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The h-current in the substantia Nigra pars compacta neurons: a re-examination.

ABSTRACT: The properties of the hyperpolarization-activated cation current (I(h)) were investigated in rat substantia nigra - pars compacta (SNc) principal neurons using patch-clamp recordings in thin slices. A reliable identification of single dopaminergic neurons was made possible by the use of a transgenic line of mice expressing eGFP under the tyrosine hydroxylase promoter. The effects of temperature and different protocols on the I(h) kinetics showed that, at 37°C and minimizing the disturbance of the intracellular milieu with perforated patch, this current actually activates at potentials more positive than what is generally indicated, with a half-activation potential of -77.05 mV and with a significant level of opening already at rest, thereby substantially contributing to the control of membrane potential, and ultimately playing a relevant function in the regulation of the cell excitability. The implications of the known influence of intracellular cAMP levels on I(h) amplitude and kinetics were examined. The direct application of neurotransmitters (DA, 5-HT and noradrenaline) physiologically released onto SNc neurons and known to act on metabotropic receptors coupled to the cAMP pathway modify the I(h) amplitude. Here, we show that direct activation of dopaminergic and of 5-HT receptors results in I(h) inhibition of SNc DA cells, whereas noradrenaline has the opposite effect. Together, these data suggest that the modulation of I(h) by endogenously released neurotransmitters acting on metabotropic receptors -mainly but not exclusively linked to the cAMP pathway- could contribute significantly to the control of SNc neuron excitability.

SUBMITTER: Gambardella C

PROVIDER: S-EPMC3528748 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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The h-current in the substantia Nigra pars compacta neurons: a re-examination.

Gambardella Cristina C Pignatelli Angela A Belluzzi Ottorino O

PloS one 20121221 12

The properties of the hyperpolarization-activated cation current (I(h)) were investigated in rat substantia nigra - pars compacta (SNc) principal neurons using patch-clamp recordings in thin slices. A reliable identification of single dopaminergic neurons was made possible by the use of a transgenic line of mice expressing eGFP under the tyrosine hydroxylase promoter. The effects of temperature and different protocols on the I(h) kinetics showed that, at 37°C and minimizing the disturbance of th ...[more]

PMID: 23284989

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Project description:This article gives a detailed description of a protocol using density gradient centrifugation for the enrichment of neuromelanin granules and synaptosomes from low amounts (?0.15g) of human substantia nigra pars compacta tissue. This has a great advantage compared to already existing methods as it allows for the first time (i) a combined enrichment of neuromelanin granules and synaptosomes and (ii) just minimal amounts of tissue necessary to enable donor specific analysis. Individual specimens were classified as control or diseased according to clinical evaluation and neuropathological examination. For the enrichment of synaptosomes and neuromelanin granules from the same tissue sample density gradient centrifugations using Percoll® and Iodixanol were performed. The purity of resulting fractions was checked by transmission electron microscopy. We were able to establish a reproducible and easy to handle protocol combining two different density gradient centrifugations: using an Iodixanol gradient neuromelanin granules were enriched and in parallel, from the same sample, a fraction of synaptosomes with high purity using a Percoll® gradient was obtained. Our subfractionation strategy will enable a subsequent in depth proteomic characterization of neurodegenerative processes in the substantia nigra pars compacta in patients with Parkinson's disease and dementia with Lewy bodies compared to appropriate controls.Key features of Parkinson's disease are the degeneration of dopaminergic neurons in the substantia nigra pars compacta, an associated loss of the brain pigment neuromelanin and a resulting impairment of the neuronal network. The accumulation of iron binding neuromelanin granules is age- and disease-dependent and disease specific alterations could affect the neuronal iron homeostasis leading to oxidative stress induced cell death. The focus of the described method is the analysis of neuromelanin granules as well as axonal cell-endings of nerve cells (synaptosomes) of individual donors (control and diseased). It is the basis for the identification of disease-relevant changes in the iron homeostasis and the generation of new insight into altered protein compositions or regulations which might lead to disturbed communications between nerve cells resulting in pathogenic processes.

Dataset Information

The h-current in the substantia Nigra pars compacta neurons: a re-examination.

Publications

The h-current in the substantia Nigra pars compacta neurons: a re-examination.

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