| Abstract |
Ontogenesis of the central nervous system includes processes such as cell-birth, migration, cell-fate determination, synapse formation and elimination. Similar plastic processes, i.e. synaptogenesis and neural circuit modification, occur following nervous system injury or during learning. Neurotransmitters, such as nitric oxide free radical (ΝΟ.) and noradrenaline, participate in nervous system normal development as well as in its reactions to environmental factors. ΝΟ. is a recently identified, non-typical neurotransmitter: it is a small molecule, very reactive, with a low half-life, readily diffusible in aqueous media and through lipid bilayers. It exerts its actions, mainly interacting with guanylyl cyclase. In contrast, noradrenaline is a well conserved neurotransmitter. Its action is mediated by membrane receptors coupled to G-proteins. Different types of noradrenaline receptors have been characterized (α1, α2, β1, β2, β3), with α2 receptors localized pre-synaptically, post-synaptically and exo-synaptically. We investigated the role of ΝΟ. and α2 noradrenergic receptors in avian (quail and chicken) brain, during normal development, following injury and in learning and memory. The activity of ΝΟ. biosynthetic enzyme (NOS) has been determined by the histochemical method of NADPH-diaphorase. Cell-birth has been studied by the immuno-cytochemical method of bromo-deoxy-uridine. The distribution of α2 noradrenergic receptors has been determined by in vitro quantitative autoradiographic binding of radio-labeled ligand. In quail cerebellum transient NOS expression was observed in Purkinje cells, which is correlated with the selective elimination of supernumerary afferent projections. Granule cells showed gradually increasing NOS activity, which they retain in adult life, possibly due to their participation in neural circuits storing kinetic memory. In the external granular layer, NOS positive (NOS+) fibers were found during the developmental stages when cell proliferation rate was high. This localization probably enables ΝΟ. to participate in the control of cell-divisions or cell-migration. Cells were still born in the external granular layer after hatching. Density of α2 noradrenergic receptors was very low in quail embryonic cerebellum. Optic tectum layers showed differential NOS activity during embryonic life. Positive cells were present in stratum griseum periventriculare from embryonic day 6 (E6) until mature stages. Bipolar cells in layer i, which synapse with the optic fibers, showed transient NOS activity, with a peak during the period synapses are formed and eliminated. During similar developmental stages, transient NOS activity was observed in other visual nuclei in brain stem (isthmic nuclei) and thalamus (nucleus dorsolateralis anterior thalami). Throughout the embryonic period studied, higher levels of α2 noradrenergic receptors were found in the outer layers of optic tectum (SGFSs) than in the deeper ones (SGFSd, SGC, SAC, SGP). Moreover, in all stages studied, a topographic gradient in α2 noradrenergic receptor density was observed (both rostro-caudally and dorso-ventrally). Significantly higher levels of α2 receptors were found at E16. Their topographic distribution and developmental profile suggests a role in synapse formation. Levels of α2 noradrenergic receptors were very low in other visual areas of quail brain during embryonic life. Suprachiasmatic nucleus was an exception as it showed high α2 noradrenergic receptor density from E12. Two types of NOS positive cells were found in chicken forebrain: with intense staining and apparent processes (type I) and smaller, granule cells, with light staining and no-obvious processes (type II). Type I cells attained their mature distribution pattern around hatching, with higher numbers in basal ganglia and neostriatum where ΝΟ. is probably involved in dopaminergic neurotransmition. During embryonic development, transient NOS expression was found in type II cells in nucleus basalis, ectostriatum, hyperstriatum ventrale, archeostriatum and parts of neostriatum, where ΝΟ. participates possibly in synapse formation and/or elimination. The localization of α2 noradrenergic receptors in quail forebrain exhibited topographic and developmental differences. Higher levels were present in the hippocampus, septum, nucleus basalis, basal ganglia and parts of neostriatum. The density of α2 noradrenergic receptors increased during development, with the exception of hippocampus where it gradually decreased. Unilateral lesion of the right optic tectum at E9 did not affect the distribution of NOS+ cells in quail brain. On the contrary, α2 noradrenergic receptor density was modified. Three days after the lesion, transient changes were present in localized optic tectum sectors as well as in the ectostriatum and area parahippocampalis; five days after the lesion, a large number of other brain areas was affected, which mainly belong to the limbic system. Unilateral transection of the left optic nerve in chicken hatchlings (N1) altered α2 noradrenergic receptor levels in specific parts of the optic tectum as well as in many other brain areas, mainly of the limbic system. Unilateral lesion of the right caudal-dorsal forebrain at E14 did not affect NOS activity in quail brain. Conversely, the expression of α2 noradrenergic receptors was modified in areas spanning the entire brain, including parts of the limbic system. In the intermediate part of hyperstriatum ventrale, α2 noradrenergic receptors participate in memory storage of aversive stimuli. Their number increased following the memory task (one-trial passive avoidance training) while their deactivation by an antagonist (rauwolscine) caused amnesia. In summary, ΝΟ. and α2 noradrenergic receptors take part in plastic processes during normal development (synaptogenesis, maturation of neural connections). ΝΟ. is probably part of the mechanism that controls cell cycle and/or cell-migration. α2 noradrenergic receptors participate in nervous system re-organization following injury as well as during learning and memory.
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