Alessandro Gozzi
email: alessandro.gozzi@iit.it

Michela Tessari

Lisa Dacome

Federica Agosta

Stefano Lepore

Anna Lanzoni

Patrizia Cristofori

Emilio Merlo Pich

Mauro Corsi

Angelo Bifone

Gozzi

Neuroimaging evidence of altered fronto-cortical and striatal function after prolonged cocaine self-administration in the rat

Keywords: Cocaine, fMRI, phMRI, dopamine, addiction, rat

Cocaine addiction is often modeled in experimental paradigms where rodents learn to selfadminister the drug. However, the extent to which these models replicate the functional alterations observed in clinical neuroimaging studies of cocaine addiction remains unknown.

We used Magnetic Resonance Imaging (MRI) to assess basal and evoked brain function in rats subjected to a prolonged, extended-access cocaine self-administration scheme. Specifically, we measured basal cerebral blood volume (bCBV), an established correlate of basal metabolism, and assessed the reactivity of the dopaminergic system by mapping the pharmacological MRI (phMRI) response evoked by the dopamine-releaser amphetamine.

Cocaine-exposed subjects exhibited reduced bCBV in fronto-cortical areas, nucleus accumbens, ventral hippocampus and thalamus. The cocaine group also showed an attenuated functional response to amphetamine in ventro-striatal areas, an effect that was significantly correlated with total cocaine intake. An inverse relationship between bCBV in the reticular thalamus and the frontal response elicited by amphetamine was found in control subjects but not in the cocaine group, suggesting that the inhibitory interplay within this attentional circuit may be compromised by the drug. Importantly, histopathological analysis did not reveal significant alterations of the microvascular bed in the brain of cocaine-exposed subjects, suggesting that the imaging findings cannot be merely ascribed to cocaine-induced vascular damage.

These results document that chronic, extended-access cocaine SA in the rat produces focal fronto-cortical and striatal alterations that serve as plausible neuro-biological substrate for the behavioral expression of compulsive drug-intake in laboratory animals.

Introduction

Chronic cocaine use produces long-lasting neurobiological changes that are thought to underlie the loss of control over drug intake that defines cocaine dependence [START_REF] Koob | Neuroscience of addiction[END_REF]. Human neuroimaging studies have started to shed light on the nature of these changes and their relationship with specific behaviors or symptoms. Reduced fronto-striatal perfusion and metabolism in abstinent cocaine abusers have been reported by multiple investigators [START_REF] Strickland | Cerebral perfusion and neuropsychological consequences of chronic cocaine use[END_REF]; [START_REF] London | Brain imaging studies of cocaine abuse: implications for medication development[END_REF][START_REF] Volkow | Long-term frontal brain metabolic changes in cocaine abusers[END_REF]). The disrupted function of frontal regions has been linked to the persistent neuropsychological deficits and impaired control over drug taking that frequently triggers relapse [START_REF] Strickland | Cerebral perfusion and neuropsychological consequences of chronic cocaine use[END_REF][START_REF] Kalivas | Glutamate systems in cocaine addiction[END_REF]). Positron emission tomography (PET) studies with selective D 2 dopamine (DA) ligands have demonstrated that subjects with cocaine addiction show persistent reduction in D 2 DA receptor availability [START_REF] Volkow | Decreased dopamine D2 receptor availability is associated with reduced frontal metabolism in cocaine abusers[END_REF]; [START_REF] Martinez | Cocaine Dependence and D2 Receptor Availability in the Functional Subdivisions of the Striatum: Relationship with Cocaine-Seeking Behavior[END_REF]) and decreased dopaminergic responsivity in the nucleus accumbens and other components of the "reward circuit" [START_REF] Volkow | Decreased striatal dopaminergic responsiveness in detoxified cocaine-dependent subjects[END_REF], consistent with a decreased sensitivity to natural reinforcers observed in these subjects [START_REF] Volkow | Dopamine in Drug Abuse and Addiction: Results of Imaging Studies and Treatment Implications[END_REF]). Recent research suggests that altered functional connectivity of catecholamine circuits may underlie the impaired inhibition of cortical function observed in cocaine abusers, a finding that portrays novel pathways for the neuroadaptational processes associated to addictive states [START_REF] Tomasi | Disrupted Functional Connectivity with Dopaminergic Midbrain in Cocaine Abusers[END_REF][START_REF] Gu | Mesocorticolimbic circuits are impaired in chronic cocaine users as demonstrated by resting-state functional connectivity[END_REF]).

Cocaine abuse is often modelled pre-clinically in experimental paradigms where rats are trained to self-administer (SA) the drug. By employing different SA patterns, experimenters have been able to reproduce several hallmark features of drug addiction, including compulsive drug seeking [START_REF] Vanderschuren | Drug Seeking Becomes Compulsive After Prolonged Cocaine Self-Administration[END_REF], uncontrolled drug use [START_REF] Ahmed | Transition from Moderate to Excessive Drug Intake: Change in Hedonic Set Point[END_REF]) and increased Gozzi et al.,[START_REF] Barroso-Moguel | Brain lesions induced by chronic cocaine administration to rats[END_REF] motivation to self-administer the drug [START_REF] Paterson | Increased motivation for self-administered cocaine after escalated cocaine intake[END_REF]. These features make these models an experimental tool of excellent face-validity to investigate the neuroplastic events associated to voluntary drug-intake [START_REF] Roberts | How to make a rat addicted to cocaine[END_REF]). However, specific clinical correlates of cocaine addiction, such as the blunted DA responsivity of striatal areas observed in PET studies [START_REF] Volkow | Decreased dopamine D2 receptor availability is associated with reduced frontal metabolism in cocaine abusers[END_REF]; [START_REF] Martinez | Cocaine Dependence and D2 Receptor Availability in the Functional Subdivisions of the Striatum: Relationship with Cocaine-Seeking Behavior[END_REF], do not appear to be adequately modelled by traditional short-term, limited-access cocaine SA paradigms, where instead "sensitized" (i.e. increased) dopaminergic responses are typically observed [START_REF] Narendran | Cocaine abuse and sensitization of striatal dopamine transmission: A critical review of the preclinical and clinical imaging literature[END_REF]. Moreover, the extent to which these models replicate the multiple neuro-functional alterations observed in human neuroimaging studies remains unknown.

In the present study, we used Magnetic Resonance Imaging (MRI) to map basal and evoked brain function in a rat model of cocaine SA. A prolonged (52 days), extended-access (12 hours) SA protocol was employed to model the characteristics of high-dose, chronic cocaine abuse in humans [START_REF] Gawin | Cocaine and Other Stimulants[END_REF][START_REF] Briand | Persistent Alterations in Cognitive Function and Prefrontal Dopamine D2 Receptors Following Extended, but Not Limited, Access to Self-Administered Cocaine[END_REF]. Repeated abstinence periods were introduced to minimize the acute toxic effects of the drug and to ensure sustained motivation to self-administer high doses of cocaine [START_REF] Roberts | How to make a rat addicted to cocaine[END_REF]). After a 10-day detoxification period, we measured microvascular basal cerebral blood volume (bCBV), an indirect indicator of resting brain function (Gaisler-Salomon et al. 2009; [START_REF] Small | Imaging correlates of brain function in monkeys and rats isolates a hippocampal subregion differentially vulnerable to aging[END_REF]), and assessed the reactivity of dopaminergic system by mapping the functional response elicited by the DA-releaser amphetamine using a CBV-based pharmacological MRI (phMRI) protocol [START_REF] Gozzi | Antagonism at serotonin 5HT2a receptros modulates functional activity of fonto-hippocampal circuit[END_REF][START_REF] Schwarz | Selective dopamine D(3) receptor antagonist SB-277011-A potentiates phMRI response to acute amphetamine challenge in the rat brain[END_REF]). Correlation analysis between resting (bCBV) and amphetamine-evoked (rCBV) responses were performed in an attempt to identify disregulation in circuits that control the recruitment and functional responsiveness of specific brain areas. Finally, post mortem histo-Gozzi et al., 5 pathological examinations were carried out to assess the potential contribution of direct vascular and neurotoxic effects of prolonged cocaine SA to the imaging findings.

Gozzi et al., 6

Methods

Experiments were carried out in accordance with Italian regulations governing animal welfare and protection. Protocols were also reviewed by a local animal care committee, in accordance with the guidelines of the Principles of Laboratory Animal Care (NIH publication 86-23, revised 1985).

Cocaine self administration

Apparatus for Cocaine SA

Rats that underwent cocaine SA were tested in operant chambers as previously described [START_REF] Moretti | A comparative study of the effects of the intravenous self-administration or subcutaneous minipump infusion of nicotine on the expression of brain neuronal nicotinic receptor subtypes[END_REF]). Each experimental chamber (Med Associates Inc., St. Albans, VT) was fitted with a cue light placed above each lever, and with a 2900-Hz tone module. An infusion pump was connected via an external catheter to a single-channel liquid swivel (Instech Laboratories Inc., Plymouth Meeting, PA). Data acquisition and operant-schedule parameters were controlled by a Med-PC software (Med Associates Inc.).

Cocaine SA procedure

Thirty male Lister-Hooded rats (Charles-River, UK) weighing 275-300 g were individually housed in a temperature and humidity controlled room with water available ad libitum. Animals were food-restricted throughout the experiment to maintain a constant body weight of 300 g (± 10 g).

Gozzi et al., 7

After their arrival, rats were acclimatized for one week and subsequently implanted with a catheter in the jugular vein as previously described [START_REF] Moretti | A comparative study of the effects of the intravenous self-administration or subcutaneous minipump infusion of nicotine on the expression of brain neuronal nicotinic receptor subtypes[END_REF]). After a 7-day recovery period, rats were transported to the operant chamber. Cocaine self-administration procedure was initiated under a fixed ratio (FR) 1 schedule of reinforcement. Each press on the active lever was associated with a 0.1 ml infusion of a cocaine hydrochloride solution (3 mg/ml, corresponding to 300 μg per infusion and 1 mg/Kg in rats weighing 300 g) plus the simultaneous illumination of the stimulus (cue) light and extinction of the chamber light for 20 s. Presses on the "inactive" lever had no programmed consequences. Each drug infusion ("reward delivery") was followed by a 20-s lever retraction. The first three "training" sessions were terminated after either 50 infusions or 2 h from the start of the session. In the subsequent 30 sessions the cocaine access time was extended to 12 hours (6.00 p.m. -6 a.m.), the unit dose reduced to 0.150 μg/infusion, (0.1 ml of 1.5 mg/ml cocaine solution, corresponding to 0.5 mg/kg in rats weighing 300 g) and FR gradually increased to 3 (sessions 4-6) and eventually to 5 (remaining 27 sessions).

Subjects that lost catheter patency or appeared unhealthy (i.e. showed signs of infection) were removed from the study (eleven subjects altogether). Repeated 48-72 hrs abstinence periods were introduced on days 16 (session 14, 72 hrs), 23 (session 18, 72 hrs), 31 (session 23, 48 hrs) to minimize the risk of acute cocaine-induced intoxication. Session 30 was followed by a longer (5 days) binge abstinence followed by two additional sessions. Such intervals were introduced due to the necessity to harmonize the timing of MRI scan and self-administration protocol over the relatively large number of subjects employed. A 10-day detoxification period within the home cage was introduced prior to the imaging experiment.

Vehicle SA procedure

Gozzi et al., 8

A group of fourteen rats was used as baseline reference group. The subjects were implanted with a jugular catheter and subjected to the same training and SA procedures (including number, duration of SA sessions and abstinence) as described above, except for the use of vehicle (saline, 0.1 ml) instead of cocaine during operant sessions.

Magnetic resonance Imaging

Animal preparation

Imaging studies were performed 10 days after the last SA session. Animal preparation and MRI acquisition parameters have been previously described in greater detail [START_REF] Gozzi | Antagonism at serotonin 5HT2a receptros modulates functional activity of fonto-hippocampal circuit[END_REF][START_REF] Schwarz | Selective dopamine D(3) receptor antagonist SB-277011-A potentiates phMRI response to acute amphetamine challenge in the rat brain[END_REF]). Briefly, rats were anaesthetized with 3% halothane, tracheotomized and artificially ventilated with a mechanical respirator. A femoral artery and vein were cannulated and the animals were paralyzed with D-Tubocurarine. After surgery halothane level was set to 0.8%. The body temperature of all subjects was maintained within physiological range and mean arterial blood pressure (MABP) was monitored continually through the femoral artery.

MR Image acquisition

Anatomical and fMRI timeseries were acquired on a Bruker Avance 4.7 Tesla system. The animals were positioned prone in a custom-made holding support, and a "Rat Brain" curved quadrature two-loop receive coil (Bruker, Ettlingen, Germany) was mounted on top of the animal skull and fixed to the animal holder. The animal holder was then fitted into a 72 mm birdcage resonator (Bruker, Ettlingen, Germany) which was used for radiofrequency transmit only. Both coils are standard components provided by the manufacturer.

Gozzi et al., 9

A T 2 -weighted anatomical volume was acquired using the RARE sequence (TR= 5461ms, TE eff = 72 ms, RARE factor 8, FOV 40mm, 256x256 matrix, 20 contiguous 1mm slices) followed by a time series acquisition (TR eff = 2700 ms, TE eff = 111 ms, RARE factor 32, dt=27) with same spatial coverage, yielding a functional pixel volume of ≈ 1 mm 3 . Total MRI time-series acquisition time was 58-min (128 repetitions) for both groups.

Following five reference images, 2.67ml/kg of the contrast agent Endorem (Guerbet, France) was injected to make the fMRI signal changes sensitive to cerebral blood volume (rCBV) [START_REF] Mandeville | Dynamic functional imaging of relative cerebral blood volume during rat forepaw stimulation[END_REF][START_REF] Schwarz | Functional MRI using intravascular contrast agents: detrending of the relative cerebrovascular (rCBV) time course[END_REF]). D-amphetamine (0.5 mg/kg) was administered intravenously 25 min after contrast agent injection, and MRI data were acquired over a period of 25 min following the challenge. The dose of D-amphetamine was chosen based on previous in vivo studies [START_REF] Schwarz | Selective dopamine D(3) receptor antagonist SB-277011-A potentiates phMRI response to acute amphetamine challenge in the rat brain[END_REF][START_REF] Gozzi | Functional Magnetic Resonance Imaging Reveals Different Neural Substrates for the Effects of Orexin-1 and Orexin-2 Receptor Antagonists[END_REF]). The dose ensures robust brain activation, does not produce "ceiling" rCBV responses [START_REF] Micheli | 1,2,4-triazol-3-yl-thiopropyl-tetrahydrobenzazepines: a series of potent and selective dopamine D(3) receptor antagonists[END_REF]) and elicits transient MABP responses that are homeostatically compensated under halothane anesthesia [START_REF] Gozzi | A multimodality investigation of cerebral haemodynamics and autoregulation in phMRI[END_REF][START_REF] Zaharchuk | Cerebrovascular dynamics of autoregulation and hypoperfusion. An MRI study of CBF and changes in total and microvascular cerebral blood volume during hemorrhagic hypotension[END_REF]).

Data analysis

Basal CBV (bCBV)

bCBV time series image data for each experiment were analyzed within the framework of the general linear model [START_REF] Worsley | A three-dimensional statistical analysis for CBF activation studies in human brain[END_REF]). Individual subjects were spatially normalized to a stereotaxic rat brain MRI template set (Schwarz et al. 2006a). Signal intensity changes were converted into basal cerebral blood volume (bCBV(t)) on a pixel-wise basis as previously described [START_REF] Chen | Improved Mapping of Pharmacologically Induced Neuronal Activation Using the IRON Technique with Superparamagnetic Blood Pool Agents[END_REF][START_REF] Mandeville | Dynamic functional imaging of relative cerebral blood volume during rat forepaw stimulation[END_REF]). bCBV time series were calculated over a 4.5 minute time-window starting 6.8 min after contrast agent injection. Mean bCBV volumes for Gozzi et al., 10 individual subjects were created by averaging the 10 time-points time-wise. Linear detrending was introduced to account for contrast agent washout [START_REF] Schwarz | Functional MRI using intravascular contrast agents: detrending of the relative cerebrovascular (rCBV) time course[END_REF]. Voxel-wise group statistics was carried out using FSL [START_REF] Smith | Advances in functional and structural MR image analysis and implementation as FSL[END_REF]) using multi-level Bayesian inference, with 0.7 mm spatial smoothing, a Z threshold > than 1.6 and a corrected cluster significance threshold of p=0.01.

phMRI response to D-amphetamine

MRI signal intensity changes were converted into fractional CBV (rCBV) as previously described [START_REF] Mandeville | Dynamic functional imaging of relative cerebral blood volume during rat forepaw stimulation[END_REF]) and detrended to account for contrast agent elimination from the blood-pool [START_REF] Schwarz | Functional MRI using intravascular contrast agents: detrending of the relative cerebrovascular (rCBV) time course[END_REF]. Unsmoothed, rCBV time series for amphetamine challenge were calculated covering 12.5 minute pre-challenge and 24 minutes post-challenge window.

Voxel-wise statistics was carried out using FEAT with 0.7 mm spatial smoothing and using a model function (Figure S1) capturing the temporal profile of amphetamine-induced rCBV response (Schwarz et al. 2006b). Higher-level group comparisons were carried out with multilevel Bayesian inference and thresholded at Z>1.6 with a corrected cluster significance threshold of p=0.01. In order to specifically test the hypothesis of an altered striatal reactivity to Damphetamine in cocaine rats, a 3D binary mask of major subcortical area (striatum, thalamus, hippocampus, hypothalamus the striatum, ventral pallidum, BNST, amygdala) was generated using a digital reconstruction of the rat brain atlas (Schwarz et al. 2006a) and used to prethreshold rCBV time series prior to higher-level FSL analysis. This procedure increases the statistical power of the analysis by reducing the number of multiple comparisons [START_REF] Huettel | Functional Magnetic Resonance Imaging[END_REF]). To investigate the regional specificity of the effect in a hypothesis-free manner and rule out generalized reductions in amphetamine response throughout the brain, the same analysis was Gozzi et al., 11 repeated on non-masked rCBV datasets (Figure S5). Volume of interest (VOI) mean bCBV values and time courses for the amphetamine challenge were extracted as previously described (Schwarz et al. 2006a;[START_REF] Gozzi | Differential Effects of Antipsychotic and Glutamatergic Agents on the phMRI Response to Phencyclidine[END_REF]). Statistical differences in mean bCBV were assessed using a one-way ANOVA test followed by Fisher's test for multiple comparisons.

Correlation analysis

Maps of correlated bCBV and D-amphetamine induced rCBV responses across subjects were calculated within the GLM framework at the group level with reference to bCBV in representative regions using FSL (Schwarz et al. 2007a;Schwarz et al. 2007b). A number of representative VOIs were selected based on the results of the inter-group bCBV maps (medial prefrontal, insular, orbitofrontal, somatosensory cortex, caudate putamen, nucleus accumbens, reticular thalamus, posteroventral thalamus). For each VOI, the design matrix comprised a regressor capturing the group mean bCBV signal in the anatomical structure and another containing the zero-meaned bCBV vector across the N subjects in the group from the selected reference structure. Z statistic images were calculated via contrasts capturing positive and negative correlations with the reference response, and were thresholded with Z>1.6 and a corrected cluster significance threshold of p=0.01. Linear regression plots of correlated bCBV and rCBV responses were calculated by plotting bCBV and mean rCBV response to amphetamine across individual subjects, the latter being expressed as mean response over a 20 minute (4-24 min post injection) time-window.

Histopathology

Gozzi et al., 12

Histopathological evaluation was performed on 10 cocaine subject and 8 randomly-chosen controls as previously described (Barroso-Moguel et al. 2002). After the MRI experiment, rats were maintained under deep anesthesia (halothane 5%), and a 15 min aortic perfusion of fixative media (10% buffered formalin) was performed, preceded by a 5 minutes infusion of saline.

Perfused brains were removed and stored in fixative solution for further 24-72 hours. Brain trimming was then performed using a brain matrix (ASI-Instruments®) designed for rats weighing 200-400 g. Tissue samples were paraffin-embedded, sectioned into 5 μm-thin slices, and stained with a combination of Haematoxylin-eosin and Luxol Fast Blue [START_REF] Scholtz | Quantitative histochemistry of myelin using Luxol Fast Blue MBS[END_REF]. The slice and brain regions analyzed where the cingulate and prefrontal cortex, caudate putamen, corpus callosum, hippocampus (C2), cerebellum (purkinje cells) and substantia nigra. The examination was performed by two study-blind veterinary pathologists.

Gozzi et al., 13

Results

Chronic cocaine self-administration (SA)

All the subjects completed the 33 cocaine SA sessions successfully over a time period of 52 days. The SA schedule used ensured a prolonged and sustained intake of cocaine throughout the study (Figure 1). The average cumulative intake of SA cocaine per subject was 1138.4 ±33.3 mg/rat. Both active level presses and cocaine intake appeared to be rather stable throughout the course of the experiment, although linear regression highlighted a weak but significant (p<0.03, F=4.62) trend towards an overall increased cocaine intake over time when all homogeneous sessions were compared (sessions 4-31, FR 3-5, binge abstinence intervals 48-72 hrs) (Figure S2).

bCBV

In order to investigate the effect of chronic cocaine administration on basal brain function, we measured bCBV in cocaine SA and control subjects and mapped the regions exhibiting statistically significant differences between groups. Rats the self-administered cocaine showed significantly reduced bCBV in several brain areas compared to control rats (Figure 2 and3). The effect was prominent in the medial-prefrontal, cingulate, orbitofrontal cortex, septum, ventral hippocampus, in the core region of the nucleus accumbens as well as raphe nuclei and reticular thalamic areas. No difference in total CBV between groups was observed (p=0.23, student's t test). No correlation was found between bCBV and total cocaine intake in all the VOIs examined (P>0.16, all VOIs).

Functional response to D-amphetamine

Gozzi et al., 14

In order to probe striatal dopaminergic reactivity, cocaine SA and control rats were challenged with the DA releaser amphetamine, and the presence of functional alterations in the magnitude of the rCBV response elicited by the drug was assessed via voxel-wise statistics. Consistent with previous studies [START_REF] Schwarz | Selective dopamine D(3) receptor antagonist SB-277011-A potentiates phMRI response to acute amphetamine challenge in the rat brain[END_REF]) amphetamine produced robust activation of sub-cortical and cortical areas in both groups of subjects (Figure S3). Rats chronically exposed to cocaine exhibited an attenuated functional response to amphetamine in the striatum compared to control rats (Figure 4, Figure S3). The effect was also apparent in un-detrended rCBV time-profiles (Figure S4). In rats that self administered cocaine, the magnitude of the striatal response to amphetamine was found to be inversely correlated with cumulative cocaine intake (p=0.03, Figure 4). Additional foci of reduced functional response to amphetamine were observed in sensory-motor and orbitofrontal cortex (Figure S3 and S5).

Administration of amphetamine produced transient increases in MABP (Figure S6). The effect was not temporally correlated with the functional response, and well within the blood flow autoregulatory range within which vasopressive responses are homeostatically compensated without producing significant rCBV alterations [START_REF] Gozzi | A multimodality investigation of cerebral haemodynamics and autoregulation in phMRI[END_REF][START_REF] Zaharchuk | Cerebrovascular dynamics of autoregulation and hypoperfusion. An MRI study of CBF and changes in total and microvascular cerebral blood volume during hemorrhagic hypotension[END_REF].

Arterial blood gases (p a CO 2 and p a O 2 ) were measured prior to and after the fMRI timeseries (Table SI). No statistically significant difference in mean pre-or post-acquisition p a CO 2 values between groups was found (p>0.1, all groups; one-way ANOVA).

Correlation between basal and evoked activity

In an attempt to establish a correlation between basal and evoked functional activity and to investigate dysregulations in the control of these two states, we measured the correlation between bCBV and amphetamine-induced response in control and animals that self-administered cocaine.

Gozzi et al., 15

No correlation between bCBV and amphetamine-induced rCBV responses was found in either group in any of the regions examined, with exception of the reticular and posterior-ventral thalamus which revealed, in control subjects, an inverse relationship with amphetamine-induced rCBV in fronto-cortical areas (Figures S7 andS8). No such correlation was present in the cocaine SA group (Figure S8).

Histopathology

A histopathological evaluation of brain white and grey matter, glial and interstitial compartments, as well as macro-and micro-vascular, ependymal and meningeal structures did not highlight any neurocellular, interstitial or microvascular lesions in either group. Specifically, no signs of cellular pyknosis or atrophy, fiber alteration, necrosis, interstitial edema, were observed in any of the brain regions examined., nor alterations of the microvascular and capillary bed (i.e., basal membrane dilation or rupture, hemorrhage, endothelial thickening or wall fibrosis, thrombi or occlusions, necrosis or vacuolation of endothelial cells).

Gozzi et al., 16

Discussion

The present study documents that chronic, extended-access cocaine SA in the rat produces neuroimaging alterations that closely mimic hallmark imaging findings in human cocaine addicts. Specifically, we observed significantly reduced bCBV, a marker of resting brain function, in regions that play a key contribution in higher cognitive functions and inhibitory control (fronto-cortical areas), craving and anticipation (fronto-hippocampal areas) and reward (mesolimbic areas). Moreover, cocaine SA was associated with reduced striatal reactivity to dopaminergic stimulation, and the presence of putative functional alterations in the inhibitory interplay between reticular thalamus and the activation of fronto-cortical areas. Our results provide neuroimaging evidence of multiple alterations in rat brain function following chronic and voluntary cocaine-intake that serve as plausible neuro-biological substrate for the behavioral expression of compulsive drug-intake in laboratory animals. neuroimaging studies, thus maximizing the translational relevance of our findings. Moreover, the use of extended-access to cocaine (i.e. ≥ 6 hours) is known to specifically model specific neurobehavioral features of addiction, such as persistent alterations in cognitive functions [START_REF] Briand | Persistent Alterations in Cognitive Function and Prefrontal Dopamine D2 Receptors Following Extended, but Not Limited, Access to Self-Administered Cocaine[END_REF][START_REF] George | Extended Access to Cocaine Self-Administration Produces Long-Lasting Prefrontal Cortex-Dependent Working Memory Impairments[END_REF]), increased motivation for cocaine [START_REF] Paterson | Increased motivation for self-administered cocaine after escalated cocaine intake[END_REF], and escalation in drug intake [START_REF] Ahmed | Transition from Moderate to Excessive Drug Intake: Change in Hedonic Set Point[END_REF]. Repeated periods of forced abstinence were introduced to reduce the acute toxic effects of the drug and to ensure sustained motivation to self-administer high doses of cocaine [START_REF] Roberts | How to make a rat addicted to cocaine[END_REF]). Although the total cocaine intake achieved with the present protocol is higher than that observed with short-access paradigms, the values attained are sufficiently distant from the limit of acute toxicity [START_REF] Mantsch | Effects of extended access to high versus low cocaine doses on self-administration, cocaine-induced reinstatement and brain mRNA levels in rats[END_REF][START_REF] Wee | Effects of Dose and Session Duration on Cocaine Self-Administration in Rats[END_REF]), which explains the lack of lethality observed in this study.

Compared to unlimited access protocols, where drug intake exhibits high and low numbers of infusions on alternating days [START_REF] Wilson | Heterogeneous subregional binding patterns of 3H-WIN 35,428 and 3H-GBR 12,935 are differentially regulated by chronic cocaine self-administration[END_REF] Focal bCBV reductions were also observed in reticular thalamic and raphe nuclei. The former finding is consistent with human neuroimaging studies showing altered GABAergic neurotransmission in the thalamus of abstinent cocaine abusers [START_REF] Volkow | Enhanced Sensitivity to Benzodiazepines in Active Cocaine-Abusing Subjects: A PET Study[END_REF]) and recent electrophysiological evidence of a state of protracted over-inhibition of reticular thalamic areas following binge administration of cocaine [START_REF] Urbano | Cocaine Acute "Binge" Administration Results in Altered Thalamocortical Interactions in Mice[END_REF]). Interestingly, as serotonin exerts a direct excitatory action on GABAergic neurons in the reticular thalamus (McCormick and Wang 1991), the reduced activity of this nuclei and that observed in regions of the raphe may be functionally interrelated and part of a single defective circuit. Gozzi et al.,[START_REF] Goldstein | Oral methylphenidate normalizes cingulate activity in cocaine addiction during a salient cognitive task[END_REF] No correlation between total cocaine intake and bCBV was found in any of the VOIs examined.

The lack of correlation could reflect different individual susceptibility to the effect of the drug, or could be related to the high amount of cocaine self-administered which might exceed the quantity required to produce maximal bCBV alterations.

In attempt to identify an fMRI correlate of the decreased striatal dopaminergic responsivity observed in human PET studies [START_REF] Volkow | Effects of chronic cocaine abuse on postsynaptic dopamine receptors[END_REF][START_REF] Volkow | Decreased dopamine D2 receptor availability is associated with reduced frontal metabolism in cocaine abusers[END_REF][START_REF] Martinez | Cocaine Dependence and D2 Receptor Availability in the Functional Subdivisions of the Striatum: Relationship with Cocaine-Seeking Behavior[END_REF]), we also mapped the functional response elicited by the DA-releaser amphetamine using a phMRI protocol [START_REF] Schwarz | Selective dopamine D(3) receptor antagonist SB-277011-A potentiates phMRI response to acute amphetamine challenge in the rat brain[END_REF] an effect that can be later restored after fetal or stem cell transplantation [START_REF] Bjorklund | Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model[END_REF][START_REF] Chen | Detection of dopaminergic cell loss and neural transplantation using pharmacological MRI, PET and behavioural assessment[END_REF]). Thus, the sum of these data indicates that amphetamine-induced rCBV responses can be reliably used as a marker of striatal DA neurotransmission. Within this framework, the presence of an attenuated striatal rCBV response to amphetamine in the cocaine SA group points towards a reduced-responsivity of ventro-striatal dopaminergic function analogous to what observed in PET studies in humans [START_REF] Narendran | Cocaine abuse and sensitization of striatal dopamine transmission: A critical review of the preclinical and clinical imaging literature[END_REF]. This finding provides for the first time a plausible preclinical neuroimaging correlate of one of the most replicated clinical manifestation of cocaine addiction, which is believed to play a key Gozzi et al., 21 contribution to the "hypohedonia" and amotivation reported by drug-addicted subjects during protracted withdrawal [START_REF] Volkow | Decreased striatal dopaminergic responsiveness in detoxified cocaine-dependent subjects[END_REF]. This result documents a potentially important correspondence between clinical and preclinical neuro-adaptational changes induced by cocaine on DA systems, an aspect that does not appear to be adequately modeled by traditional cocaine exposure paradigms, where "sensitized" (i.e. increased) dopaminergic responses are typically observed (reviewed by [START_REF] Narendran | Cocaine abuse and sensitization of striatal dopamine transmission: A critical review of the preclinical and clinical imaging literature[END_REF]. As similarly attenuated striatal responses were not observed in rodent neuroimaging studies using short-term (5-days) drug administration protocols [START_REF] Febo | The neural consequences of repeated cocaine exposure revealed by functional MRI in awake rats[END_REF]Reese et al. 2004, Gozzi A. unpublished results), our data suggest that, for this characteristic to be modeled in rodents, prolonged and extended access to high doses of cocaine may be required. Importantly, no appreciable microscopic lesions in the vascular, neurocellular and interstitial compartments of brains exposed to cocaine were observed. This result is important, as it permits to rule out a potential contribution of abnormal cerebrovascular processes on the haemodynamic measures of brain function performed (i.e. bCBV and rCBV).

Correlation analysis between resting and amphetamine-evoked (rCBV) responses revealed an inverse relationship between bCBV in reticular thalamic areas and amphetamine-induced frontal activation in control subjects, but not in cocaine group. Previous studies have demonstrated that inhibition of reticular thalamic activity can enhance fronto-cortical dopaminergic neurotransmission [START_REF] Jones | Dopamine function in the prefrontal cortex of the rat is sensitive to a reduction of tonic GABA-mediated inhibition in the thalamic mediodorsal nucleus[END_REF]), a finding consistent with the functional connectivity of these regions [START_REF] Paxinos | The Rat Nervous System[END_REF]) and the high GABAergic density in of reticular thalamic nucleus [START_REF] Paxinos | The Rat Nervous System[END_REF]. As prefrontal projections to the thalamic reticular nucleus play a unique circuit for attentional mechanisms [START_REF] Zikopoulos | Prefrontal Projections to the Thalamic Reticular Nucleus form a Unique Circuit for Attentional Mechanisms[END_REF], we hypothesize that the loss of Gozzi et al.,[START_REF] Gozzi | A multimodality investigation of cerebral haemodynamics and autoregulation in phMRI[END_REF] correlation between basal and evoked function observed in the cocaine SA group may be related to the attentional deficits observed in rats allowed extended access to cocaine [START_REF] Briand | Persistent Alterations in Cognitive Function and Prefrontal Dopamine D2 Receptors Following Extended, but Not Limited, Access to Self-Administered Cocaine[END_REF][START_REF] George | Extended Access to Cocaine Self-Administration Produces Long-Lasting Prefrontal Cortex-Dependent Working Memory Impairments[END_REF]. A putative role for thalamo-frontal dysfunctions in cocaine addiction is supported by recent neuroimaging studies showing altered thalamo-cortical connectivity in cocaine abusers under resting conditions [START_REF] Gu | Mesocorticolimbic circuits are impaired in chronic cocaine users as demonstrated by resting-state functional connectivity[END_REF]) and when performing a cognitive task [START_REF] Tomasi | Thalamo-cortical dysfunction in cocaine abusers: Implications in attention and perception[END_REF]). However, as correlation measurements do not reflect causal association, further research is warranted to elucidate the exact nature of this finding.

In summary, we provide evidence of altered brain function in rats that underwent prolonged and extended access cocaine self-administration. Consistent with clinical neuroimaging findings, cocaine-exposed animals revealed reduced basal brain function in fronto-cortical and thalamic areas, and attenuated responsivity in striatal regions upon challenge with the DA releaser amphetamine, an effect that was significantly correlated with the total cocaine intake. The consistency of these findings with neuroimaging measures in cocaine-addicted patients supports the use of prolonged and extended-access SA paradigms in the rat to investigate the neuroadaptations underlying cocaine-addiction.

Disclosure / Conflict of interest

All the authors are employees of GlaxoSmithKline. The authors declare that, except for income received from their primary employer, no financial support or compensation has been received from any individual or corporate entity over the past three years for research or professional service and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest.

  Chronic cocaine abuse is often modeled in behavioral paradigms where rodents are trained to voluntarily self-administer the drug. Here we implemented a prolonged, extended-access cocaine SA protocol with repeated binge-abstinence periods[START_REF] Parsons | Serotonin dysfunction in the nucleus accumbens of rats during withdrawal after unlimited access to intravenous cocaine[END_REF][START_REF] Wilson | Heterogeneous subregional binding patterns of 3H-WIN 35,428 and 3H-GBR 12,935 are differentially regulated by chronic cocaine self-administration[END_REF][START_REF] Wilson | The Vesicular Monoamine Transporter, in Contrast to the Dopamine Transporter, Is Not Altered by Chronic Cocaine Self-Administration in the Rat[END_REF] to mimic the characteristics of high-dose, chronic cocaine abuse in humans. Prolonged SA paradigms have been reported to reproduce key clinical features of cocaine-addiction including compulsive drug-use despite the presence of environmental adversities[START_REF] Vanderschuren | Drug Seeking Becomes Compulsive After Prolonged Cocaine Self-Administration[END_REF], and high propensity to relapse to drug seeking (Deroche-Gamonet et al. 2004). The chronic protocol employed (covering ≈ 10% of the adult life-span of a rat) (Sharp and La Regina 1998) permits to mimic patient populations with a significant history (> 6 months) of cocaine addiction like those typically enrolled in human Gozzi et al., 17

  , the extended-access protocol used here ensured sustained SA of high doses of cocaine. In contrast to what reported by other groups[START_REF] Ahmed | Transition from Moderate to Excessive Drug Intake: Change in Hedonic Set Point[END_REF][START_REF] Ferrario | Neural and behavioral plasticity associated with the transition from controlled to escalated cocaine use[END_REF];[START_REF] Wee | Effects of Dose and Session Duration on Cocaine Self-Administration in Rats[END_REF]), we did not observe unequivocal evidence of dose escalation, although a trend towards an increased cocaine intake over consecutive sessions was apparent (FigureS2).One limitation of the model employed is that it did not include behavioral measurements of drug use despite adverse consequences (e.g. "resistance to punishment" Deroche-Gamonet et al.2004), a behavioral trait that is considered an essential diagnostic criteria of addiction in humans (V.A. 2008). Because this feature is present in ca. 20% of rats exposed to cocaine (Deroche-Gamonet et al. 2004; Ahmed 2010), the imaging alterations mapped in the present work are likely to include contributions from subsets of subjects exhibiting this behavior. However, Gozzi et al., 18 whether this trait is characterized by specific functional alterations separate from those highlighted in this study, remains to be determined.A ten-day washout period was introduced prior to the imaging study to rule out acute carry-over effects of cocaine and minimize the potential interference of acute abstinence symptoms on the measures of brain function. Most of the neurochemical and behavioral alterations that can be related to acute withdrawal have an almost immediate onset, peak between 6 and 72 h after termination of drug access and generally cease within 2-7 days from the last cocaine session. (Baumann and Rothman 1998; Harris and Aston-Jones 1993; Malin et al. 2000; Mutschler and Miczek 1998; Markou and Koob 1992). It is therefore unlikely that the imaging findings contain major perturbations from transient neuro-biological phenomena related to acute cocaine abstinence. On the other hand, the observed functional alterations are expected to contain contributions from longer-lasting neuroadaptational processes (i.e. incubation of cocaine craving) which have been shown to build up after cocaine discontinuation (Lu et al. 2004), and that are of translational relevance as they can be related to propensity to relapse. MRI measures of bCBV allow high resolution mapping of resting brain function that tightly correlate with regional energy metabolism and cerebral blood flow (Gaisler-Salomon et al. 2009; Hyder et al. 2001; Gonzalez et al. 1995). Our data showed the presence of reduced bCBV in the cingulated gyrus, pre-frontal and orbito-frontal cortex, as well as in striatal and hippocampal areas of cocaine SA subjects. The fronto-striatal effect is in excellent agreement with clinical neuroimaging research of cocaine addiction, where reduced frontal and striatal activity have been consistently observed (Strickland et al. 1993; Tumeh et al. 1990; London et al. 1999; Volkow et al. 1992; Volkow et al. 1988) and found to correlate with the cognitive impairments, compulsion Gozzi et al., 19 and loss of inhibitory control over drug taking which may lead to relapse (Goldstein et al. 2010; Kalivas et al. 2005; Kalivas 2004; Hong et al. 2010; Strickland et al. 1993). Importantly, cognitive deficits have been observed in rats allowed extended (but not limited) access to cocaine (Briand et al. 2008; George et al. 2007), a phenomenon that involved working memory and sustained attention tasks (two prefrontal-cortex-dependent tasks) as well as object recognition measures (a hippocampus dependent task). The involvement of hippocampal systems is also consistent with the role played by this brain structure in contextual conditioning and memory, two functions that are altered by cocaine use and are believed to play a role in cue-elicited craving (reviewed by Koob and Volkow 2010). Likewise, the reduced bCBV in the nucleus accumbens was not unexpected, given the established interconnection between fronto-cortical activity and ventro-striatal DA cell firing and release (Kalivas et al. 2005; Peoples et al. 2007). In keeping with this, recent PET imaging studies showed lower levels of endogenous DA in cocaine addicts relative to comparison subjects (Martinez et al. 2009) and primate research revealed reduced glucose utilization in the striatal areas upon chronic cocaine use, a feature that became more pronounced with increased cocaine exposure (Porrino et al. 2007).

  ; Bifone and Gozzi 2010). Several phMRI studies have provided compelling evidence that the striatal haemodynamic response produced by amphetamine reflect primarily dopaminergic effects (reviewed in Knutson and Gibbs 2007). For example, amphetamine has been shown to elicit BOLD or rCBV increases in DA-rich ventro-striatal areas that are linearly-correlated to synaptic DA concentrations (Dixon et al. 2005; Ren et al. 2009; Choi et al. 2006; Schwarz et al. 2007b; Preece et al. 2007). Moreover, amphetamine-induced rCBV responses are abolished in the DA denervated areas (Chen et al. 1999; Chen et al. 1997),

Figure 1 A:

 1 Figure 1

Figure 2

 2 Figure 2

Figure 3

 3 Figure 3

Figure 4

 4 Figure 4

Acknowledgments

The authors would like to thank Valerio Crestan and Giuliano Turrini for their excellent technical support to the phMRI measures, and Pamela Rodegher from Histolab Verona, Italy for the histological preparations.

Gozzi et al., 24