Increased level of neurokinin-1 tachykinin receptor gene expression during early postnatal development of rat brain

Abstract

Substance P is known to elicit diverse actions via activating multiple subtypes of tachykinin receptors, and these actions appear to be involved not only in synaptic transmission but also in synaptic plasticity during development of the mammalian cental nervous system. The availability of sensitive quantitation of individual tachykinin receptor subtypes is crucial for elucidating the physiological function specifically mediated by activation of a particular receptor subtype. We thus attempted to develop an assay to determine the level of messenger RNA molecule encoding the neurokinin-1-type tachykinin receptor and apply it for assessment of developmental changes in the neurokinin-1 receptor gene expression in the rat brain to explore the role of tachykinin receptors during ontogeny. The assay was designed to use a competitive reverse transcription–polymerase chain reaction co-amplifying endogenous neurokinin-1 receptor messenger RNA and internal standard, which enabled specific quantification of the number of neurokinin-1 receptor transcripts, ranging from 3.1 × 10 3 to 1.3 × 10 5 molecules/μg total RNA. The levels of neurokinin-1 receptor gene expression were examined in three different brain regions of the rat aged 0–56 days after birth. The order of neurokinin-1 receptor messenger RNA expression was hippocampus > cerebral cortex ≫ cerebellum at all ages examined except postnatal day 0, where its expression was more abundant in the cerebral cortex than in the hippocampus. From postnatal day 3 onward, the hippocampus contained 140–160% of the cortical levels. Although the tachykinin receptor expression in the cerebellum was too low to be accurately assessed by conventional techniques, our assay enabled us to determine the amount of cerebellar neurokinin-1 receptor messenger RNA that changed in the range 7–23% of the cortical level during postnatal development. A prominent feature revealed by this assay is that the neurokinin-1 receptor gene expression in the rat brain is developmentally regulated. The hippocampus displayed a transient peak of neurokinin-1 receptor messenger RNA at postnatal day 3 and a subsequent gradual decrease. In the cerebral cortex, the amount of the message was highest at birth, and was followed by a moderate decrease during postnatal development. At 56 days after birth, the expression levels in both brain regions were down-regulated to approximately 50% of their maximal levels. The transitory pattern of gene expression was also observed in the cerebellum. The results of this study demonstrate that the reverse transcription–polymerase chain reaction-based assay is useful to quantitate precisely the neurokinin-1 tachykinin receptor message in limited tissue samples derived from discrete brain regions. Together with previous findings, the increased level of neurokinin-1 receptor messenger RNA expression in immature rat brain shown by the present analysis suggests that the neurokinin-1-type tachykinin receptor may play a role in the synaptic plasticity associated with morphological and functional development of the mammalian CNS.