mg/kg, i.p) was given 45 min before the test and (RS)-8-OH-DPAT other case, in certain schizophrenia patients, increased levels of 5-HT1A was administered subcutaneously (s.c) 15 min before the test. The receptors were observed; however, the function of this receptor is control groups of animals were injected with appropriate vehicles. The linked predominantly with cognitive dysfunction. schedule of administration and doses of drugs were adapted from our The modified forced swim test is regarded as a test for the animal earlier studies. For WAY100635 and (RS)-8-OH-DPAT see the papers model of schizophrenia and is supposed to reflect depressive-like ne- Woźniak et al., 2017; Wierońska et al., 2013, 2015a, and for GABAB gative symptoms of the disease (Noda et al., 1995, 1997, 2000). The ligands see the papers Wierońska et al., 2011, 2015b. The doses of construct validity of the model is based on the chronic administration of racBHFF and SFF were partially taken from Gannon and Millan, 2011 or NMDA antagonist, MK-801 (0.4 mg/kg; 13 days). The prolonged NMDA Frankowska et al., 2009, and were established experimentally. receptor blockade induces an increased time of immobility in the forced swim test, which is reversed by the administration of neuroleptics 2.3. Determination of the neurotransmitter levels (HPLC) (Noda et al., 1995; Langen et al., 2012). The predictive validity of the model is well-established, as the administration of atypical anti- All measurements were performed with the use of the P680 HPLC psychotic drugs but no other drugs (antidepressants, anxiolytics) re- system (Dionex, Sunnyvale, CA, USA) equipped