Circadian rhythm disruptions associated with opioid use disorder in synaptic proteomes of human dorsolateral prefrontal cortex and nucleus accumbens.

Opioid craving and relapse vulnerability is associated with severe and persistent sleep and circadian rhythm disruptions. Understanding the neurobiological underpinnings of circadian rhythms and opioid use disorder (OUD) may prove valuable for developing new treatments for opioid addiction. Previous work indicated molecular rhythm disruptions in the human brain associated with OUD, highlighting synaptic alterations in the dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc)-key brain regions involved in cognition and reward, and heavily implicated in the pathophysiology of OUD. To provide further insights into the synaptic alterations in OUD, we used mass-spectrometry based proteomics to deeply profile protein expression alterations in bulk tissue and synaptosome preparations from DLPFC and NAc of unaffected and OUD subjects. We identified 55 differentially expressed (DE) proteins in DLPFC homogenates, and 44 DE proteins in NAc homogenates, between unaffected and OUD subjects. In synaptosomes, we identified 161 and 56 DE proteins in DLPFC and NAc, respectively, of OUD subjects. By comparing homogenate and synaptosome protein expression, we identified proteins enriched specifically in synapses that were significantly altered in both DLPFC and NAc of OUD subjects. Across brain regions, synaptic protein alterations in OUD subjects were primarily identified in glutamate, GABA, and circadian rhythm signaling. Using time-of-death (TOD) analyses, where the TOD of each subject is used as a time-point across a 24-h cycle, we were able to map circadian-related changes associated with OUD in synaptic proteomes associated with vesicle-mediated transport and membrane trafficking in the NAc and platelet-derived growth factor receptor beta signaling in DLPFC. Collectively, our findings lend further support for molecular rhythm disruptions in synaptic signaling in the human brain as a key factor in opioid addiction.

Integrative multi-dimensional characterization of striatal projection neuron heterogeneity in adult brain.

Striatal projection neurons (SPNs) are traditionally segregated into two subpopulations expressing dopamine (DA) D1-like or D2-like receptors. However, this dichotomy is challenged by recent evidence. Functional and expression studies raise important questions: do SPNs co-express different DA receptors, and do these differences reflect unique striatal spatial distributions and expression profiles? Using RNAscope in mouse striatum, we report heterogenous SPN subpopulations distributed across dorsal-ventral and rostral-caudal axes. SPN subpopulations co-express multiple DA receptors, including D1 and D2 (D1/2R) and D1 and D3. Our integrative approach using single-nuclei multi-omics analyses provides a simple consensus to describe SPNs across diverse datasets, connecting it to complementary spatial mapping. Combining RNAscope and multi-omics shows D1/2R SPNs further separate into distinct subtypes according to spatial organization and conserved marker genes. Each SPN cell type contributes uniquely to genetic risk for neuropsychiatric diseases. Our results bridge anatomy and transcriptomics to offer new understandings of striatal neuron heterogeneity.

Circadian rhythm disruptions associated with opioid use disorder in the synaptic proteomes of the human dorsolateral prefrontal cortex and nucleus accumbens.

Opioid craving and relapse vulnerability is associated with severe and persistent sleep and circadian rhythm disruptions. Understanding the neurobiological underpinnings of circadian rhythms and opioid use disorder (OUD) may prove valuable for developing new treatments for opioid addiction. Previous work indicated molecular rhythm disruptions in the human brain associated with OUD, highlighting synaptic alterations in the dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc)-key brain regions involved in cognition and reward, and heavily implicated in the pathophysiology of OUD. To provide further insights into the synaptic alterations in OUD, we used mass-spectrometry based proteomics to deeply profile protein expression alterations in bulk tissue and synaptosome preparations from DLPFC and NAc of unaffected and OUD subjects. We identified 55 differentially expressed (DE) proteins in DLPFC homogenates, and 44 DE proteins in NAc homogenates, between unaffected and OUD subjects. In synaptosomes, we identified 161 and 56 DE proteins in DLPFC and NAc, respectively, of OUD subjects. By comparing homogenate and synaptosome protein expression, we identified proteins enriched specifically in synapses that were significantly altered in both DLPFC and NAc of OUD subjects. Across brain regions, synaptic protein alterations in OUD subjects were primarily identified in glutamate, GABA, and circadian rhythm signaling. Using time-of-death (TOD) analyses, where the TOD of each subject is used as a time-point across a 24- hour cycle, we were able to map circadian-related changes associated with OUD in synaptic proteomes related to vesicle-mediated transport and membrane trafficking in the NAc and platelet derived growth factor receptor beta signaling in DLPFC. Collectively, our findings lend further support for molecular rhythm disruptions in synaptic signaling in the human brain as a key factor in opioid addiction.

Chronic Morphine Modulates PDGFR-ß and PDGF-B Expression and Distribution in Dorsal Root Ganglia and Spinal Cord in Male Rats.

The analgesic effect of opioids decreases over time due to the development of analgesic tolerance. We have shown that inhibition of the platelet-derived growth factor beta (PDGFR-ß) signaling eliminates morphine analgesic tolerance in rats. Although the PDGFR-ß and its ligand, the platelet-derived growth factor type B (PDGF-B), are expressed in the substantia gelatinosa of the spinal cord (SG) and in the dorsal root ganglia (DRG), their precise distribution within different cell types of these structures is unknown. Additionally, the impact of a tolerance-mediating chronic morphine treatment, on the expression and distribution of PDGF-B and PDGFR-ß has not yet been studied. Using immunohistochemistry (IHC), we found that in the spinal cord, PDGFR-ß and PDGF-B were expressed in neurons and oligodendrocytes and co-localized with the mu-opioid receptor (MOPr) in opioid naïve rats. PDGF-B was also found in microglia and astrocytes. Both PDGFR-ß and PDGF-B were detected in DRG neurons but not in spinal primary afferent terminals. Chronic morphine exposure did not change the cellular distribution of PDGFR-ß or PDGF-B. However, PDGFR-ß expression was downregulated in the SG and upregulated in the DRG. Consistent with our previous finding that morphine caused tolerance by inducing PDGF-B release, PDGF-B was upregulated in the spinal cord. We also found that chronic morphine exposure caused a spinal proliferation of oligodendrocytes. The changes in PDGFR-ß and PDGF-B expression induced by chronic morphine treatment suggest potential mechanistic substrates underlying opioid tolerance.

A Spatial Atlas of Wnt Receptors in Adult Mouse Liver.

Hepatic zonation is critical for most metabolic functions in liver. Wnt signaling plays an important role in establishing and maintaining liver zonation. Yet, the anatomic expression of Wnt signaling components, especially all 10 Frizzled (Fzd) receptors, has not been characterized in adult liver. To address this, the spatial expression of Fzd receptors was quantitatively mapped in adult mouse liver via multiplex fluorescent in situ hybridization. Although all 10 Fzd receptors were expressed within a metabolic unit, Fzd receptors 1, 4, and 6 were the highest expressed. Although most Wnt signaling occurs in zone 3, expression of most Fzd receptors was not zonated. In contrast, Fzd receptor 6 was preferentially expressed in zone 1. Wnt2 and Wnt9b expression was highly zonated and primarily found in zone 3. Therefore, the current results suggest that zonated Wnt/ß-catenin signaling at baseline occurs primarily due to Wnt2 and Wnt9b rather than zonation of Fzd mRNA expression. Finally, the study showed that Fzd receptors and Wnts are not uniformly expressed by all hepatic cell types. Instead, there is broad distribution among both hepatocytes and nonparenchymal cells, including endothelial cells. Overall, this establishment of a definitive mRNA expression atlas, especially of Fzd receptors, opens the door to future functional characterization in healthy and diseased liver states.

Mu-opioid receptor and receptor tyrosine kinase crosstalk: Implications in mechanisms of opioid tolerance, reduced analgesia to neuropathic pain, dependence, and reward.

Despite the prevalence of opioid misuse, opioids remain the frontline treatment regimen for severe pain. However, opioid safety is hampered by side-effects such as analgesic tolerance, reduced analgesia to neuropathic pain, physical dependence, or reward. These side effects promote development of opioid use disorders and ultimately cause overdose deaths due to opioid-induced respiratory depression. The intertwined nature of signaling via µ-opioid receptors (MOR), the primary target of prescription opioids, with signaling pathways responsible for opioid side-effects presents important challenges. Therefore, a critical objective is to uncouple cellular and molecular mechanisms that selectively modulate analgesia from those that mediate side-effects. One such mechanism could be the transactivation of receptor tyrosine kinases (RTKs) via MOR. Notably, MOR-mediated side-effects can be uncoupled from analgesia signaling via targeting RTK family receptors, highlighting physiological relevance of MOR-RTKs crosstalk. This review focuses on the current state of knowledge surrounding the basic pharmacology of RTKs and bidirectional regulation of MOR signaling, as well as how MOR-RTK signaling may modulate undesirable effects of chronic opioid use, including opioid analgesic tolerance, reduced analgesia to neuropathic pain, physical dependence, and reward. Further research is needed to better understand RTK-MOR transactivation signaling pathways, and to determine if RTKs are a plausible therapeutic target for mitigating opioid side effects.

Sex-specific role of the circadian transcription factor NPAS2 in opioid tolerance, withdrawal and analgesia.

Opioids like fentanyl remain the mainstay treatment for chronic pain. Unfortunately, opioid's high dependence liability has led to the current opioid crisis, in part, because of side-effects that develop during long-term use, including analgesic tolerance and physical dependence. Both tolerance and dependence to opioids may lead to escalation of required doses to achieve previous therapeutic efficacy. Additionally, altered sleep and circadian rhythms are common in people on opioid therapy. Opioids impact sleep and circadian rhythms, while disruptions to sleep and circadian rhythms likely mediate the effects of opioids. However, the mechanisms underlying these bidirectional relationships between circadian rhythms and opioids remain largely unknown. The circadian protein, neuronal PAS domain protein 2 (NPAS2), regulates circadian-dependent gene transcription in structure of the central nervous system that modulate opioids and pain. Here, male and female wild-type and NPAS2-deficient (NPAS2-/-) mice were used to investigate the role of NPAS2 in fentanyl analgesia, tolerance, hyperalgesia and physical dependence. Overall, thermal pain thresholds, acute analgesia and tolerance to a fixed dose of fentanyl were largely similar between wild-type and NPAS2-/- mice. However, female NPAS2-/- exhibited augmented analgesic tolerance and significantly more behavioral symptoms of physical dependence to fentanyl. Only male NPAS2-/- mice had increased fentanyl-induced hypersensitivity, when compared with wild-type males. Together, our findings suggest sex-specific effects of NPAS2 signaling in the regulation of fentanyl-induced tolerance, hyperalgesia and dependence.

Brain region- and sex-specific transcriptional profiles of microglia.

Microglia are resident macrophages of the brain, performing roles related to brain homeostasis, including modulation of synapses, trophic support, phagocytosis of apoptotic cells and debris, as well as brain protection and repair. Studies assessing morphological and transcriptional features of microglia found regional differences as well as sex differences in some investigated brain regions. However, markers used to isolate microglia in many previous studies are not expressed exclusively by microglia or cannot be used to identify and isolate microglia in all contexts. Here, fluorescent activated cell sorting was used to isolate cells expressing the microglia-specific marker TMEM119 from prefrontal cortex (PFC), striatum, and midbrain in mice. RNA-sequencing was used to assess the transcriptional profile of microglia, focusing on brain region and sex differences. We found striking brain region differences in microglia-specific transcript expression. Most notable was the distinct transcriptional profile of midbrain microglia, with enrichment for pathways related to immune function; these midbrain microglia exhibited a profile similar to disease-associated or immune-surveillant microglia. Transcripts more highly expressed in PFC isolated microglia were enriched for synapse-related pathways while microglia isolated from the striatum were enriched for pathways related to microtubule polymerization. We also found evidence for a gradient of expression of microglia-specific transcripts across the rostral-to-caudal axes of the brain, with microglia extracted from the striatum exhibiting a transcriptional profile intermediate between that of the PFC and midbrain. We also found sex differences in expression of microglia-specific transcripts in all 3 brain regions, with many selenium-related transcripts more highly expressed in females across brain regions. These results suggest that the transcriptional profile of microglia varies between brain regions under homeostatic conditions, suggesting that microglia perform diverse roles in different brain regions and even based on sex.

A role for the circadian transcription factor NPAS2 in the progressive loss of non-rapid eye movement sleep and increased arousal during fentanyl withdrawal in male mice.

RATIONALE: Synthetic opioids like fentanyl are contributing to the rise in rates of opioid use disorder and drug overdose deaths. Sleep dysfunction and circadian rhythm disruption may worsen during opioid withdrawal and persist during abstinence. Severe and persistent sleep and circadian alterations are putative factors in opioid craving and relapse. However, very little is known about the impact of fentanyl on sleep architecture and sleep-wake cycles, particularly opioid withdrawal. Further, circadian rhythms regulate sleep-wake cycles, and the circadian transcription factor, neuronal PAS domain 2 (NPAS2) is involved in the modulation of sleep architecture and drug reward. Here, we investigate the role of NPAS2 in fentanyl-induced sleep alterations. OBJECTIVES: To determine the effect of fentanyl administration and withdrawal on sleep architecture, and the role of NPAS2 as a factor in fentanyl-induced sleep changes. METHODS: Electroencephalography (EEG) and electromyography (EMG) was used to measure non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS) at baseline and following acute and chronic fentanyl administration in wild-type and NPAS2-deficient male mice. RESULTS: Acute and chronic administration of fentanyl led to increased wake and arousal in both wild-type and NPAS2-deficient mice, an effect that was more pronounced in NPAS2-deficient mice. Chronic fentanyl administration led to decreased NREMS, which persisted during withdrawal, progressively decreasing from day 1 to 4 of withdrawal. The impact of fentanyl on NREMS and arousal was more pronounced in NPAS2-deficient mice. CONCLUSIONS: Chronic fentanyl disrupts NREMS, leading to a progressive loss of NREMS during subsequent days of withdrawal. Loss of NPAS2 exacerbates the impact of fentanyl on sleep and wake, revealing a potential role for the circadian transcription factor in opioid-induced sleep changes.

Lateralized Decrease of Parvalbumin+ Cells in the Somatosensory Cortex of ASD Models Is Correlated with Unilateral Tactile Hypersensitivity.

Inhibitory control of excitatory networks contributes to cortical functions. Increasing evidence indicates that parvalbumin (PV+)-expressing basket cells (BCs) are a major player in maintaining the balance between excitation (E) and inhibition (I). Disruption of E/I balance in cortical networks is believed to be a hallmark of autism spectrum disorder (ASD). Here, we report a lateralized decrease in the number of PV+ BCs in L2/3 of the somatosensory cortex in the dominant hemisphere of Shank3-/- and Cntnap2-/- mouse models of ASD. The dominant hemisphere was identified during a reaching task to establish each animal's dominant forepaw. Double labeling with anti-PV antibody and a biotinylated lectin (Vicia villosa lectin [VVA]) showed that the number of BCs was not different but rather, some BCs did not express PV (PV-), resulting in an elevated number of PV- VVA+ BCs. Finally, we showed that dominant hindpaws had higher mechanical sensitivity when compared with the other hindpaws. This mechanical hypersensitivity in the dominant paw strongly correlated with the decrease in the number of PV+ interneurons and reduced PV expression in the corresponding cortex. Together, these results suggest that the hypersensitivity in ASD patients could be due to decreased inhibitory inputs to the dominant somatosensory cortex.

Spinal Cord Stimulation Reduces Ventricular Arrhythmias by Attenuating Reactive Gliosis and Activation of Spinal Interneurons.

OBJECTIVES: This study investigated spinal cord neuronal and glial cell activation during cardiac ischemia-reperfusion (IR)-triggered ventricular arrhythmias and neuromodulation therapy by spinal cord stimulation (SCS). BACKGROUND: Myocardial ischemia induces changes in cardiospinal neural networks leading to sudden cardiac death. Neuromodulation with SCS decreases cardiac sympathoexcitation; however, the molecular mechanisms remain unknown. METHODS: Yorkshire pigs (n = 16) were randomized to Control, IR, or IR+SCS groups. A 4-pole SCS lead was placed in the T1-T4 epidural space with stimulation for 30 minutes before IR (50 Hz, 0.4-ms duration, 90% motor threshold). Cardiac electrophysiological mapping and Ventricular Arrhythmia Score (VAS) were recorded. Immunohistochemistry of thoracic spinal sections was used to map and identify Fos-positive neuronal and glial cell types during IR with and without SCS. RESULTS: IR increased cardiac sympathoexcitation and arrhythmias (VAS = 6.2 ± 0.9) that were attenuated in IR + SCS (VAS = 2.8 ± 0.5; P = 0.017). IR increased spinal cellular Fos expression (#Fos+ cells Control = 23 ± 2 vs IR = 88 ± 5; P < 0.0001) in T1-T4, with the greatest increase localized to T3, and the greatest %Fos+ cells being microglia and astrocytes. Fos expression was attenuated by IR + SCS (62 ± 4; P < 0.01), primarily though a reduction in Fos+ microglia and astrocytes, as SCS also led to increase in Fos+ neurons in deep dorsal laminae. CONCLUSIONS: In a porcine model, cardiac IR was associated with astrocyte and microglial cell activation. Our results suggest that preemptive thoracic SCS decreased IR-induced cardiac sympathoexcitation and ventricular arrhythmias through attenuation of reactive gliosis and activation of inhibitory interneurons in the dorsal horn of spinal cord.

EGFR Signaling Causes Morphine Tolerance and Mechanical Sensitization in Rats.

The safety and efficacy of opioids are compromised as analgesic tolerance develops. Opioids are also ineffective against neuropathic pain. Recent reports have suggested that inhibitors of the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK), may have analgesic effects in cancer patients suffering from neuropathic pain. It has been shown that the platelet-derived growth factor receptor-ß (PDGFR-ß), an RTK that has been shown to interact with the EGFR, mediates opioid tolerance but does not induce analgesia. Therefore, we sought to determine whether EGFR signaling was involved in opioid tolerance and whether EGFR and PDGFR signaling could induce pain in rats. We found that gefitinib, an EGFR antagonist, eliminated morphine tolerance. In addition, repeated EGF administration rendered animals unresponsive to subsequent analgesic doses of morphine, a phenomenon we call "pre-tolerance." Using a nerve injury model, we found that gefitinib alone was not analgesic. Rather, it reversed insensitivity to morphine analgesia (pre-tolerance) caused by the release of EGF by injured nerves. We also showed that repeated, but not acute EGF or PDGF-BB administration induced mechanical hypersensitivity in rats. EGFR and PDGFR-ß signaling interacted to produce this sensitization. EGFR was widely expressed in primary sensory afferent cell bodies, demonstrating a neuroanatomical substrate for our findings. Taken together, our results suggest a direct mechanistic link between opioid tolerance and mechanical sensitization. EGFR antagonism could eventually play an important clinical role in the treatment of opioid tolerance and neuropathic pain that is refractory to opioid treatment.

Growth Factor Signaling Regulates Mechanical Nociception in Flies and Vertebrates.

Mechanical sensitization is one of the most difficult clinical pain problems to treat. However, the molecular and genetic bases of mechanical nociception are unclear. Here we develop a Drosophila model of mechanical nociception to investigate the ion channels and signaling pathways that regulate mechanical nociception. We fabricated von Frey filaments that span the subthreshold to high noxious range for Drosophila larvae. Using these, we discovered that pressure (force/area), rather than force per se, is the main determinant of aversive rolling responses to noxious mechanical stimuli. We demonstrated that the RTK PDGF/VEGF receptor (Pvr) and its ligands (Pvfs 2 and 3) are required for mechanical nociception and normal dendritic branching. Pvr is expressed and functions in class IV sensory neurons, whereas Pvf2 and Pvf3 are produced by multiple tissues. Constitutive overexpression of Pvr and its ligands or inducible overexpression of Pvr led to mechanical hypersensitivity that could be partially separated from morphological effects. Genetic analyses revealed that the Piezo and Pain ion channels are required for mechanical hypersensitivity observed upon ectopic activation of Pvr signaling. PDGF, but not VEGF, peptides caused mechanical hypersensitivity in rats. Pharmacological inhibition of VEGF receptor Type 2 (VEGFR-2) signaling attenuated mechanical nociception in rats, suggesting a conserved role for PDGF and VEGFR-2 signaling in regulating mechanical nociception. VEGFR-2 inhibition also attenuated morphine analgesic tolerance in rats. Our results reveal that a conserved RTK signaling pathway regulates baseline mechanical nociception in flies and rats.SIGNIFICANCE STATEMENT Hypersensitivity to touch is poorly understood and extremely difficult to treat. Using a refined Drosophila model of mechanical nociception, we discovered a conserved VEGF-related receptor tyrosine kinase signaling pathway that regulates mechanical nociception in flies. Importantly, pharmacological inhibition of VEGF receptor Type 2 signaling in rats causes analgesia and blocks opioid tolerance. We have thus established a robust, genetically tractable system for the rapid identification and functional analysis of conserved genes underlying mechanical pain sensitivity.

Platelet-derived growth factor activates nociceptive neurons by inhibiting M-current and contributes to inflammatory pain.

Endogenous inflammatory mediators contribute to the pathogenesis of pain by acting on nociceptors, specialized sensory neurons that detect noxious stimuli. Here, we describe a new factor mediating inflammatory pain. We show that platelet-derived growth factor (PDGF)-BB applied in vitro causes repetitive firing of dissociated nociceptor-like rat dorsal root ganglion neurons and decreased their threshold for action potential generation. Injection of PDGF-BB into the paw produced nocifensive behavior in rats and led to thermal and mechanical pain hypersensitivity. We further detailed the biophysical mechanisms of these PDGF-BB effects and show that PDGF receptor-induced inhibition of nociceptive M-current underlies PDGF-BB-mediated nociceptive hyperexcitability. Moreover, in vivo sequestration of PDGF or inhibition of the PDGF receptor attenuates acute formalin-induced inflammatory pain. Our discovery of a new pain-facilitating proinflammatory mediator, which by inhibiting M-current activates nociceptive neurons and thus contributes to inflammatory pain, improves our understanding of inflammatory pain pathophysiology and may have important clinical implications for pain treatment.

A "tail" of opioid receptor variants.

Opioids are the gold-standard treatment for severe pain. However, potentially life-threatening side effects decrease the safety and effectiveness of these compounds. The addiction liability of these drugs has led to the current epidemic of opioid abuse in the US. Extensive research efforts have focused on trying to dissociate the analgesic properties of opioids from their undesirable side effects. Splice variants of the mu opioid receptor (MOR), which mediates opioid actions, have unique pharmacological properties and anatomic distributions that make them attractive candidates for therapeutic pain relief. In this issue of the JCI, Xu et al. show that specific C-terminal regions of the MOR can modulate side effects without altering analgesia. This discovery greatly improves our understanding of opioid side effects and suggests intriguing therapeutic approaches that could improve both the safety and long-term effectiveness of opioids.

Temporal regulation of peripheral BDNF levels during cocaine and morphine withdrawal: comparison with a natural reward.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is a neurotrophin that has long been studied in the field of addiction and its importance in regulating drug addiction-related behavior has been widely demonstrated. The aim of our study was to analyze the consequences of a repeated exposure to drugs of abuse or natural reward on plasma BDNF levels during withdrawal. METHODS: Rats were chronically injected with morphine (subcutaneously, 5mg/kg) or cocaine (intraperitoneally, 20mg/kg) or fed with a butter biscuit (per os, 4g) once per day for 14 days. Blood collection was performed on the 1st (withdrawal day 1 or WD1) or on (WD14), either at the same time point rats had been exposed to drugs or natural reward or at a different time point (used to quantify basal brain-derived neurotrophic factor levels). RESULTS: Cocaine treatment led to a rapid (WD1) and persistent (WD14) decrease of basal BDNF levels compared with saline-treated animals, whereas morphine induced an increase on WD14 without any alteration on WD1. On the contrary, the natural reward induced a significant increase of basal brain-derived neurotrophic factor levels only on WD1. The analysis of BDNF levels at the usual time point at which animals had been exposed showed that both drugs, but not the natural reward, increased BDNF levels compared with basal levels. CONCLUSION: Our data highlight that only drugs of abuse are able to persistently alter BDNF levels and to induce specific variations of this neurotrophic factor at the usual hour of injection.

Influence of cocaine administration patterns on dopamine receptor regulation.

RATIONALE: Chronic exposure to drugs of abuse induces important modifications on neuronal systems. Increasing evidence shows that the consequences to chronic cocaine exposure can be different depending on the administration pattern. OBJECTIVES: The aim of the present study was to evaluate the consequences of two cocaine administration patterns on dopaminergic receptor regulation. METHODS: Male Sprague-Dawley rats were injected with cocaine (20 mg/kg, i.p.) for 14 days according to an intermittent (one daily injection) or a binge (three daily injections) pattern. By autoradiography, we compared the modifications of dopamine D1 and D2 receptor densities in the dopaminergic systems (mesocorticolimbic and nigrostriatal) 1 (WD1) and 14 (WD14) days after the last cocaine injection. RESULTS: On WD1, we observed modifications of D1 receptors after the binge cocaine treatment pattern while no modification was observed after the intermittent pattern, suggesting that multiple daily injections are needed to induce early D1 receptor modifications. On the contrary, densities of the D2 receptors were modified by both cocaine administration patterns, and interestingly, they were opposite depending on the administration pattern. On WD14, we observed different modifications of D1 and D2 receptors depending on the administration pattern, suggesting that the cocaine administration pattern promoted long-term regulations of the dopaminergic system. CONCLUSION: Two cocaine administration patterns induce different modifications of the dopaminergic receptor densities.