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1.
Learn Mem ; 24(10): 524-531, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28916627

RESUMO

Working memory is involved in the maintenance and manipulation of information essential for complex cognition. While the neural substrates underlying working memory capacity have been studied in humans, considerably less is known about the circuitry mediating working memory capacity in rodents. Therefore, the present experiments tested the involvement of medial prefrontal cortex (mPFC) and dorsal striatum (STR) in the odor span task (OST), a task proposed to assay working memory capacity in rodents. Initially, Long Evans rats were trained to dig in scented sand for food following a serial delayed nonmatching-to-sample rule. Temporary inactivation of dorsomedial (dm) STR significantly reduced span in well trained rats. Inactivation of mPFC or contralateral disconnection of the mPFC and dmSTR also reduced span. Infusing the GluN2B-containing NMDA receptor antagonist Ro 25-6981 into mPFC did not affect span; however, span was significantly reduced following bilateral Ro 25-6981 infusions into dmSTR or contralateral disconnection of mPFC (inactivation) and dmSTR (Ro 25-6981). These results suggest that span capacity in rats depends on GluN2B-containing NMDA receptor-dependent interactions between the mPFC and the dmSTR. Therefore, interventions targeting this circuit may improve the working memory capacity impairments in patients with schizophrenia, Alzheimer's disease, and Parkinson's disease.


Assuntos
Corpo Estriado/metabolismo , Memória de Curto Prazo/fisiologia , Percepção Olfatória/fisiologia , Córtex Pré-Frontal/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Cateteres de Demora , Corpo Estriado/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Agonistas de Receptores de GABA-A/farmacologia , Masculino , Memória de Curto Prazo/efeitos dos fármacos , Muscimol/farmacologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Testes Neuropsicológicos , Odorantes , Percepção Olfatória/efeitos dos fármacos , Fenóis/farmacologia , Piperidinas/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Ratos Long-Evans , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores
2.
J Neurosci ; 33(19): 8202-15, 2013 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-23658159

RESUMO

Activation of the nerve growth factor (NGF) receptor trkA and tissue acidosis are critically linked to inflammation-associated nociceptor sensitization. This study explored how increased acidity is linked to sensory neuron sensitization to NGF. Adult Wistar rat primary sensory neurons grown at physiological pH 7.4, then either kept at pH 7.4 or challenged for 30 min in pH 6.5 medium, provided a model of acidosis. Nonpermeabilizing trkA immunofluorescence revealed a significant increase in trkA mobilization to the plasma membrane from intracellular stores in response to proton challenge. This was confirmed using a surface protein biotinylation assay and Brefeldin A disruption of the rough endoplasmic reticulum-Golgi-trans-Golgi network. Mobilization of trkA to the membrane at pH 6.5 was abolished in neurons treated with the acid-sensitive ion channel blocker, amiloride. While elevated levels of NGF-independent trkA phosphorylation occurred at pH 6.5 alone, the level of activation was significantly increased in response to NGF challenge. Exposure of sensory neurons to pH 6.5 medium also resulted in strong calcium (Ca(2+)) transients that were reversible upon reintroduction to physiological pH. The pH 6.5-induced mobilization of trkA to the membrane was Ca(2+) dependent, as BAPTA-AM Ca(2+) chelation abrogated the response. Interestingly, KCl-induced depolarization was sufficient to induce mobilization of trkA to the cell surface at pH 7.4, but did not augment the response to pH 6.5. In conclusion, increased mobilization of trkA to neuronal membranes in response to either acidosis or neuronal depolarization provides two novel mechanisms by which sensory neurons can rapidly sensitize to NGF and has important implications for inflammatory pain states.


Assuntos
Líquido Extracelular/metabolismo , Receptor trkA/metabolismo , Células Receptoras Sensoriais/metabolismo , Acidose/fisiopatologia , Animais , Anticorpos/farmacologia , Biotinilação , Brefeldina A/farmacologia , Cálcio/metabolismo , Células Cultivadas , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Líquido Extracelular/efeitos dos fármacos , Gânglios Espinais/citologia , Concentração de Íons de Hidrogênio , Isótopos de Iodo/farmacocinética , Masculino , Fator de Crescimento Neural/imunologia , Fator de Crescimento Neural/farmacocinética , Cloreto de Potássio/farmacologia , Ligação Proteica/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos Wistar , Células Receptoras Sensoriais/efeitos dos fármacos
3.
Breast Cancer Res Treat ; 140(2): 273-84, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23881522

RESUMO

One of the major obstacles in human epidermal growth factor receptor 2 (HER2)-specific trastuzumab antibody immunotherapy of HER2-positive breast cancer is the development of trastuzumab resistance, warranting the search for other therapeutic strategies. Using mouse models, we previously demonstrated that ovalbumin (OVA)-specific dendritic cell (DC)-released exosome (EXOOVA)-targeted CD4(+) T cell-based (OVA-TEXO) vaccine stimulates efficient cytotoxic T lymphocyte (CTL) responses via exosomal peptide/major histocompatibility complex (pMHC)-I, exosomal CD80 and endogenous IL-2 signaling; and long-term CTL memory by means of via endogenous CD40L signaling. In this study, using two-photon microscopy, we provide the first visual evidence on targeting OVA-TEXO to cognate CD8(+) T cells in vivo via exosomal pMHC-I complex. We prepared HER2/neu-specific Neu-TEXO and HER2-TEXO vaccines using adenoviral vector (AdVneu and AdVHER2)-transfected DC (DCneu and DCHER2)-released EXOs (EXOneu and EXOHER2), and assessed their stimulatory effects on HER2/neu-specific CTL responses and antitumor immunity. We demonstrate that Neu-TEXO vaccine is capable of stimulating efficient neu-specific CTL responses, leading to protective immunity against neu-expressing Tg1-1 breast cancer in all 6/6 transgenic (Tg) FVBneuN mice with neu-specific self-immune tolerance. We also demonstrate that HER2-TEXO vaccine is capable of inducing HER2-specific CTL responses and protective immunity against transgene HLA-A2(+)HER2(+) BL6-10A2/HER2 B16 melanoma in 2/8 double Tg HLA-A2/HER2 mice with HER2-specific self-immune tolerance. The remaining 6/8 mice had significantly prolonged survival. Furthermore, we demonstrate that HER2-TEXO vaccine stimulates responses of CD8(+) T cells capable of not only inducing killing activity to HLA-A2(+)HER2(+) BL6-10A2/HER2 melanoma and trastuzumab-resistant BT474A2 breast cancer cells in vitro but also eradicating 6-day palpable HER2(+) BT474A2 breast cancer (3-4 mm in diameter) in athymic nude mice. Therefore, the novel T cell-based HER2-TEXO vaccine may provide a new therapeutic alternative for women with HER2(+) breast cancer, especially for trastuzumab-resistant HER2(+) breast cancer patients.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Neoplasias da Mama/imunologia , Vacinas Anticâncer/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/imunologia , Antígeno HLA-A2/imunologia , Linfócitos T Citotóxicos/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Exossomos/imunologia , Feminino , Humanos , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Camundongos Nus , Camundongos Transgênicos , Receptor ErbB-2/imunologia , Linfócitos T Citotóxicos/imunologia , Trastuzumab
4.
J Neurosci ; 30(50): 16855-63, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21159956

RESUMO

Endothelin (ET-1) given centrally has many reported actions on hormonal and autonomic outputs from the CNS. However, it is unclear whether these effects are due to local ischemia via its vasoconstrictor properties or to a direct neuromodulatory action. ET-1 stimulates the release of oxytocin (OT) and vasopressin (VP) from supraoptic magnocellular (MNCs) neurons in vivo; therefore, we asked whether ET-1 modulates the excitatory inputs onto MNCs that are critical in sculpting the activity of these neurons. To investigate whether ET-1 modulates excitatory synaptic transmission, we obtained whole-cell recordings and analyzed quantal glutamate release onto MNCs in the supraoptic nucleus (SON). Neurons identified as VP-containing neurosecretory cells displayed a decrease in quantal frequency in response to ET-1 (10-100 pm). This decrease was mediated by ET(A) receptor activation and production of a retrograde messenger that targets presynaptic cannabinoid-1 receptors. In contrast, neurons identified as OT-containing MNCs displayed a transient increase in quantal glutamate release in response to ET-1 application via ET(B) receptor activation. Application of TTX to block action potential-dependent glutamate release inhibited the excitatory action of ET-1 in OT neurons. There were no changes in quantal amplitude in either MNC type, suggesting that the effects of ET-1 were via presynaptic mechanisms. A gliotransmitter does not appear to be involved as ET-1 failed to elevate astrocytic calcium in the SON. Our results demonstrate that ET-1 differentially modulates glutamate release onto VP- versus OT-containing MNCs, thus implicating it in the selective regulation of neuroendocrine output from the SON.


Assuntos
Endotelina-1/farmacologia , Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Ocitocina/metabolismo , Núcleo Supraóptico/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Vasopressinas/metabolismo , Animais , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Endotelina-1/administração & dosagem , Endotelina-1/antagonistas & inibidores , Masculino , Microinjeções , Neurônios/fisiologia , Técnicas de Patch-Clamp/métodos , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina A/fisiologia , Receptor de Endotelina B/fisiologia , Núcleo Supraóptico/metabolismo , Núcleo Supraóptico/fisiologia , Transmissão Sináptica/fisiologia , Tetrodotoxina/farmacologia
5.
J Clin Immunol ; 31(5): 811-26, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21674137

RESUMO

Both CD4(+) Th17-cells and CD8(+) cytotoxic T lymphocytes (CTLs) are involved in type 1 diabetes and experimental autoimmune encephalomyelitis (EAE). However, their relationship in pathogenesis of these autoimmune diseases is still elusive. We generated ovalbumin (OVA)- or myelin oligodendrocyte glycoprotein (MOG)-specific Th17 cells expressing RORγt and IL-17 by in vitro co-culturing OVA-pulsed and MOG(35-55) peptide-pulsed dendritic cells (DC(OVA) and DC(MOG)) with CD4(+) T cells derived from transgenic OTII and MOG-T cell receptor mice, respectively. We found that these Th17 cells when transferred into C57BL/6 mice stimulated OVA- and MOG-specific CTL responses, respectively. To assess the above question, we adoptively transferred OVA-specific Th17 cells into transgenic rat insulin promoter (RIP)-mOVA mice or RIP-mOVA mice treated with anti-CD8 antibody to deplete Th17-stimulated CD8(+) T cells. We demonstrated that OVA-specific Th17-stimulated CTLs, but not Th17 cells themselves, induced diabetes in RIP-mOVA. We also transferred MOG-specific Th17 cells into C57BL/6 mice and H-2K(b-/-) mice lacking of the ability to generate Th17-stimulated CTLs. We further found that MOG-specific Th17 cells, but not Th17-activated CTLs induced EAE in C57BL/6 mice. Taken together, our data indicate a distinct role of Th17 cells and Th17-stimulated CTLs in the pathogenesis of TID and EAE, which may have great impact on the overall understanding of Th17 cells in the pathogenesis of autoimmune diseases.


Assuntos
Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Encefalomielite Autoimune Experimental/imunologia , Linfócitos T Citotóxicos/metabolismo , Células Th17/metabolismo , Transferência Adotiva , Animais , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/patologia , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas da Mielina/genética , Proteínas da Mielina/imunologia , Proteínas da Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito , Ovalbumina/genética , Ovalbumina/imunologia , Ovalbumina/metabolismo , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia , Células Th17/imunologia , Células Th17/patologia , Células Th17/transplante
6.
Cancer Immunol Immunother ; 60(10): 1473-84, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21660450

RESUMO

CD4(+) Th17 cells induce antitumor immunity leading to the eradication of established tumors. However, the mechanism of antitumour immunity and CTL activation by Th17 cells and the distinct role of Th17 and Th17-activated CTLs in antitumor immunity are still elusive. In this study, we generated ovalbumin (OVA)-specific Th17 cells by cultivating OVA-pulsed dendritic cells with CD4(+) T cells derived from transgenic OTII mice in the presence of IL-6, IL-23, TGF-ß, and anti-IFN-γ antibody. We demonstrated that Th17 cells acquired major histocompatibility complex/peptide (pMHC)-I and expressed RORγt, IL-17, and IL-2. Th17 cells did not have any direct in vitro tumor cell-killing activity. However, Th17 cells were able to stimulate CD8(+) CTL responses via IL-2 and pMHC I, but not IL-17 signaling, which play a major role in Th17-induced preventive immunity against OVA-expressing B16 melanoma. Th17 cells stimulated the expression of CCL2 and CCL20 in lung tumor microenvironments promoting the recruitment of various inflammatory leukocytes (DCs, CD4(+), and CD8(+) T cells) stimulating more pronounced therapeutic immunity for early-stage (5-day lung metastases or 3 mm, s.c.) tumor than for well-established (6 mm, s.c.) tumor. The therapeutic effect of Th17 cells is associated with IL-17 and is mediated by Th17-stimulated CD8(+) CTLs and other inflammatory leukocytes recruited into B16 melanoma via Th17-stimulated CCL20 chemoattraction. Taken together, our data elucidate a distinct role of Th17 and Th17-stimulated CD8(+) CTLs in the induction of preventive and therapeutic antitumor immunity, which may greatly impact the development of Th17-based cancer immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica/imunologia , Ativação Linfocitária/imunologia , Neoplasias Experimentais/imunologia , Células Th17/imunologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologia , Microambiente Tumoral/imunologia
7.
J Immunol ; 182(1): 193-206, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19109150

RESUMO

T cell-T cell Ag presentation is increasingly attracting attention. We previously showed that the in vitro OVA-pulsed dendritic cell (DC(OVA))-activated CD4(+) Th cells acquired OVA peptide/MHC (pMHC) class I and costimulatory molecules such as CD54 and CD80 from DC(OVA) and acted as CD4(+) Th-APC capable of stimulating OVA-specific CD8(+) CTL responses. In this study, we further applied the OVA-specific TCR-transgenic OT I and OT II mice with deficiency of various cytokines or costimulatory molecule genes useful for studying the molecular mechanisms underlying in Th-APC's stimulatory effect. We demonstrated that DC(OVA)-stimulated OT II CD4(+) Th-APC also acquired costimulatory molecules such as CD40, OX40L, and 4-1BBL and the functional pMHC II complexes by DC(OVA) activation. CD4(+) Th-APC with acquired pMHC II and I were capable of stimulating CD4(+) Th1 and central memory CD8(+)44(+)CD62L(high)IL-7R(+) T cell responses leading to antitumor immunity against OVA-expressing mouse B16 melanoma. Their stimulatory effect on CD8(+) CTL responses and antitumor immunity is mediated by IL-2 secretion, CD40L, and CD80 signaling and is specifically targeted to CD8(+) T cells in vivo via acquired pMHC I. In addition, CD4(+) Th-APC expressing OVA-specific TCR, FasL, and perforin were able to kill DC(OVA) and neighboring Th-APC expressing endogenous and acquired pMHC II. Taken together, we show that CD4(+) Th-APC can modulate immune responses by stimulating CD4(+) Th1 and central memory CD8(+) T cell responses and eliminating DC(OVA) and neighboring Th-APC. Therefore, our findings may have great impacts in not only the antitumor immunity, but also the regulatory T cell-dependent immune tolerance in vivo.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Antígenos de Histocompatibilidade Classe I/fisiologia , Memória Imunológica , Ativação Linfocitária/imunologia , Fragmentos de Peptídeos/fisiologia , Células Th1/imunologia , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Testes Imunológicos de Citotoxicidade , Receptores de Hialuronatos/biossíntese , Selectina L/biossíntese , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Receptores de Interleucina-7/biossíntese , Células Th1/metabolismo
8.
Nature ; 431(7005): 195-9, 2004 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-15356633

RESUMO

Cerebral blood flow (CBF) is coupled to neuronal activity and is imaged in vivo to map brain activation. CBF is also modified by afferent projection fibres that release vasoactive neurotransmitters in the perivascular region, principally on the astrocyte endfeet that outline cerebral blood vessels. However, the role of astrocytes in the regulation of cerebrovascular tone remains uncertain. Here we determine the impact of intracellular Ca(2+) concentrations ([Ca(2+)](i)) in astrocytes on the diameter of small arterioles by using two-photon Ca(2+) uncaging to increase [Ca(2+)](i). Vascular constrictions occurred when Ca(2+) waves evoked by uncaging propagated into the astrocyte endfeet and caused large increases in [Ca(2+)](i). The vasoactive neurotransmitter noradrenaline increased [Ca(2+)](i) in the astrocyte endfeet, the peak of which preceded the onset of arteriole constriction. Depressing increases in astrocyte [Ca(2+)](i) with BAPTA inhibited the vascular constrictions in noradrenaline. We find that constrictions induced in the cerebrovasculature by increased [Ca(2+)](i) in astrocyte endfeet are generated through the phospholipase A(2)-arachidonic acid pathway and 20-hydroxyeicosatetraenoic acid production. Vasoconstriction by astrocytes is a previously unknown mechanism for the regulation of CBF.


Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Encéfalo/irrigação sanguínea , Sinalização do Cálcio/fisiologia , Circulação Cerebrovascular/fisiologia , Ácido Egtázico/análogos & derivados , Vasoconstrição/fisiologia , Animais , Ácido Araquidônico/metabolismo , Arteríolas/efeitos dos fármacos , Arteríolas/fisiologia , Astrócitos/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Ácido Egtázico/farmacologia , Ácidos Hidroxieicosatetraenoicos/metabolismo , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Norepinefrina/farmacologia , Técnicas de Patch-Clamp , Fosfolipases A/metabolismo , Fotólise , Fótons , Ratos , Vasoconstrição/efeitos dos fármacos
9.
Glia ; 57(15): 1610-8, 2009 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-19382211

RESUMO

Microglia cells exhibit two forms of motility, constant movement of filopodia probing surrounding brain tissue, and outgrowth of larger processes in response to nearby damage. The mechanisms and functions of filopodia sensing and process outgrowth are not well characterized but are likely critical for normal immune function in the brain. Using two photon laser scanning microscopy we investigated microglia process outgrowth in response to damage, and explored the relationship between process outgrowth and filopodia movement. Further, we examined the roles of Cl(-) or K(+) channel activation, as well as actin polymerization in these two distinct processes, because mechanistic understanding could provide a strategy to modulate microglia function. We found that volume sensitive Cl(-) channel blockers (NPPB, tamoxifen, DIDS) prevented the rapid process outgrowth of microglia observed in response to damage. In contrast, filopodia extension during sensing was resistant to Cl(-) channel inhibitors, indicating that these motile processes have different cellular mechanisms. However, both filopodia sensing and rapid process outgrowth were blocked by inhibition of actin polymerization. Following lesion formation under control conditions, rapidly outgrowing processes contacted the damaged area and this was associated with a 37% decrease in lesion volume. Inhibition of process outgrowth by Cl(-) channel block, prevention of actin polymerization, or by selectively ablating microglia all allowed lesion volume to increase and spread into the surrounding tissue. Therefore, process outgrowth in response to focal brain damage is beneficial by preventing lesion expansion and suggests microglia represent a front line defence against damage in the brain.


Assuntos
Lesões Encefálicas/patologia , Canais de Cloreto/fisiologia , Microglia/patologia , Microglia/fisiologia , Pseudópodes/fisiologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Lesões Encefálicas/genética , Receptor 1 de Quimiocina CX3C , Canais de Cloreto/antagonistas & inibidores , Proteínas de Fluorescência Verde/genética , Hipocampo/patologia , Técnicas In Vitro , Lasers/efeitos adversos , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Nitrobenzoatos/farmacologia , Pseudópodes/efeitos dos fármacos , Pseudópodes/genética , Receptores de Quimiocinas/genética , Tamoxifeno/farmacologia , Fatores de Tempo
10.
Sci STKE ; 2006(357): pe42, 2006 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-17047222

RESUMO

Because the brain is encased by the skull, the ability to control cell volume in the brain is crucial, and pathological conditions that disturb cell volume homeostasis may severely compromise neural function and survival. Astrocytes are the main cell type to show swelling in response to pathological conditions. More recently, a role for swelling-induced neurotransmitter release from astrocytes under nonpathological conditions has been reported. Astrocytes express a volume-regulated anion channel (VRAC) that is involved in volume homeostasis. In addition to transporting chloride, VRACs allow the efflux of chloride and amino acids such as taurine, glutamate, and aspartate. Glutamate and aspartate are potent activators of neuronal glutamate receptors. Therefore, this nonsynaptic form of cellular communication may modulate neuronal excitability and synaptic activity.


Assuntos
Astrócitos/fisiologia , Tamanho Celular , Sistema Nervoso Central/citologia , Canais Iônicos/fisiologia , Neurônios/fisiologia , Neurotransmissores/metabolismo , Transmissão Sináptica/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Ácido Aspártico/metabolismo , Astrócitos/efeitos dos fármacos , Encéfalo/citologia , Química Encefálica , Cálcio/fisiologia , Tamanho Celular/efeitos dos fármacos , Quelantes/farmacologia , Canais de Cloreto/fisiologia , Cloretos/fisiologia , Furosemida/farmacologia , Ácido Glutâmico/metabolismo , Humanos , Nitrobenzoatos/farmacologia , Concentração Osmolar , Inibidores de Proteínas Quinases/farmacologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia
11.
Neuropharmacology ; 93: 267-73, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25721395

RESUMO

While µ-opioid receptor (MOR) agonists remain the most powerful analgesics for the treatment of severe pain, serious adverse side effects that are secondary to their central nervous system actions pose substantial barriers to therapeutic use. Preclinical and clinical evidence suggest that peripheral MORs play an important role in opioid analgesia, particularly under inflammatory conditions. However, the mechanisms of peripheral MOR signaling in primary afferent pain fibres remain to be established. We have recently introduced a novel ex vivo optical imaging approach that, for the first time, allows the study of physiological functioning within individual peripheral nociceptive fibre free nerve endings in mice. In the present study, we found that MOR activation in selectively identified, primary afferent CGRP nociceptive terminals caused inhibition of N-type Ca(2+) channel signaling and suppression of action potential-evoked Ca(2+) fluorescent transients mediated by 'big conductance' Ca(2+)-activated K(+) channels (BKCa). In the live animal, we showed that the peripherally acting MOR agonist HS-731 produced analgesia and that BKCa channels were the major effectors of the peripheral MOR signaling. We have identified two key molecular transducers of MOR activation that mediate significant inhibition of nociceptive signaling in primary afferent terminals. Understanding the mechanisms of peripheral MOR signaling may promote the development of pathway selective µ-opioid drugs that offer improved therapeutic profiles for achieving potent analgesia while avoiding serious adverse central side effects.


Assuntos
Potenciais de Ação/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Sinalização do Cálcio/fisiologia , Fibras Nervosas Amielínicas/fisiologia , Nociceptores/metabolismo , Receptores Opioides mu/metabolismo , Potenciais de Ação/efeitos dos fármacos , Vias Aferentes/citologia , Analgésicos Opioides/farmacologia , Animais , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Compostos de Epóxi/farmacologia , Olho/inervação , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Morfinanos/farmacologia , Fibras Nervosas Amielínicas/metabolismo , Medição da Dor , Receptores Opioides mu/antagonistas & inibidores , Nervo Trigêmeo/fisiologia
13.
PLoS One ; 7(11): e51124, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23226475

RESUMO

Astrocytes, the major type of non-neuronal cells in the brain, play an important functional role in extracellular potassium ([K(+)](o)) and pH homeostasis. Pathological brain states that result in [K(+)](o) and pH dysregulation have been shown to cause astrocyte swelling. However, whether astrocyte volume changes occur under physiological conditions is not known. In this study we used two-photon imaging to visualize real-time astrocyte volume changes in the stratum radiatum of the hippocampus CA1 region. Astrocytes were observed to swell by 19.0±0.9% in response to a small physiological increase in the concentration of [K(+)](o) (3 mM). Astrocyte swelling was mediated by the influx of bicarbonate (HCO(3-)) ions as swelling was significantly decreased when the influx of HCO(3-) was reduced. We found: 1) in HCO(3-) free extracellular solution astrocytes swelled by 5.4±0.7%, 2) when the activity of the sodium-bicarbonate cotransporter (NBC) was blocked the astrocytes swelled by 8.3±0.7%, and 3) in the presence of an extracellular carbonic anhydrase (CA) inhibitor astrocytes swelled by 11.4±0.6%. Because a significant HCO(3-) efflux is known to occur through the γ-amino-butyric acid (GABA) channel, we performed a series of experiments to determine if astrocytes were capable of HCO(3-) mediated volume shrinkage with GABA channel activation. Astrocytes were found to shrink -7.7±0.5% of control in response to the GABA(A) channel agonist muscimol. Astrocyte shrinkage from GABA(A) channel activation was significantly decreased to -5.0±0.6% of control in the presence of the membrane-permeant CA inhibitor acetazolamide (ACTZ). These dynamic astrocyte volume changes may represent a previously unappreciated yet fundamental mechanism by which astrocytes regulate physiological brain functioning.


Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Bicarbonatos/metabolismo , Tamanho Celular , Animais , Transporte Biológico , Imageamento Tridimensional , Canais Iônicos/metabolismo , Masculino , Potássio/metabolismo , Ratos , Ratos Sprague-Dawley , Ácido gama-Aminobutírico/metabolismo
14.
Neuropharmacology ; 63(3): 362-7, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22691374

RESUMO

The selective 5-HT1 receptor agonist sumatriptan is an effective therapeutic for migraine pain yet the antimigraine mechanisms of action remain controversial. Pain-responsive fibres containing calcitonin gene-related peptide (CGRP) densely innervating the cranial dura mater are widely believed to be an essential anatomical substrate for the development of migraine pain. 5-HT1 receptors in the dura colocalize with CGRP fibres in high density and thus provide a possible peripheral site of action for sumatriptan. In the present study, we used high-resolution optical imaging selectively within individual mouse dural CGRP nociceptive fibre terminations and found that application of sumatriptan caused a rapid, reversible dose-dependent inhibition in the amplitude of single action potential evoked Ca²âº transients. Pre-application of the 5-HT1 antagonist GR 127935 or the selective 5-HT(1D) antagonist BRL 15572 prevented inhibition while the selective 5-HT(1B) antagonist SB 224289 did not, suggesting this effect was mediated selectively through the 5-HT(1D) receptor subtype. Sumatriptan inhibition of the action potential evoked Ca²âº signaling was mediated selectively through N-type Ca²âº channels. Although the T-type Ca²âº channel accounted for a greater proportion of the Ca²âº signal it did not mediate any of the sumatriptan inhibition. Our findings support a peripheral site of action for sumatriptan in inhibiting the activity of dural pain fibres selectively through a single Ca²âº channel subtype. This finding adds to our understanding of the mechanisms that underlie the clinical effectiveness of 5-HT1 receptor agonists such as sumatriptan and may provide insight for the development of novel peripherally targeted therapeutics for mitigating the pain of migraine.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina/fisiologia , Bloqueadores dos Canais de Cálcio , Canais de Cálcio Tipo N/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Fibras Nervosas/efeitos dos fármacos , Terminações Pré-Sinápticas/efeitos dos fármacos , Agonistas do Receptor de Serotonina/farmacologia , Sumatriptana/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Dura-Máter/efeitos dos fármacos , Estimulação Elétrica , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Dor/fisiopatologia , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT1 de Serotonina/farmacologia
15.
J Leukoc Biol ; 92(2): 289-300, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22544940

RESUMO

CD4(+) T cell help contributes critically to DC-induced CD8(+) CTL immunity. However, precisely how these three cell populations interact and how CD4(+) T cell signals are delivered to CD8(+) T cells in vivo have been unclear. In this study, we developed a novel, two-step approach, wherein CD4(+) T cells and antigen-presenting DCs productively engaged one another in vivo in the absence of cognate CD8(+) T cells, after which, we selectively depleted the previously engaged CD4(+) T cells or DCs before allowing interactions of either population alone with naïve CD8(+) T cells. This protocol thus allows us to clearly document the importance of CD4(+) T-licensed DCs and DC-primed CD4(+) T cells in CTL immunity. Here, we provide direct in vivo evidence that primed CD4(+) T cells or licensed DCs can stimulate CTL response and memory, independent of DC-CD4(+) T cell clusters. Our results suggest that primed CD4(+) T cells with acquired pMHC-I from DCs represent crucial "immune intermediates" for rapid induction of CTL responses and for functional memory via CD40L signaling. Importantly, intravital, two-photon microscopy elegantly provide unequivocal in vivo evidence for direct CD4-CD8(+) T cell interactions via pMHC-I engagement. This study corroborates the coexistence of direct and indirect mechanisms of T cell help for a CTL response in noninflammatory situations. These data suggest a new "dynamic model of three-cell interactions" for CTL immunity derived from stimulation by dissociated, licensed DCs, primed CD4(+) T cells, and DC-CD4(+) T cell clusters and may have significant implications for autoimmunity and vaccine design.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Ligante de CD40/fisiologia , Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/fisiologia , Memória Imunológica , Transdução de Sinais/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Feminino , Marcação de Genes/métodos , Antígenos de Histocompatibilidade Classe I/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos
16.
J Neurosci Methods ; 198(2): 274-9, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21530587

RESUMO

Here we introduce a simple experimental approach for studying afferent pain fibre physiology. We have developed a mouse en bloc dural-skull preparation for optical microfluorometric imaging to directly study the physiological functioning in selectively identified, individual nociceptive fibre free nerve endings. Functional optical imaging using widefield epifluorescence microscopy was combined with electrophysiological stimulations, pharmacological manipulations, and the UV photolysis of caged compounds. For the first time, we show high-resolution functional imaging of single action potential-evoked fluorescent transients, as well as sub- and supra-threshold calcium signaling events within individual nociceptive fibre terminations. This novel experimental approach opens up a new window for studying nociceptive fibre physiology and pathophysiology.


Assuntos
Vias Aferentes/fisiopatologia , Microscopia de Fluorescência/métodos , Neurônios Aferentes/fisiologia , Nociceptores/fisiologia , Fotólise , Potenciais de Ação/fisiologia , Animais , Camundongos , Camundongos Transgênicos , Dor/fisiopatologia , Percepção da Dor/fisiologia
17.
Neuron ; 58(4): 557-70, 2008 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-18498737

RESUMO

Dopamine signaling through D1 receptors in the prefrontal cortex (PFC) plays a critical role in the maintenance of higher cognitive functions, such as working memory. At the cellular level, these functions are predicated to involve alterations in neuronal calcium levels. The dendrites of PFC neurons express D1 receptors and N-type calcium channels, yet little information exists regarding their coupling. Here, we show that D1 receptors potently inhibit N-type channels in dendrites of rat PFC neurons. Using coimmunoprecipitation, we demonstrate the existence of a D1 receptor-N-type channel signaling complex in this region, and we provide evidence for a direct receptor-channel interaction. Finally, we demonstrate the importance of this complex to receptor-channel colocalization in heterologous systems and in PFC neurons. Our data indicate that the N-type calcium channel is an important physiological target of D1 receptors and reveal a mechanism for D1 receptor-mediated regulation of cognitive function in the PFC.


Assuntos
Canais de Cálcio Tipo N/fisiologia , Cálcio/metabolismo , Dendritos/metabolismo , Neurônios/citologia , Receptores de Dopamina D1/fisiologia , Animais , Animais Recém-Nascidos , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Dendritos/efeitos dos fármacos , Dopamina/farmacologia , Dopaminérgicos/farmacologia , Interações Medicamentosas , Estimulação Elétrica/métodos , Regulação da Expressão Gênica/fisiologia , Humanos , Imunoprecipitação , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Microscopia Confocal , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Córtex Pré-Frontal/citologia , Ratos , Transfecção/métodos
18.
Glia ; 55(12): 1214-1221, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17659528

RESUMO

The control of cerebral vessel diameter is of fundamental importance in maintaining healthy brain function because it is critical to match cerebral blood flow (CBF) to the metabolic demand of active neurons. Recent studies have shown that astrocytes are critical players in the regulation of cerebral blood vessel diameter and that there are several molecular pathways through which astrocytes can elicit these changes. Increased intracellular Ca(2+) in astrocytes has demonstrated a dichotomy in vasomotor responses by causing the constriction as well as the dilation of neighboring blood vessels. The production of arachidonic acid (AA) in astrocytes by Ca(2+) sensitive phospholipase A(2) (PLA(2)) has been shown to be common to both constriction and dilation mechanisms. Constriction results from the conversion of AA to 20-hydroxyeicosatetraenoic acid (20-HETE) and dilation from the production of prostaglandin E(2) (PGE2) or epoxyeicosatrienoic acid (EET) and the level of nitric oxide (NO) appears to dictate which of these two pathways is recruited. In addition the activation of Ca(2+) activated K(+) channels in astrocyte endfeet and the efflux of K(+) has also been suggested to modify vascular tone by hyperpolarization and relaxation of smooth muscle cells (SMCs). The wide range of putative pathways indicates that more work is needed to clarify the contributions of astrocytes to vascular dynamics under different cellular conditions. Nonetheless it is clear that astrocytes are important albeit complicated regulators of CBF.


Assuntos
Astrócitos/fisiologia , Circulação Cerebrovascular/fisiologia , Animais , Capilares/fisiologia , Humanos , Músculo Liso Vascular/fisiologia , Neurotransmissores/fisiologia , Potássio/fisiologia , Canais de Potássio/fisiologia , Vasodilatação/fisiologia
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