Supplementary Information


Methods

C-Fos immunohistochemistry
C-Fos immunohistochemistry was used to examine the effect of repeated THC exposure on risperidone and clozapine-induced neuronal activity. Mice were anaesthetized with isoflourane and transcardially perfused with 4% paraformaldehyde 90 minutes after the antipsychotic challenge (1 mg/kg risperidone or 3 mg/kg clozapine). Brain tissue was extracted and 40 μm sections were stained as previously described (Boucher et al, 2007; Boucher et al, 2011a; Todd and Arnold, 2016) using a c-Fos primary antibody (Santa Cruz Biotechnology, USA) and an anti-rabbit IgG secondary antibody (Vector Laboratories, USA). The peroxidase reaction was visualised with nickel diaminobenzidine and glucose oxidase and sections were mounted slides and coverslipped. The mouse brain atlas by Paxinos and Franklin (2004) was used to identify regions of interest at Bregma levels +1.98 (prefrontal cortex); +0.98 (dorsomedial caudate putamen, cingulate cortex, ventrolateral septum, nucleus accumbens shell and core); -0.94 (paraventricular nucleus of the hypothalamus/thalamus) and -1.58 (CA3 hippocampus). A blind observer quantified the cells as previously described under 20X magnification (Boucher et al, 2007; Boucher et al, 2011a; Todd et al, 2016).  

Prepulse inhibition of startle (PPI) and open field (OF) test 
We also examined whether THC exposure reduces the effects of antipsychotic drugs on prepulse inhibition of startle (PPI) and locomotor activity. 30 min after challenge injection with risperidone (0.3 and 1 mg/kg) and clozapine (3 mg/kg), mice were placed in the PPI chamber and tested before being placed in an open field chamber for locomotor activity testing. 
PPI is a phenomenon whereby exposure to a weak auditory stimulus (prepulse) reduces the extent of the startle reaction to a subsequent startling auditory stimulus (Arnold et al, 2012; Boucher et al, 2011b; Garcia-Sanchez et al, 2011). Schizophrenia patients display PPI deficits which are normalised by antipsychotic drug treatments due to their ability to facilitate PPI (Garcia-Sanchez et al, 2011; Kumari and Sharma, 2002; Oranje et al, 2002; Quednow et al, 2006; Vollenweider et al, 2006). Mice were tested using a modified protocol of (Boucher et al, 2011b; Chohan et al, 2014a; Duncan et al, 2006a; Duncan et al, 2006b; Long et al, 2010). Briefly, mice were exposed to a test session of 42 trials where the trials presented included no stimulus, acoustic startle stimulus alone (120 dB for 40 ms) and PPI trials, where a prepulse stimulus was presented 100 ms before the 120 dB startle stimulus. Prepulse stimuli were 20 ms in duration at the intensities of 74, 78, 82 and 86 dB. The trials were presented in pseudorandomized order, with an average intertrial interval of 15 s (range 10 - 20 s).  Startle response was measured as the average mean amplitude across all trials. Locomotor activity was measured by placing mice into a plexiglass open field activity test chamber (43 cm x 43 cm x 25 cm; Motion Mensura, NSW, Australia) (Boucher et al, 2011a; Chohan et al, 2014a; Spencer et al, 2012). The animal’s horizontal movement was monitored using infrared motion-tracking software as outlined by Dielenberg et al (2006). The total distance travelled (m) in a period of 60 minutes was measured.

D2 and 5-HT2A receptor autoradiography
We wished to examine whether THC pretreatment might influence the efficacy of antipsychotic drugs via a pharmacodynamic mechanism. As both D2 and 5-HT2A receptors have been implicated in the antipsychotic mechanism of action of risperidone and clozapine, we examined their binding and expression with D2 and 5-HT2A receptor autoradiography using well-validated radiolabelled ligands, [3H] raclopride and [3H] ketanserin respectively (Dalton and Zavitsanou, 2010; du Bois et al, 2006). Mice were injected with 1 mg/kg THC or vehicle, daily for 14 days. Following a 14 day washout period, animals were sacrificed and the whole brain was snap frozen in liquid nitrogen. 20 μm sections of mouse brain mounted onto polysine slides as previously described by Chohan et al (2014b). Sections for 5-HT2A receptor analyses were incubated in Tris-HCl buffer containing 10 nM [3H]ketanserin (specific activity 67.0 Ci/mmol, Perkin Elmer, USA) for 1 h (du Bois et al, 2006). Non-specific binding was determined by incubating adjacent sections in the presence of 10 μM spiperone (Sigma-Aldrich, NSW, Australia). Sections for D2 receptor analysis were incubated in TrisHCl buffer containing 5 nM [3H]raclopride (specific activity 62.2 Ci/mmol, Perkin Elmer, USA) for 1 h (Dalton et al, 2010). Non-specific binding was determined by incubating adjacent sections with in the presence of 10 μM (+)-butaclamol (Sigma-Aldrich, NSW, Australia). Following the binding assays, all sections were placed on Kodak BioMax MR film along with a [3H] autoradiographic standard (Amersham, UK). Films were developed using Kodak GBX developer/fixer (Sigma-Aldrich, NSW, Australia) and scanned using a BioRad GS-800 calibrated densitometer. The average optical density of six brain sections for total binding and nonspecific binding was quantified using ImageJ (NIH, USA). Specific in vitro binding of [3H] ketanserin or [3H] raclopride was calculated by subtraction of non-specific from total binding values, and converted into fmol per mg tissue equivalent (fmol/mg). The mouse brain atlas by Paxinos et al (2004) was used to identify brain the same regions quantified for c-Fos immunohistochemistry.

Quantification of risperidone and 9-hydroxy risperidone in brain and blood samples 
To examine a pharmacokinetic mechanism of THC-induced modulation of risperidone efficacy we determined the concentration of risperidone and its metabolite 9-hydroxy risperidone in brain samples collected from mice pretreated with THC. Mice were treated for 14 days with VEH or 1 mg/kg THC and after the two week washout period brain samples were collected. The risperidone quantification procedure was conducted as outlined in detail by (Brzozowska et al, 2016). In brief, mouse brains were homogenised. Methyl-risperidone (10 μM) was added to all samples as an internal standard. A standard curve in brain plasma was constructed using linear concentrations of 1-100 nM of risperidone and 9-hydroxy risperidone over methyl-risperidone. All quantification was performed using triple quadrupole liquid chromatography-mass spectrometry (Agilent 6460A). Our next aim was to confirm that risperidone and its metabolite are indeed excellent substrates of the ABC transporter P-glycoprotein. This was done by measuring the brain and plasma concentrations of these drugs in samples collected from P-gp knockout mice (Abcb1a/b-/-). Mice were injected with 3 mg/kg risperidone and brain and plasma was collected at 1 and 3 h post-injection. Risperidone brain quantification was carried out as described above (Brzozowska et al, 2016). 

Detection of Δ9-THC and Δ9-THC-COOH in blood samples 
Blood samples were collected from mice pretreated with THC daily for 14 days to determine if THC or its terminal metabolite 11-nor-9-carboxydelta-9-tetrahydrocannabinol (Δ9-THC-COOH) was present in blood after the 14 day washout period. The THC blood quantification procedure was conducted as outlined in detail by (Gunasekaran et al, 2009; Quinn et al, 2007; Spiro et al, 2012). Determination of Δ9-THC and Δ9-THC-COOH was conducted by gas chromatography-mass spectrometry (GC-MS) using selected ion monitoring with deuterium labeled D3-THC and D3-THC-COOH as the internal standards. Spiked blood samples were used to construct a calibration curve (Δ9-THC, 5-50 ng/mL; Δ9-THC-COOH, 10-100 ng/mL) for calculation of the unknown Δ9-THC and Δ9-THC-COOH concentrations.

P-glycoprotein immunofluorescence
To determine if THC pretreatment was increasing P-gp transporters expression we performed immunofluorescence analysis of P-gp in mice. Mice were dosed daily for 14 days with 1 mg/kg THC or VEH and following the two week washout period they underwent transcardial perfusion with 4% paraformaldehyde. Brain tissue was extracted, cryoprotected and then sliced to yield 40 μm sections. For unmasking of P-gp epitopes brain sections were pretreated with a 34% acetic acid and 66% ethanol solution for 20 min at -20°C (Volk et al, 2005). To prevent unspecific antibody binding, tissue was incubated with a blocking solution containing 1% BSA and 2% normal rabbit serum in PBS including 0.1% Tween 20 overnight. The tissue was then incubated with the primary monoclonal mouse antibody C219 (Merck Millipore, VIC, Australia) overnight before the secondary rabbit anti-mouse IgG Alexa Fluor 594 (Abcam, VIC, Australia) antibody for 60 min. Sections were mounted onto slides and coverslipped using ProLong Gold Antifade (Invitrogen, Australia). 
P-gp immunofluorescence was acquired using an Olympus LSM 710 confocal microscope (Olympus, Germany). Z-stacks of 30 μm were acquired at 0.50 μm, using a 20X water immersion objective (NA 0.95) at 1024 x 1024 resolution. A 561 nm laser was utilised for excitation and emission was collected between 585 and 733 nm. Data was then exported to Volocity (Perkin Elmer, United States of America) where the data was batch processed as follows; three-dimensional rendering of the Z-stacks were done and then all images were thresholded using the same levels. Particle analysis was performed to yield the cumulative amount of tissue stained for P-gp in a fixed field of view (1024 x 1024 pixels). The mouse brain atlas by Paxinos et al (2004) was used to identify brain the same regions quantified for c-Fos immunohistochemistry and autoradiography experiments.














































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