Developmental regulation of nicotinic acetylcholine receptors within midbrain dopamine neurons

Abstract

We have combined anatomical and functional methodologies to provide a comprehensive analysis of the properties of nicotinic acetylcholine receptors (nAChRs) on developing dopamine (DA) neurons of Sprague-Dawley rats. Double-labeling in situ hybridization was used to examine the expression of nAChR subunit mRNAs within developing midbrain DA neurons. As brain maturation progressed there was a change in the pattern of subunit mRNA expression within DA neurons, such that alpha3 and alpha4 subunits declined and alpha6 mRNA increased. Although there were strong similarities in subunit mRNA expression in substantia nigra (SNc) and ventral tegmental area (VTA), there was higher expression of alpha4 mRNA in SNc than VTA at gestational day (G) 15, and of alpha5, alpha6 and beta3 mRNAs during postnatal development. Using a superfusion neurotransmitter release paradigm to functionally characterize nicotine-stimulated release of [(3)H]DA from striatal slices, the properties of the nAChRs on DA terminals were also found to change with age. Functional nAChRs were detected on striatal terminals at G18. There was a decrease in maximal release in the first postnatal week, followed by an increase in nicotine efficacy and potency during the second and third postnatal weeks. In the transition from adolescence (postnatal days (P) 30 and 40) to adulthood, there was a complex pattern of functional maturation of nAChRs in ventral, but not dorsal, striatum. In males, but not females, there were significant changes in both nicotine potency and efficacy during this developmental period. These findings suggest that nAChRs may play critical functional roles throughout DA neuronal maturation.

Figure 1

Developmental profiles of α3, α4, α5, α6, α7, β2, β3 and β4 mRNAs within SNc and VTA. Negative values on the x-axis indicate prenatal periods G15 (−7) and G18 (−4). P0 indicates birth (G22). Values represent mean ± SEM from 2-10 animals (males and females). *p<0.05, **p<0.01, ***p<0.001, significantly different from the VTA, t-test with Bonferroni correction. ###p<0.001, significantly different from adult, Dunnett's post-hoc.

Figure 2

Photomicrographs of emulsion-dipped slides showing expression of α3, α4, α5, α6, α7, β2, β3 and β4 mRNAs (silver grains) in SNc and VTA at G15, P1, P21 and P60. Dark cells represent TH Dig -labeled DAergic neurons. In all panels, SN is on the left hand side and VTA on the right. Scale bar = 200μm.

Figure 3

Photomicrographs of α6 mRNA expression within midbrain DA neurons at P21 (A) and adult (B). Note the higher number of silver grains per DA neuron (dark cells) in P21 as compared to the adult. The peak of expression of this subunit at P21 is, therefore, due to higher level of mRNA per DA neuron. Scale bar = 50μm.

Figure 4

Autoradiograms and developmental profiles of (A) [¹²⁵I]α-BTX and (B) [³H]NIC binding within midbrain DA areas. Dig-labeled TH labeling (middle panel) is shown to indicate areas analyzed for binding. Values for [³H]NIC and [¹²⁵I]α-BTX represent mean ± SEM from one male and one female per age.

Figure 5

Developmental profile of (A) average total [³H]DA uptake and (B) 15 mM K⁺-induced [³H]DA release throughout development. Data from males and females are shown separately for ages P1-60. Because males and females were pooled at G17-18, values are shown as a single point. Values are mean ± SEM from 3-7 independent experiments. *p<0.05, **p<0.01, significantly different from P21 using Dunnett's post-hoc analysis.

Figure 6

Nicotine-stimulated [³H]DA release from males during the first 3 postnatal weeks. (A) nicotine-stimulated [³H]DA release from prenatal and perinatal striatum. (B) Nicotine stimulated [³H]DA release at end of first and second postnatal weeks. Release from P21 animals is shown for comparison. At all postnatal ages, maximum release is significantly lower than at P21, a developmental period when nicotine-stimulated [³H]DA release has reached adult levels Values are mean ± SEM from 3-4 independent experiments.

Figure 7

Dose-response curves for nicotine-stimulated [³H]DA release for P30, P40 and adult female (A) and male (B) ventral and female (C) and male (D) dorsal striatal slices. Release is significantly higher from P30 male ventral striatum when compared to either P40 or adult, whereas in the female ventral striaum, the release is similar at all three ages. There is no age-dependent difference in either sex in the dorsal striatum. Values are mean ± SEM from 3-6 independent experiments. *p<0.05, Dunnett's post-hoc analysis with adult as control.