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Excitation of rat cerebellar Golgi cells by ethanol: further characterization of the mechanism.

ABSTRACT: Studies with rodents suggest that acute ethanol exposure impairs information flow through the cerebellar cortex, in part, by increasing GABAergic input to granule cells. Experiments suggest that an increase in the excitability of specialized GABAergic interneurons that regulate granule cell activity (i.e., Golgi cells [GoCs]) contributes to this effect. In GoCs, ethanol increases spontaneous action potential firing frequency, decreases the afterhyperpolarization amplitude, and depolarizes the membrane potential. Studies suggest that these effects could be mediated by inhibition of the Na(+)/K(+) ATPase. The purpose of this study was to characterize the potential role of other GoC conductances in the mechanism of action of ethanol.Computer modeling techniques and patch-clamp electrophysiological recordings with acute slices from rat cerebella were used for these studies.Computer modeling suggested that modulation of subthreshold Na(+) channels, hyperpolarization-activated currents, and several K(+) conductances could explain some but not all actions of ethanol on GoCs. Electrophysiological studies did not find evidence consistent with a contribution of these conductances. Quinidine, a nonselective blocker of several types of channels (including several K(+) channels) that also antagonizes the Na(+)/K(+) ATPase, reduced the effect of ethanol on GoC firing.These findings further support that ethanol increases GoC excitability via modulation of the Na(+)/K(+) ATPase and suggest that a quinidine-sensitive K(+) channel may also play a role in the mechanism of action of ethanol.

SUBMITTER: Botta P

PROVIDER: S-EPMC3525106 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Excitation of rat cerebellar Golgi cells by ethanol: further characterization of the mechanism.

Botta Paolo P Simões de Souza Fabio M FM Sangrey Thomas T De Schutter Erik E Valenzuela C Fernando CF

Alcoholism, clinical and experimental research 20111017 4

<h4>Background</h4>Studies with rodents suggest that acute ethanol exposure impairs information flow through the cerebellar cortex, in part, by increasing GABAergic input to granule cells. Experiments suggest that an increase in the excitability of specialized GABAergic interneurons that regulate granule cell activity (i.e., Golgi cells [GoCs]) contributes to this effect. In GoCs, ethanol increases spontaneous action potential firing frequency, decreases the afterhyperpolarization amplitude, and ...[more]

PMID: 22004123

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Project description:It is known that ethanol (EtOH) and its metabolites have a negative effect on protein glycosylation. The fragmentation of the Golgi apparatus induced by alteration of the structure of largest Golgi matrix protein, giantin, is the major consequence of damaging effects of EtOH-metabolism on the Golgi; however, the link between this and abnormal glycosylation remains unknown. Because previously we have shown that Golgi morphology dictates glycosylation, we examined the effect EtOH administration has on function of Golgi residential enzymes involved in N-glycosylation.HepG2 cells transfected with mouse ADH1 (VA-13 cells) were treated with 35 mM EtOH for 72 hours. Male Wistar rats were pair-fed Lieber-DeCarli diets for 5 to 8 weeks. Characterization of Golgi-associated mannosyl (?-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (MGAT1), ?-1,2-mannosidase (Man-I), and ?-mannosidase II (Man-II) were performed in VA-13 cells and rat hepatocytes followed by three-dimensional structured illumination microscopy (3D SIM).First, we detected that EtOH administration results in the loss of sialylated N-glycans on asialoglycoprotein receptor; however, the high-mannose-type N-glycans are increased. Further analysis by 3D SIM revealed that EtOH treatment despite Golgi disorganization does not change cis-Golgi localization for Man-I, but does induce medial-to-cis relocation of MGAT1 and Man-II. Using different approaches, including electron microscopy, we revealed that EtOH treatment results in dysfunction of ADP-ribosylation factor 1 (Arf1) GTPase followed by a deficiency in COPI vesicles at the Golgi. Silencing beta-COP or expression of GDP-bound mutant Arf1(T31N) mimics the EtOH effect on retaining MGAT1 and Man-II at the cis-Golgi, suggesting that (i) EtOH specifically blocks activation of Arf1, and (ii) EtOH alters the proper localization of Golgi enzymes through impairment of COPI. Importantly, the level of MGAT1 was reduced, because likely MGAT1, contrary to Man-I and Man-II, is giantin sensitive.Thus, we provide the mechanism by which EtOH-induced Golgi remodeling may significantly modify formation of N-glycans.

Project description:Abstract Insight into the bidirectional signaling between primary human myogenic cells and neurons is lacking. For this purpose, human myogenic cells were derived from the semitendinosus and gracilis muscles of five healthy individuals and co‐cultured with cerebellar granule neurons from two litters of 7‐day‐old Wistar rat pups, in muscle medium or neural medium, alongside monocultures of myogenic cells or neurons. RT‐PCR was performed to determine human mRNA levels of GAPDH, Ki67, myogenin, and MUSK, and the acetylcholine receptor subtypes CHRNA1, CHRNB1, CHRNG, CHRND, and CHRNE, and rat mRNA levels of GAPDH, Fth1, Rack1, vimentin, Cdh13, and Ppp1r1a. Immunocytochemistry was used to evaluate neurite outgrowth (GAP43) in the presence and absence of myogenic cells. Co‐culture with primary neurons lead to higher myogenic cell gene expression levels of GAPDH, myogenin, MUSK, CHRNA1, CHRNG, and CHRND, compared to myogenic cells cultured alone. It appeared that neurons preferentially attached to myotubes and that neurite outgrowth was enhanced when neurons were cultured with myogenic cells compared to monoculture. In neural medium, rat mRNA levels of GAPDH, vimentin, Cdh13, and Ppp1r1a were greater in co‐culture, versus monoculture, whereas in muscle medium co‐culture lead to lower levels of Fth1, Rack1, vimentin, and Cdh13 than monoculture. These findings demonstrate mutually beneficial stimulatory signaling between rat cerebellar granule neurons and human myogenic cells, providing support for an active role for both the neuron and the muscle cell in stimulating neurite growth and myogenesis. Bidirectional muscle nerve signaling. These findings demonstrate mutually beneficial stimulatory signaling between rat cerebellar granule neurons and human myogenic cells. The presence of myogenic cells stimulated neurite growth. Species specific PCR of the mono‐cultures vs. co‐cultures revealed effects of co‐culture and/or medium type (statistical main effects and interactions indicated).

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Excitation of rat cerebellar Golgi cells by ethanol: further characterization of the mechanism.

Publications

Excitation of rat cerebellar Golgi cells by ethanol: further characterization of the mechanism.

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