Bio-behavioral characterisation of a selective α2C -receptor antagonist in animal models of schizophrenia and depression MM Uys 12989193 Thesis submitted for the degree Philosophiae Doctor in Pharmacology at the Potchefstroom Campus of the North-West University Promoter: Prof BH Harvey November 2016 I can do all things through Christ who strengthens me I dedicate this work to my husband and friend, Nelis Uys, and to my serendipitous neighbours and ever-supporting, ever-loving friends, Anke and Theunis Cloete. You have always been there to encourage and strengthen me. “Twee is beter as een. Saam bereik hulle meer in hulle werk. As een mens val, kan sy vriend hom ophelp. Maar as een val wat alleen is, is daar niemand om hom op te help nie. ’n Driedubbele tou breek nie maklik nie” – Pred 9:10, 12 – Abstract Purpose Schizophrenia and depression are neuropsychiatric disorders characterised by affective and cognitive dysfunction and are associated with altered monoaminergic and neurotrophic function. Social isolation rearing (SIR) is a neurodevelopmental rodent model of schizophrenia that reflects many of the behavioural and neurochemical features of schizophrenia, while the Flinders Sensitive Line (FSL) genetic rodent model of depression reflects various behavioural and neurochemical features of the human disorder. The α2-adrenoceptor (α2-AR) is a well-established neurobiological target for antipsychotic and antidepressant drug design, with a number of clinically used drugs presenting with α2-AR antagonism in their pharmacological profile. Selective α2C-adrenoceptor (α2C-AR) antagonism has been suggested to present with superior neuropsychiatric effects vs. non-selective α2-AR antagonism. The purpose of this study was to assess cognitive and antipsychotic-like effects of α2C-AR antagonism in the SIR model of schizophrenia, as well as cognition and antidepressant-like effects in the FSL model of depression. These pharmacological effects were compared to various reference agents, such as clozapine (CLOZ) and imipramine (IMI), as well as the non-selective α2-AR antagonist, idazoxan (IDAZ). Additionally, the behavioural and neurotrophic effects of augmenting D2-antagonist therapy with α2C-AR antagonism in the SIR model were investigated. Finally this study assessed the effects of α2C-AR antagonism on striatal (SIR) and hippocampal (FSL) monoamine levels and brain-derived neurotrophic factor (BDNF) in the respective animal models. Methods Three separate studies were conducted employing chronic treatment with the novel, highly selective α2C-AR antagonist ORM-10921. In the first study, male Sprague Dawley rats were either reared socially (SOC) or reared in social isolation (SIR) for 8 weeks following weaning. SIR rats received either vehicle (1 ml/kg), CLOZ 5 mg/kg , IDAZ 3 mg/kg or one of various doses of ORM-10921 (0.3 – 1mg/kg) subcutaneously (SC) once daily for 14 days, where after behaviour in the prepulse inhibition (PPI) test and novel object recognition test (NORT) were assessed. Post-mortem striatal monoamine levels were determined by high performance liquid chromatography (HPLC), while total striatal BDNF levels were determined by enzyme-linked immunosorbent assay (ELISA). In study 2, SIR animals received either vehicle, CLOZ, haloperidol (HAL) 2 mg/kg, ORM-10921 (0.01 or 0.03 mg/kg) or HAL + ORM-10921 (0.01 or 0.03 mg/kg) for 14 days SC. Again behaviour in the PPI test and NORT were assessed, where after post-mortem striatal BDNF levels were determined as above. The third study employed 11-week old i Abstract male FSL rats that were treated with either vehicle, ORM-10921 (0.01 – 1mg/kg), IMI 15 mg/kg or IDAZ 3mg/kg administered SC or intraperitoneally for 14 days. Behaviour in the forced swim test (FST) and the NORT were subsequently assessed. In a separate group of drug treated animals, post-mortem hippocampal monoamine and BDNF levels were determined as described above. Results α2C-AR-antagonism with ORM-10921 reversed SIR-induced deficits in PPI and object recognition memory comparable to CLOZ treatment but superior to the non-selective α2-antagonist, IDAZ. ORM- 10921 increased striatal noradrenaline (NA) and decreased striatal dopamine (DA) in SIR rats, while CLOZ, but not ORM-10921 or IDAZ increased striatal BDNF. Augmentation of HAL with ORM-10921 bolstered the effects of HAL on PPI and object recognition memory, while also elevating striatal BDNF, an effect not obtained with monotherapy of either drug. ORM-10921 improved object recognition memory and decreased immobility in the FST in FSL rats. These behaviours were comparable to IMI, but superior to that of IDAZ. ORM-10921 increased serotonergic-driven swimming more than noradrenergic-driven climbing behaviour in the FST. ORM-10921 did not alter hippocampal BDNF levels after 14 days of treatment, but did increase hippocampal levels of DA, NA and serotonin. Conclusions α2C-AR-antagonism with ORM-10921 presents with pro-cognitive, antipsychotic-like and antidepressant-like effects in the SIR and FSL translational models of schizophrenia and depression. These behavioural effects were associated with beneficial effects on dysfunctional monoamine levels in the striatum and hippocampus of SIR and FSL rats, respectively, albeit not with immediate effects on striatal and hippocampal BDNF levels. Furthermore, beneficial effects of α2C-AR-antagonism on the outcomes of D2-antagonist therapy on sensorimotor gating and cognition was evident, while these effects were correlated to increased striatal BDNF levels. The results thus suggest that α2C-AR- antagonism is a potentially valuable therapeutic strategy in the treatment of neuropsychiatric disorders characterised by cognitive and affective dysfunction associated with monoaminergic alterations, as evidenced in two translational models of neuropsychiatric illnesses. α2C-AR-antagonism also shows potential as augmentation strategy to typical antipsychotic treatment. Keywords: α2C-adrenoceptor, α2C-antagonism, schizophrenia, depression, social isolation rearing, Flinders Sensitive Line rat, prepulse inhibition, object recognition memory, forced swim test, hippocampus, striatum, BDNF, monoamines ii Opsomming Doelstelling Skisofrenie en depressie is neuropsigiatriese steurnis wat geassosieer word met afwykings in monoaminergiese en neurotrofiese funksies en gekenmerk word deur affektiewe en kognitiewe versteurings. Sosiale isolasie-geïnduseerde stres, oftewel, sosiale isolasie-stres (SIS) is ‘n neuro- ontwikkelingsmodel van skisofrenie in rotte, en dit reflekteer baie van die neurochemiese en gedragversteurings wat waargeneem word in skisofrenie. Aan die ander kant is die Flinders se Sensitiewe Lyn- (FSL-) rot ‘n genetiese knaagdiermodel van depressie, wat verskeie neurochemiese en gedragseienskappe van menslike depressie weerspieël. Die adrenergiese α2-reseptor (α2-AR) is ‘n goedgevestigde neurobiologiese teiken vir die ontwerp van farmakologiese middels wat as antipsigotikums en antidepressante kan optree, en ‘n aantal middels wat tans klinies aangewend word beskik oor α2-AR-antagonisme as deel van hul farmakologiese profiel. Daar word aangevoer dat selektiewe antagonisme van die adrenergiese α2C-reseptor (α2C-AR) verbeterde neuropsigiatriese effekte kan toon teenoor nieselektiewe α2-reseptorantagonisme. Die doel van hierdie studie was om die kognitiewe en antipsigotiese effekte van α2C-AR-antagonisme in die SIS-model van skisofrenie en die kognitiewe en antidepressantagtige effekte van α2C-AR-antagonisme in die FSL-model van depressie te ondersoek. Hierdie farmakologiese effekte is vergelyk met die kliniese antipsigotikum, klosapien (KLOS), die kliniese antidepressant, imipramien (IMI) en die nieselektiewe α2-AR-antagonis, idasoksaan (IDAS). Daarbenewens is die effekte van kombinasieterapie met D2-antagonism en α2C-AR-antagonisme op gedrag en neurotrofiese effekte in die SIS-model ondersoek. Laastens het hierdie studie ook die effekte van α2C-AR-antagonisme op breinmonoamienvlakke en brainafkomstige neurotrofiese faktor (BDNF) in die striatum (SIS) en hippokampus (FSL) in die onderskeie dieremodelle ondersoek. Metodes Drie afsonderlike studies het chroniese behandeling met die nuwe, hoogs selektiewe α2C-AR antagonist, ORM-10921, geïmplementeer. In die eerste studie is Sprague Dawley-mannetjiesrotte toegelaat om óf sosiaal (SOS), óf sosiaal geïsoleerd (SI) te ontwikkel na spening. SI-rotte is eenmaaldaaglikse onderhuidse soutoplossing (1ml/kg), KLOS 5 mg/kg, IDAS 3 mg/kg of een van ‘n aantal dosisse van ORM- 10921 (0.3-1mg/kg) toegedien vir 14 dae, waarna gedrag in die prepulsinhibisie-toets (PPI) en voorwerpherkenningstoets (VHT) geëvalueer is. Nadoodse striatale monoamienvlakke is bepaal d.m.v. die hoogsdoeltreffende vloeistofchromatografie (HDVC) metode en totale striatale BDNF-vlakke is iii Opsomming m.b.v. die ensiemgekoppelde immuunsorberende essai (ELISA)-metode bepaal. In die tweede studie het SI-diere vir 14 dae onderhuidse soutoplossing, KLOS, haloperidol (HAL) 2mg/kg, ORM-10921 (0.01 of 0.03 mg/kg) of HAL+ORM-10921 (0.01 of 0.03 mg/kg) ontvang. Gedrag is weereens in die PPI en VHT geëvalueer, waarna nadoodse striatale BDNF-vlakke bepaal is soos hierbo beskryf. Die derde studie het van 11-weekoue mannetjies FSL-rotte gebruik gemaak. FSL-rotte is vir 14 dae behandel met onderhuidse of intraperitoneale soutoplossing, ORM-10921 (0.01 – 1 mg/kg), IMI 15 mg/kg of IDAS 3mg/kg. Gedrag in die geforseerde swemtoets (FST) en die VHT is daarna waargeneem. In ‘n afsonderlike groep diere is nadoodse monoamien- en BDNF-vlakke in