Article
Reference
Dopamine D2/3 Receptor Availabilities and Evoked Dopamine Release in Striatum Differentially Predict Impulsivity and Novelty
Preference in Roman High- and Low-Avoidance Rats
BELLES ANO, Lidia,
et al.
Abstract
Background: Impulsivity and novelty preference are both associated with an increased propensity to develop addiction-like behaviors, but their relationship and respective underlying dopamine (DA) underpinnings are not fully elucidated. Methods: We evaluated a large cohort (n = 49) of Roman high- and low-avoidance rats using single photon emission computed tomography to concurrently measure in vivo striatal D2/3 receptor (D2/3R) availability and amphetamine (AMPH)-induced DA release in relation to impulsivity and novelty preference using a within-subject design. To further examine the DA-dependent processes related to these traits, midbrain D2/3-autoreceptor levels were measured using ex vivo autoradiography in the same animals. Results: We replicated a robust inverse relationship between impulsivity, as measured with the 5-choice serial reaction time task, and D2/3R availability in ventral striatum and extended this relationship to D2/3R levels measured in dorsal striatum. Novelty preference was positively related to impulsivity and showed inverse associations with D2/3R availability in dorsal striatum and ventral [...]
BELLES ANO, Lidia,
et al. Dopamine D2/3 Receptor Availabilities and Evoked Dopamine
Release in Striatum Differentially Predict Impulsivity and Novelty Preference in Roman High- and Low-Avoidance Rats.
International Journal of Neuropsychopharmacology, 2021, vol. 24, p. 239-251
PMID : 33151278
DOI : 10.1093/ijnp/pyaa084
Available at:
http://archive-ouverte.unige.ch/unige:146984
Disclaimer: layout of this document may differ from the published version.
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Supplementary methods
Five-choice serial time task (5-CSRTT)
Rats were trained to the 5-CSRTT in 11 modular operating chambers (Med Associates Inc., St.
Albans, VT). Each chamber contained a house-light and was equipped with a curved wall including five equally spaced response holes raised 2.5 cm from the floor. Each response holes was provided with a yellow cue light and head-entry detectors. The opposite wall was equipped with a food receptacle containing a light and connected to an external dispenser. At the beginning of each session, the house light was illuminated and a food pellet (45-mg purified rodent tablets, TestDiet, Sandown Scientific, UK) was delivered. Its collection by the rat initiated the first trial of the session.
After an inter-trial interval (ITI) of 2 seconds, a light stimulus appeared pseudo-randomly into one of the 5 response holes for a period of 30 seconds. A nose-poke into the correct location of the visual stimulus was rewarded with a pellet delivered in the receptacle. A nose-poke into any other response hole counted as incorrect and was punished with a time out (TO) period of 5 seconds during which the house light was extinguished and no food delivered. Missed trials (i.e. omissions) were also recorded and resulted in a TO period. Responses made before the onset of the visual stimulus during the ITI were considered as premature and also resulted in a TO period, and subsequent re-setting of the trial.
Throughout the training period, and with improvement of the animal performances, the level of difficulty progressively increased by shortening the light stimulus duration from 30 seconds to 1.5 seconds, shortening the time available for the rat to respond (limited hold) from 30 seconds to 5 seconds and increasing the duration of the ITI from 2 to 7 seconds over 8 training phases. The criteria for level change, i.e. more than 80% accuracy and less than 30% omissions, were to be maintained over 2 consecutive sessions. Rats were trained until the final set of task parameters (stimulus duration, 1.5 seconds; limited hold, 5 seconds; ITI, 7 seconds) and fulfilled the criterion performance (>80% accuracy and <30% omissions) and stable baseline measures (<10% variation in accuracy over 3 consecutive days). Testing was carried out daily (5 days/week), and the session lasted for 100 trials or 40 min, whichever came first.
SPECT imaging Radiotracer preparation
[123I]IBZM was prepared as described previously (Tsartsalis et al., 2018). Briefly, a mixture containing 5 μl of BZM precursor (ABX, Germany, 24 nmol/ μl in ethanol), 2 μl of glacial acetic acid, 1 μl of 30%
H2O2 and 10 mCi of carrier-free 123I sodium iodide (Perkin Elmer, Basel, Switzerland) in 0.05 M NaOH was incubated at 68°C for 15 min. The radiotracer was isolated by linear gradient HPLC run (from 5%
acetonitrile, ACN, to 95% ACN, 10 mM H3PO4, in 10 min). HPLC was equipped with a reverse-phase column (Phenomenex Bonclone C18, Phenomenex, Schlieren, Switzerland) and radiotracer was eluted at a flow of 3 ml/min. Fractions containing [123I]IBZM were diluted in water and loaded on a Sep- Pak cartridge (Sep-Pak C18, Waters, Switzerland). [123I]IBZM was eluted with 0.5 ml of 95% ACN, 10 mM H3PO4 and concentrated using a rotary evaporator, and the final product was diluted in saline prior to animal administration.
SPECT imaging system
Rats were scanned using the ultra-high-resolution multipinhole SPECT scanner (U-SPECT II, Milabs, Utrecht, The Netherlands). The U-SPECT II has been described elsewhere (Deleye et al., 2013;
Goorden et al., 2013). Briefly, U-SPECT II is a stationary system with three detectors (9.5-mm thick NaI(TI) crystal). Only the animal bed is translated into 3 dimensions using the XYZ stage to acquire data in the defined field of view. A collimator with 75 pinholes (pinhole size 1.0 mm) with reconstructed resolution of <0.8 mm and sensitivity >700 cps/MBq was used for focused rat imaging.
SPECT imaging
Rats were anaesthetized with 2.5% Isoflurane and gently positioned in a custom-build head-holder (MiLabs, Utrecht, The Netherlands) to ensure reproducible positioning of the animal’s head within the field of view of the SPECT system. SPECT data acquisition started at the time of the i.v. injection of
[123I]IBZM and lasted for 132 minutes. There was no significant difference (p>0.05) between either the injected dose of [123I]IBZM (RHA = 37.4±9.4 MBq ; RLA = 41.9±9.4 MBq ; p>0.05 ; Student’s t-test) or the specific radioactivity at the time of injection (RHA = 151.4±38.2 GBq/μmol ; RLA = 169.5±32.9 GBq/μmol ; p>0.05) for the RHA and RLA rat groups. Body temperature was monitored during the scan and maintained at 37±1 °C using a thermostatically controlled heating blanket.
SPECT image reconstruction was performed using a pixel ordered subsets expectation maximization (P-OSEM, 0.4 mm voxels, 4 iterations, 6 subsets) algorithm using MiLabs image reconstruction software. Radioactive decay correction was performed while correction for attenuation or scatter was not.
SPECT data analysis
Reconstructed SPECT images were processed with the dedicated software PMOD V3.8 (PMOD Technologies Ltd, Zurich, Switzerland). Summation SPECT images were generated over the first 70- minutesof dynamic [123I]IBZM data and coregistered to a magnetic resonance imaging (MRI) atlas of the rat brain (Schweinhardt et al., 2003). The resulting transformation was applied to the SPECT dynamic images, mapping all rat brains into the same reference space.
A region of interest (ROI) template was defined on the MRI atlas, using the stereotaxic rat brain atlas of Paxinos and Watson (Paxinos and Watson, 1998) and previous guidelines described for analyzing rodent PET data (Dalley et al., 2007). The ROI template included three brain regions: the dorsolateral striatum (DST; wherein the CPu is located), the ventral striatum (VST; wherein the NAcc is located), and the cerebellum. ROIs consisted of fixed size circular ROIs placed bilaterally over the DST and VST (2mm in diameter). To minimize partial voluming effect, ROIs were placed on the central planes on which the structures appeared. ROIs delineating the contours of the cerebellum were drawn to cover most of the cerebellar cortex. The ROI template was applied to the dynamic images to produce time-activity curves (TACs) for the target-rich (DST and VST) and reference (cerebellum) regions.
TACs for [123I]IBZM were analyzed using the linear extension of the simplified reference region model
(LSSRM; (Alpert et al., 2003)). The cerebellum, which contains negligible D2/3R density, was used as the reference region.
Nonlinear least squares fitting (NLSF) analyses using the Marquardt-Levenberg algorithm and based on the LSSRM were applied to the regional TACs of [123I]IBZM binding to estimate BPND as an index of D2/3R availability and γ as an index of AMPH-induced DA release in the DST and VST. The standard error of each parameter as estimated by the NLSF procedure was given by the diagonal of the measurement error covariance matrix, expressed in percent of the parameter value (percent standard error, %SE), and was used to assess the parameter identifiability (Carson, 1986). A smaller %SE indicates better identifiability.
Ex vivo autoradiography of [3H]-(+)-PHNO binding
At one week following SPECT scanning, part of the animals (15 RHA and 15 RLA rats) were injected with 3.7 MBq [3H]-(+)-PHNO i.v (1.7 GBq/μmol; Movarek Biochemical, Brea, USA). Rats were sacrificed by decapitation at 60 min post-radiotracer injection, their brains quickly removed and stored at -80°C until further processing. Series of five 20 μM-thick adjacent sections, taken at 300 μm interval, were cut with a cryostat. Sections were selected to cover most of the rostro-caudal extension of the caudate-putamen (CPu), nucleus accumbens (NAcc), substantia nigra (SN), ventral tegmental area (VTA) and cerebellum. The first sections were stained for acetylcholinesterase and used as histological reference. The second sections were exposed with 3H standards (3H-microscale;
Amersham) onto 3H-sensitive phosphor-imaging plates (BAS-TR2025) for 12 weeks to reveal brain distribution of [3H]-(+)-PHNO binding. Autoradiograms were analyzed with the Fujifilm BAS-1800II phosphorimager system using AIDA software V4.06 (Raytest Isotopenmessgeräte GmbH, Germany).
ROIs were delineated on histological references and transferred onto adjacent autoradiographic sections. Background film signal values were subtracted from the ROI signal and a mean radioactivity measure was calculated for each ROI. For each region, results of multiple measurements across several sections and across both hemispheres were averaged for each rat.
Ex vivo autoradiography of [35S]GTPγS binding
Sections adjacent to those used for [3H]-(+)-PHNO autoradiography were assayed for D2/3R- stimulated G-protein activation using [35S]GTPγS autoradiography (Bailey et al., 2008; Blume et al., 2013). Sections were preincubated for 10 min at room temperature in 50 mM Tris-HCl containing 100 mM NaCl, 0.2 mm EGTA, and 3 mM MgCl2, pH 7.4, followed by 15 min in the same buffer supplemented with 2 mM GDP (Sigma Aldrich, Buchs, Switzerland) and 20 μM DPCPX (Tocris Bioscience, Bristol, UK) an adenosine A1 receptor antagonist used to block [35S]GTPγS binding mediated by these receptors (Laitinen and Jokinen, 1998). Adjacent sections were then incubated in parallel for 120 min in the same solution containing 0.04 nM [35S]GTPγS (1250 Ci/mmol; PerkinElmer, Schwerzenbach, Switzerland) with (stimulated condition) or without (basal condition) 1 mM of the D2 receptor agonist quinelorane (Bio-Techne AG, Zug, Switzerland). Non-specific [35S]GTPγS binding was assessed in the presence of 10 µM unlabeled GTPγS. Sections were subsequently washed twice for 5 min in ice-cold 50 mM Tris buffer (pH 7.4) followed by a final rinse (30 sec) in ice-cold distilled water. Sections were dried and then apposed with [14C] standards to Kodak BioMax MR films.
Autoradiograms were analyzed using AIDA software V4.06 (Raytest Isotopenmessgeräte GmbH, Germany). Total and non-specific binding were first both determined by subtracting background film signal from each autoradiogram. For each brain region examined, [35S]GTPγS specific binding was then calculated by subtracting the optical density of non-specific binding sections from the optical density of agonist-stimulated and basal-binding sections. Agonist stimulated binding in brain regions was then expressed as the percentage increase in [35S]GTPγS binding induced by the agonist relative to that observed under basal (GDP only) conditions. The regions analyzed include the DST, VST, SN and VTA. For each region, results of multiple measurements across several sections and across both hemispheres were averaged for each rat.
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