Development of Muscarinic Receptor Binding in Spinal Cord Cell Cultures and Its Reduction by Glutamic and Kainic Acids

Abstract

The density of [³H]quinuclidinyl benzilate ([³H]QNB) binding sites in dissociated cell cultures of mouse spinal cord increased almost 4-fold between the 5th and 21st days of maturation in vitro. The mean specific binding of [³H]QNB in 21-day cultures was 340 pmol/g protein. Up to 79% of.[³H]QNB binding sites were lost within a few days after exposure of the cultures to 1 mM glutamate. This neurotoxic action of glutamate showed a steep concentration dependence in the range 0.1–1 mM and did not vary with the duration of exposure of the cultures from 10 min to 24 h. Kainic acid was only moderately more potent than glutamate in reducing [³H]QNB binding. The susceptibility of 8-day cultures to glutamate-induced loss of [³H]QNB binding was less than that of 12- and 19-day cultures. These findings suggest that 80% or more of muscarinic binding sites in the spinal cord cell cultures are on neurons and that the sensitivity of these neurons to the toxic action of glutamate increases with their maturation in vitro.

Glutamate neurotoxicity in cortical cell culture

“…The neuronal swelling seen after only 90 set of glutamate application is comparable to the rapid swelling of locust leg muscle reported within 5 min of exposure to glutamate (Duce et al, The potency ratio of glutamate relative to kainate as a neurotoxin in the present experiments was dramatically increased compared with that customarily found in vivo. This finding is similar to that reported by Brookes and Burt (1980) for spinal cord cells in culture, and by Garthwaite (1985) for cerebellar cells in culture but not with cerebellar cells in slice. As discussed above, a likely explanation for the finding is a heightened neurotoxic effect of glutamate in cell culture, due to the free access of bath glutamate to neuronal membranes in culture.…”

“…The observed ED,, of 50-100 PM is near the ED,, of 100-300 PM reported by Brookes and Burt (1980) for GNT on spinal cord neurons. The concentration of glutamate required to kill a large percentage of the in vitro cortical neuronal population after 5 min of exposure (100 PM) is only 11100th of the 10 mM concentration normally present in whole cortex (Waelsch,195 l), and presumably a smaller fraction of the actual glutamate concentrations present in intracellular compartments.…”

Electrical development in spinal cord cell culture

“…This roughly simultaneous development adds support to the proposal that glycine is the neurotransmitter of the majority of the inhibitory synapses in this preparation (Nelson et al, 1977). The appearance of muscarinic binding sites and kainic acid sensitivity in the same culture system follows a similar time course (Brookes and Burt, 1980). It will be important for future research to address the question of whether there is coordination in the development of these various processes.…”

“…This dip seen in Figure 5 may represent a pause between periods when different generators of electrical activity are in control. Linking this observation to the development of IPSPs, or to muscarinic receptors (Brookes and Burt, 1980), is another intriguing possibility.…”

Specificity and Reversibility of the Inhibition by HgCl2 of Glutamate Transport in Astrocyte Cultures

“…At least two areas of uncertainty make this proposed neurotoxic mechanism speculative. First, the quantitative contributions of glial glutamate transport and synaptosomal glutamate transport [which differ in affinity and capacity, but not in substrate specificity (Debler and Lajtha, 1987)] in maintaining extracellular glutamate below the acutely neurotoxic level of about 100 pA4 (Brookes and Burt, 1980) are unknown. Second, local brain Hg2+ concentrations associated with mercury vapor intoxication are not known.…”