Regional distribution of nicotinic receptors during prenatal development of human brain and spinal cord

doi:10.1016/S0165-3806(98)00046-7

Developmental Brain Research

Volume 108, Issues 1–2, 15 June 1998, Pages 147-160

https://doi.org/10.1016/S0165-3806(98)00046-7 Get rights and content

Abstract

The development of nicotinic acetylcholine receptors (nAChRs) in brains from human fetuses of 4–12 weeks gestational age was studied. The expression of nAChR subunit mRNAs was analyzed using reverse transcriptase-polymerase chain reaction. Expression of α3, α4, α5, α7, β2, β3 and β4 mRNA were all detected in the prenatal spinal cord, medulla oblongata, pons, cerebellum, mesencephalon, subcortical forebrain and cortex during first trimester development. Relative quantification of mRNA showed that the highest levels for α3, α4 and α7 were expressed in the spinal cord, α5 was most abundant in the cortex and β3 was highest in the cerebellum. β4 seemed to be equally distributed in all regions whereas β2 was high in the cortex and cerebellum. A comparison of expression of nAChR subunit mRNAs in the cortex and cerebellum of prenatal and aged (54–81 years) brain showed that mRNA levels for α4, α5, α7, β2 and β4 were significantly higher in the prenatal cortex and cerebellum than in aged brain, whereas the level of α3 transcript was similar, and β3 significantly higher in aged cortex. Specific binding of [ $^{3} H$ ]-epibatidine to prenatal brain membranes was detected as early as 4–5 weeks of gestation in the spinal cord, medulla oblongata, pons and subcortical forebrain. A positive correlation between gestational age and [ $^{3} H$ ]-epibatidine and [ $^{3} H$ ]-cytisine binding was found in several brain regions. The highest specific binding of [ $^{3} H$ ]-epibatidine and [ $^{3} H$ ]-cytisine was detected in the spinal cord, pons and medulla oblongata and the lowest in the cortex. Saturation analysis of [ $^{3} H$ ]-cytisine binding in both prenatal and aged brain were best fit by a model for a single site, whereas binding data for [ $^{3} H$ ]-epibatidine revealed two classes of binding sites. The early presence of nAChR proteins and gene transcripts shown in the present study suggests an important role for nAChRs in modulating dendritic outgrowth, establishment of neuronal connections and synaptogenesis during development.

Introduction

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels comprised of α and β subunits. Eleven nAChR subunit genes (α2–α9; β2–β4) have been identified to date in the rat and chicken nervous system, while human α2–α7 and β2–β4 nAChR subunits have been cloned 14, 21, 36, 47. Thus, assembly of the different subunits can generate many subtypes of nAChRs with different functional and pharmacological properties. Precise subunit composition of most subtypes is still unclear and the role of the many potential nAChR subtypes in brain remains to be fully elucidated. Several distinct classes of nAChRs can be distinguished in brain by radioligand binding studies. The most abundant nAChR subtype, which accounts for most of the high affinity nicotine binding, at least in rats, is made up of the α4 and β2 subunits and has high affinity for epibatidine, (−)-nicotine and cytisine. Epibatidine has been shown to be the most potent nicotine agonist in several nAChR preparations yet reported and is a full agonist to the human α4β2 subtype relative to nicotine as well as to α3 to which nicotine show less affinity 19, 20, 26, 29. The α7 containing nAChRs account for most of the high affinity α-bungarotoxin binding. A third class is the complex subtype of α3 subunits together with α5, β4 or β2. It has been suggested that nAChRs are localized mostly presynaptically [54]. In view of the accumulating evidence that nAChRs at presynaptic sites can modulate synaptic transmission by regulating the release of a number of neurotransmitters, including acetylcholine, dopamine, noradrenaline, serotonin, GABA and glutamate, it has been considered that a significant role of nAChRs in CNS may be to modulate, in addition to mediate, synaptic transmission 3, 54.

The nAChRs appear to have a functional role during brain development, since periods of transient high receptor density have been reported in the frontal cortex, hippocampus, cerebellum and brain stem in humans during mid-gestation and neonatal periods 10, 11, 12, 30. The presence of nAChRs in the brain during development is of considerable interest, especially in view of the adverse effects of maternal smoking on fetal development including behavioural abnormalities and cognitive deficits 18, 51, as a risk factor for the sudden infant death syndrome [23], but probably also for dependence to nicotine later in life. Nicotine is readily transferred to the fetal compartment throughout pregnancy and the fetuses of smoking mothers to be are in fact exposed to higher nicotine concentrations than the mother [35]. The early presence of both choline acetyltransferase (ChAT) for synthesizing acetylcholine and nAChRs during early development suggest important roles for nicotinic signalling in brain development. ChAT activity has been detected as early as 8 weeks of gestation 5, 42while the earliest reported binding of [ $^{3} H$ ]-nicotine so far has been demonstrated in whole brain homogenates from 12 week old fetuses [4].

Few data is available concerning the ontogenesis of nAChRs in brain during the first trimester. Moreover, there are no reports on their regional distribution and the gene expression of the different nAChR subunits. In this study we describe the distribution of nAChRs and mRNA levels for α3, α4, α5, α7, β2, β3 and β4 subunits in the brain from 4–12 gestational weeks. In addition, data from first trimester cortex and cerebellum have been compared with those obtained from aged human brain. Since nAChR gene transcripts are present at relatively low levels and with the limited amounts of tissue that could be obtained from fetal brain, we choose to use the sensitive reverse transcriptase-polymerase chain reaction technique (RT-PCR) to measure nAChR mRNAs.

Section snippets

Brain tissues

First trimester brain tissue from humans (4–12 weeks gestation) was obtained following routine abortions by vacuum aspiration. The collection of residual tissue was approved by the Human Ethics Committee of the Huddinge Hospital and was in accordance with guidelines of the Swedish Society of Medicine and the US Public Health Service, including informed consent from the pregnant women [40]. Dissections of brains were performed within 1–1.5 h of surgical tissue retrieval and samples were weighed

nAChR Gene expression in prenatal brain during first trimester development

RT-PCR experiments were performed to determine which subunits of the nAChRs were expressed in developing prenatal brain. Control reactions were carried out where samples not reverse transcribed to cDNA were amplified to ensure that the detected product was not the result of genomic DNA contamination. No products were detected from these negative controls or when PCR amplification was performed with sterile water instead of cDNA. A representative example of PCR products obtained after performing

Discussion

We have demonstrated that nAChR proteins and gene transcripts are present at least as early as 4–5 weeks of gestation in the human spinal cord, medulla oblongata, pons and subcortical forebrain. Although the period of development investigated (4–12 weeks) was necessarily restricted, clear age-related alterations in number of nAChRs in prenatal brain and spinal cord were apparent. Correlations between gestational age and [ $^{3} H$ ]-epibatidine binding were positive and significant in all regions

Acknowledgements

This work was supported by grants from the Swedish Medical Research Council (Projects No. 05817, 06555), Stiftelsen Gamla Tjänarinnor, Åke Wiberg's Foundation, Loo and Hans Osterman's Foundation, KI Foundations and Swedish Match. We thank Dr. Ritva Ravid at the Netherlands Brain Bank for providing the aged human brain tissue.

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Research reportRegional distribution of nicotinic receptors during prenatal development of human brain and spinal cord

Abstract

Introduction

Section snippets

Brain tissues

nAChR Gene expression in prenatal brain during first trimester development

Discussion

Acknowledgements

Brain Res.

Neurosci. Lett.

Dev. Brain Res.

Dev. Brain. Res.

Dev. Brain Res.

Eur. J. Pharmacol.

Progr. Neurobiol.

Int. J. Dev. Neurosci.

Brain Res.

Neuroscience

Dev. Brain Res.

Trend. Neurol. Sci.

J. Biol. Chem.

Dev. Brain Res.

Neuron

Brain Res.

J. Biol. Chem.

Mol. Brain Res.

Trends Neurosci.

Neurochem. Int.

Life Sci.

Int. J. Dev. Neurosci.

Brain Res.

Evidence that tobacco smoking increases the density of (−)[3H] nicotine binding sites in human brain

J. Neurochem.

Effect of smoking history on [3H] nicotine binding in human postmortem brain

J. Pharmacol. Exp. Ther.

The pharmacology of (−)-nicotine and novel cholinergic channel modulators

Adv. Pharmacol.

Single-step method of RNA isolation by acid guanidium thiocyanate–phenol–chloroform extraction

Anal. Biochem.

Expression of neuronal acetylcholine nicotinic receptor α4 and β2 subunits during postnatal development of the rat brain

J. Neural. Transm.

Age associated decline in high affinity nicotinic binding in human brain frontal cortex does not correlate with changes in choline acetyltransferase activity

Neurosci. Res. Commun.

Research report
Regional distribution of nicotinic receptors during prenatal development of human brain and spinal cord

Evidence that tobacco smoking increases the density of (−)[ $^{3} H$ ] nicotine binding sites in human brain

Effect of smoking history on [ $^{3} H$ ] nicotine binding in human postmortem brain