Effect of 5-HT2A receptor antagonism on levels of D2/3 receptor occupancy and adverse behavioral side-effects induced by haloperidol: a SPECT imaging study in the rat

Several studies suggested that 5-HT2A receptor (5-HT2AR) blockade may provide a more favorable efficacy and side-effect profile to antipsychotic treatment. We hypothesized that a combined haloperidol (a D2/3 receptor (D2/3R) antagonist) and MDL-100,907 (a 5-HT2AR antagonist) treatment would reverse the side effects and the neurochemical alterations induced by haloperidol alone and would potentialize its efficacy. We thus chronically treated male Mdr1a knock-out rats with several doses of haloperidol alone or in combination with a saturating dose of a MDL-100,907. Receptor occupancy at clinically relevant levels was validated with a dual-radiotracer in-vivo SPECT imaging of D2/3R and 5-HT2AR occupancy. Experimental tests of efficacy (dizocilpine-disrupted prepulse inhibition (PPI) of the startle reflex) and side effects (catalepsy, vacuous chewing movements) were performed. Finally, a second dual-radiotracer in-vivo SPECT scan assessed the neurochemical changes induced by the chronic treatments. Chronic haloperidol failed to reverse PPI disruption induced by dizocilpine, whilst administration of MDL-100,907 along with haloperidol was associated with a reversal of the effect of dizocilpine. Haloperidol at 0.5 mg/kg/day and at 1 mg/kg/day induced catalepsy that was significantly alleviated (by ~50%) by co-treatment with MDL-100,907 but only at 0.5 mg/kg/day dose of haloperidol. Chronic haloperidol treatment, event at doses as low as 0.1 mg/kg/day induced a significant upregulation of the D2/3R in the striatum (by over 40% in the nucleus accumbens and over 20% in the caudate-putamen nuclei), that was not reversed by MDL-100,907. Finally, an upregulation of 5-HT2AR after chronic haloperidol treatment at a moderate dose only (0.25 mg/kg/day) was demonstrated in frontal cortical regions and the ventral tegmental area. Overall, a partial contribution of a 5-HT2AR antagonism to the efficacy and side-effect profile of antipsychotic agents is suggested.

T209. EFFECTS OF CHRONIC HALOPERIDOL EXPOSURE ON [3H]RO15-4513 AND [3H]FLUMAZENIL GABA-A RECEPTOR BINDING SITES

Background Post-mortem studies in schizophrenia (SZ) have reported abnormalities in the GABAergic system, more specifically, on αGABA-A receptors (αGABA-A R) (Benes, Kwok, Vincent, & Todtenkopf, 1998; Beneyto, Abbott, Hashimoto, & Lewis, 2011). Consistently, animal models of SZ have also reported changes in these receptor subtypes (Lodge, Behrens, & Grace, 2009; Richetto, Calabrese, Riva, & Meyer, 2014). Preclinical work in the methylazoxymethanol acetate (MAM) animal model of SZ has shown that administration of an α5GABA-A modulating drug rescues the psychosis-like phenotype (hyperdopaminergia) (Gill, Lodge, Cook, Aras, & Grace, 2011). However, this rescue does not occur if the animals are previously administered the first-generation antipsychotic haloperidol (Gill, Cook, Poe, & Grace, 2014). A recent study in patients with first-episode psychosis found increased baseline GABA levels as measured by magnetic resonance spectroscopy (de la Fuente-Sandoval et al., 2017). After 4 weeks of treatment with the antipsychotic risperidone, these GABA levels normalized in the medial prefrontal cortex and decreased at a trend level in the dorsal caudate (de la Fuente-Sandoval et al., 2017). All in all these studies suggest that antipsychotic medication besides directly regulating the dopaminergic system may also be acting on the GABAergic system, but the underlying mechanisms remain unknown. The aim of our study was thus to investigate, for the first time, the effects of clinically-relevant dosages of haloperidol on α5GABA-A R and benzodiazepine-sensitive α1–3;5GABA-A R in healthy rats. Methods A total of 36 male Sprague-Dawley rats were treated with vehicle, haloperidol 0.5 mg/kg/day or 2 mg/kg/day with an osmotic minipump for 28 days, representing clinically-relevant dosages of antipsychotic administration (Kapur, Vanderspek, Brownlee, & Nobrega, 2003). Quantitative autoradiography was conducted with [3H]Ro15-4513 and [3H]flumazenil, to assess the effects of haloperidol on α5GABA-A R and overall α1–3;5GABA-A R. Optical density measurements from cortical and subcortical regions were obtained from the autoradiographs using the MCID Software and converted into radioactivity values using a robust linear regression on GraphPad Prism. All data were analyzed using a mixed-effects model ANOVA on the same software and considered significant at p<0.05 with FDR correction for multiple comparisons. Results Administration of chronic haloperidol showed a dose-specific increase in [3H]Ro15-4513 in the dorsal CA1 area of the hippocampus, showing an increase in binding in the 0.5 mg/kg/day vs. vehicle (p<0.01; q<0.01) and a decrease in the 2 mg/kg/day vs. the 0.5 group (p<0.05; q<0.01). In the nucleus accumbens, there was a significant reduction in [3H]Ro15-4513 binding in the higher-dose group compared to the 0.5 mg/kg/day group (p<0.001; q<0.001). In contrast, haloperidol exposure relayed a general increase in [3H]flumazenil binding across all the regions of interest explored (p<0.05). Discussion This is the first study to investigate, in healthy rats, the effects of clinically-relevant doses of haloperidol on GABA-A R binding in a receptor-subtype specific manner. Our results show that exposure to haloperidol causes changes in α5GABA-A R predominantly in the dorsal CA1 and the nucleus accumbens. An overall change in benzodiazepine-sensitive α1–3;5GABA-A R is also observed, irrespective of region. These results suggest that exposure to antipsychotic medication impacts the GABAergic system and should be considered when testing new pharmacological strategies in humans that have been exposed to antipsychotic medication.

S77. ROLE OF DOPAMINE AND GLUTAMATE TRANSPORTERS IN GENERATION OF ANTIPSYCHOTIC EFFICACY

Background Antipsychotic drugs are the first line intervention to treat psychosis in schizophrenia and D2 receptor blockade is thought to be their primary mechanism of action. However, multiple lines of evidence from human and animal studies show that D2 receptor blockade is not always correlated with markers of antipsychotic efficacy. We previously demonstrated that reduced antipsychotic efficacy occurs after chronic antipsychotic administration in rodents despite stable D2 blockade, examined using PET imaging. Instead, we found that changes in expression of the dopamine transporter (DAT) were associated with decreases in endogenous dopamine and dopamine-mediated autoinhibition. These studies have led us to examine the DAT as a critical player in generation of an antipsychotic response. Methods Using antisense morpholino oligonucleotides, administered for 3 consecutive days using Long Evans rats, we selectively blocked translation of DAT or GLT-1 mRNA in the core of the nucleus accumbens, a brain region critical for motor outputs in response to salient stimuli. Baseline locomotion was monitored prior to and after an acute i.p. injection of haloperidol. Next, locomotion was monitored in response to a tail pinch or acute i.p. administration of cocaine. Transporter expression was quantified during acute or chronic haloperidol treatment using confocal microscopy. Results We found that DAT knockdown enhanced tail pinch-induced locomotion after acute haloperidol administration. Additionally, knockdown of the glutamate transporter GLT-1 strongly enhanced locomotion induced by tail pinch or cocaine injection after antipsychotic treatment. Confocal analysis of GLT-1 expression after acute or chronic haloperidol revealed significant GLT-1 up-regulation during a time period associated with antipsychotic efficacy. Discussion Our findings demonstrate a cause/effect relationship between reduced DAT and the behavioral response to an acute injection of antipsychotics in rodents. In all, our data point to the importance of both dopamine and glutamate uptake in the efficacy of antipsychotic drugs and argue against a D2-centric hypothesis of antipsychotic action.

Region-specific and dose-specific effects of chronic haloperidol exposure on [3H]-Flumazenil and [3H]-Ro15-4513 GABAA receptor binding sites in the rat brain

Post-mortem studies suggest that schizophrenia is associated with abnormal expression of specific GABAA receptor (GABAAR) α subunits, including α5GABAAR. Positron emission tomography (PET) measures of GABAAR availability in schizophrenia, however, have not revealed consistent alterations in vivo. Animal studies using the GABAAR agonist [3H]-muscimol provide evidence that antipsychotic drugs influence GABAAR availability, in a region-specific manner, suggesting a potential confounding effect of these drugs. No such data, however, are available for more recently developed subunit-selective GABAAR radioligands. To address this, we therefore combined a rat model of clinically relevant antipsychotic drug exposure with quantitative receptor autoradiography. Haloperidol (0.5 and 2 mg/kg/day) or drug vehicle were administered continuously to adult male Sprague-Dawley rats via osmotic mini-pumps for 28 days. Quantitative receptor autoradiography was then performed post-mortem using the GABAAR subunit-selective radioligand [3H]-Ro15-4513 and the non-subunit selective radioligand [3H]-flumazenil. Chronic haloperidol exposure increased [3H]-Ro15-4513 binding in the CA1 sub-field of the rat dorsal hippocampus (p<0.01; q<0.01; d = +1.3), which was not dose-dependent. [3H]-flumazenil binding also increased in most rat brain regions (p<0.05; main effect of treatment), irrespective of the haloperidol dose. These data confirm previous findings that chronic haloperidol exposure influences the specific binding of non-subtype selective GABAAR radioligands and is the first to demonstrate a potential effect of haloperidol on the binding of a α1/5GABAAR-selective radioligand. Although caution should be exerted when extrapolating results from animals to patients, our data support a view that exposure to antipsychotics may be a confounding factor in PET studies of GABAAR in the context of schizophrenia.