RESUMO
Collagen VI (Col-VI) is an extracellular matrix protein primarily known for its bridging role in connective tissues that has been suggested to play a neuroprotective role. In the present study we report increased mRNA and protein expression of Col-VI in the hippocampus and cortex at a late stage of epileptogenesis in a post-status epilepticus (SE) model of epilepsy and in brain tissue from patients with epilepsy. We further present a novel finding that exposure of mouse hippocampal slices to Col-VI augments paired-pulse facilitation in Schaffer collateral-CA1 excitatory synapses indicating decreased release probability of glutamate. In line with this finding, lack of Col-VI expression in the knock-out mice show paired-pulse depression in these synapses, suggesting increased release probability of glutamate. In addition, we observed dynamic changes in Col-VI blood plasma levels in rats after Kainate-induced SE, and increased levels of Col-VI mRNA and protein in autopsy or postmortem brain of humans suffering from epilepsy. Thus, our data indicate that elevated levels of ColVI following seizures leads to attenuated glutamatergic transmission, ultimately resulting in less overall network excitability. Presumably, increased Col-VI may act as part of endogenous compensatory mechanism against enhanced excitability during epileptogenic processes in the hippocampus, and could be further investigated as a potential functional biomarker of epileptogenesis, and/or a novel target for therapeutic intervention.
Assuntos
Colágeno Tipo VI , Camundongos Knockout , Convulsões , Transmissão Sináptica , Animais , Humanos , Masculino , Camundongos , Ratos , Colágeno Tipo VI/metabolismo , Colágeno Tipo VI/genética , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/metabolismo , Ácido Caínico/toxicidade , Camundongos Endogâmicos C57BL , Ratos Sprague-Dawley , Convulsões/metabolismo , Convulsões/fisiopatologia , Convulsões/induzido quimicamente , Transmissão Sináptica/fisiologiaRESUMO
Gene therapy with AAV vectors carrying genes for neuropeptide Y and its receptor Y2 has been shown to inhibit seizures in multiple animal models of epilepsy. It is however unknown how the AAV serotype or the sequence order of these two transgenes in the expression cassette affects the actual parenchymal gene expression levels and the seizure-suppressant efficacy. To address these questions, we compared three viral vector serotypes (AAV1, AAV2 and AAV8) and two transgene sequence orders (NPY-IRES-Y2 and Y2-IRES-NPY) in a rat model of acutely induced seizures. Wistar male rats were injected bilaterally with viral vectors and 3 weeks later acute seizures were induced by a subcutaneous injection of kainate. The latency until 1st motor seizure, time spent in motor seizure and latency to status epilepticus were measured to evaluate the seizure-suppressing efficacy of these vectors compared to an empty cassette control vector. Based on the results, the effect of the AAV1-NPY-IRES-Y2 vector was further investigated by in vitro electrophysiology, and its ability to achieve transgene overexpression in resected human hippocampal tissue was evaluated. The AAV1-NPY-IRES-Y2 proved to be better to any other serotype or gene sequence considering both transgene expression and ability to suppress induced seizures in rats. The vector also demonstrated transgene-induced decrease of glutamate release from excitatory neuron terminals and significantly increased both NPY and Y2 expression in resected human hippocampal tissue from patients with drug-resistant temporal lobe epilepsy. These results validate the feasibility of NPY/Y2 receptor gene therapy as a therapeutic opportunity in focal epilepsies.
Assuntos
Epilepsia , Convulsões , Ratos , Masculino , Humanos , Animais , Sorogrupo , Ratos Wistar , Convulsões/genética , Convulsões/terapia , Epilepsia/terapia , Neuropeptídeo Y/genética , Neuropeptídeo Y/metabolismo , Terapia Genética/métodos , Hipocampo/metabolismoRESUMO
Progressive degeneration of dopaminergic neurons, immune activation, and α-synuclein pathology characterize Parkinson's disease (PD). We previously reported that unilateral intranigral injection of recombinant adeno-associated viral (rAAV) vectors encoding wild-type human α-synuclein produced a rat model of early PD with dopamine terminal dysfunction. Here we tested the hypothesis that decreases in dopamine result in increased postsynaptic dopamine D2/D3 receptor expression, neuroinflammation, and reduced synaptic vesicle glycoprotein 2A (SV2A) density. Rats were injected with rAAV encoding α-synuclein or green fluorescent protein and subjected to non-pharmacological motor tests, before euthanization at 12 weeks post-injection. We performed: (1) in situ hybridization of nigral tyrosine hydroxylase mRNA, (2) HPLC of striatal dopamine content, and (3) autoradiography with [3H]raclopride, [3H]DTBZ, [3H]GBR12935, [3H]PK11195, and [3H]UCB-J to measure binding at D2/3 receptors, vesicular monoamine transporter 2, dopamine transporters, mitochondrial translocator protein, and SV2A, respectively. rAAV-α-synuclein induced motor asymmetry and reduced tyrosine hydroxylase mRNA and dopamine content in ipsilateral brain regions. This was paralleled by elevated ipsilateral postsynaptic dopamine D2/3 receptor expression and immune activation, with no changes to synaptic SV2A density. In conclusion, α-synuclein overexpression results in dopaminergic degeneration that induced compensatory increases in D2/3 binding and immune activation, recapitulating many of the pathological characteristics of PD.
RESUMO
OBJECTIVE: The goal of this study was to investigate the importance of central hormone-sensitive lipase (HSL) expression in the regulation of food intake and body weight in mice to clarify whether intracellular lipolysis in the mammalian hypothalamus plays a role in regulating appetite. METHODS: Using pharmacological and genetic approaches, we investigated the role of HSL in the rodent brain in the regulation of feeding and energy homeostasis under basal conditions during acute stress and high-fat diet feeding. RESULTS: We found that HSL, a key enzyme in the catabolism of cellular lipid stores, is expressed in the appetite-regulating centers in the hypothalamus and is activated by acute stress through a mechanism similar to that observed in adipose tissue and skeletal muscle. Inhibition of HSL in rodent models by a synthetic ligand, global knockout, or brain-specific deletion of HSL prevents a decrease in food intake normally seen in response to acute stress and is associated with the increased expression of orexigenic peptides neuropeptide Y (NPY) and agouti-related peptide (AgRP). Increased food intake can be reversed by adeno-associated virus-mediated reintroduction of HSL in neurons of the mediobasal hypothalamus. Importantly, metabolic stress induced by a high-fat diet also enhances the hyperphagic phenotype of HSL-deficient mice. Specific deletion of HSL in the ventromedial hypothalamic nucleus (VMH) or AgRP neurons reveals that HSL in the VMH plays a role in both acute stress-induced food intake and high-fat diet-induced obesity. CONCLUSIONS: Our results indicate that HSL activity in the mediobasal hypothalamus is involved in the acute reduction in food intake during the acute stress response and sensing of a high-fat diet.
Assuntos
Apetite/fisiologia , Homeostase , Hipotálamo/metabolismo , Esterol Esterase/metabolismo , Proteína Relacionada com Agouti/metabolismo , Animais , Peso Corporal , Dieta Hiperlipídica/efeitos adversos , Ingestão de Alimentos , Metabolismo Energético , Feminino , Hiperfagia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Neuropeptídeo Y/metabolismo , Obesidade/metabolismo , Fatores de Processamento de RNA , Esterol Esterase/genética , Estresse Fisiológico/genética , TranscriptomaRESUMO
Neuropeptide S (NPS) has shown anxiolytic-like effects in rodents after acute administration, but its long-term effects remain unknown. Gene therapy enables the targeted delivery of DNA to cell nuclei, and recombinant adeno-associated viral (rAAV) vectors have been identified as suitable tools for stable overexpression. Thus, to explore the effects of long-term expression of NPS, the present study examined anxiety- and depressive-like effects after rAAV-mediated NPS overexpression in the rat amygdala. Compared to rats injected with an empty control vector (rAAV-Empty), rAAV-NPS treatment was associated with reduced anxiety-like behavior in the elevated plus maze and light-dark box, but did not affect depressive-like behavior in the forced swim test. Importantly, rAAV-NPS did not cause confounding effects on locomotion or bodyweight as opposed to currently used anxiolytic drugs. Immunohistochemical stainings revealed NPS-positive cells in the central and basolateral region of the amygdala in rAAV-NPS but not rAAV-Empty rats, indicating successful transduction. Our study provides novel evidence for sustained anxiolytic-like properties of NPS by transgenic overexpression. These data suggest that rAAV-NPS application deserves further attention as a potential treatment strategy for anxiety in humans.
Assuntos
Tonsila do Cerebelo/metabolismo , Ansiedade/metabolismo , Ansiedade/fisiopatologia , Comportamento Animal/fisiologia , Depressão/metabolismo , Neuropeptídeos/metabolismo , Animais , Peso Corporal/fisiologia , Dependovirus , Modelos Animais de Doenças , Vetores Genéticos , Locomoção/fisiologia , Masculino , Ratos , Ratos WistarRESUMO
Attention-deficit/hyperactivity disorder (ADHD) is a psychiatric disorder characterized by inattention, aberrant impulsivity, and hyperactivity. Although the underlying pathophysiology of ADHD remains unclear, dopamine and norepinephrine signaling originating from the ventral tegmental area (VTA) and locus coeruleus (LC) is thought to be critically involved. In this study, we employ Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) together with the mouse 5-Choice Serial Reaction Time Task (5-CSRTT) to investigate the necessary roles of these catecholamines in ADHD-related behaviors, including attention, impulsivity, and motivation. By selective inhibition of tyrosine hydroxylase (TH)-positive VTA dopamine neurons expressing the Gi-coupled DREADD (hM4Di), we observed a marked impairment of effort-based motivation and subsequently speed and overall vigor of responding. At the highest clozapine N-oxide (CNO) dose tested (i.e. 2â¯mg/kg) to activate hM4Di, we detected a reduction in locomotor activity. DREADD-mediated inhibition of LC norepinephrine neurons reduced attentional performance in a variable stimulus duration test designed to increase task difficulty, specifically by increasing trials omissions, reducing mean score, and visual processing speed. These findings show that VTA dopamine and LC norepinephrine neurons differentially affect attention, impulsive and motivational control. In addition, this study highlights how molecular genetic probing of selective catecholamine circuits can provide valuable insights into the mechanisms underlying ADHD-relevant behaviors.
Assuntos
Dopamina/metabolismo , Neurônios/metabolismo , Norepinefrina/metabolismo , Animais , Atenção/efeitos dos fármacos , Atenção/fisiologia , Técnicas Genéticas , Comportamento Impulsivo/efeitos dos fármacos , Comportamento Impulsivo/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Motivação/efeitos dos fármacos , Motivação/fisiologia , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Neurônios/efeitos dos fármacos , Testes Neuropsicológicos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/metabolismoRESUMO
Pain remains a major concern in patients suffering from metastatic cancer to the bone and more knowledge of the condition, as well as novel treatment avenues, are called for. Neuropeptide Y (NPY) is a highly conserved peptide that appears to play a central role in nociceptive signaling in inflammatory and neuropathic pain. However, little is known about the peptide in cancer-induced bone pain. Here, we evaluate the role of spinal NPY in the MRMT-1 rat model of cancer-induced bone pain. Our studies revealed an up-regulation of NPY-immunoreactivity in the dorsal horn of cancer-bearing rats 17â¯days after inoculation, which could be a compensatory antinociceptive response. Consistent with this interpretation, intrathecal administration of NPY to rats with cancer-induced bone pain caused a reduction in nociceptive behaviors that lasted up to 150â¯min. This effect was diminished by both Y1 (BIBO3304) and Y2 (BIIE0246) receptor antagonists, indicating that both receptors participate in mediating the antinociceptive effect of NPY. Y1 and Y2 receptor binding in the spinal cord was unchanged in the cancer state as compared to sham-operated rats, consistent with the notion that increased NPY results in a net antinociceptive effect in the MRMT-1 model. In conclusion, the data indicate that NPY is involved in the spinal nociceptive signaling of cancer-induced bone pain and could be a new therapeutic target for patients with this condition.
Assuntos
Dor do Câncer/metabolismo , Dor Musculoesquelética/metabolismo , Neuropeptídeo Y/metabolismo , Nociceptividade/efeitos dos fármacos , Corno Dorsal da Medula Espinal/metabolismo , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Comportamento Animal/efeitos dos fármacos , Benzazepinas/farmacologia , Dor do Câncer/tratamento farmacológico , Feminino , Masculino , Dor Musculoesquelética/tratamento farmacológico , Neuropeptídeo Y/farmacologia , Neuropeptídeo Y/uso terapêutico , Medição da Dor , Ratos , Ratos Sprague-Dawley , Receptores de Neuropeptídeo Y/antagonistas & inibidoresRESUMO
Ghrelin receptor (Ghr-R) signaling in neurons of the ventral tegmental area (VTA) can modulate dopaminergic function and the reward-related effects of both palatable foods and drugs of abuse. In this study, we re-introduced the Ghr-R in VTA neurons in Ghr-R knockout mice (Ghr-RVTA mice) to specifically study the importance of the constitutively active Ghr-R for VTA neuronal signaling. Our results showed that re-introduction of the Ghr-R in the VTA had no impact on body weight or food intake under basal conditions. However, during novel environment stress Ghr-RVTA mice showed increased food intake and energy expenditure compared to Ghr-R knockout mice, demonstrating the significance of Ghr-R signaling in the response to stress. Ghr-RVTA mice also showed increased cocaine-induced locomotor activity compared to Ghr-R knockout mice, highlighting the importance of ghrelin signaling for the reward-related effects of activation of VTA neurons. Overall, our data suggest that re-introduction of the Ghr-R in the mesolimbic reward system of Ghr-R knockout mice increases the level of activation induced by both cocaine and novelty stress.
Assuntos
Comportamento Animal/fisiologia , Receptores de Grelina/metabolismo , Área Tegmentar Ventral/metabolismo , Animais , Peso Corporal , Dependovirus/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Ingestão de Alimentos , Metabolismo Energético , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Locomoção , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Consumo de Oxigênio , Receptores de Dopamina D2/metabolismo , Receptores de Grelina/deficiência , Receptores de Grelina/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
It is well known that physical activity has several health benefits, yet many people do not exercise. Dopamine levels in the striatum of the brain are thought to be important for the motivation to exercise. Conversely, we hypothesized that muscle quality can affect the motivation to exercise through alterations of the brain dopamine levels specifically in the striatal region. To test this hypothesis, transgenic mice overexpressing an inactivatable dominant negative α2 AMPK construct (AMPK α2 KD) in muscles and littermate wildtype (WT) mice were tested. AMPK α2 KD mice have impaired running capacity and display reduced voluntary wheel running activity. Striatal content of dopamine and its metabolites were measured under basal physiological conditions and after cocaine-induced dopamine efflux from the ventral striatum by in vivo microdialysis. Moreover, cocaine-induced locomotor activity was tested in an open field test. Furthermore, we investigated maximal running capacity and voluntary running over a period of 19days. AMPK α2 KD mice ran 30% less in daily distance compared to WT. Furthermore, AMPK α2 KD mice showed significantly decreased locomotor activity in the open field test compared to WT when treated with saline or cocaine, respectively, but the increase induced by cocaine was similar in AMPK α2 KD and WT mice. The efflux of dopamine in ventral striatum after cocaine treatment increased similarly by 2.5-fold in the two genotypes, and basal levels of dopamine and its metabolites DOPAC and HVA were also similar between genotypes. These findings show that decreased AMPK activity in muscle leads to decreased voluntary activity which is not due to secondary abnormalities in dopamine levels in the ventral striatum or sensitivity to cocaine. Thus, decreased voluntary activity in AMPK muscle deficient mice is most likely unrelated to regulation of brain dopamine content and metabolism.
Assuntos
Proteínas Quinases Ativadas por AMP/deficiência , Corpo Estriado/metabolismo , Dopamina/metabolismo , Músculo Esquelético/metabolismo , Corrida/fisiologia , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Cocaína/farmacologia , Corpo Estriado/efeitos dos fármacos , Inibidores da Captação de Dopamina/farmacologia , Ácido Homovanílico/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , MutaçãoRESUMO
Optogenetics is one of the most powerful tools in neuroscience, allowing for selective control of specific neuronal populations in the brain of experimental animals, including mammals. We report, for the first time, the application of optogenetic tools to human brain tissue providing a proof-of-concept for the use of optogenetics in neuromodulation of human cortical and hippocampal neurons as a possible tool to explore network mechanisms and develop future therapeutic strategies.
Assuntos
Encéfalo/citologia , Encéfalo/fisiologia , Neurônios/fisiologia , Optogenética , Channelrhodopsins , Potenciais Evocados/efeitos da radiação , Antagonistas GABAérgicos/farmacologia , Expressão Gênica , Ácido Glutâmico/metabolismo , Humanos , Luz , Receptores de GABA/metabolismo , Técnicas de Cultura de TecidosRESUMO
Although novel treatment strategies based on the gene therapy approach for epilepsy has been encouraging, there is still a gap in demonstrating a proof-of-concept in a clinically relevant animal model and study design. In the present study, a conceptually novel framework reflecting a plausible clinical trial for gene therapy of temporal lobe epilepsy was explored: We investigated (i) whether the post intrahippocampal kainate-induced status epilepticus (SE) model of chronic epilepsy in rats could be clinically relevant; and (ii) whether a translationally designed neuropeptide Y (NPY)/Y2 receptor-based gene therapy approach targeting only the seizure-generating focus unilaterally can decrease seizure frequency in this chronic model of epilepsy. Our data suggest that the intrahippocampal kainate model resembles the disease development of human chronic mesial temporal lobe epilepsy (mTLE): (i) spontaneous seizures originate in the sclerotic hippocampus; (ii) only a part of the animals develops chronic epilepsy; (iii) animals show largely variable seizure frequency that (iv) tends to progressively increase over time. Despite significant hippocampal degeneration caused by the kainate injection, the use of MRI allowed targeting the recombinant adeno-associated viral (rAAV) vectors encoding NPY and Y2 receptor genes to the remaining dorsal and ventral hippocampal areas ipsilateral to the kainate injection. Continuous video-EEG monitoring demonstrated not only prevention of the progressive increase in seizure frequency in rAAV-NPY/Y2 treated animals as compared to the controls, but even 45% decrease of seizure frequency in 80% of the epileptic animals. This translationally designed study in a clinically relevant model of epilepsy suggests that simultaneous overexpression of NPY and Y2 receptors unilaterally in the seizure focus is a relevant and promising approach that can be further validated in more extensive preclinical studies to develop a future treatment strategy for severe, often pharmacoresistant focal epilepsy cases that cannot be offered alternative therapeutic options.
Assuntos
Córtex Cerebral/fisiopatologia , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/terapia , Terapia Genética/métodos , Receptores de Neuropeptídeo Y/genética , Animais , Córtex Cerebral/efeitos dos fármacos , Dependovirus/genética , Eletroencefalografia , Epilepsia do Lobo Temporal/induzido quimicamente , Vetores Genéticos/administração & dosagem , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Ácido Caínico/administração & dosagem , Masculino , Ratos , Ratos Wistar , Pesquisa Translacional BiomédicaRESUMO
BACKGROUND: Besides the well-known effects of ghrelin on adiposity and food intake regulation, the ghrelin system has been shown to regulate aspects of behavior including anxiety and stress. However, the effect of virus-mediated overexpression of the ghrelin receptor in the amygdala has not previously been addressed directly. METHODS: First, we examined the acute effect of peripheral ghrelin administration on anxiety- and depression-like behavior using the open field, elevated plus maze, forced swim, and tail suspension tests. Next, we examined the effect of peripheral ghrelin administration and ghrelin receptor deficiency on stress in a familiar and social environment using the Intellicage system. Importantly, we also used a novel approach to study ghrelin receptor signaling in the brain by overexpressing the ghrelin receptor in the amygdala. We examined the effect of ghrelin receptor overexpression on anxiety-related behavior before and after acute stress and measured the modulation of serotonin receptor expression. RESULTS: We found that ghrelin caused an anxiolytic-like effect in both the open field and elevated plus maze tests. Additionally, it attenuated air-puff-induced stress in the social environment, while the opposite was shown in ghrelin receptor deficient mice. Finally, we found that overexpression of the ghrelin receptor in the basolateral division of the amygdala caused an anxiolytic-like effect and decreased the 5HT1a receptor expression. CONCLUSIONS: Ghrelin administration and overexpression of the ghrelin receptor in the amygdala induces anxiolytic-like behavior. Since the ghrelin receptor has high constitutive activity, ligand-independent signaling in vivo may be important for the observed anxiolytic-like effects. The anxiolytic effects seem to be mediated independently from the HPA axis, potentially engaging the central serotonin system.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Ansiolíticos/farmacologia , Ansiedade/prevenção & controle , Comportamento Animal/efeitos dos fármacos , Grelina/farmacologia , Receptores de Grelina/agonistas , Transdução de Sinais/efeitos dos fármacos , Tonsila do Cerebelo/metabolismo , Tonsila do Cerebelo/fisiopatologia , Animais , Ansiedade/genética , Ansiedade/metabolismo , Ansiedade/psicologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Elevação dos Membros Posteriores , Humanos , Locomoção/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Receptor 5-HT1A de Serotonina/metabolismo , Receptores de Grelina/genética , Receptores de Grelina/metabolismo , Comportamento Social , Estresse Psicológico/complicações , Estresse Psicológico/metabolismo , Estresse Psicológico/psicologia , Natação , Fatores de TempoRESUMO
Development of novel disease-modifying treatment strategies for neurological disorders, which at present have no cure, represents a major challenge for today's neurology. Translation of findings from animal models to humans represents an unresolved gap in most of the preclinical studies. Gene therapy is an evolving innovative approach that may prove useful for clinical applications. In animal models of temporal lobe epilepsy (TLE), gene therapy treatments based on viral vectors encoding NPY or galanin have been shown to effectively suppress seizures. However, how this translates to human TLE remains unknown. A unique possibility to validate these animal studies is provided by a surgical therapeutic approach, whereby resected epileptic tissue from temporal lobes of pharmacoresistant patients are available for neurophysiological studies in vitro. To test whether NPY and galanin have antiepileptic actions in human epileptic tissue as well, we applied these neuropeptides directly to human hippocampal slices in vitro. NPY strongly decreased stimulation-induced EPSPs in dentate gyrus and CA1 (up to 30 and 55%, respectively) via Y2 receptors, while galanin had no significant effect. Receptor autoradiographic binding revealed the presence of both NPY and galanin receptors, while functional receptor binding was only detected for NPY, suggesting that galanin receptor signaling may be impaired. These results underline the importance of validating findings from animal studies in human brain tissue, and advocate for NPY as a more appropriate candidate than galanin for future gene therapy trials in pharmacoresistant TLE patients.
Assuntos
Epilepsia/patologia , Galanina/farmacologia , Hipocampo/efeitos dos fármacos , Neuropeptídeo Y/farmacologia , Sinapses/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Adolescente , Adulto , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Hipocampo/patologia , Humanos , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos , Pessoa de Meia-Idade , Técnicas de Patch-Clamp , Ensaio Radioligante , Receptores de Galanina/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Isótopos de Enxofre/farmacocinética , Adulto JovemRESUMO
Electroconvulsive therapy (ECT) remains one of the most effective treatments of major depression. It has been suggested that the mechanisms of action involve gene expression. In recent decades there have been several investigations of gene expression following both acute and chronic electroconvulsive stimulation (ECS). These studies have focused on several distinct gene targets but have generally included only few time points after ECS for measuring gene expression. Here we measured gene expression of three types of genes: Immediate early genes, synaptic proteins, and neuropeptides at six time points following an acute ECS. We find significant increases for c-Fos, Egr1, Neuritin 1 (Nrn 1), Bdnf, Snap29, Synaptotagmin III (Syt 3), Synapsin I (Syn 1), and Psd95 at differing time points after ECS. For some genes these changes are prolonged whereas for others they are transient. Npy expression significantly increases whereas the gene expression of its receptors Npy1r, Npy2r, and Npy5r initially decreases. These decreases are followed by a significant increase for Npy2r, suggesting anticonvulsive adaptations following seizures. In summary, we find distinct changes in mRNA quantities that are characteristic for each gene. Considering the observed transitory and inverse changes in expression patterns, these data underline the importance of conducting measurements at several time points post-ECS.
Assuntos
Eletroconvulsoterapia/efeitos adversos , Genes Precoces/genética , Hipocampo/metabolismo , Membranas Sinápticas/metabolismo , Animais , Perfilação da Expressão Gênica , Masculino , Modelos Animais , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/biossíntese , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Receptores de Neuropeptídeo Y/biossíntese , Receptores de Neuropeptídeo Y/genética , Receptores de Neuropeptídeo Y/metabolismo , Convulsões/genética , Membranas Sinápticas/genética , TranscriptomaRESUMO
Neural stem cells of the subventricular zone (SVZ) represent a potentially important source of surrogate cells for the treatment of brain damage. Proper use of these cells for neuronal replacement depends on the ability to drive neuronal differentiation. Several neuromodulators stimulate neurogenesis. Here we examined the effects of the neuropeptide galanin, on neuronal differentiation in murine SVZ cultures. SVZ neurospheres obtained from early postnatal mice were treated with 10 nM to 2 µM galanin. Galanin promoted neuronal differentiation, increasing numbers of NeuN-, vesicular GABA transporter- and tyrosine hydroxylase-expressing neurons. In contrast, galanin neither affected cell proliferation assessed by BrdU incorporation nor cell death evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Neuronal differentiation was further confirmed at the functional level by measuring [Ca(2+)]i variations in single SVZ cells after KCl and histamine stimulations to distinguish neurons from immature cells. Galanin treatment increased the numbers of neuronal-like responding cells compared to immature cells. Using selective agonists (M617, AR-M1896) and antagonists (galantide, M871) for galanin receptors 1 and 2, we showed that both galanin receptors mediated neuronal differentiation. Early proneuronal effects of galanin included positive regulation of the transcription factor neurogenin-1 (Ngn1). In addition, galanin promoted axonogenesis and dendritogenesis, increasing both the length of phosphorylated stress-activated protein kinase- and Tau-positive axons and the numbers of microtubule associated protein-2 (MAP-2)-labelled dendrites. Moreover, galanin inhibited SVZ cell migration in the transwell assay. Our results show a proneurogenic effect of galanin and open new perspectives for future applications in stem cell-based therapies for neuronal replacement.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Galanina/farmacologia , Células-Tronco Neurais/metabolismo , Receptor Tipo 1 de Galanina/metabolismo , Receptor Tipo 2 de Galanina/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Técnicas de Cultura de Células , Morte Celular/efeitos dos fármacos , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ventrículos Cerebrais/citologia , Ventrículos Cerebrais/efeitos dos fármacos , Proteínas de Ligação a DNA , Galanina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Proteínas Nucleares/biossíntese , Fragmentos de Peptídeos/farmacologia , Tirosina 3-Mono-Oxigenase/biossíntese , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/biossínteseRESUMO
Electroconvulsive stimulation (ECS) remains one of the most effective treatments of major depression. However, the underlying molecular changes still remain to be elucidated. Since ECS causes rapid and significant changes in gene expression we have looked at epigenetic regulation of two important immediate early genes that are both induced after ECS: c-Fos and Arc. We examined Arc and c-Fos protein expression and found Arc present over 4 h, in contrast to c-Fos presence lasting only 1 h. Both genes had returned to baseline expression at 24 h post-ECS. Histone H4 acetylation (H4Ac) is one of the important epigenetic marks associated with gene activation. We show increased H4Ac at the c-Fos promoter at 1 h post-ECS. Surprisingly, we also observed a significant increase in DNA methylation of the Arc gene promoter at 24 h post-ECS. DNA methylation, which is responsible for gene silencing, is a rather stable covalent modification. This suggests that Arc expression has been repressed and may consequently remain inhibited for a prolonged period post-ECS. Arc plays a critical role in the maintenance phase of long-term potentiation (LTP) and consolidation of memory in the rat brain. Thus, this study is one of the first to demonstrate DNA methylation as a regulator of ECS-induced gene expression and it provides a molecular link to the memory deficits observed after ECS.
Assuntos
Proteínas do Citoesqueleto/genética , Eletrochoque , Epigênese Genética/genética , Hipocampo/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas c-fos/genética , Animais , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/fisiologia , Metilação de DNA/genética , Eletrochoque/métodos , Masculino , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/fisiologia , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-fos/fisiologia , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
RATIONALE: Several studies suggest a role for neuropeptide Y (NPY) in addiction to drugs of abuse, including cocaine. However, the NPY receptors mediating addiction-related effects remain to be determined. OBJECTIVES: To explore the potential role of Y5 NPY receptors in cocaine-induced behavioural effects. METHODS: The Y5 antagonist L-152,804 and Y5-knockout (Y5-KO) mice were tested in two models of cocaine addiction-related behaviour: acute self-administration and cocaine-induced hyperactivity. We also studied effects of Y5 receptor antagonism on cocaine-induced c-fos expression and extracellular dopamine with microdialysis as well as dopamine transporter-mediated uptake of dopamine in vitro. Immunocytochemistry was used to determine whether dopamine neurons express Y5-like immunoreactivity. RESULTS: In self-administration, L-152,804 prominently decreased nose-poking for the peak dose of cocaine and shifted the dose-response curve for cocaine downward. Y5-KO mice also showed modestly attenuated self-administration. Cocaine-induced hyperactivity was attenuated by L-152,804 and in Y5-KO mice. Cocaine failed to increase c-fos expression in the nucleus accumbens and striatum of L-152,804-treated mice, indicating that the Y5 antagonist could act by influencing neural activity in these regions. Accordingly, the cocaine-induced increase in accumbal extracellular dopamine was attenuated by L-152,804 and in Y5-KO mice, suggesting that Y5 antagonism influences cocaine-induced behaviour by regulating dopamine. Consistent with this concept, dopamine neurons in the ventral tegmental area appeared to contain Y5 receptors. In contrast, neither L-152,804 nor NPY influenced dopamine transporter-mediated dopamine uptake. CONCLUSIONS: The present data indicate that Y5 antagonism may attenuate cocaine-induced behavioural effects, suggesting that Y5 receptors could be a potential therapeutic target in cocaine addiction.
Assuntos
Cocaína/antagonistas & inibidores , Cicloexanos/farmacologia , Cicloexanos/uso terapêutico , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Xantenos/farmacologia , Xantenos/uso terapêutico , Animais , Animais não Endogâmicos , Cocaína/administração & dosagem , Cocaína/farmacologia , Transtornos Relacionados ao Uso de Cocaína/tratamento farmacológico , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Feminino , Hipercinese/induzido quimicamente , Hipercinese/tratamento farmacológico , Masculino , Camundongos , Camundongos Knockout , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Receptores de Neuropeptídeo Y/genética , Reforço Psicológico , AutoadministraçãoRESUMO
We recently demonstrated that recombinant adeno-associated viral vector-induced hippocampal overexpression of neuropeptide Y receptor, Y2, exerts a seizure-suppressant effect in kindling and kainate-induced models of epilepsy in rats. Interestingly, additional overexpression of neuropeptide Y in the hippocampus strengthened the seizure-suppressant effect of transgene Y2 receptors. Here we show for the first time that another neuropeptide Y receptor, Y5, can also be overexpressed in the hippocampus. However, unlike Y2 receptor overexpression, transgene Y5 receptors in the hippocampus had no effect on kainate-induced motor seizures in rats. However, combined overexpression of Y5 receptors and neuropeptide Y exerted prominent suppression of seizures. This seizure-suppressant effect of combination gene therapy with Y5 receptors and neuropeptide Y was significantly stronger as compared to neuropeptide Y overexpression alone. These results suggest that overexpression of Y5 receptors in combination with neuropeptide Y could be an alternative approach for more effective suppression of hippocampal seizures.
Assuntos
Hipocampo/metabolismo , Excitação Neurológica/genética , Neuropeptídeo Y/genética , Receptores de Neuropeptídeo Y/genética , Convulsões/genética , Animais , Ácido Caínico , Excitação Neurológica/metabolismo , Masculino , Neurônios/metabolismo , Neuropeptídeo Y/metabolismo , Ratos , Ratos Transgênicos , Ratos Wistar , Receptores de Neuropeptídeo Y/metabolismo , Convulsões/induzido quimicamente , Convulsões/metabolismoRESUMO
OBJECTIVE: There is clear evidence of a genetic component in major depression, and several studies indicate that neuropeptide Y (NPY) could play an important role in the pathophysiology of the disease. A well-known polymorphism encoding the substitution of leucine to proline in the signal peptide sequence of NPY (Leu7Pro variation) was previously found to protect against depression. Our study aimed at replicating this association in a large Danish population with major depression. METHOD: Leu7Pro was studied in a sample of depressed patients and ethnically matched controls, as well as psychiatric disease controls with schizophrenia. Possible functional consequences of Leu7Pro were explored in vitro. RESULTS: In contrast to previous studies, Pro7 appeared to be a risk allele for depression, being significantly more frequent in the depression sample (5.5%, n = 593; p = 0.009; odds ratio, OR: 1.46) as compared to ethnically matched controls (3.8%, n = 2912), while schizophrenia patients (4.1%, n = 503) did not differ. In vitro, the Pro7 substitution appeared to be associated with reduced levels of NPY without affecting its mRNA level. CONCLUSION: The Leu7Pro variation may increase the risk of major depression, possibly by affecting the biosynthesis of NPY.
RESUMO
Gene therapy using recombinant adeno-associated viral vectors overexpressing neuropeptide Y in the hippocampus exerts seizure-suppressant effects in rodent epilepsy models and is currently considered for clinical application in patients with intractable mesial temporal lobe epilepsy. Seizure suppression by neuropeptide Y in the hippocampus is predominantly mediated by Y2 receptors, which, together with neuropeptide Y, are upregulated after seizures as a compensatory mechanism. To explore whether such upregulation could prevent seizures, we overexpressed Y2 receptors in the hippocampus using recombinant adeno-associated viral vectors. In two temporal lobe epilepsy models, electrical kindling and kainate-induced seizures, vector-based transduction of Y2 receptor complementary DNA in the hippocampus of adult rats exerted seizure-suppressant effects. Simultaneous overexpression of Y2 and neuropeptide Y had a more pronounced seizure-suppressant effect. These results demonstrate that overexpression of Y2 receptors (alone or in combination with neuropeptide Y) could be an alternative strategy for epilepsy treatment.