AbstractINTRODUCTIONSeed amplification assays (SAAs) demonstrate remarkable diagnostic performance in alpha‐synucleinopathies. However, existing protocols lack accessibility in routine laboratories, mainly due to the requirement for in‐house production of recombinant alpha‐synuclein (aSyn). This study proposes a cerebrospinal fluid (CSF) aSyn‐SAA protocol using solely commercial reagents to facilitate its clinical implementation.METHODSRoutine clinical care CSF samples from 126 patients, comprising 47 with Lewy body diseases (LBD) (41 with dementia with Lewy bodies, six with Parkinson's disease), 37 without alpha‐synucleinopathy, and 42 with Alzheimer's disease (AD), underwent assessment for aSyn‐SAA activity.RESULTSCSF aSyn‐SAA showed a sensitivity of 72.3% and a specificity of 100% when distinguishing clinically diagnosed LBD patients from those without alpha‐synucleinopathy. In AD patients, 14.3% were tested positive for aSyn.DISCUSSIONThe commercial‐only CSF aSyn‐SAA protocol exhibited excellent specificity when applied to a real‐life cohort, signaling progress toward the accessibility of an aSyn biomarker in clinical settings.Highlights Diagnosis of LBD through aSyn‐SAA lacks accessibility. This commercial‐only aSyn‐SAA has satisfactory performance in a real‐life cohort. A negative aSyn‐SAA does not completely exclude a synucleinopathy. Some technical points must be considered when developing aSyn‐SAA. aSyn‐SAA must be confined to expert laboratories due to prion‐like risk management.

Le diagnostic des α-synucléinopathies (A-syn) par la détection des formes agrégées d'alpha-synucléine semble prometteur. Cependant, les techniques proposées manquent de standardisation et nécessitent d'être validées avant leur utilisation en routine. Nous proposons un protocole de détection des formes agrégées par « Real Time-Quaking Induced Conversion » (RT-QuIC) utilisant uniquement des réactifs commerciaux pour le rendre accessible aux laboratoires de routine. Vingt-deux liquides cérébrospinaux (LCS) de patients atteints d'A-syn (démence à Corps de Lewy [DCL]) (n = 11), maladie de Parkinson (Park) (n = 6), DCL avec co-pathologies (n = 5) et 28 contrôles ont été retenus dans cette étude rétrospective. Le diagnostic a été établi lors de réunions de concertation pluridisciplinaires. La mise au point a été réalisée sur tissu cérébral post-mortem puis validée sur LCS sur l'automate FluoSTAR OMEGA (BMG Labtech®). Les réactifs utilisés sont uniquement issus du commerce. La sensibilité et la spécificité de notre protocole sur tissu cérébral sont de 100 %. Le protocole développé pour la détection dans le LCS a permis d'amplifier les formes agrégées d'alpha-synucléine avec une sensibilité de 77,3 % (n = 22) et une spécificité de 100 % (n = 28). Les sensibilités respectives par étiologie sont de 72,7 % pour les DCL, 100 % pour les Park, 60 % pour les DCL avec co-pathologie Alzheimer (n = 5) (Fig. 1, Fig. 2). La détection sur tissu cérébral est excellente pour les patients atteints d'A-syn et absente des contrôles. Dans le LCS, la méthode reste très spécifique mais avec une sensibilité légèrement moins élevée que dans la littérature, probablement du fait de l'analyse d'une cohorte en vie réelle. L'étude de LCS de patients confirmés neuropathologiquement permettrait de consolider nos premiers résultats. La détection d'A-syn agrégée dans le LCS par notre protocole RT-QuIC est fonctionnelle et très spécifique. Une étude multicentrique permettra de confirmer ces performances avant d'envisager son utilisation en routine.

Amyloid fibrils are self-assembled mesoscopic protein aggregates, which can accumulate to form deposits or plaques in the brain. In vitro amplification of fibrils can be achieved with real-time quaking-induced conversion (RT-QuIC). However, this emerging technique would benefit from a complementary method to assess structural properties of the amplification products. This work demonstrates the feasibility of nanospray-charge-detection-mass-spectrometry (CDMS) performed on α-synuclein (αSyn) fibrils amplified from human brains with Parkinson's disease (PD) or Dementia with Lewy bodies (DLB) and its synergistic combination with RT-QuIC.

Les maladies à prions sont des maladies neurodégénératives rares d’issue toujours fatale. Elles sont caractérisées par l’accumulation principalement cérébrale d’une isoforme pathologique (PrPsc) d’une protéine cellulaire normale, la PrPc. Du fait de leur caractère transmissible d’homme à homme et animal-homme, ces maladies sont étroitement surveillées par les autorités de santé et à déclaration obligatoire dès leur suspicion. Leur diagnostic est donc un enjeu majeur de santé publique alors même que le diagnostic de certitude reste l’examen neuropathologique, très majoritairement réalisé en post-mortem. Les marqueurs biologiques jouent un rôle important dans la démarche diagnostique du vivant du patient et dans la surveillance de ces maladies. En effet, la détection de la protéine 1433 dans le liquide cérébrospinal (LCS), marqueur non étiologique, permet d’augmenter la probabilité diagnostique. De nombreux efforts ont été menés pour mesurer directement la PrPsc dans les fluides biologiques. Seuls des progrès technologiques récents reposant sur des méthodes innovantes d’amplification des protéines ont permis de la mesurer dans le LCS. Cependant, ces technologies nécessitent une mise en œuvre dans des environnements de sécurité microbiologique de niveau 3. Ces techniques d’avenir, réservées à des centres de référence experts, sont de réalisation délicate, manquent de robustesse et de standardisation avant de pouvoir être proposées comme outils diagnostiques en pratique courante. Prion diseases are fatal neurodegenerative disorders which are characterized by aggregations of a misfolded protein (called PrPsc) coming from its cellular isoform PrPc, mainly in brain tissue. Due to the transmissibility of this agent, every suspected case has to be monitored and reported to health authorities. Prion diseases diagnosis is a major public health issue, while the definite diagnostic is still based on post-mortem neuropathological examination. CSF biomarkers are an integral part of the diagnostic criteria and are important for the disease surveillance. The 14-3-3 detection is the first CSF surrogate biomarker helping in probable CJD classification. Despite many efforts for specific PrPsc detection, only recent technological development of assays based on protein amplification allowed to detect it in the CSF. However, reference centers have to manage this innovative technology in high security facilities (level 3). Then, nowadays, these protein amplification assays need to be optimized and standardized before their use in current practice.

To compare CSF biomarkers’ levels in patients suffering from anti-Leucine-rich Glioma-Inactivated 1 (LGI1) encephalitis to neurodegenerative [Alzheimer’s disease (AD), Creutzfeldt–Jakob’s disease (CJD)] and primary psychiatric (PSY) disorders. Patients with LGI1 encephalitis were retrospectively selected from the French Reference Centre database between 2010 and 2019 and enrolled if CSF was available for biomarkers analysis including total tau (T-tau), phosphorylated tau (P-tau), amyloid-beta Aβ1-42, and neurofilaments light chains (Nf L). Samples sent for biomarker determination as part of routine practice, and formally diagnosed as AD, CJD, and PSY, were used as comparators. Twenty-four patients with LGI1 encephalitis were compared to 39 AD, 20 CJD and 20 PSY. No significant difference was observed in T-tau, P-tau, and Aβ1-42 levels between LGI1 encephalitis and PSY patients. T-Tau and P-Tau levels were significantly lower in LGI1 encephalitis (231 and 43 ng/L) than in AD (621 and 90 ng/L, p < 0.001) and CJD patients (4327 and 55 ng/L, p < 0.001 and p < 0.01). Nf L concentrations of LGI1 encephalitis (2039 ng/L) were similar to AD (2,765 ng/L) and significantly higher compared to PSY (1223 ng/L, p < 0.005), but significantly lower than those of CJD (13,457 ng/L, p < 0.001). Higher levels of Nf L were observed in LGI1 encephalitis presenting with epilepsy (3855 ng/L) compared to LGI1 without epilepsy (1490 ng/L, p = 0.02). No correlation between CSF biomarkers’ levels and clinical outcome could be drawn. LGI encephalitis patients showed higher Nf L levels than PSY, comparable to AD, and even higher when presenting epilepsy suggesting axonal or synaptic damage linked to epileptic seizures.

Objectives: To estimate the attributable fraction of nursing home placement associated with cognitive impairment, neuropsychiatric symptoms, behavioral disorders, functional limitations, and caregiver burden. Design: Longitudinal study conducted on the "MEMORA cohort" linked with both regional public health insurance and hospital discharge databases. Setting: Memory center at the University Hospital of Lyon, France. Participants: A sample of 2456 outpatients attending the memory center between 2012 and 2017. Measures: Cognitive impairment, functional limitations, neuropsychiatric symptoms/behavioral disorders, and caregiver burden were measured with the Mini-Mental State Examination, the Instrumental Activities of Daily Living scale, the Neuropsychiatric Inventory (NPI), and a short version of the Zarit Burden Inventory, respectively. Sociodemographics characteristics were collected during the first visit. Comorbidities were gathered from the hospital discharge database. Dates of nursing home placement were obtained from the public health insurance database. Results: More than 38% of nursing home placements were attributable to caregiver burden, and the attributable fraction associated with functional limitations exceeded 35%. Between 20% and 25% of nursing home placements were due to cognitive impairment whereas less than 16% were attributable to neuropsychiatric symptoms or behavioral disorders. The associations between anxiety or agitation and nursing home placement were mediated by caregiver burden. Apathy or aberrant motor behaviors were associated with a higher risk of nursing home placement independently of caregiver burden. Conclusions/Implications: Our findings suggest that a high proportion of nursing home placements are attributable to caregiver burden and functional limitations in outpatients attending a memory center. Cognitive impairment and neuropsychiatric symptoms or behavioral disorders contribute less to nursing home placements. Interventions directed to delay nursing home placement should emphasize actions toward reducing caregiver burden and functional limitations of patients. (C) 2019 AMDA - The Society for Post-Acute and Long-Term Care Medicine.

The serotonergic system plays a key modulatory role in the brain. This system is critical in many drug developments for brain disorders via interactions with the 14 subtypes of 5-HT receptors or through reuptake blockade. Positron emission tomography (PET) is an efficient tool for in vivo studies of physiological and pathological processes. Because of its pertinent radiochemical properties, fluorine-18 is one of the most used radioisotopes in PET imaging. This chapter will propose an overview of the 18F-radioligands targeting serotonin receptors, which have been developed over the last few years. Both radiosyntheses and pharmacological properties of these radiotracers will be described, with a specific emphasis on their potential medical applications.

The identification of factors associated with functional impairment, in particular those which are potentially modifiable, may help to delay the advanced stages of functional dependence in patients with neurocognitive disorders such as Alzheimer’s disease and related disorders. The objectives of the MEMORA cohort are to investigate the factors associated, first with functional autonomy change over time, and secondarily with the cognitive performance and behavioral disorders changes over time. The MEMORA study is a multicenter prospective cohort study carried out throughout the patient’s care pathway, in Memory centers of Lyon (France). The study will include 6780 patients at all stages of memory disorders in 6 years. The follow-up for each patient is planned for 3 years. The main outcome is the functional autonomy level change as assessed by the instrumental abilities of daily living (IADL) score. Patient characteristics include sociodemographic and clinical features, neuropsychological performance, pharmaceutical and non-pharmaceutical therapy. This study conducted in a context of routine care may help to identify the factors associated with functional impairment related to progressive neurocognitive disorders. Subsequently, interventions on potentially modifiable factors could be proposed to the patients to improve their management and delay functional dependence. NCT02302482 , registered 27 November 2014.

The accumulation of aggregated alpha-synuclein ( α -syn) in multiple brain regions is a neuropathological hallmark of synucleinopathies. Multiple system atrophy (MSA) is a synucleinopathy characterized by the predominant cerebral accumulation of aggregated α -syn as cytoplasmic glial inclusions (CGI). A premortem diagnosis tool would improve early diagnosis and help monitoring disease progression and therapeutic efficacy. One Positron Emission Tomography (PET) study suggested [ 11 C]BF-227 as a promising radiotracer for monitoring intracellular α -syn deposition in MSA patients. We sought to confirm the binding of this radiotracer to α -syn using state-of-the-art autoradiography. Medulla sections were obtained from 9 MSA patients and 9 controls (London Neurodegenerative Diseases Brain Bank). [ 18 F]BF-227, chemically identical to [ 11 C]BF-227, was used at nanomolar concentrations to perform in vitro autoradiography assays. Autoradiograms were superimposed on fluorescent staining from the conformational anti- α -syn antibody 5G4 and quantified after immunofluorescence-driven definition of regions of interest. Autoradiography showed no specific signals in MSA patients in comparison to controls despite widespread pathology detected by immunofluorescence. Autoradiography does not support a significant binding of [ 18 F]BF-227 to CGI at concentrations typically achieved in PET experiments.

Accumulation of α-synuclein (α-syn) is a neuropathological hallmark of synucleinopathies. To date, no selective α-syn positron emission tomography (PET) radiotracer has been identified. Our objective was to develop the first original, selective, and specific α-syn PET radiotracer. Chemical design inspired from three structural families that demonstrated interesting α-syn binding characteristics was used as a starting point. Bioinformatics modeling of α-syn fibrils was then employed to select the best molecular candidates before their syntheses. An in vitro binding assay was performed to evaluate the affinity of the compounds. Radiotracer specificity and selectivity were assessed by in vitro autoradiography and in vivo PET studies in animal (rodents) models. Finally, gold standard in vitro autoradiography with patients' postmortem tissues was performed to confirm/infirm the α-syn binding characteristics. Two compounds exhibited a good brain availability and bound to α-syn and Aβ fibrils in a rat model. In contrast, no signal was observed in a mouse model of synucleinopathy. Experiments in human tissues confirmed these negative results.

Objectives Several studies have established that alpha-synuclein (aSyn) fibrils made in vitro from recombinant protein can seed the aggregation of soluble aSyn in living organisms (cells, animals). Considering the prion-like properties of these synthetic fibrils, it is necessary to evaluate their stability so as to design efficient removal, disassembly and/or inactivation procedures. The objective of this study was to evaluate the comparative stability of WT and A53T-mutated fibrils and their ability to retain fibrillar structure in different solvents. Methods Human recombinant fibrils were prepared from WT and A53T aSyn in PBS. Resistance to proteolysis was evaluated under proteinase K (PK) treatment. Fibrillar conformation was assessed using spectrofluorimetry with Thioflavin T (ThT) and Transmission Electron Microscopy (TEM) after one dilution into various solvents. Results A53T fibrils were insensitive to PK treatment, in contrast to WT fibrils, which showed limited proteolysis (Fig. 1). TEM and ThT spectrofluorimetry gave concordant results, which confirmed the higher resistance of A53T vs WT fibrils to solvent denaturation (summarized in Table 1). Conclusions Among the solvents tested, only formic acid at 50% was able to fully disassemble both WT and A53T aSyn fibrils. Higher concentration SDS requires further testing.

There is increasing evidence that the serotonergic system is highly dysfunctional in Alzheimer's disease (AD), and this could be related to cognitive impairments associated with dementia. Of the various serotonin receptors, 5-HT1A receptors are relevant to AD as they are highly expressed in the human hippocampus and are known to be involved in the regulation of memory processes. This review will discuss the involvement of 5-HT1A receptors in AD at several levels (post-mortem, in-vivo imaging, animal models). The involvement of this receptor subtype in AD pathophysiology will be reviewed particularly in terms of the modulation of its expression in the hippocampal region. Hypotheses involving 5-HT1A receptors will be developed, from two points of view: 5-HT1A receptors expression regulation as being beneficial and needing to be pharmacologically stimulated; and 5-HT1A receptors expression modulation as deleterious and needing to be limited. Finally, we will propose perspectives for future experiments that should weigh in favor of one or the other of the two hypotheses.

Evidence accumulates suggesting a complex interplay between neurodegenerative processes and serotonergic neurotransmission. We have previously reported an overexpression of serotonin 5-HT1A receptors (5-HT1AR) after intrahippocampal injections of amyloid-beta 1-40 (A beta 40) fibrils in rats. This serotonergic reactivity paralleled results from clinical positron emission tomography studies with [F-18] MPPF revealing an overexpression of 5-HT1AR in the hippocampus of patients with mild cognitive impairment. Because A beta 40 and A beta 42 isoforms are found in amyloid plaques, we tested in this study the hypothesis of a peptide-and region-specific 5-HT1AR reactivity by injecting them, separately, into the hippocampus or striatum of rats. [F-18] MPPF in vitro autoradiography revealed that A beta 40 fibrils, but not A beta 42, were triggering an overexpression of 5-HT1AR in the hippocampus and striatum of rat brains after 7 days. Immunohistochemical approaches targeting neuronal precursor cells, mature neurons, and astrocytes showed that A beta 42 fibrils caused more pathophysiological damages than A beta 40 fibrils. The mechanisms of A beta 40 fibrilseinduced 5-HT1AR expression remains unknown, but hypotheses including neurogenesis, glial expression, and axonal sprouting are discussed. (C) 2016 Elsevier Inc. All rights reserved.

PET imaging studies using 5-HT1A receptor radiotracers show a decreased density of this receptor in hippocampi of patients with Alzheimer's disease (AD) at advanced stages. However, current 5-HT1A receptor radiopharmaceuticals used in neuroimaging are antagonists, thought to bind to 5-HT1A receptors in different functional states (i.e., both the one which displays high affinity for agonists and is thought to mediate receptor activation, as well as the state which has low affinity for agonists). Comparing the PET imaging obtained using an agonist radiotracer, which binds selectively to functional receptors, with the PET imaging obtained using an antagonist radiotracer would therefore provide original information on 5-HT1A receptor impairment during AD. Quantitative autoradiography using [F-18] F13640 and [F-18]MPPF, a 5-HT1A agonist and antagonist, respectively, was measured in hippocampi of patients with AD (n = 25, at different Braak stages) and control subjects (n = 9). The neuronal density was measured in the same tissues by NeuN immunohistochemistry. The specific binding of both radiotracers was determined by addition of WAY-100635, a selective 5-HT1A receptor antagonist. The autoradiography distribution of both 5-HT1A, PET radiotracers varied across hippocampus regions. The highest binding density was in the pyramidal layer of CA1. Incubation with Gpp(NH)p, a non-hydrolysable analogue of GTP, reduced significantly [F-18]F13640 binding in hippocampal regions, confirming its preferential interaction with G-coupled receptors, and slightly increased [F-18]MPPF binding. In the CA1 subfield, [F-18]F13640 binding was significantly decreased at Braak stages I/II (-19%), Braak stages III/IV (-23%), and Braak stages V/VI (-36%) versus control. In contrast, [F-18]MPPF binding was statistically reduced only at the most advanced Braak stages V/VI compared to control (-33%).Since [F-18]F13640 and [F-18]MPPF can be used in vivo in humans, this neuropharmacological paradigm supports testing the concept of functional imaging using agonist radiopharmaceuticals in future clinical studies. (C)2016 Elsevier Ltd. All rights reserved.

Evidence suggests that there is a link between the endocannabinoid system (ECS) and neuropsychiatric illnesses, including schizophrenia. Whilst the ECS has been shown to be involved in immune system regulation in various ways, it is known that infections during pregnancy can modulate the immune system of the mother and increase the risk for schizophrenia in offspring. In animal studies, maternal immune activation following administration of viral or bacterial mimics has been shown to reproduce many key structural, behavioural, and pharmacological abnormalities in offspring that resemble schizophrenia. In the present study, we used Positron Emission Tomography (PET) and [18F]MK-9470, a selective high-affinity inverse agonist radioligand for cannabinoid type 1 receptors (CB1R), to longitudinally assess CB1R expression in the progeny of female rats exposed to the viral mimic polyriboinosinic–polyribocytidilic acid (poly I:C) (4mg/kg i.v.) or vehicle at gestational day 15 (GD 15). PET scans were performed in offspring at postnatal days (PND) 32–42 (adolescence) and in the same animals again at PNDs 75–79 (adulthood). Sixteen regions of interest were assessed, encompassing the whole rat brain. At adolescence, offspring exposed prenatally to poly I:C had significantly lower CB1R relative Standard Uptake Values (rSUV) compared to controls in the globus pallidus (p=0.046). In adulthood, however, poly I:C exposed offspring had higher levels of CB1R rSUV in sensory cortex (p=0.034) and hypothalamus (p=0.032) compared to controls. Our results suggest that prenatal poly I:C leads to long term alterations in the integrity of the ECS that are age and region-specific. The increased CB1R expression in adulthood following poly I:C mirrors the increased CB1R observed in patients with schizophrenia in post-mortem and in vivo PET studies.

Alpha-synuclein (α-syn) aggregation is a neuropathological hallmark of many neurodegenerative diseases, collectively termed synucleinopathies. There is currently no pre-mortem diagnosis tool for these diseases. Although some compounds have been described as potential ligands for α-syn aggregates, no specific PET radiotracer of aggregated α-syn is currently available. Recently, [(18)F]BF227 has been proposed as an α-syn PET radiotracer in the absence of other specific candidates. We proposed here, for the first time, to use this radiotracer in an accelerated mouse model of synucleinopathy presenting α-syn depositions in brainstem and thalamus. Our in vivo and in vitro studies showed that [(18)F]BF227 does not bind to α-syn aggregates. These results highlight the fact that [(18)F]BF227 PET has no suitable characteristics for monitoring this experimental synucleinopathy, justifying the need to develop alternative α-syn PET radiotracers.

Although imaging has long been indispensable in clinical practice, in vivo imaging of small laboratory animals has emerged more recently as an important component of preclinical biomedical research. Small animal imaging provides a noninvasive means of exploring biological structures and functions in vivo, revealing qualitative and quantitative information on normal and diseased tissues in a given spatial and temporal location. The success of imaging is mainly explained by its noninvasive nature, allowing longitudinal assay of animal models of human diseases, from diagnosis to progression of the pathology, and monitoring of treatment efficacy. Importantly, and unlike alternative cell or tissue culture-based protocols, studies in intact animals, and particularly in the brain, take account of all the interacting physiological factors – neurochemical, neurophysiological, neurohormonal, nutritional, and so on – present in the complex organ in situ. Intact whole-animal models accessible to imaging thus facilitate investigation of systemic aspects of disease, which are almost impossible to fully replicate in in vitro or ex vivo systems. Another key advantage of in vivo imaging is the ability to perform multiple types of studies in the same animal. Because animal model-based research has in recent years driven the growth of small animal MRI (magnetic resonance imaging) and PET (positron emission tomography) for brain exploration, we will focus on these two imaging approaches. In this chapter, we will highlight the advantages and limitations of MRI and PET in the translational use of preclinical imaging.

BACKGROUND:GABA(A) receptors (GABA(A)R) are composed of several subunits that determine sensitivity to drugs, synaptic localisation and function. Recent studies suggest that agonists targeting selective GABA(A)R subunits may have therapeutic value against the cognitive impairments observed in schizophrenia. In this study, we determined whether GABA(A)R binding deficits exist in the dorsolateral prefrontal cortex (DLPFC) of people with schizophrenia and tested if changes in GABA(A)R binding are related to the changes in subunit mRNAs. The GABA orthosteric and the benzodiazepine allosteric binding sites were assessed autoradiographically using [(3)H]Muscimol and [(3)H]Flumazenil, respectively, in a large cohort of individuals with schizophrenia (n = 37) and their matched controls (n = 37). We measured, using qPCR, mRNA of β (β1, β2, β3), γ (γ1, γ2, γ2S for short and γ2L for long isoform, γ3) and δ subunits and used our previous measurements of GABA(A)R α subunit mRNAs in order to relate mRNAs and binding through correlation and regression analysis.RESULTS:Significant increases in both [(3)H]Muscimol (p = 0.016) and [(3)H]Flumazenil (p = 0.012) binding were found in the DLPFC of schizophrenia patients. Expression levels of mRNA subunits measured did not show any significant difference in schizophrenia compared to controls. Regression analysis revealed that in schizophrenia, the [(3)H]Muscimol binding variance was most related to α4 mRNA levels and the [(3)H]Flumazenil binding variance was most related to γ2S subunit mRNA levels. [(3)H]Muscimol and [(3)H]Flumazenil binding were not affected by the lifetime anti-psychotics dose (chlorpromazine equivalent).CONCLUSIONS:We report parallel increases in orthosteric and allosteric GABA(A)R binding sites in the DLPFC in schizophrenia that may be related to a "shift" in subunit composition towards α4 and γ2S respectively, which may compromise normal GABAergic modulation and function. Our results may have implications for the development of treatment strategies that target specific GABA(A)R receptor subunits.

Background/Aims: Functional linkages between the cannabinoid CB1 and the dopaminergic systems have been reported although the observations and the mechanisms hypothesizing their interactions at the G protein-coupled receptor (GPCR) functionality level are conflicting. Methods: Administration of a potent cannabinoid agonist, HU210, at various doses (25–100 µg/kg) and treatment regimens (1- to 14-day treatment) in rats was carried out to investigate the effect of HU210 treatment on the CB1 and D2-like agonist-mediated GPCR activation. Results: The desensitizations (reduced coupling) of both D2 agonist- and CB1 agonist-mediated GPCR activation was found to be treatment duration dependent and region specific, suggesting implication of receptor tolerance and adaptation due to the cannabinoid treatment. The effect of HU210 on the CB1 agonist-mediated GPCR desensitization in all treatment groups was not dose dependent. Conclusions: The desensitization of D2-like receptors found after a cannabinoid treatment in this study strengthens the evidence that the two neurotransmitter systems interact at the intercellular level; this interaction might occur via multiple mechanisms, which also vary according to region.