Elsevier

Brain Research

Volume 760, Issues 1–2, 20 June 1997, Pages 22-33
Brain Research

Research report
Catecholaminergic regulation of proliferation and survival in rat forebrain paraventricular germinal cells

https://doi.org/10.1016/S0006-8993(97)00272-2Get rights and content

Abstract

We have investigated the possible role of α1-adrenoreceptors in regulating the germination of progenitor cells cultured from embryonic rat neocortex. High binding levels of the α1-selective radioligand 3[H]prazosin were detected in the forebrain of the rat embryo at E13, and the greatest density of binding sites was localized to the ventricular and subventricular zones. Catecholamine-containing axon terminals were present in these zones in the same period. Germinal neuroepithelial cells retained specific 3[H]prazosin binding in culture. ≈25% of cells in culture displayed complex intracellular Ca2+ transients in response to phenylephrine, many of which were abolished with the α1B antagonist, chloroethylclonidine. Cultures exhibited concentration-dependent catecholamine stimulation of DNA synthesis mediated by α1 receptors in serum-limited conditions. Neuroepithelial cells were labelled via their ventricular processes by intraventrivcular injection of Fast blue in E13 embryos prior to transfer of the neocortex to dissociated cell culture. Many of labelled cells were present in culture in germinal foci. Some cells which migrated from these foci underwent apoptosis, as determined by TUNEL in situ hybridization. During a transitory period of up to 48 h in culture, α1-adrenoreceptor activation by phenylephrine or noradrenaline increased the number of surviving cells. Apoptosis was observed in vivo in both ventricular and subventricular zones of the neocortex from E13 to E15 in increasing numbers. We propose that both the supply of noradrenaline to forebrain germinal cells, and the expression of α1-adrenoreceptors on their surface could act to determine whether they die or continue to proliferate.

Introduction

The forebrain preventricular germinal cell layer proliferates continuously during the major period of growth of the neopallium. Germinal activity begins in the rat during E9 and continues until after birth. Mitosis within the ventricular zone (VZ) increases its lateral dimensions, and hence ventricular volume [4], and also produces neural progenitor cells which migrate into the subventricular zone (SVZ) prior to commitment. The size of the germinal cell population of the VZ, and the proportion which gives rise to progenitors, is regulated by factors that control the duration of the cell cycle in G1 [9], but may also be influenced by cell survival controls. Programmed cell death in the developing brain may occur both in post-mitotic differentiating neurons [28]and also through loss of cell cycle control in dividing stem cells, where the apoptotic signal may lead to failure of cells to progress from G1- to S-phase 17, 20. Apoptosis occurs predominantly in post-mitotic cells within the outer layers of the cerebral cortex of the developing rat forebrain during late fetal life 14, 17. However, there is recent evidence that cells in the SVZ of the perinatal rat neopallium undergo apoptosis in G1-phase of the cell cycle [45]. It is not certain how soon apoptosis commences after the onset of germinal cell mitosis in the embryonic VZ.

Survival and proliferation throughout the period of neuroepithelial germinal activity are under the control of growth factors, such as EGF, TGFα and FGF-2 22, 38. However, neurotransmitters can also support the survival and regulate the development of neural cells (reviewed in [27]). Controlled local delivery of neurotransmitters via axon terminal varicosities and synapses may permit trophic control over cell number and cell commitment to be targetted locally to functionally organized cell clusters in discrete subregions of the developing cerebral cortex. Neurons expressing a number of neurotransmitters are present in the neopallium at early stages of embryonic development in the rat 10, 35, 40, 48, 49, although until recently there has been direct evidence for transmitter receptors only in the outer cortical layers of the embryo and not in the VZ and SVZ 29, 41. However [30], provided the first evidence that functional receptors for GABA and glutamate can regulate cell division in small groups of germinal cells in the VZ and SVZ, pointing to the possibility of cell cycle control through the mediation of neurotransmitter microenvironments.

Tyrosine hydroxylase undergoes transient expression in rodent forebrain neurons of the catecholamine lineage commencing at E10 [44]. Monoamine fibres from the brainstem enter the rodent forebrain at ≈E13 and ramify within the neocortex during the subsequent 4–5 days 8, 18, 24, 34. Although monoamine-containing fibres in the E13–E15 neuroepithelium have been presumed to be catecholaminergic, this has not been established unequivocally since studies with antibodies to tyrosine hydroxylase and dopamine β-hydroxylase immunocytochemistry place the earliest appearance of cortical catecholamine fibres at E17 [5]. Catecholamines may participate in the development of the forebrain since the adrenoreceptor antagonist isoproterenol alters the rate of proliferation and differentiation of neuronal precursors [43]. Trophic responses of non-neural cells to α1- and β-adrenoreceptors are qualitatively different [32]. α1 receptors promote cell division in both neural and non-neural cell types [12]. High levels of adrenoreceptors are present in the developing primate cortex [25]. α and β receptors are distributed in non-overlapping strata with highest levels of α1 receptors in the germinal ventricular and subplate zones [26]. The liberation of monoamine neurotransmitters in the developing rat forebrain may also be linked to the control of cell survival, reducing the numbers of cells which undergo apoptosis [39]. α1-Adrenoreceptor occupancy is believed to be critical for maintaining the signal function of the high molecular mass G-protein, Ghα, a bifunctional G-protein which, when uncoupled from the ligand-unbound receptor, functions as a type 2 transglutaminase [33], whose activity is associated with the apoptotic pathway [15]. It is therefore of interest to determine whether survival and apoptosis are also associated with α-adrenoreceptor occupancy in VZ cells.

In this study, we show that functional α1-adrenoreceptors are present in the VZ ans SVZ of the rat embryo forebrain and that their presence coincides with catecholamine innervation of these layers. Furthermore, we present evidence that α1 receptors may be implicated in controlling cell proliferation and survival in germinal cells cultured from the neuroepithelium. Part of these studies has appeared in a preliminary report [6].

Section snippets

Histology

Pregnant Wistar rat dams from the University of Bristol breeding colony were mated overnight and gestational age was determined from the timing of post-copulatory plugs and external features of the embryonic brain using the atlas of Altman and Bayer [2]. Dams were killed by rapid CO2 narcosis and cervical dislocation. Embryos were washed in three changes of phosphate-buffered saline pH 7.3. For immunochemistry, whole embryos (E13) or heads alone (older ages) were immersion-fixed in ice-cold

Localization of α1-adrenoreceptors and catecholamine fibers to the embryonic forebrain periventricular zones

We investigated the distribution of α1 receptor-binding sites in the rat embryo at embryonic days 13–15 (E13–E15) using the radioligand 3[H]prazosin (Fig. 1). At E13, the liver, heart and brain (neopallium and mesencephalon) of the embryo contained the highest levels of specific binding (Fig. 1A). Specific labelling of the forebrain at E13, was uniformly high within the sections when the entire thickness of the neopallium still comprises the neuroepithelium (Fig. 1B). At E14, labelling was more

α1-Adrenoreceptors are implicated in cell growth regulation in the forebrain neuroepithelium

Adrenoreceptors of both α and β subtypes have been linked to the control of DNA synthesis and cell replication [32]. β receptors downregulate cell proliferation in developing brain both in vitro and in vivo 3, 43through adenylate cyclase activation. α1 receptors signal predominantly through Gq/1111, 50but also through Gh7, 13, both of which are coupled to phospholipase C. Although α1- and β-adrenoreceptors have been shown to display counter-regulation of DNA synthesis in smooth muscle and

Acknowledgements

Supported by the Motor Neurone Disease Association.

References (53)

  • P. Levitt et al.

    Development of the noradrenergic innervation of neocortex

    Brain. Res.

    (1979)
  • S.A. Lipton et al.

    Neurotransmitter regulation of neuronal outgrowth, plasticity and survival

    Trends Neurosci.

    (1989)
  • J.J. Lo Turco et al.

    GABA and Glutamate depolarize cortical progenitor cells and inhibit DNA synthesis

    Neuron

    (1995)
  • O. Meucci et al.

    α1B but not α1A adrenoreceptor activates calcium influx through the stimulation of a tyrosine kinase/phosphotyrosine pathway following noradrenaline-induced emptying of IP3 sensitive calcium stores in PC C13 rat thyroid cell line

    Biochem. Biophys. Res. Commun.

    (1995)
  • M.J.M. Perry et al.

    Transglutaminase C in cerebellar granule neurons: regulation and localization of substrate cross-linking

    Neuroscience

    (1995)
  • A.G. Sadile et al.

    Adrenergic receptor systems and unscheduled DNA synthesis in rat brain

    Brain Res. Bull.

    (1995)
  • T.A. Slotkin et al.

    Adrenergic control of DNA synthesis in developing rat brain regions: effects of intracisternal administration of isoproterenol

    Brain Res. Bull.

    (1988)
  • J.H. Son et al.

    Early ontogeny of catecholaminergic cell lineage in brain and peripheral neurons monitored by tyrosine hydroxylase-lacZ transgene

    Mol. Brain. Res.

    (1996)
  • J.A. Wallace et al.

    Development of the serotonergic system in the rat embryo: an immunocytochemical study

    Brain Res. Bull.

    (1983)
  • D. Wu et al.

    Activation of phospholipase C by α1 adrenergic receptors is mediated by the a subunits of Gq family

    J. Biol. Chem.

    (1992)
  • G.M. Yan et al.

    Depolarization or glutamate receptor activation blocks apoptotic death of cultured cerebellar granule neurons

    Brain Res.

    (1994)
  • T.J.G. Allen et al.

    Detection and modulation of acetylcholine release from neurites of rat basal forebrain cells in culture

    J. Physiol.

    (1996)
  • J. Altman and S.A. Bayer, Atlas of Prenatal Rat Brain Development, CRC, Boca Raton, FL,...
  • O. Barochovsky et al.

    Effect of central nervous system-acting drugs in brain cell replication in vitro

    Neurosci. Res.

    (1982)
  • B. Berger, C. Verney and P.S. Goldman-Rakic, Prenatal monoaminergic innervation of the cerebral cortex: differences...
  • H. Brennan et al.

    Trophic control of survival and proliferation in cultured rat embryo forebrain germinal cells by α-adrenoreceptors

    J. Physiol.

    (1995)
  • Cited by (10)

    • Section III. The Norepinephrine System

      2005, International Review of Neurobiology
    • Developmental regulation of catecholamine levels during sea urchin embryo morphogenesis

      2004, Comparative Biochemistry and Physiology - A Molecular and Integrative Physiology
    • Survival and mitogenesis of neuroepithelial cells are influenced by noradrenergic but not cholinergic innervation in cultured embryonic rat neopallium

      2000, Brain Research
      Citation Excerpt :

      Serotonergic innervation contributes to the pathways leading to differentation of embryonic progenitors to excitatory neurons [19] but not to the control of progenitor cell numbers. We have demonstrated that cells of the rat cortical neuroepithelium at E13 possess functional α1 adrenoreceptors which stimulate mitogenesis and increase survival [12, 30], and in this study we show that noradrenergic innervation can fulfil the same function in organized cultures. These data indicate non-overlapping roles for catecholaminergic and indoleaminergic systems in the embryonic CNS.

    View all citing articles on Scopus
    View full text