RESUMO
AMPA glutamate receptors (AMPARs) play a pivotal role in excitatory neurotransmission, particularly in the hippocampus where the TARP γ-8 subunit is enriched and serves as a target for emerging anti-epileptic drugs. To enable inâ vivo visualization of TARP γ-8 distribution and expression by positron emission tomography (PET), this study focuses on the development of novel 18 F-labeled TARP γ-8 inhibitors and their corresponding precursors, stemming from the azabenzimidazole scaffold. The resulting radioligands [18 F]TARP-2204 and [18 F]TARP-2205 were successfully synthesized with acceptable radiochemical yield, high molar activity, and excellent radiochemical purity. In vitro autoradiography demonstrates high level of specific binding of [18 F]TARP-2205 to TARP γ-8 in both rat and nonhuman primate brain tissues. However, unexpected radiodefluorination in PET imaging studies of rodents emphasizes the need for further structural refinement. This work serves as an excellent starting point for the development of future 18 F-labeled TARP γ-8 PET tracers, offering valuable insights into medicinal chemistry design, radiosynthesis and subsequent PET evaluation.
Assuntos
Tomografia por Emissão de Pósitrons , Receptores de AMPA , Ratos , Animais , Receptores de AMPA/metabolismo , Tomografia por Emissão de Pósitrons/métodos , HipocampoRESUMO
Dysregulation of microtubules is commonly associated with several psychiatric and neurological disorders, including addiction and Alzheimer's disease. Imaging of microtubules in vivo using positron emission tomography (PET) could provide valuable information on their role in the development of disease pathogenesis and aid in improving therapeutic regimens. We developed [11C]MPC-6827, the first brain-penetrating PET radiotracer to image microtubules in vivo in the mouse brain. The aim of the present study was to assess the reproducibility of [11C]MPC-6827 PET imaging in non-human primate brains. Two dynamic 0-120 min PET/CT imaging scans were performed in each of four healthy male cynomolgus monkeys approximately one week apart. Time activity curves (TACs) and standard uptake values (SUVs) were determined for whole brains and specific regions of the brains and compared between the "test" and "retest" data. [11C]MPC-6827 showed excellent brain uptake with good pharmacokinetics in non-human primate brains, with significant correlation between the test and retest scan data (r = 0.77, p = 0.023). These initial evaluations demonstrate the high translational potential of [11C]MPC-6827 to image microtubules in the brain in vivo in monkey models of neurological and psychiatric diseases.
Assuntos
Encéfalo , Radioisótopos de Carbono , Microtúbulos/metabolismo , Tomografia por Emissão de Pósitrons , Quinazolinas/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Macaca fascicularis , MasculinoRESUMO
BACKGROUND: Neuroactive steroids such as (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone) are potent neuromodulators that enhance GABAergic neurotransmission and produce inhibitory neurobehavioral and anti-inflammatory effects. Chronic ethanol (EtOH) consumption reduces 3α,5α-THP levels in human plasma, but has brain region- and species-specific effects on central nervous system levels of 3α,5α-THP. We explored the relationship between 3α,5α-THP levels in the hippocampus and voluntary EtOH consumption in the cynomolgus monkey following daily self-administration of EtOH for 12 months and further examined the relationship with hypothalamic-pituitary-adrenal (HPA) axis function prior to EtOH exposure. We simultaneously explored hippocampus levels of monocyte chemoattractant protein 1 (MCP-1), a pro-inflammatory cytokine that plays an important role in the neuroimmune response to EtOH, following chronic self-administration. METHODS: Monkeys were subjected to scheduled induction of water and EtOH consumption (0 to 1.5 g/kg) over 4 months, followed by free access to EtOH or water for 22 h/d over 12 months. Immunohistochemistry was performed using an anti-3α,5α-THP or anti-MCP-1 antibody. Prolonged voluntary drinking resulted in individual differences in EtOH consumption that ranged from 1.2 to 4.2 g/kg/d over 12 months. RESULTS: Prolonged EtOH consumption increased cellular 3α,5α-THP immunoreactivity by 12 ± 2% (p < 0.05) and reduced MCP-1 immunoreactivity by 23 ± 9% (p < 0.05) in the hippocampus CA1. In both cases, the effect of EtOH was most pronounced in heavy drinkers that consumed ≥3 g/kg for ≥20% of days. 3α,5α-THP immunoreactivity was positively correlated with average daily EtOH intake (Spearman r = 0.76, p < 0.05) and dexamethasone inhibition of HPA axis function (Spearman r = 0.9, p < 0.05). In contrast, MCP-1 immunoreactivity was negatively correlated with average daily EtOH intake (Spearman r = -0.78, p < 0.05) and dexamethasone suppression of HPA axis function (Spearman r = -0.76, p < 0.05). Finally, 3α,5α-THP and MCP-1 immunoreactivity were inversely correlated with each other (Spearman r = -0.68, p < 0.05). CONCLUSIONS: These data indicate that voluntary, long-term EtOH consumption results in higher levels of 3α,5α-THP, while decreasing levels of MCP-1 in the CA1 hippocampus, and that both changes may be linked to HPA axis function and the magnitude of voluntary EtOH consumption.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Região CA1 Hipocampal/metabolismo , Quimiocina CCL2/metabolismo , Pregnanolona/metabolismo , Consumo de Bebidas Alcoólicas/psicologia , Animais , Biomarcadores/metabolismo , Macaca fascicularisRESUMO
BACKGROUND: Recent work with long-term ethanol (EtOH) self-administration in nonhuman primate models has revealed a complex array of behavioral and physiological effects that closely mimic human alcohol abuse. Detailed neurophysiological analysis in these models suggests a myriad of pre- and postsynaptic neurobiological effects that may contribute to the behavioral manifestations of long-term EtOH drinking. The molecular mechanisms regulating presynaptic effects of this chronic EtOH exposure are largely unknown. To this end, we analyzed the effects of long-term EtOH self-administration on the levels of presynaptic SNARE complex proteins in Macaca mulatta basolateral amygdala, a brain region known to regulate both aversive and reward-seeking behaviors. METHODS: Basolateral amygdala samples from control and EtOH-drinking male and female monkeys were processed. Total basolateral amygdala protein was analyzed by Western blotting using antibodies directed against both core SNARE and SNARE-associated proteins. We also performed correlational analyses between protein expression levels and a number of EtOH drinking parameters, including lifetime grams of EtOH consumed, preference, and blood alcohol concentration. RESULTS: Significant interactions or main effects of sex/drinking were seen for a number of SNARE core and SNARE-associated proteins. Across the range of EtOH-drinking phenotypes, SNAP25 and Munc13-1 proteins levels were significantly different between males and females, and Munc13-2 levels were significantly lower in animals with a history of EtOH drinking. A separate analysis of very heavy-drinking individuals revealed significant decreases in Rab3c (females) and complexin 2 (males). CONCLUSIONS: Protein expression analysis of basolateral amygdala total protein from controls and animals following long-term EtOH self-administration suggests a number of alterations in core SNARE or SNARE-associated components that could dramatically alter presynaptic function. A number of proteins or multiprotein components were also correlated with EtOH drinking behavior, which suggest a potentially heritable role for presynaptic SNARE proteins.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Consumo de Bebidas Alcoólicas/tendências , Complexo Nuclear Basolateral da Amígdala/efeitos dos fármacos , Complexo Nuclear Basolateral da Amígdala/metabolismo , Etanol/administração & dosagem , Proteínas SNARE/biossíntese , Consumo de Bebidas Alcoólicas/efeitos adversos , Animais , Complexo Nuclear Basolateral da Amígdala/química , Etanol/efeitos adversos , Feminino , Macaca mulatta , Masculino , Proteínas SNARE/análise , Autoadministração , Fatores de TempoRESUMO
This is the first description of the relationship between chronic ethanol self-administration and the brain transcriptome in a non-human primate (rhesus macaque). Thirty-one male animals self-administered ethanol on a daily basis for over 12 months. Gene transcription was quantified with RNA-Seq in the central nucleus of the amygdala (CeA) and cortical Area 32. We constructed coexpression and cosplicing networks, and we identified areas of preservation and areas of differentiation between regions and network types. Correlations between intake and transcription included largely distinct gene sets and annotation categories across brain regions and between expression and splicing; positive and negative correlations were also associated with distinct annotation groups. Membrane, synaptic and splicing annotation categories were over-represented in the modules (gene clusters) enriched in positive correlations (CeA); our cosplicing analysis further identified the genes affected only at the exon inclusion level. In the CeA coexpression network, we identified Rab6b, Cdk18 and Igsf21 among the intake-correlated hubs, while in the Area 32, we identified a distinct hub set that included Ppp3r1 and Myeov2. Overall, the data illustrate that excessive ethanol self-administration is associated with broad expression and splicing mechanisms that involve membrane and synapse genes.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Encéfalo/metabolismo , Depressores do Sistema Nervoso Central/administração & dosagem , Etanol/administração & dosagem , Consumo de Bebidas Alcoólicas/metabolismo , Animais , Calcineurina/genética , Núcleo Central da Amígdala/metabolismo , Córtex Cerebral/metabolismo , Quinases Ciclina-Dependentes/genética , Perfilação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Macaca mulatta , Masculino , Proteínas do Tecido Nervoso/genética , Splicing de RNA , Autoadministração , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Neuroactive steroids such as (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone) enhance the gamma-aminobutyric acid (GABA)-ergic effects of ethanol and modulate excessive drinking in rodents. Moreover, chronic ethanol consumption reduces 3α,5α-THP levels in human plasma, rat hippocampus and mouse limbic regions. We explored the relationship between 3α,5α-THP levels in limbic brain areas and voluntary ethanol consumption in the cynomolgus monkey following daily self-administration of ethanol for 12 months and further examined the relationship to hypothalamic-pituitary-adrenal (HPA) axis function prior to ethanol exposure. Monkeys were subjected to scheduled induction of ethanol consumption followed by free access to ethanol or water for 22 h/day over 12 months. Immunohistochemistry was performed using an anti-3α,5α-THP antibody. Prolonged voluntary drinking resulted in individual differences in ethanol consumption that ranged from 1.2 to 4.2 g/kg/day over 12 months. Prolonged ethanol consumption reduced cellular 3α,5α-THP immunoreactivity by 13 ± 2 percent (P < 0.05) in the lateral amygdala and 17 ± 2 percent (P < 0.05) in the basolateral amygdala. The effect of ethanol was most pronounced in heavy drinkers that consumed ≥3 g/kg ≥ 20 percent of days. Consequently, 3α,5α-THP immunoreactivity in both the lateral and basolateral amygdala was inversely correlated with average daily ethanol intake (Spearman r = -0.87 and -0.72, respectively, P < 0.05). However, no effect of ethanol and no correlation between drinking and 3α,5α-THP immunoreactivity were observed in the basomedial amygdala. 3α,5α-THP immunoreactivity following ethanol exposure was also correlated with HPA axis function prior to ethanol exposure. These data indicate that voluntary ethanol drinking reduces amygdala levels of 3α,5α-THP in non-human primates and that amygdala 3α,5α-THP levels may be linked to HPA axis function.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Pregnanolona/metabolismo , Tonsila do Cerebelo/metabolismo , Animais , Comportamento Animal , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Depressores do Sistema Nervoso Central/administração & dosagem , Relação Dose-Resposta a Droga , Etanol/administração & dosagem , Imuno-Histoquímica , Macaca fascicularis , Masculino , AutoadministraçãoRESUMO
Alcohol is a human carcinogen. A causal link has been established between alcohol drinking and cancers of the upper aerodigestive tract, colon, liver and breast. Despite this established association, the underlying mechanisms of alcohol-induced carcinogenesis remain unclear. Various mechanisms may come into play depending on the type of cancer; however, convincing evidence supports the concept that ethanol's major metabolite acetaldehyde may play a major role. Acetaldehyde can react with DNA forming adducts which can serve as biomarkers of carcinogen exposure and potentially of cancer risk. The major DNA adduct formed from this reaction is N (2)-ethylidenedeoxyguanosine, which can be quantified as its reduced form N (2)-ethyl-dG by LC-ESI-MS/MS. To investigate the potential use of N (2)-ethyl-dG as a biomarker of alcohol-induced DNA damage, we quantified this adduct in DNA from the oral, oesophageal and mammary gland tissues from rhesus monkeys exposed to alcohol drinking over their lifetimes and compared it to controls. N (2)-Ethyl-dG levels were significantly higher in the oral mucosa DNA of the exposed animals. Levels of the DNA adduct measured in the oesophageal mucosa of exposed animals were not significantly different from controls. A correlation between the levels measured in the oral and oesophageal DNA, however, was observed, suggesting a common source of formation of the DNA adducts. N (2) -Ethyl-dG was measured in mammary gland DNA from a small cohort of female animals, but no difference was observed between exposed animals and controls. These results support the hypothesis that acetaldehyde induces DNA damage in the oral mucosa of alcohol-exposed animals and that it may play role in the alcohol-induced carcinogenic process. The decrease of N (2)-ethyl-dG levels in exposed tissues further removed from the mouth also suggests a role of alcohol metabolism in the oral cavity, which may be considered separately from ethanol liver metabolism in the investigation of ethanol-related cancer risk.
Assuntos
Acetaldeído/toxicidade , Consumo de Bebidas Alcoólicas/efeitos adversos , Adutos de DNA/análise , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análise , Mucosa Bucal/efeitos dos fármacos , Acetaldeído/farmacologia , Animais , Cromatografia Líquida de Alta Pressão , Dano ao DNA , Mucosa Esofágica/química , Mucosa Esofágica/efeitos dos fármacos , Feminino , Macaca mulatta , Masculino , Glândulas Mamárias Animais/química , Glândulas Mamárias Animais/efeitos dos fármacos , Mucosa Bucal/química , Espectrometria de Massas em TandemRESUMO
Alterations in hypothalamic-pituitary-adrenal axis function contribute to many of the adverse behavioral effects of chronic voluntary alcohol drinking, including alcohol dependence and mood disorders; limbic brain structures such as the bed nucleus of the stria terminalis (BNST) may be key sites for these effects. Here, we measured circulating levels of several steroid hormones and performed whole-cell electrophysiological recordings from acutely prepared BNST slices of male rhesus monkeys allowed to self-administer alcohol for 12 months or a control solution. Initial comparisons revealed that BNST neurons in alcohol-drinking monkeys had decreased membrane resistance, increased frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) with no change in spontaneous excitatory postsynaptic currents (sEPSCs). We then used a combined variable cluster analysis and linear mixed model statistical approach to determine whether specific factors including stress and sex hormones, age and measures of alcohol consumption and intoxication are related to these BNST measures. Modeling results showed that specific measures of alcohol consumption and stress-related hormone levels predicted differences in membrane conductance in BNST neurons. Distinct groups of adrenal stress hormones were negatively associated with the frequency of sIPSCs and sEPSCs, and alcohol drinking measures and basal neuronal membrane properties were additional positive predictors of inhibitory, but not excitatory, PSCs. The amplitude of sEPSCs was highly positively correlated with age, independent of other variables. Together, these results suggest that chronic voluntary alcohol consumption strongly influences limbic function in non-human primates, potentially via interactions with or modulation by other physiological variables, including stress steroid hormones and age.
Assuntos
Consumo de Bebidas Alcoólicas/fisiopatologia , Neurônios/fisiologia , Núcleos Septais/efeitos dos fármacos , Animais , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Hormônios/metabolismo , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Macaca mulatta , Masculino , Neurônios/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Núcleos Septais/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
BACKGROUND: Long-term alcohol abuse is associated with change in behavior, brain structure, and brain function. However, the nature of these changes is not well understood. In this study, we used network science to analyze a nonhuman primate model of ethanol self-administration to evaluate functional differences between animals with chronic alcohol use and animals with no exposure to alcohol. Of particular interest was how chronic alcohol exposure may affect the resting state network. METHODS: Baseline resting state functional magnetic resonance imaging was acquired in a cohort of vervet monkeys. Animals underwent an induction period where they were exposed to an isocaloric maltose dextrin solution (control) or ethanol in escalating doses over three 30-day epochs. Following induction, animals were given ad libitum access to water and a maltose dextrin solution (control) or water and ethanol for 22 h/d over 12 months. Cross-sectional analyses examined region of interests in hubs and community structure across animals to determine differences between drinking and nondrinking animals after the 12-month free access period. RESULTS: Animals were classified as lighter (<2.0 g/kg/d) or heavier drinkers (≥2.0 g/kg/d) based on a median split of their intake pattern during the 12-month ethanol free access period. Statistical analysis of hub connectivity showed significant differences in heavier drinkers for hubs in the precuneus, posterior parietal cortices, superior temporal gyrus, subgenual cingulate, and sensorimotor cortex. Heavier drinkers were also shown to have less consistent communities across the brain compared to lighter drinkers. The different level of consumption between the lighter and heavier drinking monkeys suggests that differences in connectivity may be intake dependent. CONCLUSIONS: Animals that consume alcohol show topological differences in brain network organization, particularly in animals that drink heavily. Differences in the resting state network were linked to areas that are associated with spatial association, working memory, and visuomotor processing.
Assuntos
Consumo de Bebidas Alcoólicas/fisiopatologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Etanol/administração & dosagem , Etanol/farmacologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiologia , Animais , Encéfalo/citologia , Chlorocebus aethiops , Neuroimagem Funcional , Imageamento por Ressonância Magnética , Masculino , AutoadministraçãoRESUMO
BACKGROUND: An estimated 18 million adults in the United States meet the clinical criteria for diagnosis of alcohol abuse or alcoholism, a disorder ranked as the third leading cause of preventable death. In addition to brain pathology, heavy alcohol consumption is comorbid with damage to major organs including heart, lungs, liver, pancreas, and kidneys. Much of what is known about risk for and consequences of heavy consumption derive from rodent or retrospective human studies. The neurobiological effects of chronic intake in rodent studies may not easily translate to humans due to key differences in brain structure and organization between species, including a lack of higher-order cognitive functions, and differences in underlying prefrontal cortical neural structures that characterize the primate brain. Further, rodents do not voluntarily consume large quantities of ethanol (EtOH) and they metabolize it more rapidly than primates. METHODS: The basis of the Monkey Alcohol Tissue Research Resource (MATRR) is that nonhuman primates, specifically monkeys, show a range of drinking excessive amounts of alcohol (>3.0 g/kg or a 12 drink equivalent per day) over long periods of time (12 to 30 months) with concomitant pathological changes in endocrine, hepatic, and central nervous system (CNS) processes. The patterns and range of alcohol intake that monkeys voluntarily consume parallel what is observed in humans with alcohol use disorders and the longitudinal experimental design spans stages of drinking from the EtOH-naïve state to early exposure through chronic abuse. Age- and sex-matched control animals self-administer an isocaloric solution under identical operant procedures. RESULTS: The MATRR is a unique postmortem tissue bank that provides CNS and peripheral tissues, and associated bioinformatics from monkeys that self-administer EtOH using a standardized experimental paradigm to the broader alcohol research community. CONCLUSIONS: This resource provides a translational platform from which we can better understand the disease processes associated with alcoholism.
Assuntos
Alcoolismo , Encéfalo , Glândulas Endócrinas , Fígado , Bancos de Tecidos , Animais , Biologia Computacional , Etanol/administração & dosagem , Feminino , Haplorrinos , Masculino , Autoadministração , Manejo de EspécimesRESUMO
The brain adapts to chronic ethanol intoxication by altering synaptic and ion-channel function to increase excitability, a homeostatic counterbalance to inhibition by alcohol. Delirium tremens occurs when those adaptations are unmasked during withdrawal, but little is known about whether the primate brain returns to normal with repeated bouts of ethanol abuse and abstinence. Here, we show a form of bidirectional plasticity of pacemaking currents induced by chronic heavy drinking within the inferior olive of cynomolgus monkeys. Intracellular recordings of inferior olive neurons demonstrated that ethanol inhibited the tail current triggered by release from hyperpolarization (I(tail)). Both the slow deactivation of hyperpolarization-activated cyclic nucleotide-gated channels conducting the hyperpolarization-activated inward current and the activation of Ca(v)3.1 channels conducting the T-type calcium current (I(T)) contributed to I(tail), but ethanol inhibited only the I(T) component of I(tail). Recordings of inferior olive neurons obtained from chronically intoxicated monkeys revealed a significant up-regulation in I(tail) that was induced by 1 y of daily ethanol self-administration. The up-regulation was caused by a specific increase in I(T) which (i) greatly increased neurons' susceptibility for rebound excitation following hyperpolarization and (ii) may have accounted for intention tremors observed during ethanol withdrawal. In another set of monkeys, sustained abstinence produced the opposite effects: (i) a reduction in rebound excitability and (ii) a down-regulation of I(tail) caused by the down-regulation of both the hyperpolarization-activated inward current and I(T). Bidirectional plasticity of two hyperpolarization-sensitive currents following chronic ethanol abuse and abstinence may underlie persistent brain dysfunction in primates and be a target for therapy.
Assuntos
Alcoolismo/fisiopatologia , Etanol/farmacologia , Macaca fascicularis/anatomia & histologia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Núcleo Olivar/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Feminino , Macaca fascicularis/fisiologia , Masculino , Núcleo Olivar/anatomia & histologia , Núcleo Olivar/efeitos dos fármacos , Técnicas de Patch-Clamp , Fenótipo , Síndrome de Abstinência a Substâncias/fisiopatologiaRESUMO
Appropriate animal models are critical to conduct translational studies of human disorders without variables that can confound clinical studies. Such analytic methods as patch-clamp electrophysiological and voltammetric recordings of neurons in brain slices require living brain tissue. In order to obtain viable tissue from nonhuman primate brains, tissue collection methods must be designed to preserve cardiovascular and respiratory functions for as long as possible. This paper describes a method of necropsy that has been used in three species of monkeys that satisfies this requirement. At necropsy, animals were maintained under a deep surgical plane of anesthesia while a craniotomy was conducted to expose the brain. Following the craniotomy, animals were perfused with ice-cold, oxygenated artificial cerebrospinal fluid to displace blood and to reduce the temperature of the entire brain. The brain was removed within minutes of death and specific brain regions were immediately dissected for subsequent in vitro electrophysiology or voltammetry experiments. This necropsy method also provided for the collection of tissue blocks containing all brain regions that were immediately frozen and stored for subsequent genomic, proteomic, autoradiographic and histological studies. An added benefit from the design of this necropsy method is that all major peripheral tissues were also collected and are now being utilized in a wide range of genomic, biochemical and histological assays. This necropsy method has resulted in the establishment and growth of a nonhuman primate alcohol tissue bank designed to distribute central nervous system and peripheral tissues to the larger scientific community.
Assuntos
Encéfalo/fisiologia , Coleta de Tecidos e Órgãos/métodos , Animais , Encéfalo/citologia , Mapeamento Encefálico , Craniotomia , Eletrofisiologia , Macaca fascicularis , Bancos de TecidosRESUMO
BACKGROUND: Animal studies have long been an important tool for basic research as they offer a degree of control often lacking in clinical studies. Of particular value is the use of nonhuman primates (NHPs) for neuroimaging studies. Currently, studies have been published using functional magnetic resonance imaging (fMRI) to understand the default-mode network in the NHP brain. Network science provides an alternative approach to neuroimaging allowing for evaluation of whole-brain connectivity. In this study, we used network science to build NHP brain networks from fMRI data to understand the basic functional organization of the NHP brain. We also explored how the brain network is affected following an acute ethanol (EtOH) pharmacological challenge. METHODS: Baseline resting-state fMRI was acquired in an adult male rhesus macaque (n = 1) and a cohort of vervet monkeys (n = 10). A follow-up scan was conducted in the rhesus macaque to assess network variability and to assess the effects of an acute EtOH challenge on the brain network. RESULTS: The most connected regions in the resting-state networks were similar across species and matched regions identified as the default-mode network in previous NHP fMRI studies. Under an acute EtOH challenge, the functional organization of the brain was significantly impacted. CONCLUSIONS: Network science offers a great opportunity to understand the brain as a complex system and how pharmacological conditions can affect the system globally. These models are sensitive to changes in the brain and may prove to be a valuable tool in long-term studies on alcohol exposure.
Assuntos
Encéfalo/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Rede Nervosa/efeitos dos fármacos , Animais , Chlorocebus aethiops , Neuroimagem Funcional , Macaca mulatta , Imageamento por Ressonância Magnética , Masculino , Redes Neurais de ComputaçãoRESUMO
Leucine-rich repeat kinase 2 (LRRK2) has been demonstrated to be closely involved in the pathogenesis of Parkinson's disease (PD), and pharmacological blockade of LRRK2 represents a new opportunity for therapeutical treatment of PD and other related neurodegenerative conditions. The development of an LRRK2-specific positron emission tomography (PET) ligand would enable a target occupancy study in vivo and greatly facilitate LRRK2 drug discovery and clinical translation as well as provide a molecular imaging tool for studying physiopathological changes in neurodegenerative diseases. In this work, we present the design and development of compound 8 (PF-06455943) as a promising PET radioligand through a PET-specific structure-activity relationship optimization, followed by comprehensive pharmacology and ADME/neuroPK characterization. Following an efficient 18F-labeling method, we have confirmed high brain penetration of [18F]8 in nonhuman primates (NHPs) and validated its specific binding in vitro by autoradiography in postmortem NHP brain tissues and in vivo by PET imaging studies.
Assuntos
Doença de Parkinson , Tomografia por Emissão de Pósitrons , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Leucina/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Ligantes , Doença de Parkinson/metabolismo , Tomografia por Emissão de Pósitrons/métodosRESUMO
There is an accumulating body of evidence implicating the muscarinic acetylcholine receptor 4 (M4) in schizophrenia and dementia with Lewy bodies, however, a clinically validated M4 positron emission tomography (PET) radioligand is currently lacking. As such, the aim of this study was to develop a suitable M4 PET ligand that allows the non-invasive visualization of M4 in the brain. Structure-activity relationship studies of pyrazol-4-yl-pyridine derivates led to the discovery of target compound 12 - a subtype-selective positive allosteric modulator (PAM). The radiofluorinated analogue, [18F]12, was synthesized in 28 ± 10% radiochemical yield, >37 GBq/µmol and an excellent radiochemical purity >99%. Initial in vitro autoradiograms on rodent brain sections were performed in the absence of carbachol and showed moderate specificity as well as a low selectivity of [18F]12 for the M4-rich striatum. However, in the presence of carbachol, a significant increase in tracer binding was observed in the rat striatum, which was reduced by >60% under blocking conditions, thus indicating that orthosteric ligand interaction is required for efficient binding of [18F]12 to the allosteric site. Remarkably, however, the presence of carbachol was not required for high specific binding in the non-human primate (NHP) and human striatum, and did not further improve the specificity and selectivity of [18F]12 in higher species. These results pointed towards significant species-differences and paved the way for a preliminary PET study in NHP, where peak brain uptake of [18F]12 was found in the putamen and temporal cortex. In conclusion, we report on the identification and preclinical development of the first radiofluorinated M4 PET radioligand with promising attributes. The availability of a clinically validated M4 PET radioligand harbors potential to facilitate drug development and provide a useful diagnostic tool for non-invasive imaging.
RESUMO
GluN2B subunit-containing N-methyl-d-aspartate (NMDA) receptors have been implicated in various neurological disorders. Nonetheless, a validated fluorine-18 labeled positron emission tomography (PET) ligand for GluN2B imaging in the living human brain is currently lacking. As part of our PET ligand development program, we have recently reported on the preclinical evaluation of [18F]OF-NB1 - a GluN2B PET ligand with promising attributes for potential clinical translation. However, the further development of [18F]OF-NB1 is currently precluded by major limitations in the radiolabeling procedure. These limitations include the use of highly corrosive reactants and racemization during the radiosynthesis. As such, the aim of this study was to develop a synthetic approach that allows an enantiomerically pure radiosynthesis of (R)-[18F]OF-NB1 and (S)-[18F]OF-NB1, as well as to assess their in vitro and in vivo performance characteristics for imaging the GluN2B subunit-containing NMDA receptor in rodents. A two-step radiosynthesis involving radiofluorination of the boronic acid pinacol ester, followed by coupling to the 3-benzazepine core structure via reductive amination was employed. The new synthetic approach yielded enantiomerically pure (R)-[18F]OF-NB1 and (S)-[18F]OF-NB1, while concurrently circumventing the use of corrosive reactants. In vitro autoradiograms with mouse and rat brain sections revealed a higher selectivity of (R)-[18F]OF-NB1 over (S)-[18F]OFNB1 for GluN2B-rich brain regions. In concert with these observations, blockade studies with commercially available GluN2B antagonist, CP101606, showed a significant signal reduction, which was more pronounced for (R)-[18F]OF-NB1 than for (S)-[18F]OF-NB1. Conversely, blockade experiments with sigma2 ligand, FA10, did not result in a significant reduction of tracer binding for both enantiomers. PET imaging experiments with CD1 mice revealed a higher brain uptake and retention for (R)-[18F]OF-NB1, as assessed by visual inspection and volumes of distribution from Logan graphical analyses. In vivo blocking experiments with sigma2 ligand, FA10, did not result in a significant reduction of the brain signal for both enantiomers, thus corroborating the selectivity over sigma2 receptors. In conclusion, we have developed a novel synthetic approach that is suitable for upscale to human use and allows the enantiomerically pure radiosynthesis of (R)-[18F]OF-NB1 and (S)-[18F]OF-NB1. While both enantiomers were selective over sigma2 receptors in vitro and in vivo, (R)-[18F]OF-NB1 showed superior GluN2B subunit specificity by in vitro autoradiography and higher volumes of distribution in small animal PET studies.
RESUMO
The cannabinoid type 2 receptor (CB2) has been implicated in a variety of central and peripheral inflammatory diseases, prompting significant interest in the development of CB2-targeted diagnostic and therapeutic agents. A validated positron emission tomography (PET) radioligand for imaging CB2 in the living human brain as well as in peripheral tissues is currently lacking. As part of our research program, we have recently identified the trisubstituted pyridine, [18F]RoSMA-18-d6, which proved to be highly suitable for in vitro and in vivo mapping of CB2 in rodents. The aim of this study was to assess the performance characteristics of [18F]RoSMA-18-d6 in nonhuman primates (NHPs) to pave the way for clinical translation. [18F]RoSMA-18-d6 was synthesized from the respective tosylate precursor according to previously reported procedures. In vitro autoradiograms with NHP spleen tissue sections revealed a high binding of [18F]RoSMA-18-d6 to the CB2-rich NHP spleen, which was significantly blocked by coincubation with the commercially available CB2 ligand, GW405833 (10 µM). In contrast, no specific binding was observed by in vitro autoradiography with NHP brain sections, which was in agreement with the notion of a CB2-deficient healthy mammalian brain. In vitro findings were corroborated by PET imaging experiments in NHPs, where [18F]RoSMA-18-d6 uptake in the spleen was dose-dependently attenuated with 1 and 5 mg/kg GW405833, while no specific brain signal was observed. Remarkably, we observed tracer uptake and retention in the NHP spinal cord, which was reduced by GW405833 blockade, pointing toward a potential utility of [18F]RoSMA-18-d6 in probing CB2-expressing cells in the bone marrow. If these observations are substantiated in NHP models of enhanced leukocyte proliferation in the bone marrow, [18F]RoSMA-18-d6 may serve as a valuable marker for hematopoietic activity in various pathologies. In conclusion, [18F]RoSMA-18-d6 proved to be a suitable PET radioligand for imaging CB2 in NHPs, supporting its translation to humans.
Assuntos
Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Animais , Humanos , Compostos Radiofarmacêuticos/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Ligantes , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Primatas/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Radioisótopos de Flúor/metabolismo , Mamíferos/metabolismoRESUMO
GluN2B subunit-containing N-methyl-d-aspartate (NMDA) receptors have been implicated in various neurological disorders. Nonetheless, a validated fluorine-18 labeled positron emission tomography (PET) ligand for GluN2B imaging in the living human brain is currently lacking. The aim of this study was to develop a novel synthetic approach that allows an enantiomerically pure radiosynthesis of the previously reported PET radioligands (R)-[18F]OF-NB1 and (S)-[18F]OF-NB1 as well as to assess their in vitro and in vivo performance characteristics for imaging the GluN2B subunit-containing NMDA receptor in rodents. A novel synthetic approach was successfully developed, which allows for the enantiomerically pure radiosynthesis of (R)-[18F]OF-NB1 and (S)-[18F]OF-NB1 and the translation of the probe to the clinic. While both enantiomers were selective over sigma2 receptors in vitro and in vivo, (R)-[18F]OF-NB1 showed superior GluN2B subunit specificity by in vitro autoradiography and higher volumes of distribution in the rodent brain by small animal PET studies.
Assuntos
Tomografia por Emissão de Pósitrons , Receptores de N-Metil-D-Aspartato , Animais , Humanos , Receptores de N-Metil-D-Aspartato/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Radioisótopos de FlúorRESUMO
Alterations in brain cholesterol homeostasis have been broadly implicated in neurological disorders. Notwithstanding the complexity by which cholesterol biology is governed in the mammalian brain, excess neuronal cholesterol is primarily eliminated by metabolic clearance via cytochrome P450 46A1 (CYP46A1). No methods are currently available for visualizing cholesterol metabolism in the living human brain; therefore, a noninvasive technology that quantitatively measures the extent of brain cholesterol metabolism via CYP46A1 could broadly affect disease diagnosis and treatment options using targeted therapies. Here, we describe the development and testing of a CYP46A1-targeted positron emission tomography (PET) tracer, 18F-CHL-2205 (18F-Cholestify). Our data show that PET imaging readouts correlate with CYP46A1 protein expression and with the extent to which cholesterol is metabolized in the brain, as assessed by cross-species postmortem analyses of specimens from rodents, nonhuman primates, and humans. Proof of concept of in vivo efficacy is provided in the well-established 3xTg-AD murine model of Alzheimer's disease (AD), where we show that the probe is sensitive to differences in brain cholesterol metabolism between 3xTg-AD mice and control animals. Furthermore, our clinical observations point toward a considerably higher baseline brain cholesterol clearance via CYP46A1 in women, as compared to age-matched men. These findings illustrate the vast potential of assessing brain cholesterol metabolism using PET and establish PET as a sensitive tool for noninvasive assessment of brain cholesterol homeostasis in the clinic.
Assuntos
Doença de Alzheimer , Encéfalo , Doença de Alzheimer/metabolismo , Animais , Encéfalo/metabolismo , Colesterol/metabolismo , Colesterol 24-Hidroxilase/metabolismo , Feminino , Homeostase , Humanos , Masculino , Mamíferos/metabolismo , CamundongosRESUMO
Purpose: A fundamental question for Alcohol use disorder (AUD) is how and when naïve brain networks are reorganized in response to alcohol consumption. The current study aimed to determine the progression of alcohol's effect on functional brain networks during transition from the naïve state to chronic consumption. Procedures: Resting-state brain networks of six female rhesus macaque (Macaca mulatta) monkeys were acquired using magnetoencephalography (MEG) prior to alcohol exposure and after free-access to alcohol using a well-established model of chronic heavy alcohol consumption. Functional brain network metrics were derived at each time point. Results: The average connection frequency (p < 0.024) and membership of the Rich Club (p < 0.022) changed significantly over time. Metrics describing network topology remained relatively stable from baseline to free-access drinking. The minimum degree of the Rich Club prior to alcohol exposure was significantly predictive of future free-access drinking (r = -0.88, p < 0.001). Conclusions: Results suggest naïve brain network characteristics may be used to predict future alcohol consumption, and that alcohol consumption alters functional brain networks, shifting hubs and Rich Club membership away from previous regions in a non-systematic manner. Further work to refine these relationships may lead to the identification of a high-risk drinking phenotype.