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Official websites use. Share sensitive information only on official, secure websites. Cocaine addiction is often modeled in experimental paradigms where rodents learn to self-administer SA 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 SA 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 ventrostriatal 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 neurobiological substrate for the behavioral expression of compulsive drug intake in laboratory animals. Chronic cocaine use produces long-lasting neurobiological changes that are thought to underlie the loss of control over drug intake that defines cocaine dependence Koob et al , Human neuroimaging studies have started to shed light on the nature of these changes and their relationship with specific behaviors or symptoms. Reduced frontostriatal perfusion and metabolism in abstinent cocaine abusers have been reported by multiple investigators Strickland et al , ; London et al , ; Volkow et al , The disrupted function of frontal regions has been linked to the persistent neuropsychological deficits and impaired control over drug taking that frequently triggers relapse Strickland et al , ; Kalivas, 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 with addictive states Tomasi et al , ; Gu et al , Cocaine abuse is often modeled preclinically 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 Vanderschuren and Everitt, , uncontrolled drug use Ahmed and Koob, , and increased motivation to SA the drug Paterson and Markou, These features make these models an experimental tool of excellent face validity to investigate the neuroplastic events associated with voluntary drug intake Roberts et al , Moreover, the extent to which these models replicate the multiple neurofunctional 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. 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 Roberts et al , After a day detoxification period, we measured microvascular basal cerebral blood volume bCBV , an indirect indicator of resting brain function Gaisler-Salomon et al , ; Small et al , , 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 Gozzi et al , ; Schwarz et al , Correlation analyses between resting bCBV and amphetamine-evoked rCBV responses were performed in an attempt to identify dysregulation in circuits that control the recruitment and functional responsiveness of specific brain areas. Finally, post-mortem histopathological examinations were carried out to assess the potential contribution of direct vascular and neurotoxic effects of prolonged cocaine SA to the imaging findings. Experiments were carried out in accordance with the 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 Rats that underwent cocaine SA were tested in operant chambers as previously described Moretti et al , Each experimental chamber Med Associates, St Albans, VT was fitted with a cue light placed above each lever, and with a Hz tone module. An infusion pump was connected via an external catheter to a single-channel liquid swivel Instech Laboratories, Plymouth Meeting, PA. Data acquisition and operant-schedule parameters were controlled by a Med-PC software Med Associates. After their arrival, rats were acclimatized for 1 week and subsequently implanted with a catheter in the jugular vein as previously described Moretti et al , After a 7-day recovery period, rats were transported to the operant chamber. Cocaine SA procedure was initiated under a fixed ratio FR 1 schedule of reinforcement. Each press on the active lever was associated with a 0. Subjects that lost catheter patency or appeared unhealthy ie, showed signs of infection were removed from the study 11 subjects altogether. Session 30 was followed by a longer 5 days binge abstinence followed by two additional sessions. Such intervals were introduced because of the necessity to harmonize the timing of MRI scan and SA protocol over the relatively large number of subjects employed. A day detoxification period within the home cage was introduced before the imaging experiment. A group of 14 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. Imaging studies were performed 10 days after the last SA session. Animal preparation and MRI acquisition parameters have been previously described in greater detail Gozzi et al , ; Schwarz et al , The femoral artery and vein were cannulated and the animals were paralyzed with D-tubocurarine. After surgery, halothane level was set to 0. The body temperature of all subjects was maintained within physiological range and mean arterial blood pressure MABP was monitored continually through the femoral artery. Both coils are standard components provided by the manufacturer. Following five reference images, 2. D-amphetamine 0. The dose of -amphetamine was chosen based on previous in vivo studies Schwarz et al , ; Gozzi et al , The bCBV time-series image data for each experiment were analyzed within the framework of the general linear model Worsley et al , Individual subjects were spatially normalized to a stereotaxic rat brain MRI template set Schwarz et al , a. Signal intensity changes were converted into bCBV t on a pixel-wise basis as previously described Chen et al , ; Mandeville et al , Mean bCBV volumes for individual subjects were created by averaging the 10 time points time-wise. Linear detrending was introduced to account for contrast agent washout Schwarz et al , Voxel-wise group statistics was carried out using FSL Smith et al , using multilevel Bayesian inference, with 0. MRI signal intensity changes were converted into fractional CBV rCBV as previously described Mandeville et al , and detrended to account for contrast agent elimination from the blood pool Schwarz et al , Unsmoothed, rCBV time series for amphetamine challenge were calculated covering Voxel-wise statistics was carried out using FEAT with 0. In order to specifically test the hypothesis of an altered striatal reactivity to D-amphetamine in cocaine rats, a 3D binary mask of major subcortical area striatum, thalamus, hippocampus, hypothalamus the striatum, ventral pallidum, BNST, and amygdala was generated using a digital reconstruction of the rat brain atlas Schwarz et al , a and used to prethreshold rCBV time series before higher-level FSL analysis. This procedure increases the statistical power of the analysis by reducing the number of multiple comparisons Huettel et al , 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 repeated on non-masked rCBV data sets Supplementary Figure S5. Volume of interest VOI mean bCBV values and time courses for the amphetamine challenge were extracted as previously described Schwarz et al , a ; Gozzi et al , A number of representative VOIs were selected based on the results of the intergroup bCBV maps medial prefrontal, insular, orbitofrontal, somatosensory cortex, caudate putamen, nucleus accumbens, reticular thalamus, and 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-mean bCBV vector across the N subjects in the group from the selected reference structure. Histopathological evaluation was performed on 10 cocaine subject and 8 randomly chosen controls as previously described Barroso-Moguel et al , The slice and brain regions analyzed were 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. 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 A day detoxification period within the home cage was introduced before the imaging experiment MRI. 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 with the SA cocaine showed significantly reduced bCBV in several brain areas compared with control rats Figures 2 and 3. The effect was prominent in the medial-prefrontal, cingulate, orbitofrontal cortex, septum, ventral hippocampus, core region of the nucleus accumbens, as well as in raphe nuclei and reticular thalamic areas. The rostro-caudal positions of the coronal slice boundaries in c are indicated in mm relative to z bregma. 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 Schwarz et al , , amphetamine produced robust activation of subcortical and cortical areas in both groups of subjects Supplementary Figure S3. Rats chronically exposed to cocaine exhibited an attenuated functional response to amphetamine in the striatum compared with control rats Figure 4 and Supplementary Figure S3. Additional foci of reduced functional response to amphetamine were observed in sensory-motor and orbitofrontal cortex Supplementary Figures S3 and S5. Cpu, caudate putamen. The effect was not temporally correlated with the functional response, and was well within the blood flow autoregulatory range within which vasopressive responses are homeostatically compensated without producing significant rCBV alterations Gozzi et al , ; Zaharchuk et al , 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. No correlation between bCBV and amphetamine-induced rCBV responses was found in either group in any of the regions examined, with the exception of the reticular and posterior-ventral thalamus that revealed, in control subjects, an inverse relationship with amphetamine-induced rCBV in fronto-cortical areas Supplementary Figures S7 and S8. A histopathological evaluation of brain white and gray 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, and interstitial edema were observed in any of the brain regions examined, nor alterations of the microvascular and capillary bed ie, basal membrane dilation or rupture, hemorrhage, endothelial thickening or wall fibrosis, thrombi or occlusions, and necrosis or vacuolation of endothelial cells. 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 have 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 neurobiological substrate for the behavioral expression of compulsive drug intake in laboratory animals. 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 Parsons et al , ; Wilson et al , ; Wilson and Kish, 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 Vanderschuren and Everitt, , and high propensity to relapse to drug seeking Deroche-Gamonet et al , 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 Roberts et al , 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 Mantsch et al , ; Wee et al , , which explains the lack of lethality observed in this study. Compared with unlimited access protocols, where drug intake exhibits high and low numbers of infusions on alternating days Wilson et al , , the extended-access protocol used here ensured sustained SA of high doses of cocaine. In contrast to what was reported by other groups Ahmed and Koob, ; Ferrario et al , ; Wee et al , , we did not observe unequivocal evidence of dose escalation, although a trend toward an increased cocaine intake over consecutive sessions was apparent Supplementary Figure S2. Because this feature is present in ca. However, whether this trait is characterized by specific functional alterations separate from those highlighted in this study remains to be determined. A day washout period was introduced before the imaging study to rule out acute carryover effects of cocaine and minimize the potential interference of acute abstinence symptoms on the measures of brain function. It is therefore unlikely that the imaging findings contain major perturbations from transient neurobiological phenomena related to acute cocaine abstinence. On the other hand, the observed functional alterations are expected to contain contributions from longer-lasting neuroadaptational processes ie, incubation of cocaine craving that have been shown to build-up after cocaine discontinuation Lu et al , , 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 correlates with regional energy metabolism and cerebral blood flow Gaisler-Salomon et al , ; Hyder et al , ; Gonzalez et al , Our data showed the presence of reduced bCBV in the cingulated gyrus, prefrontal cortex, orbitofrontal cortex, as well as in striatal and hippocampal areas of cocaine SA subjects. The frontostriatal effect is in excellent agreement with clinical neuroimaging research of cocaine addiction, where reduced frontal and striatal activities have been consistently observed Strickland et al , ; Tumeh et al , ; London et al , ; Volkow et al , , and found to correlate with the cognitive impairments, compulsion, and loss of inhibitory control over drug taking that may lead to relapse Goldstein et al , ; Kalivas et al , ; Kalivas, ; Hong et al , ; Strickland et al , Importantly, cognitive deficits have been observed in rats allowed extended but not limited access to cocaine Briand et al , ; George et al , , 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, Likewise, the reduced bCBV in the nucleus accumbens was not unexpected, given the established interconnection between fronto-cortical activity and ventrostriatal DA cell firing and release Kalivas et al , ; Peoples et al , In keeping with this, recent PET imaging studies showed lower levels of endogenous DA in cocaine addicts relative to comparison subjects Martinez et al , 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 , 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 Volkow et al , and recent electrophysiological evidence of a state of protracted overinhibition of reticular thalamic areas following binge administration of cocaine Urbano et al , Interestingly, as serotonin exerts a direct excitatory action on GABAergic neurons in the reticular thalamus McCormick and Wang, , the reduced activity of these nuclei and that observed in regions of the raphe may be functionally interrelated and part of a single defective circuit. 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 that might exceed the quantity required to produce maximal bCBV alterations. In an attempt to identify an fMRI correlate of the decreased striatal dopaminergic responsivity observed in human PET studies Volkow et al , , ; Martinez et al , , we also mapped the functional response elicited by the DA-releaser amphetamine using a phMRI protocol Schwarz et al , ; Bifone and Gozzi, Several phMRI studies have provided compelling evidence that the striatal hemodynamic response produced by amphetamine reflects primarily the dopaminergic effects reviewed in Knutson and Gibbs, Moreover, amphetamine-induced rCBV responses are abolished in the DA denervated areas Chen et al , , , an effect that can be later restored after fetal or stem cell transplantation Bjorklund et al , ; Chen et al , 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 toward a reduced responsivity of ventrostriatal dopaminergic function analogous to what was observed in PET studies in humans Narendran and Martinez, As similarly attenuated striatal responses were not observed in rodent neuroimaging studies using short-term 5 days drug administration protocols Febo et al , ; Reese et al , ; and A Gozzi, 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 hemodynamic measures of brain function performed ie, 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 Jones et al , , a finding consistent with the functional connectivity of these regions Paxinos, and the high GABAergic density of reticular thalamic nucleus Paxinos, As prefrontal projections to the thalamic reticular nucleus play a unique circuit for attentional mechanisms Zikopoulos and Barbas, , we hypothesize that the loss of 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 Briand et al , ; George et al , 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 Gu et al , and when performing a cognitive task Tomasi et al , 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 SA. 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. All the authors are employees of GlaxoSmithKline. 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How can I buy cocaine online in Verona