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. 2002 Sep 15;22(18):8101-9.
doi: 10.1523/JNEUROSCI.22-18-08101.2002.

Extrasynaptic alpha 7-nicotinic acetylcholine receptor expression in developing neurons is regulated by inputs, targets, and activity

Craig L Brumwell  1 , James L Johnson, Michele H Jacob
Affiliations

Extrasynaptic alpha 7-nicotinic acetylcholine receptor expression in developing neurons is regulated by inputs, targets, and activity

Craig L Brumwell et al. J Neurosci. .

Abstract

Alpha7-nicotinic acetylcholine receptors (nAChRs) are widely expressed in the vertebrate nervous system. alpha7-nAChR functions include postsynaptic transmission, modulating neurotransmitter release, reinforcing nicotine addiction, and a role in neurological disorders, such as schizophrenia and Alzheimer's disease. In chick parasympathetic ciliary ganglion (CG) neurons, alpha7-nAChRs are excluded from the synapse and localize perisynaptically. Despite their extrasynaptic distribution, the highly Ca2+-permeable alpha7-nAChRs have important synapse-related Ca2+-dependent signaling functions in the CG. We show here that the synaptic partners regulate alpha7-nAChR expression during synapse formation in embryonic CG neurons in situ. The absence of inputs and target tissues cause reductions in alpha7-nAChR mRNA and protein levels that primarily resemble those seen for synaptic alpha3-nAChRs. However, there is a difference in their regulation. alpha7-nAChR levels are downregulated by reduced activity, whereas alpha3-nAChR levels are not. We propose that the activity-dependent regulation of extrasynaptic alpha7-nAChR levels may be an important mechanism for postsynaptic CG neurons to detect changes in presynaptic activity levels and respond with Ca2+-dependent plasticity changes in gene expression.

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Figures

Fig. 1.
Fig. 1.
α7 subunit transcript levels increase during preganglionic and postganglionic synapse formation and then plateau in embryonic CG neurons in situ. Absolute amounts of α7 subunit mRNAs were measured in individual ganglia at landmark stages of synaptogenesis by using quantitative RT-PCR with a mutated α7 cRNA internal standard. Values are normalized to the number of transcript copies per neuron to account for developmental changes in neuron number. Each value represents the mean ± SEM of the number of separate determinations indicated for each time point. For comparison, synaptic α3 subunit mRNA levels were measured at three key stages of preganglionic and postganglionic synapse formation. α7 transcript levels increase ninefold from E5 to E15 and are twofold greater than α3 mRNA levels at all stages of synaptogenesis examined.
None
Comparison of the decreases in extrasynaptic α7 subunit mRNA levels relative to those seen for synaptic α3, β4, and α5 subunit mRNAs in CG neurons deprived of input, targets, and both synaptic partners. Data are expressed as the fold change in the absolute levels of nAChR subunit mRNA per neuron in the test condition relative to controls. For input-deprived CGs and both input- and target-deprived CGs, the sham-operated age-matched CGs are used as controls, whereas target-deprived CGs are compared with the contralateral control CG from the same embryo. The α7 mRNA data are from Figure 2, and the α3, β4, and α5 mRNA values are from Levey et al. (1995). —, Not significantly different from control values; Student'st test.
Fig. 2.
Fig. 2.
α7 transcript levels are reduced in CG neurons that have developed in the absence versus the presence of innervation, target tissues, or both synaptic partners. The absolute amounts of α7 subunit mRNA were determined in individual ganglia from operated (gray) and control (black) embryos at E8 and E12–E14 using quantitative RT-PCR. The values are expressed as the number of transcript copies per neuron to normalize for changes in neuron number after the surgeries. Results represent the mean ± SEM. The number of ganglia assayed is shown above eachbar. Ganglia deprived of inputs and both inputs and targets are compared with age-matched, sham-operated ganglia, whereas ganglia deprived of targets are compared with their contralateral control ganglia. Asterisks indicate statistically significant differences based on the Student's two-sidedt test; **p < 0.01; ***p < 0.001.
Fig. 3.
Fig. 3.
α7-nAChR staining is specifically reduced in CG neurons deprived of synaptic partners compared with control neurons. Cryostat sections of CGs from operated and control embryos at E12–E14 were incubated with biotinylated α-Bgt followed by streptavidin–HRP. The sections were then reacted for peroxidase activity and examined by bright-field microscopy. A, Sham-operated E14 CG; B, input-deprived E14 CG;C, contralateral control E12 CG; D, target tissue-deprived E12 CG; E, staining control E14 CG; F, both input- and target-deprived E12 CG. Most of the neuronal somata are intensely stained in the unoperated control ganglion sections (A, C). The interiors of the somata are filled with HRP reaction product deposits, with the exception of the nuclei, which, when visible, are not stained above background levels (E). In contrast, the majority of the neuronal somata are moderately stained in the input-deprived ganglion section (B), only lightly stained in the target-deprived ganglion section (D), and just slightly stained above background levels in the input- and target-deprived ganglion section (F). Specific α-Bgt staining is demonstrated by the absence of HRP reaction product in the sham-operated E14 ganglion section that was incubated with PBS in place of biotinylated α-Bgt (C). In contrast to the declines in α7-nAChRs, similar relative levels of MAP2 immunolabeling are present in the input- and target-deprived E8 ganglion section (H) and the sham-operated E8 ganglion section (G). Intense MAP2 immunoperoxidase labeling fills the soma and dendrites of the developing CG neurons in G and H. The decreases in α7-nAChR levels are specific. Scale bar:A–F, 30 μm; G, H, 35 μm.
Fig. 4.
Fig. 4.
Visual deprivation downregulates α7-nAChR levels, but not α3-nAChRs, in newly hatched chick CG neurons. Visual deprivation was used to reduce activity in retinal inputs to CG neurons. Chicks were either dark reared immediately after hatching, or a light-tight occluder was applied over one eye, with the contralateral uncovered eye serving as an internal control. Dark-reared chick CGs are compared with diurnally reared chick CGs at matched ages. To control for nonspecific effects of covering the eye with a plastic goggle, a clear occluder was applied on separate chicks. The total number of α7-nAChRs per ganglion was determined by the specific binding of125I-α-Bgt in ganglionic detergent extracts. Results represent the mean ± SEM of the specific activity (the number of binding sites per ganglionic protein) of the test, with both the mean and SEM values normalized to the mean of the appropriate control. The number of separate determinations is indicated above eachbar. The hatched line represents the control diurnally reared chick CG value. For comparison with α7-nAChRs, the total number of α3-nAChRs was assayed in separate ganglia using 125I-mAb-35. α7-nAChR levels are reduced in visually deprived chick CGs, whereas α3-nAChR levels are not.Asterisks indicate statistically significant differences in the raw data of Bgt-specific activities in test versus control CGs based on the Student's t test for dark-reared chicks and the Student's paired t test for monocular occluded chicks; **p < 0.05 and *p < 0.025, respectively.
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