Abstract:
The vitamin A metabolite, retinoic acid (RA) is known to play an important role in the
development, patterning and regeneration of nervous tissue, both in the embryo and in the adult.
Classically, RA is known to mediate the transcription of target genes through the binding and
activation ofits nuclear receptors: the retinoic acid receptors (RARs) and retinoid X receptors
(RXRs). Recently, mounting evidence from many animal models has implicated a number of
RA-mediated effects operating independently of gene transcription, and thus highlights nove~
nongenornic actions of RA. For example, recent work utilizing cultured neurons from the pond
snaa Lymnaea stagnalis, has shown that RA can elicit a regenerative response, growth cone
turning, independently of "classical" transcriptional activation While this work illustrates a
novel regeneration-inducing effect in culture, it is currently -unknown whether RA also induces
regeneration in situ. This study has sought to determine RA's regenerative effucts at the
morphological and molecular levels by utilizing an in situ approach focusing on a single
identified dopaminergic neuron which possesses a known "mapped" morphology within the
CNS. These studies show, for the first time in an invertebrate, that RA can increase neurite
outgrowth of dopaminergic cells that have undergone a nerve-crush injury. Utilizing Western
blot analysis, it was shown that this effect appears to be independent of any changes in whole
CNS expression levels of either the RAR or RXR. Additionally, utilizing immunohistochemistry,
to examine protein localization, there does not appear to be any obvious changes in the RXR
expression level at the crush site. Changes in cell morphology such as neurity extension are
known to be modulated by changes in neuronal firing activity. It has been previously shown that
exposure to RA over many days can lead to changes in the electrophysiological properties of
cultured Lymnaea neurons; however, no studies have investigated whether short-term exposure
to RA can elicit electrophysiological changes and/or changes in firing pattern of neurons in
Lymnaea or any other species. The studies performed here show, for the first time in any species, that short-tenn treatment with RA can elicit significant changes in the firing properties of both
identified dopaminergic neurons and peptidergic neurons. This effect appears to be independent
of protein synthesis, activation of protein kinase A or phospholipase C, and calcium influx but is
both dose-dependent and isomer-dependent. These studies provide evidence that the RXR, but
not RAR, may be involved, and that intracellular calcium concentrations decrease upon RAexposure
with a time course, dose-dependency and isomer-dependency that coincide with the
RA-induced electrophysiological changes. Taken together, these studies provide important
evidence highlighting RA as a multifunctional molecule, inducing morphological, molecular and
electrophysiological changes within the CNS, and highlight the many pathways through which
RA may operate to elicit its effects.
