Cultured Neurons as Model Systems for Biochemical and Pharmacological Studies on Receptors for Neurotransmitter Amino Acids

Schousboe, Arne; Drejer, Jørgen; Hansen, Gert H.; Meier, Eddi

doi:10.1159/000112294

Cited by 100 publications

(61 citation statements)

References 25 publications

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“…This may be due in part to the fact that SH-SY5Y are tumor cells, which may possess genomic or cellular changes that interact with AChE inhibit its catalytic activity. It was also surprising that CGN express such a high level of catalytic activity and protein, because CGN are primarily glutamatergic, however express cholinergic receptors, but have not previously been shown to express high levels of AChE [Schousboe et al, 1985;Fucile et al, 2004]. AChE has been shown to be involved transiently in neuritogenesis, and the CGN may represent immature neurons that express AChE prior to other more mature glutamatergic markers [Layer, 1990;Dong et al, 2004].…”

Section: Discussionmentioning

confidence: 99%

Differential localization of acetylcholinesterase in neuronal and non‐neuronal cells

Thullbery

Cox

Schule

et al. 2005

J of Cellular Biochemistry

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Acetylcholinesterase (AChE) expression is regulated in cell types at the transcriptional and translational levels. In this study, we characterized and compared AChE catalytic activity, mRNA, protein expression, and protein localization in a variety of neuronal (SH-SY5Y neuroblastoma and primary cerebellar granule neurons (CGN)) and non-neuronal (LLC-MK2, HeLa, THP-1, and primary astrocytes) cell types. All cell lines expressed AChE catalytic activity; however the levels of AChE-specific activity were higher in neuronal cells than in the non-neuronal cell types. CGN expressed significantly more AChE activity than SH-SY5Y cells. All cell lines analyzed expressed AChE protein at equivalent levels, as well as mRNA splice variants. Localization of AChE was characterized by immunofluorescence and confocal microscopy. SH-SY5Y, CGN, and nerve-growth factor-differentiated PC-12 cells exhibited a pattern of AChE localization characterized as diffuse in the cytoplasm and punctate staining along neurites and on the plasma membrane. The localization in HeLa, LLC-MK2, fibroblasts, and undifferentiated PC-12 cells was significantly different than in neuronal cells-AChE was intensely localized in the perinuclear region, without staining near or on the plasma membrane. Based on the evidence presented here, we hypothesize that the presence of AChE protein doesn't correlate with catalytic activity, and the diffuse cytoplasmic and plasma membrane localization of AChE is a property of neuronal cell types. Keywords acetylcholinesterase; confocal microscopy; cerebellar granule neuron; neuroblastoma; SH-SY5Y; HeLa; PC-12 Acetylcholinesterase (AChE) belongs to the serine hydrolase family that contains the α/β hydrolase fold as a common structural element [Ollis et al., 1992]. The catalytic active site of AChE is made up of a precise arrangement of active site functional groups that catalyze the hydrolytic breakdown of esters that bear quaternary ammonium groups. The biological importance of AChE-catalyzed hydrolysis is manifested in the rapid termination of the neuronal impulse that occurs when acetylcholine (ACh) is released into the synaptic cleft. AChE rapidly hydrolyzes ACh into acetic acid and choline at extremely fast turnover rates [Taylor and Radic, 1994].

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Section: Discussionmentioning

confidence: 99%

Differential localization of acetylcholinesterase in neuronal and non‐neuronal cells

Thullbery

Cox

Schule

et al. 2005

J of Cellular Biochemistry

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“…Brain growth and metabolic and functional development have enormous spurts between 10 and 21 days, with slower increases thereafter (Baquer et al, 1975 Yu et al (1984)), and cerebellar granule neurons obtained from B7-day-old postnatal rodents are used as a model system for glutamatergic neurons (Schousboe et al (1985); Hertz et al (1988) and cited references). Harvest age, culture duration, conditions, medium composition, and cellular development during culturing influence characteristics of cultures (Hertz et al (1998), Hertz (2004) and cited references), as well as any acquired pathophysiology during culturing (e.g., 15 to 30 mmol/L glucose causes diabetic complications; Gandhi et al (2010)).…”

Section: Properties and Physiology Of The Experimental Systemmentioning

confidence: 99%

“…a The age at which tissue was obtained for cultured cells is denoted by E = embryonic or PN = postnatal, followed by the age in days. Cerebral cortical neuronal cultures obtained from B15-day-old embryos are used as a model system for GABAergic neurons, and cerebellar granule neurons obtained from B7 day-old postnatal rodents are used as a model system for glutamatergic neurons (Yu et al (1984); Schousboe et al (1985); Hertz et al (1988) cited references). It must be noted that culture conditions and duration, composition of the culture medium, and cellular development during time in culture influence characteristics of the cultures (e.g., Hertz et al (1998);Hertz (2004) cited references).…”

Section: Cultured Neurons and Presynaptic Endings From The Adult Braimentioning

confidence: 99%

Brain Lactate Metabolism: The Discoveries and the Controversies

Dienel

2011

J Cereb Blood Flow Metab

422

434

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Potential roles for lactate in the energetics of brain activation have changed radically during the past three decades, shifting from waste product to supplemental fuel and signaling molecule. Current models for lactate transport and metabolism involving cellular responses to excitatory neurotransmission are highly debated, owing, in part, to discordant results obtained in different experimental systems and conditions. Major conclusions drawn from tabular data summarizing results obtained in many laboratories are as follows: Glutamate-stimulated glycolysis is not an inherent property of all astrocyte cultures. Synaptosomes from the adult brain and many preparations of cultured neurons have high capacities to increase glucose transport, glycolysis, and glucose-supported respiration, and pathway rates are stimulated by glutamate and compounds that enhance metabolic demand. Lactate accumulation in activated tissue is a minor fraction of glucose metabolized and does not reflect pathway fluxes. Brain activation in subjects with low plasma lactate causes outward, brain-toblood lactate gradients, and lactate is quickly released in substantial amounts. Lactate utilization by the adult brain increases during lactate infusions and strenuous exercise that markedly increase blood lactate levels. Lactate can be an 'opportunistic', glucose-sparing substrate when present in high amounts, but most evidence supports glucose as the major fuel for normal, activated brain. Keywords: astrocyte; brain activation; glucose; lactate shuttling; metabolism; neuron Glucose is the major fuel for the brain, and its metabolism by different pathways has important functions related to energetics, neurotransmission, oxidation-reduction (redox) reactions, and biosynthesis of essential brain components (Figure 1). For many decades, lactate production in the brain was viewed as a consequence of inadequate oxygen delivery, disruption of oxidative metabolism, or mismatch between glycolytic and oxidative rates (Siesjö, 1978), but more recently, the conceptual role of lactate metabolism and function in the normal brain have undergone major changes, shifting from developmental fuel and glycolytic waste product to include its use as a supplemental fuel and signaling molecule. Starting in the 1970s to 1980s studies carried out in different laboratories with diverse experimental interests related to brain function brought attention to upregulation of glycolysis, lactate production, lactate release into the blood, the possibility of lactate shuttling among cell types within the brain, lactate fueling adult brain during exercise, and roles of lactate in the regulation of blood flow; some of these topics are controversial and highly debated. The experimental paradigm and physiologic status of subjects are critical for interpretation of data, and this review first presents a brief historical overview of studies related to brain lactate transport and metabolism, then compares sets of data to provide a perspective and context within which the consistency of simi...

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“…Primary cultures of cerebellar granule cells were prepared from 7-day-old Wistar rats according to the method of Schousboe [32], with slight modifications as described previously [33,34]. The use of rat pups was in accordance with the ethical rules and was approved by the Local Care of Experimental Animals Committee and with the European Communities Council Directive.…”

Section: Cell Culturementioning

confidence: 99%

Neurotoxic effects of indocyanine green -cerebellar granule cell culture viability study

Toczyłowska

Ziemińska

Goch

et al. 2014

Biomed. Opt. Express

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Abstract:The aim of this study was to examine neurotoxicity indocyanine green (ICG). We assessed viability of primary cerebellar granule cell culture (CGC) exposed to ICG to test two mechanisms that could be the first triggers causing neuronal toxicity: imbalance in calcium homeostasis and the degree of oligomerization of ICG molecules. We have observed this imbalance in CGC after exposure to 75-125μΜ ICG and dose and application sequence dependent protective effect of Gadovist on surviving neurons in vitro when used with ICG. Spectroscopic studies suggest the major cause of toxicity of the ICG is connected with oligomers formation. ICG at concentration of 25 μM (which is about 4 times higher than the highest concentration of ICG in the brain applied in in-vivo human studies) is not neurotoxic in the cell culture. Orlock, and C. A. Puliafito, "Adverse reactions due to indocyanine green," Ophthalmology 101(3), 529-533 (1994). 2. B. F. Hochheimer, "Angiography of the retina with indocyanine green," Arch. Ophthalmol. 86(5), 564-565 (1971). 3. J. Fishbaugh, "Retina: indocyanine green (ICG) angiography," Insight 19(3), 30-32 (1994). 4. J. Caesar, S. Shaldon, L. Chiandussi, L. Guevara, and S. Sherlock, "The use of indocyanine green in the measurement of hepatic blood flow and as a test of hepatic function," Clin. Sci. 21, 43-57 (1961). 5. A. El-Desoky, A. M. Seifalian, M. Cope, D. T. Delpy, and B. R. Davidson, "Experimental study of liver dysfunction evaluated by direct indocyanine green clearance using near infrared spectroscopy," Br. J. Surg. 1005-1011 (1999). 6. T. Ishizawa, N. Fukushima, J. Shibahara, K. Masuda, S. Tamura, T. Aoki, K. Hasegawa, Y. Beck, M. Fukayama, and N. Kokudo, "Real-time identification of liver cancers by using indocyanine green fluorescent imaging," Cancer 115(11), 2491-2504 (2009 4998-5003 (2006). 17. T. L. Jackson, "Indocyanine green accused," Br. J. Ophthalmol. 89(4), 395-396 (2005). Reson. Imaging 14(6), 619-623 (1996). 77. D. E. Ray, J. B. Cavanagh, C. C. Nolan, and S. C. Williams, "Neurotoxic effects of gadopentetate dimeglumine: behavioral disturbance and morphology after intracerebroventricular injection in rats," AJNR Am. J. Neuroradiol. 17(2), 365-373 (1996). 78. X. Feng, Q. Xia, L. Yuan, X. Yang, and K. Wang, "Impaired mitochondrial function and oxidative stress in rat cortical neurons: implications for gadolinium-induced neurotoxicity," Neurotoxicology 31(4), 391-398 (2010). 79. M. P. Mattson and Y. Goodman, "Different amyloidogenic peptides share a similar mechanism of neurotoxicity involving reactive oxygen species and calcium," Brain Res. 676(1), 219-224 (1995). 86(8),

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Cultured Neurons as Model Systems for Biochemical and Pharmacological Studies on Receptors for Neurotransmitter Amino Acids

Cited by 100 publications

References 25 publications

Differential localization of acetylcholinesterase in neuronal and non‐neuronal cells

Differential localization of acetylcholinesterase in neuronal and non‐neuronal cells

Brain Lactate Metabolism: The Discoveries and the Controversies

Neurotoxic effects of indocyanine green -cerebellar granule cell culture viability study

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