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1.
J Physiol ; 592(7): 1505-18, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24366265

RESUMO

Huntingtin-associated protein 1 (HAP1) was initially established as a neuronal binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking and cell signalling. In this study, we establish that HAP1 is important in several steps of exocytosis in adrenal chromaffin cells. Using carbon-fibre amperometry, we measured single vesicle exocytosis in chromaffin cells obtained from HAP1(-/-) and HAP1(+/+) littermate mice. Numbers of Ca(2+)-dependent and Ca(2+)-independent full fusion events in HAP1(-/-) cells are significantly decreased compared with those in HAP1(+/+) cells. We observed no change in the frequency of 'kiss-and-run' fusion events or in Ca(2+) entry. Whereas release per full fusion event is unchanged in HAP1(-/-) cells, early fusion pore duration is prolonged, as indicated by the increased duration of pre-spike foot signals. Kiss-and-run events have a shorter duration, indicating opposing roles for HAP1 in the stabilization of the fusion pore during full fusion and transient fusion, respectively. We use electron microscopy to demonstrate a reduction in the number of vesicles docked at the plasma membrane of HAP1(-/-) cells, where membrane capacitance measurements reveal the readily releasable pool of vesicles to be reduced in size. Our study therefore illustrates that HAP1 regulates exocytosis by influencing the morphological docking of vesicles at the plasma membrane, the ability of vesicles to be released rapidly upon stimulation, and the early stages of fusion pore formation.


Assuntos
Medula Suprarrenal/metabolismo , Membrana Celular/metabolismo , Células Cromafins/metabolismo , Exocitose , Fusão de Membrana , Proteínas do Tecido Nervoso/metabolismo , Vesículas Secretórias/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Catecolaminas/metabolismo , Células Cultivadas , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Via Secretória , Fatores de Tempo
2.
J Comp Neurol ; 459(1): 25-43, 2003 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-12629665

RESUMO

Neurons in prevertebral sympathetic ganglia receive convergent synaptic inputs from peripheral enteric neurons in addition to inputs from spinal preganglionic neurons. Although all inputs are functionally cholinergic, inputs from these two sources have distinctive neurochemical and functional profiles. We used multiple-labeling immunofluorescence, quantitative confocal microscopy, ultrastructural immunocytochemistry, and intracellular electrophysiologic recordings to examine whether populations of inputs to the guinea pig coeliac ganglion express different levels of synaptic proteins that could influence synaptic strength. Boutons of enteric intestinofugal inputs, identified by immunoreactivity to vasoactive intestinal peptide, showed considerable heterogeneity in their immunoreactivity to synaptosome-associated protein of 25 kDa (SNAP-25), synapsin, synaptophysin, choline acetyltransferase, and vesicular acetylcholine transporter. Mean levels of immunoreactivity to these proteins were significantly lower in terminals of intestinofugal inputs compared with terminals of spinal preganglionic inputs. Nevertheless, many boutons with undetectable levels of SNAP-25 immunoreactivity formed morphologically normal synapses with target neurons. Treatment with botulinum neurotoxin type A (20-50 nM for 2 hours in vitro) generated significant cleavage of SNAP-25 and produced similar dose- and time-dependent inhibitions of synaptic transmission from all classes of inputs, regardless of their mean level of SNAP-25 expression. The simplest interpretation of these results is that only synaptic boutons with detectable levels of SNAP-25 immunoreactivity contribute significantly to fast cholinergic transmission. Consequently, the low synaptic strength of intestinofugal inputs to final motor neurons in sympathetic pathways may be due in part to the low proportion of their boutons that express SNAP-25 and other synaptic proteins.


Assuntos
Fibras Adrenérgicas/metabolismo , Proteínas de Membrana/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Vesículas Sinápticas/metabolismo , Fibras Adrenérgicas/química , Animais , Colina O-Acetiltransferase/análise , Colina O-Acetiltransferase/biossíntese , Feminino , Gânglios Simpáticos/química , Gânglios Simpáticos/metabolismo , Regulação da Expressão Gênica/fisiologia , Cobaias , Masculino , Potenciais da Membrana/fisiologia , Proteínas de Membrana/análise , Proteínas do Tecido Nervoso/análise , Terminações Pré-Sinápticas/química , Terminações Pré-Sinápticas/metabolismo , Sinapsinas/análise , Sinapsinas/biossíntese , Vesículas Sinápticas/química , Proteína 25 Associada a Sinaptossoma
3.
J Comp Neurol ; 455(3): 285-98, 2003 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-12483682

RESUMO

Prevertebral sympathetic ganglia contain a unique population of final motor neurons receiving convergent synaptic inputs not only from spinal preganglionic neurons, but also from peripheral intestinofugal neurons projecting from the gut. We used quantitative confocal and ultrastructural immunohistochemistry to determine how this increased synaptic convergence is accommodated by sympathetic final motor neurons in the celiac ganglion of guinea pigs. Terminals of intestinofugal neurons were identified by their immunoreactivity to vasoactive intestinal peptide. Stereologic analyses were based on transects and point counts at confocal and ultrastructural levels. The relative amount of dendritic neuropil in the medial regions of the ganglion was approximately 2.5 times greater than in the lateral regions of the ganglion, consistent with the 2 to 3 times difference in average dendritic field size of neurons in these regions. The total numbers of boutons and synaptic profiles showed significant positive correlations with the relative amount of neuropil in a region. However, the overall density of synaptic boutons was twice as high in the medial region of the ganglion compared with the lateral regions. Because the relative density of preganglionic synapses was similar in each region, this difference was due to the selective projection of intestinofugal inputs to neurons in the medial celiac ganglion, where they provided 45% of synaptic contacts. These results show that, compared with vasoconstrictor neurons, sympathetic neurons regulating gastrointestinal activity support a higher number of convergent inputs in two ways: in addition to having larger dendritic fields, they also have a twofold higher density of synapses.


Assuntos
Dendritos/ultraestrutura , Gânglios Simpáticos/citologia , Neurônios/ultraestrutura , Neurópilo/ultraestrutura , Sinapses/ultraestrutura , Animais , Colina O-Acetiltransferase/análise , Dendritos/fisiologia , Eletrofisiologia , Feminino , Gânglios Simpáticos/química , Gânglios Simpáticos/fisiologia , Gânglios Simpáticos/ultraestrutura , Cobaias , Imuno-Histoquímica , Masculino , Microscopia Confocal , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/análise , Neurônios/fisiologia , Neurônios Aferentes/fisiologia , Neurônios Aferentes/ultraestrutura , Neuropeptídeo Y/análise , Neurópilo/fisiologia , Sinapses/fisiologia , Peptídeo Intestinal Vasoativo/análise
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