Neurite extension of developing noradrenergic neurons is impaired in genetically epilepsy-prone rats (GEPR-3s): an in vitro study on the locus coeruleus

Abstract 

A primary determinant of seizure susceptibility and severity in genetically epilepsy-prone rats (GEPRs), is a generalized deficiency in the central noradrenergic system of these animals. In particular, this deficiency includes reduced numbers of norepinephrine (NE) synaptic terminals in several brain areas and distinctly fewer NE axons within the auditory tectum. Two strains of GEPRs have been developed: GEPR-3s that have moderately severe clonic seizures and GEPR-9s that have severe tonic seizures culminating in complete hindlimb extension. Seizures in animals of each substrain are preceded by a brief episode of wild running. The developmental profile of NE axonal growth in GEPRs compared to control rats is not known, but may be causally related to NE deficiencies in this seizure model. The present study compared developmental neurite extension of fetal NE neurons in vitro between GEPR-3s and Sprague-Dawley control rats, the strain from which GEPR-3s were originally derived. Neurite arborization of individual NE neurons was assessed by quantitative morphometry following immunocytochemical identification of tyrosine hydroxylase (TH). Preliminary studies using explant and dispersed-cell cultures of control-rat tissues showed that optimal culture parameters to support neuritogenesis of LC neurons included the use of dispersed-cell cultures, Pronectin-F substrate, day-14 gestation donor-tissue, no use of cytosine-arabinofuranoside (ARA-c, a glial mitotic inhibitor) and the presence of co-cultured tectal tissue. Compared to fetal control-rat NE neurons co-cultured with fetal control-rat tectum, NE neurons derived from fetal GEPR-3 LC in co-culture with GEPR-3 tectum exhibited only 30% of the neurite extension of control-rat LC neurons and GEPR-3 LC neurons had a similarly deficient amount of branching. This study suggests, but does not prove, that deficiency in tectal NE in GEPR-3s involves a developmental deficiency in neurite extension from GEPR-3 LC neurons. Hypothetically, this deficiency may also contribute to the well described NE deficiency in other regions of the adult GEPR brain. Supported by SIUSOM and UICOM-P.

Keywords:  Norepinephrine, Seizures, Epilepsy, Rats, Cell culture