The negative health effects of THC(Tetrahydrocannabinol) on memory function of human brain

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Neuropharmacology 117 (2017) 273e281

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Neuropharmacology

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Effects of tetrahydrocannabinol on glucose uptake in the rat brain

I. Miederer a, *, K. Uebbing b, J. R€ohrich b, S. Maus a, N. Bausbacher a, K. Krauter b, V. Weyer-Elberich c, B. Lutz d, M. Schreckenberger a, 1, R. Urban b, 1

a Department of Nuclear Medicine, University Medical Center of the Johannes Gutenberg University Mainz, Langenbeckstraße 1, 55131 Mainz, Germany b Institute of Legal Medicine, University Medical Center of the Johannes Gutenberg University Mainz, Am Pulvertum 3, 55131 Mainz, Germany c Institute of Medical Biostatistics, Epidemiology and Informatics, University Medical Center of the Johannes Gutenberg University Mainz, Obere Zahlbacher Straße 69, 55131 Mainz, Germany d Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Duesbergweg 6, 55128 Mainz, Germany

a r t i c l e i n f o

Article history: Received 15 September 2016 Received in revised form 24 January 2017 Accepted 12 February 2017 Available online 20 February 2017

Chemical compounds studied in this article: Dronabinol (PubChem CID:16078) [18F]-fluoro-2-deoxy-D-glucose (PubChem CID: 3232583)

Keywords: [18F]FDG THC microPET Rat

* Corresponding author. Department of Nuclear M Center of the Johannes Gutenberg University Mainz Mainz, Germany.

E-mail address: isabelle.miederer@unimedizin-ma 1 Both authors contributed equally to this work.

http://dx.doi.org/10.1016/j.neuropharm.2017.02.011 0028-3908/© 2017 Elsevier Ltd. All rights reserved.

a b s t r a c t

D9-Tetrahydrocannabinol (THC) is the psychoactive component of the plant Cannabis sativa and acts as a partial agonist at cannabinoid type 1 and type 2 receptors in the brain. The goal of this study was to assess the effect of THC on the cerebral glucose uptake in the rat brain. 21 male Sprague Dawley rats (12 e13 w) were examined and received five different doses of THC ranging from 0.01 to 1 mg/kg. For data acquisition a Focus 120 small animal PET scanner was used and 24.1e28.0 MBq of [18F]-fluoro-2-deoxy-D- glucose were injected. The data were acquired for 70 min and arterial blood samples were collected throughout the scan. THC, THC-OH and THC-COOH were determined at 55 min p.i. Nine volumes of interest were defined, and the cerebral glucose uptake was calculated for each brain region. Low blood THC levels of < 1 ng/ml (injected dose: � 0.01 mg/kg) corresponded to an increased glucose uptake (6 e30 %), particularly in the hypothalamus (p ¼ 0.007), while blood THC levels > 10 ng/ml (injected dose: � 0.05 mg/kg) coincided with a decreased glucose uptake (�2 to �22 %), especially in the cerebellar cortex (p ¼ 0.008). The effective concentration in this region was estimated 2.4 ng/ml. This glucose PET study showed that stimulation of CB1 receptors by THC affects the glucose uptake in the rat brain, whereby the effect of THC is regionally different and dependent on dose e an effect that may be of relevance in behavioural studies.

© 2017 Elsevier Ltd. All rights reserved.

1. Introduction

Cannabinoid type-1 (CB1) receptors are classified as seven transmembrane G-protein coupled receptors and are particularly present in the central and peripheral nervous systems. In the brain, a high density of receptors is found in regions involved in motor control (e.g., the cerebellum and basal ganglia), memory function (e.g. hippocampus and amygdala), and the regulation of the auto- nomic nervous system (e.g. the hypothalamus). CB1 receptors are found at low concentrations in the brain stem and spinal cord. They also occur, though at rather low concentrations, in cells of periph- eral organs, e.g. adipocytes, hepatocytes or endothelial cells. CB1

edicine, University Medical , Langenbeckstraße 1, 55131

inz.de (I. Miederer).

receptors in the brain are mainly located at the pre-synapsis, and their endogenous ligands act as retrograde neurotransmitters. The stimulation of the CB1 receptor causes inhibition of other neuro- transmitters, such as glutamate or GABA (Lutz et al., 2015).

The phytocannabinoid (�)-D9-tetrahydrocannabinol (THC) is the main psychoactive component of the plant cannabis sativa. It has a high lipophilicity and acts as a partial agonist to the CB1 re- ceptor. THC is metabolised mainly in the liver to 11-hydroxy THC (THC-OH), which is also psychoactive, and further oxidised to THC carboxylic acid (THC-COOH). It has been shown that THC affects a variety of physiological and psychological processes, such as impairment of motor coordination and short-time memory func- tion and increase in appetite (Volkow et al., 2014).

The positron emission tomography (PET) tracer [18F]-fluoro-2- deoxy-D-glucose ([18F]FDG) is a glucose analogue widely-used to image glucose uptake of tissue. The glucose uptake was formerly commonly known as the cerebral metabolic rate for glucose (CMRglc) (Phelps et al., 1979). As a surrogate marker for neuronal

I. Miederer et al. / Neuropharmacology 117 (2017) 273e281274

activity, it can be assessed using dynamic acquisition of [18F]FDG accumulation by PET together with pharmacokinetic modelling analyses.

A number of 2-deoxyglucose (2-DG) autoradiographic studies have reported effects of THC on brain glucose uptake (Margulies and Hammer, 1991; Freedland et al., 2002; Whitlow et al., 2002). [18F]FDG-PET studies in animals using der CB1 receptor agonist HU210 (Nguyen et al., 2012) and in humans using THC (Volkow et al., 1991) even showed the impact on the activation of the CB1 receptor system, but results were inconsistent. To date, no in vivo studies have been conducted on rats in order to assess the effect of THC on the brain glucose uptake. Moreover, recent ex vivo animal studies were analysed with regard to the administered dose rather than blood THC concentration. Human studies have shown, how- ever, that THC metabolism underlies differences, and blood THC concentrations may vary considerably within one group (Huestis, 2007).

The current study was subdivided into two parts. Firstly, we investigated the pharmacokinetics of THC and its metabolites THC- OH and THC-COOH in blood serum at two doses (0.001 and 0.01 mg/kg). Based on these blood THC concentrations, the dosing scheme for ensuing studies were determined, thereby considering a physiological plausible range. Secondly, we hypothesized that, THC has an effect on the cerebral glucose uptake in the rat brain as a function of the blood THC concentration. We particularly assumed to find effects of treatment in a brain region with high concentra- tions of CB1 receptors, such as the cerebellar cortex. For this reason, we assessed the cerebral glucose uptake by means of [18F]FDG-PET in the rat brain at different THC doses, ranging from 0.01 to 1.0 mg/ kg THC. In the light of human blood THC concentrations of cannabis smokers (Huestis, 2007), we assumed that first effects on brain glucose uptake are found at blood THC concentrations in the range of 1e10 ng/ml. In view of previous autoradiographic studies (Margulies and Hammer, 1991; Freedland et al., 2002; Whitlow et al., 2002), we expected high THC blood concentrations to be attended by decreased glucose uptake.

2. Materials and methods

2.1. Animals

The study cohort comprised 29 male Sprague Dawley rats (12e13 weeks of age, weighting 320e480 g; Charles River, Sulzfeld, Germany). Eight rats were examined with regard to pharmacoki- netics of THC, THC-OH and THC-COOH. Four of them received 0.001 mg/kg THC (international nonproprietary name (INN): dro- nabinol, THC Pharm GmbH, Frankfurt a. M., Deutschland); other four received 0.01 mg/kg THC. The other remaining 21 male Spra- gue Dawley rats were examined by PET. The control group con- sisted of 6 animals, the other 5 groups of 3 animals each. They received the following doses of THC: 0, 0.01, 0.05, 0.1, 0.5, 1 mg/kg. The control group received a vehicle. During the PET scan, THC, THC-OH and THC-COOH were determined 55 min p.i. All animals fasted from at least 2 h prior to the beginning of the study.

2.2. Preparation of THC

THC was obtained as a solid resin in a glass syringe. A portion of it was dissolved in ethanol and then suspended in a 1:4:1 ratio with pluronic F-68 detergent, ethanol and saline according to (Freedland et al., 2002). The THC solution was diluted with saline to a volume of 1 ml and injected intravenously over 90 s.

2.3. Liquid chromatography tandem mass chromatography (LC-MS/ MS)

A procedure according to previously published methods was used for the determination of THC, THC-OH and THC-COOH (Rohrich et al., 2010a, 2010b; Eckart et al., 2015) by means of LC- MS/MS. Serum (200 mL) was purified by solid phase extraction (Bakerbond SPE C18, 500 mg, Avantor Performance Materials, Center Valley, PA) after dilution with 6 ml of phosphate buffer (0.1 M, pH 6) and addition of 50 mL of the deuterated internal standard mixture (methanolic solution of 0.02 ng/mL each of THC- D3 and THC-OH-D3 and 0.2 ng/mL of THC-COOH-D9) using an automated Gilson Aspec GX 271Workstation (Middleton,WI). After washing with 2� 2ml water, 2� 2ml water/methanol (80:20; v/v) and 1 ml of 0.1 M acetic acid, the cannabinoids were eluted with 3 ml dichloromethane/acetone (50:50; v/v). After evaporation of the solvent (nitrogen, 40 �C), the dry residue was dissolved in 50 mL of acetonitrile/methanol/water (3:3:2 v/v/v) for injection. The LC- MS/MS system consisted of an Agilent UPLC 1290 Infinity Liquid Chromatograph (binary pump, automated sampler, degasser) coupled with G 6490 AA Triple Quadrupole Mass Spectrometer (Agilent Technologies, Santa Clara, CA) using Mass Hunter B.05.00 software. An Agilent Zorbax RRHP Eclipse Plus C18 column (2.1 � 100 mm, 1.8 mm) was utilized. The Column temperature was 50 �C, injection volume 5 mL and flow rate 1.0 ml/min. The gradient started with 40 % solution A (water/ammonium formate, 0.005 M) for the first 0.5 min. Mobile phase B (acetonitrile/formic acid, 0.1 %) increased until 2.5 min to 100 %. Solution B was hold at 100 % until 3 min. Starting conditions were reconstituted by setting solvent A to 40 % water within 0.2 min. This was hold for further 0.4 min (run time 3.6 min). The MS/MS measurements were performed in ESI positive MRM mode (gas temperature 250 �C, gas flow 15 l/min, nebulizer pressure 20 psi, sheath gas temperature 400 �C, sheath gas flow 12 l/min, capillary voltage 4000 V, nozzle voltage 1000 V, cell accelerator voltage 5 V, fragmentor voltage 380 V, polarity positive, scan segments 0e0.7, 0.7e1.9 and 1.9e2.5 min). Detailed data are presented in Table 1. For quantification the peak areas of the transitions specified as “target” (T) were used. Quantification was based on peak area ratios relative to the respective internal standard (IS). Target to qualifier (Q) ratios as well as retention times were used as identification criteria. A six-point calibration curve was used for each compound. The different calibration levels were obtained by spiking the blank matrix with methanolic solutions containing appropriate amounts of the analytes. The calibration levels were 0.5, 1, 2, 3, 4 and 5 ng/ml for THC and THC-OH as well as 5, 10, 20, 30, 40 and 50 ng/ml for THC-COOH. The method was validated according to current standards (Peters et al., 2007). The method validation was performed by using the Microsoft Excel- based validation program Valistat (Schmitt et al., 2003). Valida- tion data are given in Table 2.

2.4. Pharmacokinetic analyses of THC, THC-OH and THC-COOH

The animals were placed in head-first supine position and anesthetised by 2e2.5 % isoflurane vaporized in 100 % O2. They received a femoral artery catheter and blood samples were drawn. For the purpose of further LC-MS/MS analyses, a minimum blood volume of 500 ml was required. For this reason, the sampling scheme was split to draw blood from two animals at time points 0.5,1.5, 2.5, 5, 20, 50min and from the remaining two at time points 1, 2, 3,10, 35, 70min. The bloodwas centrifuged (HeraeusMegafuge 1.0 R, Thermo Scientific GmbH, Dreieich, Germany) (relative cen- trifugal force: 4.300 � g, 20 min) in order to obtain serum probes. The serum was filled into sodium-fluoride-coated crystals in order to avoid modification of THC and its metabolites, and subsequently

Table 1 Instrument settings of LC-MS/MS analyses.

Compound name Precursor ion (m/z) Product ion (m/z) Dwell (ms) Collision energy (V) Scan segment Delta EMV (V) Retention time (min)

THC-OH-D3 (IS) 334 196 40 25 2 400 1.3 THC-OH (T) 331 193 40 25 2 400 1.3 THC-OH (Q) 331 313 40 9 2 400 1.3 THC-COOH-D9 (IS) 354 336 40 13 2 400 1.4 THC-COOH (T) 345 327 40 13 2 400 1.4 THC-COOH (Q) 345 299 40 17 2 400 1.4 THC-D3 (IS) 318 196 60 21 3 200 2.2 THC (T) 315 193 60 21 3 200 2.2 THC (Q) 315 123 60 29 3 200 2.2

THC: (�)-D9-tetrahydrocannabinol, THC-OH: 11-hydroxy THC and THC-COOH: 11-nor-9-carboxy-THC. EMV: electron multiplier voltage, IS: internal standard, T: target, Q: target to qualifier ratio.

Table 2 Method validation data of the LC-MS/MS.

LOD (ng/mL)

LLOQ (ng/mL)

Accuracy (low) (%)

Accuracy (high) (%)

Intra-assay (low) (%)

Intra-assay (high) (%)

Inter-assay (low) (%)

Inter-assay (high) (%)

Recovery (low) (%)

Recovery (high) (%)

Matrix effect(low) (%)

THC 0.2 0.5 �1.5 �6.6 6.0 5.0 12.4 7.4 64 61 94 THC-OH 0.3 1.0 �2.2 �6.7 14.0 7.2 14.0 7.4 66 73 88 THC-COOH 1.5 5.0 �2.5 �1.7 8.3 6.7 12.1 11.1 71 78 94

LOD: limit of detection, LLOQ: lower limit of quantification, THC: (�)-D9-tetrahydrocannabinol, THC-OH: 11-hydroxy THC, THC-COOH: 11-nor-9-carboxy-THC.

I. Miederer et al. / Neuropharmacology 117 (2017) 273e281 275

frozen. The following parameters were calculated for characteri- sation of the pharmacokinetic properties of THC and its metabolites in the rat blood serum: 1) maximum concentration (Cmax), 2) time between administration of THC and Cmax (Tmax), 3) elimination half-life, i.e. time after which half of the maximum concentration is reached (T1/2), and 4) the area under the curve (AUC(0e70min)).

2.5. PET acquisition

For data acquisition a Focus 120 small animal PET scanner (Siemens/CTI, Knoxville, TN, USA) was used. It offers lutetium oxyorthosilicate detectors for coincidence detection (timing win- dow: 6 ns) with a size of 1.5 � 1.5 � 10 mm3 and a resolution � 1.4 mm at the centre of the field of view. The animals were placed in head-first supine position and anesthetised by 2e2.5 % isoflurane vaporised in 100 % O2. Prior to the emission scan, a 10-min 57Co transmission scan was performed. Emission scans in list mode data format were acquired for 70 min. 24.1e28.0 MBq of [18F]-fluoro-2-deoxy-D-glucose (18F-FDG) (PET Net GmbH, Erlan- gen, Germany) in a volume of 1 ml were injected into a tail vain over 90 s. Arterial blood samples from a femoral artery (Meyer et al., 2006) were collected throughout the scan at 0.5, 1, 1.5, 2, 2.5, 3, 5, 10, 20, 35, 50, 70 min. Fluid was replaced during the scan. Radio- activity values in blood were determined in a gamma-counter (2470 WIZARD2 Automatic Gamma Counter, PerkinElmer, Wal- tham, MA, USA). THC, THC-OH and THC-COOH were determined 55 min p.i., corresponding to the middle of the time interval of the PET analyses. Furthermore, blood glucose levels were determined (Accu Chek Aviva, Roche, Basel, Switzerland) for PET data quanti- fication at 12 and 45 min and averaged.

2.6. PET data analysis

PET list mode datawas rebinned into frames of 3� 20 s, 3� 60 s, 3� 120,12� 300 s and reconstructed using filtered back-projection (ramp filter, cut-off¼ 0.5) into 95 slices of 0.80-mm thickness (pixel size: 0.87� 0.87mm2) and amatrix of 128� 128 pixels. Corrections were applied for dead time, randoms, attenuation, scatter and radioactive decay. A 70-min summed PET image was coregistered to anMR T2 rat template as provided by the PMOD software (PMOD

v. 3.4; Zurich, Switzerland) and transformation parameters were applied to the dynamic data set. Nine brain regions were selected from the PMOD rat brain VOI template and projected onto the PET images: somatosensory cortex (143 mm3), motor cortex (65 mm3) caudate-putamen (87 mm3), thalamus (61 mm3), hippocampus (70 mm3), hypothalamus (37 mm3), amygdala (42 mm3), cerebellar cortex (150 mm3) and pons (45 mm3) and time-activity-curves (TAC) were calculated. Plasma to whole blood ratios were calcu- lated according to the function f(t) ¼ 0.51 e(-ln2/4.79 t)þ0.3 e(-ln2/ 337 t)þ0.8 (Weber et al., 2002). TAC of whole blood and plasmawere decay-corrected for the time of injection. The cerebral glucose uptake, referring to the time interval of 45e70 min, was calculated as CMRglc ¼ PGLC K1k3k2þk3 in [mmol/min/100 g] as implemented in the PMOD software. PG denotes the plasma glucose level and LC the lumped constant which was set to a value of 0.625 (Toyama et al., 2004). The relation between the administered THC dose and THC concentration in blood was calculated and fitted using R (R Foun- dation for Statistical Computing, Vienna, Austria). The relation be- tween the THC concentration in blood and the PET signal CMRglc was calculated and fitted for every brain region. Here, the outcome measure was the median effective concentration EC50. It was taken into account in cases where fits to the data were significant.

2.7. Statistics

Average values were expressed as means ± standard deviation. Pearson correlation coefficients were calculated for 1) injected THC doses and measured blood THC concentrations and 2) injected THC doses and blood glucose levels using Pearson's correlation coeffi- cient in SPSS 23 (IBM, Armonk, USA). On the basis of THC concen- tration measured in blood, CMRglc values were subdivided into four groups: I) 0 ng/ml (controls), II) 0 < 1 ng/ml, III) 1 < 10 ng/ml, IV) 10 < 51 ng/ml THC in blood. This grouping was chosen as we know from behavioural human studies that first psychotropic effects occur in the range of 1e10 ng/ml blood THC concentrations, whereas significant effects are expected at blood THC concentra- tions > 10 ng/ml (Ramaekers et al., 2009). For overall group com- parisons a one-way ANOVA was calculated (factor: blood THC concentration) for every brain region. The global level of signifi- cance was defined as aglobal ¼ 0.05 and a Bonferroni correction led

I. Miederer et al. / Neuropharmacology 117 (2017) 273e281276

to local significance levels of alocal ¼ 0.006. As we expected to find effects in a brain region with high concentrations of CB1 receptors mean CMRglc values of the cerebellar cortex region were compared using Student's independent samples t-test (SPSS 23, IBM, Armonk, USA). Prior to the analysis, three main questions were defined concerning the comparison of different groups: 1) controls versus group “0 < 1 ng/ml THC in blood”, 2) controls versus group “1 < 10 ng/ml THC in blood” and 3) controls versus group “10 < 51 ng/ml THC in blood”. The global level of significance was defined as aglobal ¼ 0.05 and a Bonferroni correction led to local significance levels of alocal ¼ 0.016. Furthermore, for those brain regions in which the ANOVA also exhibited significant results, Student's independent samples t-tests were calculated. However, these analyses should be regarded as explorative.

3. Results

Fig. 1a and b shows the time courses of THC in blood serum. For both studies, concentrations of THC reached maximum values at approximately 90 s, which corresponds to the time length of intravenous THC infusion over a period of 90 s, and reached a steady state after 15 min. At THC doses of 0.001 mg/kg, the mean maximum values were 4.1 ng/ml THC in blood and T1/2 was 2:00e2:25 min, while the mean AUC(0e70min) was 18 ng/ml/min. From 15 min p.i. until the end of the measurement, mean THC concentration were <0.2 ng/ml in blood. At THC doses of 0.01 mg/ kg, maximum mean values were 64 ng/ml THC in blood, and T1/2 was 2:00 min, and themean AUC(0e70min) was 216 ng/ml/min. From 15 min p.i. until the end of the measurement, mean THC concen- trations were < 2.5 ng/ml in blood. In 2/8 animals, mean values for THC-OH could be detectedwith amaximumvalue of 1.0 ng/ml after 5 min. The factor between the mean maximum values of the two groups was 15 (64:4.1 ng/ml THC) and after 15 min, the factor be- tween the two groups ranged from 8 to 16. The factor between the mean AUC(0e70min) of the two groups was 12. When the dose was decupled Cmax and AUC(0e70min), values were nearly decupled and Tmax and T1/2 remained nearly constant for both concentrations. That is, the blood THC concentrations are shown to be directly proportional to the injected THC dose.

Fig. 2 shows the relationship between injected THC dose and the concentration of THC in blood determined at 55 min p.i. At a THC dose of 0.5 mg/kg, the data showed a high variance. The correlation coefficient for injected THC doses and the concentration of THC in blood was r ¼ 0.96.

MR images and reconstructed PET images of [18F]FDG uptake in two representative animals are shown in Fig. 3. The animal depic- ted in the middle row received 0 mg/kg and the animal depicted at

Fig. 1. Mean values of the THC concentration in blood serum. Eight rats were examined of sampling scheme was split. Blood was drawn from two animals at time points 0.5, 1.5, 2.5, 5 points were linearly interpolated.

the bottom received 1 mg/kg THC. The image in the middle shows typical patterns of [18F]FDG uptake, whereas the image at the bottom shows less signal. Blood glucose levels, as determined during the PET scan, ranged from 6.8 to 15.8 mmol/l. The correla- tion between the administered dose and blood glucose level was r ¼ �0.48. There was no correlation between blood glucose levels and the CMRglc, nor was there correlation between blood glucose levels and the blood THC concentrations.

Fits to the data were significant for the brain regions hippo- campus, hypothalamus, amygdala and cerebellar cortex. The EC50 was in the range of 2.4e2.6 ng/ml (p < 0.002) which corresponds to a dose of approximately 0.05 mg/kg THC. Fig. 4 shows the rela- tionship between the THC concentration in blood and the PET signal CMRglc, and the model fit to the data for the brain region cerebellar cortex.

Regional mean CMRglc values are given in Table 3. One data set of group IV was excluded, as the explorative analysis showed extreme values in > 50 % of the brain regions. According to SPSS, extreme values are farther than three interquartiles ranges from the 75th or 25th percentile. All regions showed an enhanced signal where blood THC values were in the range of 0e1 ng/ml. The largest in- creases were observed in thalamus (25 %), hypothalamus (30 %), amygdala (21 %), and cerebellar cortex (18 %). The mean signal values decreased in all regions for blood THC concentrations in the range between 1 and 10 ng/ml. The largest decrease was found for amygdala (�18 %). For blood THC concentrations > 10 ng/ml some regions, such as somatosensory cortex, motor cortex, caudate pu- tamen and thalamus, showed a nearly constant or slightly increased signal (�2 %e8 %). A decreased signal was observed for limbic structures such as the hippocampus (�12 %), the hypothal- amus (�14 %) and the amygdala (�21 %). Also the cerebellar cortex (�22 %) and the pons (�19 %) exhibited a decreased glucose uptake. The one-way ANOVA delivered significant results for the hypo- thalamus (p ¼ 0.005) and the cerebellar cortex (p ¼ 0.005). Investigation of the cerebellar cortex region by means of Student's independent samples t-test resulted in significant differences for the comparison of the control group and the group “10 < 51 ng/ml THC in blood” (p ¼ 0.008). The exploratory analyses of the CMRglc values of the hypothalamus yielded in p < 0.05 for the comparison of the control group and the group “0 < 1 ng/ml THC in blood” (p ¼ 0.007).

4. Discussion

The aim of this study was to assess the glucose uptake CMRglc by means of [18F]FDG-PET as a function of intravenously administered THC. To that end, the effects of five different doses, ranging from

which four received 0.001 mg/kg THC (a) and four received 0.01 mg/kg THC (b). The , 20, 50 min and from the other two animals at time points 1, 2, 3, 10, 35, 70 min. Data

Fig. 2. Relation between administered THC dose and blood THC concentration, as determined at 55 min p. i. Data points were fitted.

I. Miederer et al. / Neuropharmacology 117 (2017) 273e281 277

0.01 to 1mg/kg THC, were investigated. Our pharmacokinetic study showed that the administered dose of 0.01 mg/kg THC yielded blood THC concentrations of ~2.5 ng/ml at a steady state. In order to refer to the typical range of human blood THC concentrations (Huestis, 2007), the maximal administered dose was in our study 1 mg/kg THC.

All analyses were conducted with reference to blood THC con- centrations rather than the injected dose, since it is well known that there are differences in absorption, distribution, metabolism and elimination of THC (Huestis, 2007; Hiemke, 2008). Likewise, our study showed that the relationship between the injected THC dose and the concentration of THC in blood, as determined at 55 min p. i., can vary (cf. Fig. 2). Possible reasons for variance in the human THC metabolism are indicated by specific isoforms of the cytochrome P450 (CYP-450), i.e. CYP-2C9 and CYP-3A4. It has been shown that CYP-450 and its isoforms hold important functions in the primary and probably also in the secondary metabolism of THC (Bland et al., 2005; Stout and Cimino, 2014). However, there is ev- idence that isoforms of CYP-450 may lead to both elevations or reductions in blood THC concentrations (Sachse-Seeboth et al., 2009; Stout and Cimino, 2014). Sprague Dawley rats, which were investigated in this study, form various isoforms of CYP-450, but not isoforms CYP-2C9 and CYP-3A4 (Martignoni et al., 2006). Further investigations are needed, therefore, in order to identify those CYP-450 isoforms which metabolise THC in rats and to establish whether they add to the variance in THC metabolism.

The regional analyses (Table 3) of our study showed multiphase properties of the CMRglc for different blood THC concentrations. In the calculation of EC50 values (cf. Fig. 4), fits to regional CMRglc data, related to the inhibitory effect of THC, were significant for the hippocampus, hypothalamus, amygdala and cerebellar cortex and yielded an estimated EC50 value of ~2.5 ng/ml THC in blood. As this corresponds to a THC dose of approximately 0.05mg/kg, this means that an inhibitory effect can be induced at even low doses of THC. The effects of CB1 receptor stimulation on cerebral glucose uptake was also investigated by other groups by means of 2-deoxy-D-

glucose autoradiographic (2DG) (Margulies and Hammer, 1991; Freedland et al., 2002; Whitlow et al., 2002) and PET studies (Nguyen et al., 2012). Margulies et al. showed that the 2DG signal significantly increased at doses of 0.2 mg/kg THC in all cortical and in some limbic regions (Margulies and Hammer, 1991). In our PET study, an increased glucose uptake was observed at lower THC doses in thalamus, hypothalamus, amygdala, and cerebellar cortex and was attended by blood values in the range of 0e1 ng/ml (≙ 0.01 mg/kg THC dose). Possible underlying mechanisms at the cellular level might be given by a stimulatory effect on L-type voltage-dependent calcium channels as described for deasace- tyllevonantradol (DALN) (Rubovitch et al., 2002). In this study, it was shown that low concentrations (1e10 nM) of the cannabinoid receptor agonist DALN enhanced 45Ca2þ-uptake by N18TG2 neu- roblastoma cells. As 45Ca2þ-uptake was unaffected by pertussis toxin, it was assumed that Gi/Go proteins do not mediate this process. Rather, it was shown that the effect is mediated by Gs proteins, as demonstrated by bringing anti-Gs antibodies into cells (Bash et al., 2003), and by the cyclic adenosine monophosphate (cAMP)edependent protein kinase A, as demonstrated by blocking studies using the selective inhibitor H-89 (Rubovitch et al., 2002). At the behavioural level, low THC doses were reported to cause opposite effects compared to high THC doses. For example, the low dose of 0.001 mg/kg THC caused increased body temperature, potentiated responses to noxious stimuli and increased locomotor activity in mice (Tselnicker et al., 2007). In another study, this bimodal effect was reported for the endogenous cannabinoid anandamide. Its effect was investigated in terms of motor activity, ring catalepsy, hypothermia, and analgesia tests. It was shown that anandamide leads to enhanced activities in the open field, on the ring and aggressive behavior in timid singly housed mice. Possible underlying mechanisms were explained by the involvement of a Gs protein rather than a Gi protein (Sulcova et al., 1998). A main effect of treatment, as indicated by the statistical analyses in our study, was shown for the region hypothalamus. This region has low con- centrations of CB1 receptors (Herkenham et al., 1990; Breivogel and

Fig. 3. MR T2 template (a), distribution of the tracer [18F]FDG in a representative Sprague-Dawley rat after administration of 0 mg/kg THC (control animal) (b), distribution of the tracer [18F]FDG in another representative rat after administration of 1.0 mg/kg THC (c). The images are presented from left to right in coronal, sagittal and transaxial view and cuts were made in plane 70(120), 45(93) and 52(93). Normalized radioactivity ¼ measured radioactivity in the PET image (kBq/ml)/injected radioactivity (kBq).normalized radioactivity ¼ measured radioactivity in the PET image ðkBq=mlÞ=injected radioactivity ðkBqÞ: S1 somatosensory cortex, M1 motor cortex, CPu caudate-putamen, Th thalamus, Hi hippocampus, Hy hypothalamus, Amy amygdala, Cer cerebellar cortex.

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Childers, 1998), but they were shown to be highly efficient in terms of G-protein activation (Bermudez-Silva et al., 2012; Cardinal et al., 2012). It is well known that the hypothalamus is responsible for many functions of the autonomic nervous system; among others it regulates food intake. The effect of activation of hypothalamic CB1 receptors has been shown in a study in which injection of THC into the paraventricular nucleus of the hypothalamus induced hyper- phagia in rats (Verty et al., 2005). Whether the effect of cannabi- noids is mediated by CB1 or cannabinoid type-2 (CB2) receptors was addressed in a recent study (Kofalvi et al., 2016). It was shown that low concentrations of selective (JWH133 and GP1a) as well as non-selective (WIN55212-2) CB2 receptor agonists stimulated [18F] FDG uptake. THC is a non-selective agonist that binds to CB1 and CB2 receptors. According to the findings of K€ofalvi et al. (Kofalvi et al., 2016), it could be possible that the stimulatory effects, as shown in our study, were mediated by CB2 receptors rather than CB1 receptors. All afore mentioned ex vivo autoradiographic studies showed that THC regionally affects brain glucose uptake in the rat,

in particular in the motor and sensory system and in meso- corticolimbic and limbic structures. Furthermore, they indicated that THC doses > 1 mg/kg decreased the brain glucose uptake. In our in vivo PET study, the statistical group comparisons revealed a significant decrease of the CMRglc for the cerebellar cortex with blood values in the range of 10e51 ng/ml (≙ 0.05e1 mg/kg THC). This brain region has high concentrations of CB1 receptors (Herkenham et al., 1990) and is associatedwith important functions in motor and proprioceptive control and cognition. It has been shown that activation of CB1 receptors impairs locomotor activity (Whitlow et al., 2002), which places our in vivo findings of a decreased glucose uptake in the cerebellum in line with behav- ioural CB1 receptor studies. Other regions such as hippocampus, hypothalamus and amygdala also showed a distinct decrease (�12 % to �21 %) of the glucose uptake but did not reach statistical sig- nificance. THC is known to cause glucose intolerance (Bermudez- Siva et al., 2006), probably due to inhibition of the insulin recep- tor function (Dalton and Zavitsanou, 2010; Kim et al., 2012;

Fig. 4. Relation between blood THC concentration and the CMRglc measured with PET. Data points were fitted; the inflection point of this function defines the EC50 value which is 2.4 ng/ml.

Table 3 CMRglc values for the defined brain regions and for animal groups I-IV.

I: 0.0 ng/ml (n ¼ 6) II: 0 < 1 ng/ml (n ¼ 3) III: 1 < 10 ng/ml (n ¼ 7) IV: 10 < 51 ng/ml (n ¼ 4) Somatosensory cortex 16.9 ± 3.4 18.7 ± 1.6 16.1 ± 2.4 17.0 ± 3.3 Motor cortex 16.4 ± 4.0 17.7 ± 1.7 16.1 ± 1.7 17.7 ± 4.1 Caudate putamen 19.5 ± 5.6 21.6 ± 2.2 18.8 ± 3.4 20.7 ± 5.1 Thalamus 19.8 ± 4.4 24.8 ± 2.3 18.8 ± 3.6 19.3 ± 3.4 Hippocampus 19.8 ± 3.7 21.7 ± 1.2 17.9 ± 2.1 17.3 ± 3.3 Hypothalamus 17.9 ± 2.6 23.2 ± 1.5 16.3 ± 2.5 15.4 ± 3.2 Amygdala 17.1 ± 4.0 20.7 ± 2.8 14.0 ± 2.2 13.6 ± 3.4 Cerebellar cortex 18.9 ± 2.1 22.3 ± 2.9 17.2 ± 2.7 14.7 ± 1.6* Pons 17.3 ± 2.3 18.3 ± 4.2 16.3 ± 2.4 14.0 ± 2.7

*: significant difference (two-sided independent samples t-test) from controls (adjusted p < 0.016).

I. Miederer et al. / Neuropharmacology 117 (2017) 273e281 279

Pinheiro et al., 2016). As blood glucose levels were taken into ac- count when calculating the glucose uptake CMRglc, we assume that the PET-Signal is not decreased due to competition effects of glucose at the cell membrane but due to decreased neuronal ac- tivity. Possible underlying cellular mechanisms of inhibitory effects might be mediated by Gi/Go proteins. It was shown that the stim- ulatory effects of the cannabinoid agonist DALN disappeared at higher concentrations (1 mM) and could be reversed by application of pertussis toxin (Rubovitch et al., 2002). At high concentrations an inhibitory pathway is mediated by pertussis toxin-sensitive Gi/Go proteins, whereas at low concentrations, a stimulatory pathway is mediated by pertussis toxin-resistant Gs proteins (Bash et al., 2003). With regard to human studies, it was shown that blood THC con- centrations in the range of 2e5 ng/ml lead to first performance impairments, and that THC blood concentrations > 30 ng/ml lead to a 100% impairment in performance tests such as critical tracking task, stop signal task and tower of London task (Ramaekers et al., 2006). Moreover, even higher blood THC concentrations up to > 50 ng/ml were reported in human THC studies (Drummer et al.,

2003, 2004; Ramaekers et al., 2004). Although it must be taken into account that THC has a higher potency in humans as compared to rats (McPartland et al., 2007), blood THC concentrations in the range of 30e50 ng/ml are supposed to be on a physiological borderline and concentration effects might lead to an impairment of neuronal activity.

Other studies showed inconsistent effects of THC on the brain glucose uptake. A human [18F]FDG-PET study carried out by Volkow and co-workers at THC doses of 2 mg (y0:03 mg=kg) showed that the glucose uptake of the cerebellumwas significantly increased on average; while a number of individuals showed a decreased, others displayed an increased glucose uptake and one individual showed no changes at all (Volkow et al., 1991). Furthermore, an animal [18F] FDG-PET study by Nguyen et al. (2012) revealed results contrary to 2DC studies and our PET study. The authors administered 100 mg/kg HU210, a highly potent CB1 receptor ligand and measured the SUV by taking the blood glucose level into account (SUV ¼ activity concentration� body weight� blood glucose level=injected dose) (SUV ¼ radioactivity concentration � body

I. Miederer et al. / Neuropharmacology 117 (2017) 273e281280

weight � blood glucose level/injected radioactivity) with [18F]FDG- PET. Interestingly, this in vivo PET study showed the opposite effect, namely a global increase of the PET signal. Nguyen et al. explained their findings by a possible overstimulation of the CB1 receptor and its downstreaming signalling effects. Also antagonistic effects were observed at high THC doses (Petitet et al., 1998). This might explain the enhanced [18F]FDG uptake when a highly potent, full agonist such as HU210 is administered. These inconsistent findings prove beyond doubt that the effect of CB1 receptor activation on the ce- rebral glucose uptake needs to be further investigated. Recent studies indicated that CB1 receptors are present not only in the cell membrane but also, to some extent, in mitochondrial membranes (Benard et al., 2012; Hebert-Chatelain et al., 2014; Busquets-Garcia et al., 2015; Koch et al., 2015). Furthermore, it is supposed that endocannabinoids that activate presynaptic mitochondrial CB1 re- ceptors (mtCB1) temporarily decrease mitochondrial respiration and adenosine triphosphate (ATP) production, which is, in turn, required for neurotransmitter release (Benard et al., 2012). These underlying mechanisms of CB1 receptor signalling and energy metabolism in the cell are still unexplained.

4.1. Limitations

It must be noted that our studies were conducted with anaes- thetised rats, in contrast to the studies mentioned above. In our study, rats were scanned under isoflurane anaesthesia as arterial blood samples were collected, which is necessary for absolute quantification of the CMRglc. Isoflurane is a general inhalation anaesthetic and has been shown to affect the brain glucose uptake (Ori et al., 1986; Toyama et al., 2004). In an autoradiographic study in rats, Ori et al. showed that isoflurane displayed heterogeneous changes of glucose uptake in the brain. That is, the glucose uptake was decreased in cerebral cortical areas and regions of the so- matosensory system. Other regions e in particular parts of the limbic system e showed an increase in glucose uptake. A PET study onmice by Toyama et al. showed global decreases in glucose uptake using [18F]FDG. However, as all animals were studied under iso- flurane anaesthesia, these effects pertain to all animals similarly and therefore we assume that they can be disregarded.

5. Conclusions

This glucose PET study showed that THC primarily has an impact on limbic structures and the cerebellar cortex. Even low blood THC concentrations of < 1 ng/ml (injected dose: � 0.01 mg/kg) corre- sponded to an increased glucose uptake in particular in the hypo- thalamus; the underlying mechanisms of enhanced glucose uptake in these brain regions, however, are in need of further investigation. On the other hand, a decreased glucose uptake especially in the cerebellar cortexwas attended by high blood THC values> 10 ng/ml (injected dose: � 0.05 mg/kg) and the effective concentration in this regionwas estimated at 2.4 ng/ml. These in vivo results indicate that stimulation of CB1 receptors by THC affects the glucose uptake in the rat brain, whereby the effect of THC is regionally different and dependent on dose e a result which may well prove rich in consequences for future behavioural studies.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Funding

This work was funded by the Bund gegen Alkohol und Drogen

im Straßenverkehr e.V. (B.A.D.S.).

Conflict of interest

None.

Acknowledgements

We are very grateful to our colleague Nuse Afahaene for his technical assistance in collecting data. We are also very grateful to Parvana Hajieva and Jessie Quinlan for critical reading of the manuscript. This work is part of a thesis to be submitted by Kristina Krauter.

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