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Substance use disorder is a major concern, with few therapeutic options. Heparan sulfate (HS) and chondroitin sulfate (CS) interact with a plethora of growth factors and their receptors and have profound effects on cellular signaling. Thus, targeting these dynamic interactions might represent a potential novel therapeutic modality. In the present study, we performed mass spectrometry-based glycomic and proteomic analysis to understand the effects of cocaine and methamphetamine (METH) on HS, CS, and the proteome of two brain regions critically involved in drug addiction: the lateral hypothalamus and the striatum. We observed that cocaine and METH significantly alter HS and CS abundances as well as sulfate contents and composition. In particular, repeated METH or cocaine treatments reduced CS 4-O-sulfation and increased CS 6-O-sulfation. Since C4S and C6S exercise differential effects on axon growth, regeneration, and plasticity, these changes likely contribute to drug-induced neural plasticity in these brain regions. Notably, we observed that restoring these alterations by increasing CS 4-0 levels in the lateral hypothalamus by adeno-associated virus delivery of an shRNA to arylsulfatase B (N-acetylgalactosamine-4-sulfatase) ameliorated anxiety and prevented the expression of preference for cocaine in a novelty induced conditioned place preference test during cocaine withdrawal. Finally, proteomics analyses revealed a number of aberrant proteins in METH- and cocaine-treated versus saline-treated mice, including myelin proteolipid protein, calcium/calmodulin-dependent protein kinase type II subunit alpha, synapsin-2, tenascin-R, calnexin, annexin A7, hepatoma-derived growth factor, neurocan, and CSPG5, and oxidative phosphorylation among the top perturbed pathway. Taken together, these data support the role of HS, CS, and associated proteins in stimulants abuse and suggest that manipulation of HSPGs can represent a novel therapeutic strategy.
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Background: Recent work has demonstrated that acute administration of the novel positive allosteric modulator of the GABAB receptor, COR659, reduces several alcohol-related behaviors in rodents.Objective: To assess whether COR659 continues to lessen alcohol intake after repeated administration, a fundamental feature of drugs with therapeutic potential.Methods: Male C57BL/6J mice (n = 40) were exposed to daily 2-hour drinking sessions (20% (v/v) alcohol) under the 1-bottle "drinking in the dark" protocol and male Sardinian alcohol-preferring rats (n = 40) were exposed to daily 1-hour drinking sessions under the 2-bottle "alcohol (10%, v/v) vs water" choice regimen. COR659 (0, 10, 20, and 40 mg/kg in the mouse experiment; 0, 5, 10, and 20 mg/kg in the rat experiment) was administered intraperitoneally before 7 consecutive drinking sessions.Results: Alcohol intake in vehicle-treated mice and rats averaged 2.5-3.0 and 1.5-1.6 g/kg/session, respectively, indicative of high basal levels. In both experiments, treatment with COR659 resulted in an initial, dose-related suppression of alcohol intake (up to 70-80% compared to vehicle treatment; P < .0005 and P < .0001 in mouse and rat experiments, respectively). The magnitude of the reducing effect of COR659 on alcohol drinking diminished progressively, until vanishing over the subsequent 2-4 drinking sessions.Conclusion: COR659 effectively reduced alcohol intake in two different rodent models of excessive alcohol drinking. However, tolerance to the anti-alcohol effects of COR659 developed rapidly. If theoretically transposed to humans, these data would represent a possible limitation to the clinical use of COR659.
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Consumo de Bebidas Alcoólicas , Receptores de GABA-B , Animais , Masculino , Camundongos , Ratos , Consumo de Bebidas Alcoólicas/tratamento farmacológico , Ácido gama-Aminobutírico , Camundongos Endogâmicos C57BL , Receptores de GABA-B/efeitos dos fármacosRESUMO
Alzheimer's disease (AD) is a multifactorial disorder driven by abnormal amyloid ß-peptide (Aß) levels. In this study, we investigated the role of presenilin-like signal peptide peptidase-like 2b (SPPL2b) in AD pathophysiology and its potential as a druggable target within the Aß cascade. Exogenous Aß42 influenced SPPL2b expression in human cell lines and acute mouse brain slices. SPPL2b and its AD-related substrate BRI2 were evaluated in the brains of AppNL-G-F knock-in AD mice and human postmortem AD brains. An early high cortical expression of SPPL2b was observed, followed by a downregulation in late AD pathology in AppNL-G-F mice, correlating with synaptic loss. To understand the consequences of pathophysiological SPPL2b dysregulation, we found that SPPL2b overexpression significantly increased APP cleavage, while genetic deletion reduced APP cleavage and Aß production. Notably, postmortem AD brains showed higher levels of SPPL2b's BRI2 substrate compared to healthy control samples. These results strongly support the involvement of SPPL2b in AD pathology. The early Aß-induced upregulation of SPPL2b may enhance Aß production in a vicious cycle, further aggravating Aß pathology. Therefore, SPPL2b emerges as a potential anti-Aß drug target.
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Doença de Alzheimer , Animais , Humanos , Camundongos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Modelos Animais de DoençasRESUMO
The consumption of alcohol and caffeine affects the lives of billions of individuals worldwide. Although recent evidence indicates that caffeine impairs the reinforcing properties of alcohol, a characterization of its effects on alcohol-stimulated mesolimbic dopamine (DA) function was lacking. Acting as the pro-drug of salsolinol, alcohol excites DA neurons in the posterior ventral tegmental area (pVTA) and increases DA release in the nucleus accumbens shell (AcbSh). Here we show that caffeine, via antagonistic activity on A2A adenosine receptors (A2AR), prevents alcohol-dependent activation of mesolimbic DA function as assessed, in-vivo, by brain microdialysis of AcbSh DA and, in-vitro, by electrophysiological recordings of pVTA DA neuronal firing. Accordingly, while the A1R antagonist DPCPX fails to prevent the effects of alcohol on DA function, both caffeine and the A2AR antagonist SCH 58261 prevent alcohol-dependent pVTA generation of salsolinol and increase in AcbSh DA in-vivo, as well as alcohol-dependent excitation of pVTA DA neurons in-vitro. However, caffeine also prevents direct salsolinol- and morphine-stimulated DA function, suggesting that it can exert these inhibitory effects also independently from affecting alcohol-induced salsolinol formation or bioavailability. Finally, untargeted metabolomics of the pVTA showcases that caffeine antagonizes alcohol-mediated effects on molecules (e.g. phosphatidylcholines, fatty amides, carnitines) involved in lipid signaling and energy metabolism, which could represent an additional salsolinol-independent mechanism of caffeine in impairing alcohol-mediated stimulation of mesolimbic DA transmission. In conclusion, the outcomes of this study strengthen the potential of caffeine, as well as of A2AR antagonists, for future development of preventive/therapeutic strategies for alcohol use disorder.
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Withania somnifera (WS) is utilized in Ayurvedic medicine owing to its central and peripheral beneficial properties. Several studies have accrued indicating that the recreational amphetamine-related drug (+/-)- 3,4-methylenedioxymethamphetamine (MDMA; Ecstasy) targets the nigrostriatal dopaminergic system in mice, inducing neurodegeneration and gliosis, causing acute hyperthermia and cognitive impairment. This study aimed to investigate the effect of a standardized extract of W. somnifera (WSE) on MDMA-induced neurotoxicity, neuroinflammation, memory impairment and hyperthermia. Mice received a 3-day pretreatment with vehicle or WSE. Thereafter, vehicle- and WSE-pretreated mice were randomly divided into four groups: saline, WSE, MDMA alone, WSE plus MDMA. Body temperature was recorded throughout treatment, and memory performance was assessed by a novel object recognition (NOR) task at the end of treatment. Thereafter, immunohistochemistry was performed to evaluate in the substantia nigra pars compacta (SNc) and striatum the levels of tyrosine hydroxylase (TH), as marker of dopaminergic degeneration, and of glial fibrillary acidic protein (GFAP) and TMEM119, as markers of astrogliosis or microgliosis, respectively. MDMA-treated mice showed a decrease in TH-positive neurons and fibers in the SNc and striatum respectively, an increase in gliosis and body temperature, and a decrease in NOR performance, irrespective of vehicle or WSE pretreatment. Acute WSE plus MDMA counteracted the modifications in TH-positive cells in SNc, GFAP-positive cells in striatum, TMEM in both areas and NOR performance, as compared to MDMA alone, while no differences were observed as compared to saline. Results indicate that WSE acutely administered in combination with MDMA, but not as pretreatment, protects mice against the noxious central effects of MDMA.
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Hipertermia Induzida , N-Metil-3,4-Metilenodioxianfetamina , Síndromes Neurotóxicas , Withania , Animais , Camundongos , N-Metil-3,4-Metilenodioxianfetamina/toxicidade , Doenças Neuroinflamatórias , Gliose , Síndromes Neurotóxicas/tratamento farmacológico , Síndromes Neurotóxicas/etiologia , Síndromes Neurotóxicas/prevenção & controle , CogniçãoRESUMO
BACKGROUND: Docosanyl ferulate (DF) is a behaviourally active GABAA receptor complex (GABAAR) agonist, recently isolated from the standardized methanolic extract of Withania somnifera Dunal (WSE) root. Previous studies have shown that WSE prevents both ethanol- and morphine-dependent acquisition and expression of conditioned place preference (CPP) and stimulation of dopamine release in the nucleus accumbens shell (AcbSh). AIMS: The study aimed at determining (a) whether DF contributes to WSE's ability to affect the acquisition and expression of ethanol- and morphine-elicited CPP and, given that phosphorylation of extracellular signal-regulated kinase (pERK) in the AcbSh is involved in associative learning and motivated behaviours, (b) whether WSE and DF may affect ethanol- and morphine-induced ERKs phosphorylation in the AcbSh. METHODS: In adult male CD1 mice, DF's effects on the acquisition and expression of ethanol- and morphine-elicited CPP were evaluated by a classical place conditioning paradigm, whereas the effects of WSE and DF on ethanol- and morphine-elicited pERK in the AcbSh were evaluated by immunohistochemistry. RESULTS AND CONCLUSIONS: The study shows that DF, differently from WSE, affects only the acquisition but not the expression of ethanol- and morphine-induced CPP. Moreover, the study shows that both WSE and DF can prevent ethanol- and morphine-elicited pERK expression in the AcbSh. Overall, these results highlight subtle but critical differences for the role of GABAARs in the mechanism by which WSE affects these ethanol- and morphine-dependent behavioural and molecular/cellular responses and support the suggestion of WSE and DF for the control of different components of drug addiction.
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Withania , Animais , Etanol/farmacologia , Camundongos , Morfina/farmacologia , Núcleo Accumbens , Fosforilação , Extratos Vegetais/farmacologiaRESUMO
BACKGROUND: Clinical and experimental studies support the therapeutic potential of Withania somnifera (WS) (L.) Dunal on anxiety disorders. This potential is attributable to components present in different plant extracts; however, the individual compound(s) endowed with specific anxiolytic effects and potential modulatory activity of the GABAA receptor complex (GABAAR) have remained unidentified until the recent isolation from a WS methanolic root extract of some GABAAR-active compounds, including the long alkyl-chain ferulic acid ester, docosanyl ferulate (DF). AIMS: This study was designed to assess whether DF (0.05, 0.25 and 2 mg/kg), similarly to diazepam (2 mg/kg), may exert anxiolytic effects, whether these effects may be significantly blocked by the benzodiazepine antagonist flumazenil (10 mg/kg) and whether DF may lack some of the benzodiazepines' typical motor, cognitive and motivational side effects. METHODS: The behavioural paradigms Elevated Plus Maze, Static Rods, Novel Object Recognition, Place Conditioning and potentiation of ethanol-induced Loss of Righting Reflex were applied on male CD-1 mice. RESULTS: Similarly to diazepam, DF exerts anxiolytic effects that are blocked by flumazenil. Moreover, at the full anxiolytic dose of 2 mg/kg, DF lacks typical benzodiazepine-like side effects on motor and cognitive performances and on place conditioning. Moreover, DF fails to potentiate ethanol's (3 g/kg) depressant activity at the ethanol-induced Loss of Righting Reflex paradigm. CONCLUSIONS: These data point to DF as an effective benzodiazepine-like anxiolytic compound that, in light of its lack of motor, mnemonic and motivational side effects, could be a suitable candidate for the treatment of anxiety disorders.
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Ansiolíticos , Extratos Vegetais , Withania , Animais , Masculino , Camundongos , Ansiolíticos/administração & dosagem , Ansiolíticos/farmacologia , Comportamento Animal/efeitos dos fármacos , Diazepam/farmacologia , Relação Dose-Resposta a Droga , Etanol/farmacologia , Flumazenil/farmacologia , Aprendizagem em Labirinto/efeitos dos fármacos , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacologia , Reflexo de Endireitamento/efeitos dos fármacos , Withania/químicaRESUMO
Abnormal consumption of ethanol, the ingredient responsible for alcoholic drinks' addictive liability, causes millions of deaths yearly. Ethanol's addictive potential is triggered through activation, by a still unknown mechanism, of the mesolimbic dopamine (DA) system, part of a key motivation circuit, DA neurons in the posterior ventral tegmental area (pVTA) projecting to the ipsilateral nucleus accumbens shell (AcbSh). The present in vivo brain microdialysis study, in dually-implanted rats with one probe in the pVTA and another in the ipsilateral or contralateral AcbSh, demonstrates this mechanism. As a consequence of the oral administration of a pharmacologically relevant dose of ethanol, we simultaneously detect a) in the pVTA, a substance, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), untraceable under control conditions, product of condensation between DA and ethanol's first by-product, acetaldehyde; and b) in the AcbSh, a significant increase of DA release. Moreover, such newly generated salsolinol in the pVTA is responsible for increasing AcbSh DA release via µ opioid receptor (µOR) stimulation. In fact, inhibition of salsolinol's generation in the pVTA or blockade of pVTA µORs prevents ethanol-increased ipsilateral, but not contralateral, AcbSh DA release. This evidence discloses the long-sought key mechanism of ethanol's addictive potential and suggests the grounds for developing preventive and therapeutic strategies against abnormal consumption.
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BACKGROUND: Epidemiological studies indicate a rise in the combined consumption of caffeinated and alcoholic beverages, which can lead to increased risk of alcoholic-beverage overconsumption. However, the effects of the combination of caffeine and ethanol in animal models related to aspects of drug addiction are still underexplored. AIMS: To characterize the pharmacological interaction between caffeine and ethanol and establish if caffeine can affect the ability of ethanol (2 g/kg) to elicit conditioned place preference and conditioned place aversion, we administered caffeine (3 or 15 mg/kg) to male CD-1 mice before saline or ethanol. Moreover, we determined if these doses of caffeine could affect ethanol (2 g/kg) elicited extracellular signal-regulated kinase phosphorylation in brain areas, nucleus accumbens, bed nucleus of stria terminalis, central nucleus of the amygdala, and basolateral amygdala, previously associated with this type of associative learning. RESULTS: In the place-conditioning paradigm, caffeine did not have an effect on its own, whereas ethanol elicited significant conditioned-place preference and conditioned-place aversion. Caffeine (15 mg/kg) significantly prevented the acquisition of ethanol-elicited conditioned-place preference and, at both doses, also prevented the acquisition of ethanol-elicited conditioned-place aversion. Moreover, both doses of caffeine also prevented ethanol-elicited extracellular signal-regulated kinase phosphorylation expression in all brain areas examined. CONCLUSIONS: The present data indicate a functional antagonistic action of caffeine and ethanol on associative learning and extracellular signal-regulated kinase phosphorylation after an acute interaction. These results could provide exciting grounds for further studies, also in a translational perspective, of their pharmacological interaction modulating other processes involved in drug consumption and addiction.
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Tonsila do Cerebelo/efeitos dos fármacos , Aprendizagem por Associação/efeitos dos fármacos , Aprendizagem da Esquiva/efeitos dos fármacos , Cafeína/farmacologia , Depressores do Sistema Nervoso Central/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Comportamento de Escolha/efeitos dos fármacos , Condicionamento Clássico/efeitos dos fármacos , Etanol/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Cafeína/administração & dosagem , Depressores do Sistema Nervoso Central/administração & dosagem , Estimulantes do Sistema Nervoso Central/administração & dosagem , Interações Medicamentosas , Etanol/administração & dosagem , Masculino , Camundongos , Núcleo Accumbens/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Núcleos Septais/efeitos dos fármacos , Percepção Espacial/efeitos dos fármacosRESUMO
A morphofunctional investigation of the different neuronal subpopulations projecting through each of the nerves IV-VI emerging bilaterally from the terminal abdominal ganglion (TAG) was correlated with the octopaminergic activity in the ganglion that controls the ovipositor movements associated with calling behavior in the female gypsy moth Lymantria dispar. Tetramethylrodamine-dextran backfills from nerve stumps resulted in a relatively low number of TAG projections, ranging from 12 to 13 for nerve pair IV, 12 to 14 for nerve pair V, and 8 to 9 for nerve pair VI. Furthermore, as assessed by electrophysiological recordings, a number of fibers within each of these nerves displays spontaneous tonic activity, also when the ganglion is fully disconnected from the ventral nerve cord (VNC). Octopamine (OA) applications to the TAG strongly enhanced the activity of these nerves, either by increasing the firing rate of a number of spontaneously firing units or by recruiting new ones. This octopaminergic activity affected calling behavior, and specifically the muscle activity leading to cycling extensions of the intersegmental membrane (IM) between segments VIII and IX (ovipositor). Our results indicate that in the female gypsy moth the octopaminergic neural activity of the TAG is coupled with extensions and retractions of IM for the purpose of releasing pheromone, where motor units innervated by nerve pair IV appear antagonistic with respect to those innervated by nerve pair V.
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Comunicação Animal , Mariposas/fisiologia , Octopamina/metabolismo , Animais , Feminino , Neurônios Motores/fisiologia , Atrativos Sexuais/metabolismoRESUMO
Findings from studies using animal models expressing amyotrophic lateral sclerosis (ALS) mutations in RNA-binding proteins, such as Transactive Response DNA-binding protein-43 (TDP-43), indicate that this protein, which is involved in multiple functions, including transcriptional regulation and pre-mRNA splicing, represents a key candidate in ALS development. This study focuses on characterizing, in a Drosophila genetic model of ALS (TDP-43), the effects of Mucuna pruriens (Mpe) and Withania somnifera (Wse). Electrophysiological and behavioural data in TDP-43 mutant flies revealed anomalous locomotion (i.e. impaired climbing with unexpected hyperactivity) and sleep dysregulation. These features, in agreement with previous findings with a different ALS model, were at least partially, rescued by treatment with Mpe and Wse. In addition, electrophysiological recordings from dorsal longitudinal muscle fibers and behavioral observations of TDP-43 flies exposed to the volatile anaesthetics, diethyl ether or chloroform, showed paradoxical responses, which were normalized upon Mpe or Wse treatment. Hence, given the involvement of some potassium channels in the effects of anaesthetics, our results also hint toward a possible dysregulation of some potassium channels in the ALS-TDP-43 Drosophila model, that might shed new light on future therapeutic strategies pertaining to ALS.
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Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/fisiopatologia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/farmacologia , Proteinopatias TDP-43/genética , Proteinopatias TDP-43/fisiopatologia , Esclerose Lateral Amiotrófica/tratamento farmacológico , Animais , Modelos Animais de Doenças , Drosophila melanogaster , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Mutação , Compostos Fitoquímicos/química , Extratos Vegetais/química , Proteinopatias TDP-43/tratamento farmacológicoRESUMO
Despite its great potentiality, little attention has been paid to modelling gastrointestinal symptoms of Parkinson's disease (PD) in Drosophila melanogaster (Dm). Our previous studies on standardized Mucuna pruriens extract (Mpe) have shown usefulness in the Drosophila model of PD. In this communication, we provide new information on the effect of Mpe on basal and serotonin treated contractions in the crop (i.e., an important and essential part of the gut) in Drosophila PD mutant for PTEN-induced putative kinase 1 (PINK1B9) gene. The effect of Mpe on PINK1B9 supplied with standard diet to larvae and/or adults, were assayed on 10-15â¯days old flies. Conversely from what we observed in the wild type flies, recordings demonstrated that exogenous applications of serotonin on crop muscles of untreated PINK1B9 affect neither the frequency nor the amplitude of the crop contraction, while the same muscle parameters are enhanced following brain injections of serotonin, thus suggesting that PINK1B9 mutants may likely have an impairment in the serotonergic pathways. Also, the mitochondrial morphology in the crop muscles is strongly compromised, as demonstrated by the transmission electron microscopy analysis. The Mpe treatment rescued the crop muscle parameters and also the mitochondrial morphology when supplied to both larvae and adults. Overall, this study strengthens the relevance of using PINK1B9 Dm as a translational model to study the gastrointestinal symptoms in PD and also confirms the useful employment of M. pruriens for PD treatment.