Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Protoc ; 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38834919

RESUMO

Neuronal pathways recruit large postsynaptic populations and maintain connections via distinct postsynaptic response types (PRTs). Until recently, PRTs were accessible as a selection criterion for single-cell RNA sequencing only through probing by low-throughput whole-cell electrophysiology. To overcome these limitations and target neurons on the basis of specific PRTs for soma collection and subsequent single-cell RNA sequencing, we developed Voltage-Seq using the genetically encoded voltage indicator Voltron in acute brain slices from mice. We also created an onsite analysis tool, VoltView, to guide soma collection of specific PRTs using a classifier based on a previously acquired database of connectomes from multiple animals. Here we present our procedure for preparing the optical path, the imaging setup and detailing the imaging and analysis steps, as well as a complete procedure for sequencing library preparation. This enables researchers to conduct our high-throughput all-optical synaptic assay and to obtain single-cell transcriptomic data from selected postsynaptic neurons. This also allows researchers to resolve the connectivity ratio of a specific pathway and explore the diversity of PRTs within that connectome. Furthermore, combining high throughput with quick analysis gives unique access to find specific connections within a large postsynaptic connectome. Voltage-Seq also allows the investigation of correlations between connectivity and gene expression changes in a postsynaptic cell-type-specific manner for both excitatory and inhibitory connections. The Voltage-Seq workflow can be completed in ~6 weeks, including 4-5 weeks for viral expression of the Voltron sensor. The technique requires knowledge of basic laboratory techniques, micromanipulator handling skills and experience in molecular biology and bioinformatics.

2.
Nat Methods ; 20(9): 1409-1416, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37474808

RESUMO

Understanding the routing of neuronal information requires the functional characterization of connections. Neuronal projections recruit large postsynaptic ensembles with distinct postsynaptic response types (PRTs). PRT is typically probed by low-throughput whole-cell electrophysiology and is not a selection criterion for single-cell RNA-sequencing (scRNA-seq). To overcome these limitations and target neurons based on specific PRTs for soma harvesting and subsequent scRNA-seq, we created Voltage-Seq. We established all-optical voltage imaging and recorded the PRT of 8,347 neurons in the mouse periaqueductal gray (PAG) evoked by the optogenetic activation of ventromedial hypothalamic (VMH) terminals. PRTs were classified and spatially resolved in the entire VMH-PAG connectome. We built an onsite analysis tool named VoltView to navigate soma harvesting towards target PRTs guided by a classifier that used the VMH-PAG connectome database as a reference. We demonstrated Voltage-seq by locating VMH-driven γ-aminobutyric acid-ergic neurons in the PAG, guided solely by the onsite classification in VoltView.


Assuntos
Conectoma , Camundongos , Animais , Transcriptoma , Neurônios/fisiologia , Substância Cinzenta Periaquedutal/fisiologia
3.
Nat Neurosci ; 26(7): 1245-1255, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37349481

RESUMO

Excitatory projections from the lateral hypothalamic area (LHA) to the lateral habenula (LHb) drive aversive responses. We used patch-sequencing (Patch-seq) guided multimodal classification to define the structural and functional heterogeneity of the LHA-LHb pathway. Our classification identified six glutamatergic neuron types with unique electrophysiological properties, molecular profiles and projection patterns. We found that genetically defined LHA-LHb neurons signal distinct aspects of emotional or naturalistic behaviors, such as estrogen receptor 1-expressing (Esr1+) LHA-LHb neurons induce aversion, whereas neuropeptide Y-expressing (Npy+) LHA-LHb neurons control rearing behavior. Repeated optogenetic drive of Esr1+ LHA-LHb neurons induces a behaviorally persistent aversive state, and large-scale recordings showed a region-specific neural representation of the aversive signals in the prelimbic region of the prefrontal cortex. We further found that exposure to unpredictable mild shocks induced a sex-specific sensitivity to develop a stress state in female mice, which was associated with a specific shift in the intrinsic properties of bursting-type Esr1+ LHA-LHb neurons. In summary, we describe the diversity of LHA-LHb neuron types and provide evidence for the role of Esr1+ neurons in aversion and sexually dimorphic stress sensitivity.


Assuntos
Habenula , Feminino , Camundongos , Animais , Habenula/fisiologia , Hipotálamo/fisiologia , Região Hipotalâmica Lateral , Neurônios/fisiologia , Afeto , Vias Neurais/fisiologia
4.
J Neurosci ; 41(14): 3120-3141, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33593856

RESUMO

Inhibitory interneurons expressing parvalbumin (PV) are central to cortical network dynamics, generation of γ oscillations, and cognition. Dysfunction of PV interneurons disrupts cortical information processing and cognitive behavior. Brain-derived neurotrophic factor (BDNF)/tyrosine receptor kinase B (trkB) signaling regulates the maturation of cortical PV interneurons but is also implicated in their adult multidimensional functions. Using a novel viral strategy for cell-type-specific and spatially restricted expression of a dominant-negative trkB (trkB.DN), we show that BDNF/trkB signaling is essential to the integrity and maintenance of prefrontal PV interneurons in adult male and female mice. Reduced BDNF/trkB signaling in PV interneurons in the medial prefrontal cortex (mPFC) resulted in deficient PV inhibition and increased baseline local field potential (LFP) activity in a broad frequency band. The altered network activity was particularly pronounced during increased activation of the prefrontal network and was associated with changed dynamics of local excitatory neurons, as well as decreased modulation of the LFP, abnormalities that appeared to generalize across stimuli and brain states. In addition, our findings link reduced BDNF/trkB signaling in prefrontal PV interneurons to increased aggression. Together our investigations demonstrate that BDNF/trkB signaling in PV interneurons in the adult mPFC is essential to local network dynamics and cognitive behavior. Our data provide direct support for the suggested association between decreased trkB signaling, deficient PV inhibition, and altered prefrontal circuitry.SIGNIFICANCE STATEMENT Brain-derived neurotrophic factor (BDNF)/tyrosine receptor kinase B (trkB) signaling promotes the maturation of inhibitory parvalbumin (PV) interneurons, neurons central to local cortical dynamics, γ rhythms, and cognition. Here, we used a novel viral approach for reduced BDNF/trkB signaling in PV interneurons in the medial prefrontal cortex (mPFC) to establish the role of BDNF/trkB signaling in adult prefrontal network activities. Reduced BDNF/trkB signaling caused pronounced morphologic alterations, reduced PV inhibition, and deficient prefrontal network dynamics. The altered network activity appeared to manifest across stimuli and brain states and was associated with aberrant local field potential (LFP) activities and increased aggression. The results demonstrate that adult BDNF/trkB signaling is essential to PV inhibition and prefrontal circuit function and directly links BDNF/trkB signaling to network integrity in the adult brain.


Assuntos
Interneurônios/metabolismo , Glicoproteínas de Membrana/metabolismo , Rede Nervosa/metabolismo , Parvalbuminas/metabolismo , Córtex Pré-Frontal/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Fatores Etários , Animais , Feminino , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Parvalbuminas/genética , Proteínas Tirosina Quinases/genética
5.
Proc Natl Acad Sci U S A ; 116(51): 25958-25967, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31796600

RESUMO

Psychostimulant use is an ever-increasing socioeconomic burden, including a dramatic rise during pregnancy. Nevertheless, brain-wide effects of psychostimulant exposure are incompletely understood. Here, we performed Fos-CreERT2-based activity mapping, correlated for pregnant mouse dams and their fetuses with amphetamine, nicotine, and caffeine applied acutely during midgestation. While light-sheet microscopy-assisted intact tissue imaging revealed drug- and age-specific neuronal activation, the indusium griseum (IG) appeared indiscriminately affected. By using GAD67gfp/+ mice we subdivided the IG into a dorsolateral domain populated by γ-aminobutyric acidergic interneurons and a ventromedial segment containing glutamatergic neurons, many showing drug-induced activation and sequentially expressing Pou3f3/Brn1 and secretagogin (Scgn) during differentiation. We then combined Patch-seq and circuit mapping to show that the ventromedial IG is a quasi-continuum of glutamatergic neurons (IG-Vglut1+) reminiscent of dentate granule cells in both rodents and humans, whose dendrites emanate perpendicularly toward while their axons course parallel with the superior longitudinal fissure. IG-Vglut1+ neurons receive VGLUT1+ and VGLUT2+ excitatory afferents that topologically segregate along their somatodendritic axis. In turn, their efferents terminate in the olfactory bulb, thus being integral to a multisynaptic circuit that could feed information antiparallel to the olfactory-cortical pathway. In IG-Vglut1+ neurons, prenatal psychostimulant exposure delayed the onset of Scgn expression. Genetic ablation of Scgn was then found to sensitize adult mice toward methamphetamine-induced epilepsy. Overall, our study identifies brain-wide targets of the most common psychostimulants, among which Scgn+/Vglut1+ neurons of the IG link limbic and olfactory circuits.


Assuntos
Mapeamento Encefálico , Encéfalo/metabolismo , Regulação da Expressão Gênica , Lobo Límbico/metabolismo , Animais , Axônios/metabolismo , Encéfalo/diagnóstico por imagem , Dendritos/metabolismo , Feminino , Glutamato Descarboxilase/genética , Humanos , Interneurônios/metabolismo , Lobo Límbico/anatomia & histologia , Lobo Límbico/efeitos dos fármacos , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Bulbo Olfatório/metabolismo , Fatores do Domínio POU/genética , Fatores do Domínio POU/metabolismo , Secretagoginas/genética , Secretagoginas/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/genética , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/genética , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Ácido gama-Aminobutírico/metabolismo
6.
Cell Rep ; 29(13): 4320-4333.e5, 2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31875543

RESUMO

The striatum is organized into two major outputs formed by striatal projection neuron (SPN) subtypes with distinct molecular identities. In addition, histochemical division into patch and matrix compartments represents an additional spatial organization, proposed to mirror a motor-motivation regionalization. To map the molecular diversity of patch versus matrix SPNs, we genetically labeled mu opioid receptor (Oprm1) expressing neurons and performed single-nucleus RNA sequencing. This allowed us to establish molecular definitions of patch, matrix, and exopatch SPNs, as well as identification of Col11a1+ striatonigral SPNs. At the tissue level, mapping the expression of candidate markers reveals organization of spatial domains, which are conserved in the non-human primate brain. The spatial markers are cell-type independent and instead represent a spatial code found across all SPNs within a spatial domain. The spatiomolecular map establishes a formal system for targeting and studying striatal subregions and SPNs subtypes, beyond the classical striatonigral and striatopallidal division.


Assuntos
Neostriado/anatomia & histologia , Neostriado/metabolismo , Animais , Colágeno Tipo XI/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Receptores Opioides mu/metabolismo
7.
Mol Psychiatry ; 24(9): 1351-1368, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30755721

RESUMO

Encoding and predicting aversive events are critical functions of circuits that support survival and emotional well-being. Maladaptive circuit changes in emotional valence processing can underlie the pathophysiology of affective disorders. The lateral habenula (LHb) has been linked to aversion and mood regulation through modulation of the dopamine and serotonin systems. We have defined the identity and function of glutamatergic (Vglut2) control of the LHb, comparing the role of inputs originating in the globus pallidus internal segment (GPi), and lateral hypothalamic area (LHA), respectively. We found that LHb-projecting LHA neurons, and not the proposed GABA/glutamate co-releasing GPi neurons, are responsible for encoding negative value. Monosynaptic rabies tracing of the presynaptic organization revealed a predominantly limbic input onto LHA Vglut2 neurons, while sensorimotor inputs were more prominent onto GABA/glutamate co-releasing GPi neurons. We further recorded the activity of LHA Vglut2 neurons, by imaging calcium dynamics in response to appetitive versus aversive events in conditioning paradigms. LHA Vglut2 neurons formed activity clusters representing distinct reward or aversion signals, including a population that responded to mild foot shocks and predicted aversive events. We found that the LHb-projecting LHA Vglut2 neurons encode negative valence and rapidly develop a prediction signal for negative events. These findings establish the glutamatergic LHA-LHb circuit as a critical node in value processing.


Assuntos
Aprendizagem da Esquiva/fisiologia , Habenula/fisiologia , Hipotálamo/fisiologia , Afeto/fisiologia , Animais , Dopamina/metabolismo , Fármacos Atuantes sobre Aminoácidos Excitatórios/metabolismo , Globo Pálido/fisiologia , Ácido Glutâmico/metabolismo , Habenula/metabolismo , Região Hipotalâmica Lateral/fisiologia , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vias Neurais/fisiologia , Neurônios/fisiologia , Recompensa
8.
J Neurosci ; 39(1): 125-139, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30413647

RESUMO

Sensory perception depends on neocortical computations that contextually adjust sensory signals in different internal and environmental contexts. Neocortical layer 1 (L1) is the main target of cortical and subcortical inputs that provide "top-down" information for context-dependent sensory processing. Although L1 is devoid of excitatory cells, it contains the distal "tuft" dendrites of pyramidal cells (PCs) located in deeper layers. L1 also contains a poorly characterized population of GABAergic interneurons (INs), which regulate the impact that different top-down inputs have on PCs. A poor comprehension of L1 IN subtypes and how they affect PC activity has hampered our understanding of the mechanisms that underlie contextual modulation of sensory processing. We used novel genetic strategies in male and female mice combined with electrophysiological and morphological methods to help resolve differences that were unclear when using only electrophysiological and/or morphological approaches. We discovered that L1 contains four distinct populations of INs, each with a unique molecular profile, morphology, and electrophysiology, including a previously overlooked IN population (named here "canopy cells") representing 40% of L1 INs. In contrast to what is observed in other layers, most L1 neurons appear to be unique to the layer, highlighting the specialized character of the signal processing that takes place in L1. This new understanding of INs in L1, as well as the application of genetic methods based on the markers described here, will enable investigation of the cellular and circuit mechanisms of top-down processing in L1 with unprecedented detail.SIGNIFICANCE STATEMENT Neocortical layer 1 (L1) is the main target of corticocortical and subcortical projections that mediate top-down or context-dependent sensory perception. However, this unique layer is often referred to as "enigmatic" because its neuronal composition has been difficult to determine. Using a combination of genetic, electrophysiological, and morphological approaches that helped to resolve differences that were unclear when using a single approach, we were able to decipher the neuronal composition of L1. We identified markers that distinguish L1 neurons and found that the layer contains four populations of GABAergic interneurons, each with unique molecular profiles, morphologies, and electrophysiological properties. These findings provide a new framework for studying the circuit mechanisms underlying the processing of top-down inputs in neocortical L1.


Assuntos
Interneurônios/fisiologia , Neocórtex/citologia , Neocórtex/fisiologia , Animais , Dendritos/fisiologia , Dendritos/ultraestrutura , Fenômenos Eletrofisiológicos/fisiologia , Feminino , Interneurônios/ultraestrutura , Masculino , Camundongos , Camundongos Transgênicos , Neocórtex/ultraestrutura , Técnicas de Patch-Clamp , Células Piramidais/fisiologia , Células Piramidais/ultraestrutura , Ácido gama-Aminobutírico/fisiologia
9.
Cereb Cortex ; 27(4): 2453-2468, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-27102657

RESUMO

Although extensively studied postnatally, the functional differentiation of cholecystokinin (CCK)-containing interneurons en route towards the cerebral cortex during fetal development is incompletely understood. Here, we used CCKBAC/DsRed mice encoding a CCK promoter-driven red fluorescent protein to analyze the temporal dynamics of DsRed expression, neuronal identity, and positioning through high-resolution developmental neuroanatomy. Additionally, we developed a dual reporter mouse line (CCKBAC/DsRed::GAD67gfp/+) to differentiate CCK-containing interneurons from DsRed+ principal cells during prenatal development. We show that DsRed is upregulated in interneurons once they exit their proliferative niche in the ganglionic eminence and remains stably expressed throughout their long-distance migration towards the cerebrum, particularly in the hippocampus. DsRed+ interneurons, including a cohort coexpressing calretinin, accumulated at the palliosubpallial boundary by embryonic day 12.5. Pioneer DsRed+ interneurons already reached deep hippocampal layers by embryonic day 14.5 and were morphologically differentiated by birth. Furthermore, we probed migrating interneurons entering and traversing the cortical plate, as well as stationary cells in the hippocampus by patch-clamp electrophysiology to show the first signs of Na+ and K+ channel activity by embryonic day 12.5 and reliable adult-like excitability by embryonic day 18.5. Cumulatively, this study defines key positional, molecular, and biophysical properties of CCK+ interneurons in the prenatal brain.


Assuntos
Diferenciação Celular/fisiologia , Córtex Cerebral/citologia , Colecistocinina/metabolismo , Interneurônios/citologia , Neurogênese/fisiologia , Animais , Movimento Celular , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Interneurônios/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Técnicas de Patch-Clamp
10.
Nat Biotechnol ; 34(2): 175-183, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26689544

RESUMO

Traditionally, neuroscientists have defined the identity of neurons by the cells' location, morphology, connectivity and excitability. However, the direct relationship between these parameters and the molecular phenotypes has remained largely unexplored. Here, we present a method for obtaining full transcriptome data from single neocortical pyramidal cells and interneurons after whole-cell patch-clamp recordings in mouse brain slices. In our approach, termed Patch-seq, a patch-clamp stimulus protocol is followed by the aspiration of the entire somatic compartment into the recording pipette, reverse transcription of RNA including addition of unique molecular identifiers, cDNA amplification, Illumina library preparation and sequencing. We show that Patch-seq reveals a close link between electrophysiological characteristics, responses to acute chemical challenges and RNA expression of neurotransmitter receptors and channels. Moreover, it distinguishes neuronal subpopulations that correspond to both well-established and, to our knowledge, hitherto undescribed neuronal subtypes. Our findings demonstrate the ability of Patch-seq to precisely map neuronal subtypes and predict their network contributions in the brain.


Assuntos
Neurônios/citologia , Neurônios/metabolismo , Técnicas de Patch-Clamp/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Eletrofisiologia , Perfilação da Expressão Gênica , Camundongos , RNA Mensageiro/análise , RNA Mensageiro/metabolismo
11.
Sci Rep ; 5: 11592, 2015 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-26118676

RESUMO

Extreme longevity requires the continuous and large-scale adaptation of organ systems to delay senescence. Naked mole rats are the longest-living rodents, whose nervous system likely undergoes life-long adaptive reorganization. Nevertheless, neither the cellular organization of their cerebral cortex nor indices of structural neuronal plasticity along extreme time-scales have been established. We find that adult neurogenesis and neuronal migration are not unusual in naked mole rat brains. Instead, we show the prolonged expression of structural plasticity markers, many recognized as being developmentally controlled, and multi-year-long postnatal neuromorphogenesis and spatial synapse refinement in hippocampal and olfactory structures of the naked mole rat brain. Neurophysiological studies on identified hippocampal neurons demonstrated that morphological differentiation is disconnected from the control of excitability in all neuronal contingents regardless of their ability to self-renew. Overall, we conclude that naked mole rats show an extremely protracted period of brain maturation that may permit plasticity and resilience to neurodegenerative processes over their decades-long life span. This conclusion is consistent with the hypothesis that naked mole rats are neotenous, with retention of juvenile characteristics to permit survival in a hypoxic environment, with extreme longevity a consequence of greatly retarded development.


Assuntos
Encéfalo/crescimento & desenvolvimento , Longevidade/fisiologia , Modelos Biológicos , Animais , Animais Recém-Nascidos , Apoptose , Fenômenos Biofísicos , Região CA1 Hipocampal/fisiologia , Proliferação de Células , Dendritos/fisiologia , Giro Denteado/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Ratos-Toupeira , Morfogênese , Neurogênese , Plasticidade Neuronal , Neurônios/citologia , Córtex Piriforme/fisiologia , Sinapses/fisiologia
12.
Nat Commun ; 5: 4421, 2014 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-25030704

RESUMO

Local environmental cues are indispensable for axonal growth and guidance during brain circuit formation. Here, we combine genetic and pharmacological tools, as well as systems neuroanatomy in human fetuses and mouse models, to study the role of endocannabinoid and Slit/Robo signalling in axonal growth. We show that excess 2-arachidonoylglycerol, an endocannabinoid affecting directional axonal growth, triggers corpus callosum enlargement due to the errant CB1 cannabinoid receptor-containing corticofugal axon spreading. This phenotype mechanistically relies on the premature differentiation and end-feet proliferation of CB2R-expressing oligodendrocytes. We further show the dependence of both axonal Robo1 positioning and oligodendroglial Slit2 production on cell-type-specific cannabinoid receptor activation. Accordingly, Robo1 and/or Slit2 manipulation limits endocannabinoid modulation of axon guidance. We conclude that endocannabinoids can configure focal Slit2/Robo1 signalling to modulate directional axonal growth, which may provide a basis for understanding impaired brain wiring associated with metabolic deficits and prenatal drug exposure.


Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Endocanabinoides/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Animais , Ácidos Araquidônicos/farmacologia , Axônios/efeitos dos fármacos , Axônios/metabolismo , Encéfalo/efeitos dos fármacos , Células Cultivadas , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/embriologia , Corpo Caloso/metabolismo , Feminino , Glicerídeos/farmacologia , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Gravidez , Receptor CB1 de Canabinoide/metabolismo , Receptores Imunológicos/genética , Proteínas Roundabout
13.
EMBO J ; 33(7): 668-85, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24469251

RESUMO

Children exposed in utero to cannabis present permanent neurobehavioral and cognitive impairments. Psychoactive constituents from Cannabis spp., particularly Δ(9)-tetrahydrocannabinol (THC), bind to cannabinoid receptors in the fetal brain. However, it is unknown whether THC can trigger a cannabinoid receptor-driven molecular cascade to disrupt neuronal specification. Here, we show that repeated THC exposure disrupts endocannabinoid signaling, particularly the temporal dynamics of CB1 cannabinoid receptor, to rewire the fetal cortical circuitry. By interrogating the THC-sensitive neuronal proteome we identify Superior Cervical Ganglion 10 (SCG10)/stathmin-2, a microtubule-binding protein in axons, as a substrate of altered neuronal connectivity. We find SCG10 mRNA and protein reduced in the hippocampus of midgestational human cannabis-exposed fetuses, defining SCG10 as the first cannabis-driven molecular effector in the developing cerebrum. CB1 cannabinoid receptor activation recruits c-Jun N-terminal kinases to phosphorylate SCG10, promoting its rapid degradation in situ in motile axons and microtubule stabilization. Thus, THC enables ectopic formation of filopodia and alters axon morphology. These data highlight the maintenance of cytoskeletal dynamics as a molecular target for cannabis, whose imbalance can limit the computational power of neuronal circuitries in affected offspring.


Assuntos
Córtex Cerebral/efeitos dos fármacos , Dronabinol/farmacologia , Hipocampo/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Psicotrópicos/farmacologia , Receptor CB1 de Canabinoide/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Proteínas de Ligação ao Cálcio , Diferenciação Celular , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Feminino , Feto/anormalidades , Feto/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Hipocampo/citologia , Hipocampo/embriologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Exposição Materna/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Gravidez , Proteômica , RNA Mensageiro/genética , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Estatmina , Fatores de Tempo
14.
Biomaterials ; 35(11): 3541-50, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24439416

RESUMO

The quantity of cells with paracrine effects for use in myocardial regeneration therapy is limited. This study investigated the effects of catheter-based endomyocardial delivery of secretome of 2.5 × 10(9) apoptotic peripheral blood mononuclear cells (APOSEC) on porcine chronic post-myocardial infarction (MI) left ventricular (LV) dysfunction and on gene expression. Closed-chest reperfused MI was induced in pigs by 90-min occlusion followed by reperfusion of the mid-LAD (day 0). At day 30, animals were randomized to receive porcine APOSEC (n = 8) or medium solution (control; n = 8) injected intramyocardially into the MI border zone using 3D NOGA guidance. At day 60, cardiac MRI with late enhancement and diagnostic NOGA (myocardial viability) were performed. Gene expression profiling of the infarct core, border zone, and normal myocardium was performed using microarray analysis and confirmed by quantitative real-time PCR. Injection of APOSEC significantly decreased infarct size (p < 0.05) and improved cardiac index and myocardial viability compared to controls. A trend towards higher LV ejection fraction was observed in APOSEC vs. controls (45.4 ± 5.9% vs. 37.4 ± 8.9%, p = 0.052). Transcriptome analysis revealed significant downregulation of caspase-1, tumor necrosis factor and other inflammatory genes in APOSEC-affected areas. rtPCR showed higher expression of myogenic factor Mefc2 (p < 0.05) and downregulated caspase genes (p < 0.05) in APOSEC-treated pigs. In conclusion, overexpression of MEF2c and repression of caspase was related to decreased infarct size and improved cardiac function in secretome-treated animals. Altered gene expression 1-month post-APOSEC treatment proved the long-acting effects of cell-free therapy with paracrine factors.


Assuntos
Apoptose , Proteínas Sanguíneas/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/transplante , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/terapia , Disfunção Ventricular Esquerda/terapia , Administração Cutânea , Animais , Apoptose/genética , Doença Crônica , Regulação da Expressão Gênica , Hemodinâmica , Imageamento por Ressonância Magnética , Infarto do Miocárdio/patologia , Isquemia Miocárdica/genética , Isquemia Miocárdica/patologia , Isquemia Miocárdica/fisiopatologia , Isquemia Miocárdica/terapia , Neovascularização Fisiológica , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sus scrofa , Disfunção Ventricular Esquerda/complicações , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologia
15.
Neuropathol Appl Neurobiol ; 40(5): 603-9, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23795719

RESUMO

AIMS: Brain ischaemia models are essential to study the pathomechanisms of stroke. Our aim was to investigate the reliability and reproducibility of our novel focal ischaemia-reperfusion model. METHODS: To induce a cortical transient ischaemic attack, we lifted the distal middle cerebral artery (MCA) with a special hook. The early changes after 2 × 15-min occlusion were observed in the somatosensory evoked responses (SERs). The histological responses to 2 × 15-min MCA occlusion and to 30-, 45- or 60-min ischaemia were examined after a 1-day survival period by 2,3,5-triphenyltetrazolium chloride (TTC) and Fluoro Jade C (FJC) staining. Another group, with 30-min ischaemia, was analysed histologically by FJC, S100 and CD11b labelling after a 5-day survival period. RESULTS: The amplitudes of the SERs decreased immediately at the beginning of the ischaemic period, and remained at a reduced level during the ischaemia. Reperfusion resulted in increasing SER amplitudes, but they never regained the control level. The short-lasting ischaemia did not lead to brain infarction when evaluated with TTC, but intense labelling was found with FJC. The 30-min ischaemia did not result in FJC labelling after 1 day, but marked labelling was observed after 5 days with FJC, S100 and CD11b in the cortical area supplied by the MCA. CONCLUSIONS: We present here a novel, readily reproducible method to induce focal brain ischaemia. The ischaemia-reperfusion results in noteworthy changes in the SERs and the appearance of conventional tissue damage markers. This method involves possibilities for precise blood flow regulation, and the setting of the required level of perfusion.


Assuntos
Isquemia Encefálica/etiologia , Modelos Animais de Doenças , Animais , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Estimulação Elétrica , Potenciais Somatossensoriais Evocados , Infarto da Artéria Cerebral Média , Masculino , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Córtex Somatossensorial/patologia , Córtex Somatossensorial/fisiopatologia
16.
Drug Des Devel Ther ; 7: 981-7, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24068867

RESUMO

Cortical spreading depression (CSD) involves a slowly-propagating depolarization wave in the cortex, which can appear in numerous pathophysiological conditions, such as migraine with aura, stroke, and traumatic brain injury. Neurons and glial cells are also depolarized transiently during the phenomena. CSD is followed by a massive increase in glutamate release and by changes in the brain microcirculation. The aim of this study was to investigate the effects of two N-methyl-D-aspartate receptor antagonists, endogenous kynurenic acid (KYNA) and dizocilpine, on CSD and the related blood-brain barrier (BBB) permeability in rats. In intact animals, KYNA hardly crosses the BBB but has some positive features as compared with its precursor L-Kynurenine, which is frequently used in animal studies (KYNA cannot be metabolized to excitotoxic agents such as 3-hydroxy-L-kynurenine and quinolinic acid). We therefore investigated the possible effects of peripherally administered KYNA. Repetitive CSD waves were elicited by the application of 1 M KCl solution to the cortex. Direct current-electrocorticograms were measured for 1 hour. Four parameters of the waves were compared. Evans blue dye and fluorescent microscopy were used to study the possible changes in the permeability of the BBB. The results demonstrated that N-methyl-D-aspartate receptor antagonists can reduce the number of CSD waves and decrease the permeability of the BBB during CSD. These results suggest that KYNA itself or its derivatives may offer a new approach in the therapy of migraines.


Assuntos
Barreira Hematoencefálica/metabolismo , Depressão Alastrante da Atividade Elétrica Cortical/efeitos dos fármacos , Maleato de Dizocilpina/farmacologia , Ácido Cinurênico/farmacologia , Animais , Eletroencefalografia , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Ácido Cinurênico/administração & dosagem , Masculino , Microscopia de Fluorescência , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/fisiopatologia , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Wistar
17.
Neuropharmacology ; 61(5-6): 1026-32, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21781978

RESUMO

Postconditioning can be induced by a broad range of stimuli within minutes to days after an ischemic cerebral insult. A special form is elicited by pharmacological intervention called second pathophysiological stress. The present study aimed to evaluate the effects of low-dose (5 mg/kg) kainate postconditioning with onsets 0, 24 and 48 h after the ischemic insult on the hippocampal synaptic plasticity in a 2-vessel occlusion model in rat. The hippocampal function was tested by LTP measurements of Schaffer collateral-CA1 pyramidal cell synapses in acute slices and the changes in density of Golgi-Cox-stained apical dendritic spines. Postconditioning 0 and 24 h after ischemia was not protective, whereas 48-h-onset postconditioning resulted in the reappearance of a normal spine density (>100,000 spines) 3 days after ischemia, in parallel with the long-term restoration of the damaged LTP function. Similar, but somewhat less effects were observed after 10 days. Our data clearly demonstrate the onset dependence of postconditioning elicited by a subconvulsant dose of kainate treatment in global ischemia, with restoration of the structural plasticity and hippocampal function.


Assuntos
Região CA1 Hipocampal/fisiologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Caínico/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Eletroencefalografia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Fenômenos Eletrofisiológicos/fisiologia , Ataque Isquêmico Transitório/mortalidade , Ataque Isquêmico Transitório/fisiopatologia , Potenciação de Longa Duração/fisiologia , Masculino , Terapia de Alvo Molecular , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Distribuição Aleatória , Ratos , Ratos Wistar , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/fisiologia
18.
Eur J Pharmacol ; 667(1-3): 182-7, 2011 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-21664350

RESUMO

Global forebrain ischemia results in damage to the pyramids in the CA1 hippocampal subfield, which is particularly vulnerable to excitotoxic processes. Morphological and functional disintegration of this area leads to a cognitive dysfunction and neuropsychiatric disorders. Treatment with N-methyl-d-aspartate receptor antagonists is a widely accepted method with which to stop the advance of excitotoxic processes and concomitant neuronal death. From a clinical aspect, competitive glycine- and polyamine-site antagonists with relatively low affinity and moderate side-effects are taken into account. Endogenous kynurenic acid acts as an antagonist on the obligatory co-agonist glycine site, and has long been at the focus of neuroprotective trials. In the present study, we estimated the neuroprotective capability of a novel kynurenic acid analog in transient global forebrain ischemia, measuring the rate of hippocampal CA1 pyramidal cell loss and the preservation of long-term potentiation at Schaffer collateral-CA1 synapses. The neuroprotective potential was reflected by a significantly diminished hippocampal CA1 cell loss and preserved long-term potentiation expression. The neuroprotective effect was robust in the event of pretreatment, and also when the drug was administered at the time of reperfusion. This result is beneficial since a putative neuroprotectant proven to be effective as post-treatment is of much greater benefit.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Artérias Carótidas/cirurgia , Ácido Cinurênico/análogos & derivados , Ácido Cinurênico/farmacologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Animais , Isquemia Encefálica/etiologia , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/patologia , Região CA1 Hipocampal/fisiopatologia , Contagem de Células , Modelos Animais de Doenças , Estimulação Elétrica , Técnicas In Vitro , Ácido Cinurênico/uso terapêutico , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Fármacos Neuroprotetores/uso terapêutico , Ratos , Ratos Wistar , Sinapses/efeitos dos fármacos , Sinapses/fisiologia
19.
Eur J Pharmacol ; 604(1-3): 51-7, 2009 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-19135048

RESUMO

Various acute brain pathological conditions are characterized by the presence of elevated glutamate concentrations in the brain interstitial fluids. It has been established that a decrease in the blood glutamate level enhances the brain-to-blood efflux of glutamate, removal of which from the brain may prevent glutamate excitotoxicity and its contribution to the long-lasting neurological deficits seen in stroke. A decrease in blood glutamate level can be achieved by exploiting the glutamate-scavenging properties of the blood-resident enzyme glutamate-oxaloacetate transaminase, which transforms glutamate into 2-ketoglutarate in the presence of the glutamate co-substrate oxaloacetate. The present study had the aim of an evaluation of the effects of the blood glutamate scavenger oxaloacetate on the impaired long-term potentiation (LTP) induced in the 2-vessel occlusion ischaemic model in rat. Transient (30-min) incomplete forebrain ischaemia was produced 72 h before LTP induction. Although the short transient brain hypoperfusion did not induce histologically identifiable injuries in the CA1 region (Fluoro-Jade B, S-100 and cresyl violet), it resulted in an impaired LTP function in the hippocampal CA1 region without damaging the basal synaptic transmission between the Schaffer collaterals and the pyramidal neurons. This impairment could be fended off in a dose-dependent manner by the intravenous administration of oxaloacetate in saline (at doses between 1.5 mmol and 0.1 mumol) immediately after the transient hypoperfusion. Our results suggest that oxaloacetate-mediated blood and brain glutamate scavenging contributes to the restoration of the LTP after its impairment by brain ischaemia.


Assuntos
Ácido Glutâmico/metabolismo , Hipocampo/efeitos dos fármacos , Ataque Isquêmico Transitório/prevenção & controle , Potenciação de Longa Duração/efeitos dos fármacos , Ácido Oxaloacético/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Animais , Artérias Carótidas/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Ácido Glutâmico/sangue , Hipocampo/patologia , Hipocampo/fisiopatologia , Ataque Isquêmico Transitório/metabolismo , Ataque Isquêmico Transitório/patologia , Ataque Isquêmico Transitório/fisiopatologia , Masculino , Ratos , Ratos Wistar
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...