Neuronal primary culture

Neuronal primary cultures are harvested from the PNS or CNS of a living organism and are maintained in culture for at least 24 h [30], The advantages of primary cultures are that many developmental key processes can be largely followed and that the functional capacity of both cell types (neuronal and glial) can be maintained. Moreover, processes that play important roles for normal and abnormal cell development, maturation, and function can be addressed, such as regional specificity, expression of receptors and neurotransmitters, and neuronal-glial interactions. 

We have recently started to analyze the cellular effects of palytoxin in primary cultures of murine neurons. Primary cultures of cerebellar granule cells constitute one of the most reliable, well-characterized, and highly sensitive models for the study of neuronal function and pathology as weU as the effects of poisonous agents." " In this system, we found that palytoxin caused a dose-dependent membrane depolarization as evaluated with the voltage-sensitive membrane probe bis(oxonol) (Figure 31.1). Signihcative differences between control and palytoxin-treated cells were found at toxin concentrations of 5 nM. 

Pomes A, Rodriguez-Farre E, Sunol C. 1994. Disruption of GABA-dependent chloride flux by cyclodienes and hexachlorocyclohexanes in primary cultures of cortical neurons. J Pharmacol Exp Ther 271(3) 1616-1623. 

The first report of the action of a chemokine on neurons was published in 1993. The study demonstrated that IL-8 could increase the survival of cultured neurons (Araujo and Cotman, 1993). However, as can be appreciated from its name, IL-8 was not known to be a chemokine at that time and was instead classed as an interleukin. Indeed, the expression of chemokine receptors by neurons was not generally appreciated until around 1997/1998 when several reports suggested this. These reports included observations of the expression of chemokine receptors by neuronal cell lines (Hesselgesser et al. 1997), primary cultures of neurons (Meucci et al. 1998 Ohtani et al. 1998), and in brain sections from HlV-1, Alzheimer s disease, and other patients (Horuk et al. 1997 Westmoreland et al. 1998 Xia et al. 1997). Furthermore, data were obtained, suggesting functions for chemokine signaling in the development of the nervous system (Zou et al. 1998) as well as in neuronal survival and communication (Giovannelli et al. 1998 Meucci et al. 1998). 

The phenothiazines, chlorpromazine and promethazine, have been described as inhibitors of CCU-induced lipid peroxidation at relatively high concentrations in rat liver microsomes (Slater, 1968). Structural modifications of chlorpromazine were undertaken to try to increase antioxidant activity and maintain molecular lipophilicity. The 2-N-N-dimethyl ethanamine methanesulphonate-substituted phenothiazine (3) was found to be a potent inhibitor of iron-dependent lipid peroxidation. It was also found to block Cu -catalysed oxidation of LDL more effectively than probucol and to protect primary cultures of rat hippocampal neurons against hydrogen peroxide-induced toxicity in vitro (Yu et al., 1992). 

Gilad GM, Gilad VH (1986) Cytotoxic effects of monodansylcadaverina and methylamine in primary cultures of rat cerebellar neurons. Int J Dev Neurosci 4(5) 401-405... 

Many MF-associated proteins [28] have been described in the nervous system (Table 8-3). In general, a good deal is known about their distribution and function in primary cultures of neurons and glia, but less is known about their role in the mature nervous system. Two that have been characterized more extensively are the major non-actin... 

A variety of different types of tissue preparation are used to study neurosecretion and synaptic transmission. A classical preparation is the frog NMJ (discussed below). The brain slice has been used for many years for biochemical studies of CNS metabolism and is a useful preparation for electrophysiological studies of synaptic transmission in the CNS. Slices can be oriented to maintain the local neuronal circuitry and can be thin, 0.3 mm, to minimize anoxia. The transverse hippocampal slice is widely used as an electrophysiological preparation to study synaptic plasticity (see Ch. 53). Primary cultures of neurons from selected CNS areas and sympathetic ganglia are also frequently used. They permit excellent visual identification of individual neurons and control of the extracellular milieu, but the normal neuronal connections are disrupted. 

Smith, D. W. and Friedmann, T. Characterization of the dopamine defect in primary cultures of dopaminergic neurons from hypoxanthine phosphoribosyltransferase knockout mice. Mol. Ther. 1 486-491,2000. 

Yu, A., Drejer, J., Hertz, L. etal. Pyruvate carboxylase activity in primary cultures of astrocytes and neurons. /. Neurochem. 41 1484-1487,1983. 

Tabernero, A., Vicario, C. and Medina, J. M. Lactate spares glucose as a metabolic fuel in neurons and astrocytes from primary culture. Neurosci. Res. 26 369-376,1996. 

Westergaard, N., Sonnewald, U., Unsglrd, G. etal. Uptake, release and metabolism of citrate in neurons and astrocytes in primary cultures. /. Neurochem. 62 1727-1733,1994. 

Bolanos, J. P., Heales, S. J., Land, J. M. etal. Effect of per-oxynitrite on the mitochondrial respiratory chain differential susceptibility of neurones and astrocytes in primary culture. /. Neurochem. 64 1965-1972,1995. 

Edmond, J., Robbins, R. A., Bergstrom, J. D. etal. Capacity for substrate utilization in oxidative metabolism by neurons, astrocytes, and oligodendrocytes from developing brain in primary culture. J. Neurosci. Res. 18 551-561, 1987. 

Electrophysiological studies on primary cultures of hypothalamic neurons and C1300 neuroblastoma cells have shown that the triphenylethylene SERMs ta-... 

Hansen, S. L Ebert, B., Fjalland, B., and Kristiansen, U. (2001) Effects of GABA(A) receptor partial agonists in primary cultures of cerebellar granule neurons and cerebral cortical neurons reflect different receptor subunit compositions. Br. J. Pharmacol. 133, 539-549. 

Hertz, L. and Schousboe, A. (1987) Primary cultures of GABAergic, and glutamatergic neurons as model systems to study neurotransmitter functions. I. Differentiated cells, in Model Systems of Development and Aging of the Nervous System (Vemadakis, A., Privat, A., Lauder, J. M., Timiras, P. S and Giacobini, E eds.), M. Nijhoff Publishing Company, Boston, MA, pp. 19-31. 

Larsson, O. M., Johnston, G. A. R., and Schousboe, A. (1983) Differences in uptake kinetics of cis-3-aminocyclohexane carboxylic acid into neurons and astrocytes in primary cultures. 

Taken together, the limited experiments conducted using neuronal cell cultures illustrate a distinct difference in the way that pyrethroids modify ion conductance and subsequent neurotransmitter release under resting and depolarized conditions. Continued efforts utilizing recent new tools like automated patch-clamp systems and MEAs to assess the effects of pyrethroids on the kinetics and voltage-dependent gating of ion channels in primary cultures or transfected cells is likely to provide new insight into the neurotoxicity of pyrethroids [79, 82]. 

Grosse G, Thiele T, Heuckendorf E, Schopp E, Merder S, Pickert G, Ahnert-Hilger G (2002) Deltamethrin differentially affects neuronal subtypes in hippocampal primary culture. Neuroscience 112 233-241... 

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