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. 2003 Jul 30;23(17):6740-7.
doi: 10.1523/JNEUROSCI.23-17-06740.2003.

Beta-amyloid regulation of presynaptic nicotinic receptors in rat hippocampus and neocortex

John J Dougherty  1 , Jianlin Wu, Robert A Nichols
Affiliations

Beta-amyloid regulation of presynaptic nicotinic receptors in rat hippocampus and neocortex

John J Dougherty et al. J Neurosci. .

Abstract

Alteration by beta-amyloid (Abeta) of signaling via nicotinic acetylcholine receptors (nAChRs) has been implicated in the early stages of Alzheimer's disease. nAChRs function both post- and presynaptically in the nervous system; however, little is known about the functional consequence of the interaction of Abeta with these receptors, particularly those on presynaptic nerve terminals. In view of the strong correlation between loss of synaptic terminals and dementia, together with the reduction in nAChRs in Alzheimer's disease, the possibility exists that presynaptic nAChRs may be targets for Abeta. To explore this possibility, we assessed the effect of Abeta peptides on nicotine-evoked changes in presynaptic Ca2+ level via confocal imaging of isolated presynaptic nerve endings from rat hippocampus and neocortex. Abeta1-42 appeared to inhibit presynaptic nAChR activation by nicotine. Surprisingly, picomolar Abeta1-42 was found to directly evoke sustained increases in presynaptic Ca2+ via nAChRs, revealing that the apparent inhibitory action of Abeta1-42 was the result of an occlusion of nicotine to further stimulate the receptors. The direct effect of Abeta was found to be sensitive to alpha-bungarotoxin, mecamylamine, and dihydro-beta-erythroidine, indicating involvement of alpha7-containing nAChRs and non-alpha7-containing nAChRs. Prior depolarization strongly attenuated subsequent Abeta-evoked responses in a manner dependent on the amplitude of the initial presynaptic Ca2+ increase, suggesting that nerve activity or Ca2+ channel density may control the impact of Abeta on presynaptic nerve terminal function. Together, these results suggest that the sustained increases in presynaptic Ca2+ evoked by Abeta may underlie disruptions in neuronal signaling via nAChRs in the early stages of Alzheimer's disease.

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Figures

Figure 1.
Figure 1.
Inhibitory effect of Aβ on nicotine-evoked increases in Ca2+ level in individual isolated hippocampal nerve endings. A, Successive stimulation with 500 nm nicotine (left) or 100 nm of the 5-HT3 agonist m-chlorophenyl biguanide (mCPBG; right) in the absence or presence of 100 nm Aβ1-42 during an intervening 10-min wash period and the second stimulation. Top, Representative successive Ca2+ responses in individual synaptosomes. Bottom, Averaged responses expressed as means ± SEM before and after incubation of Aβ1-42. Left graph, n = 45; Right graph, n = 15. Reversibility (B), concentration dependence (C), and peptide specificity (D) of the inhibitory effect of 100 nm Aβ1-42 were also examined. In D, peptides were present at 100 nm. Note that for A, B, Ca2+ responses to the second stimulation were renormalized (*), although a maintained Ca2+ increase occurred with Aβ (Fig. 3). In C,D, data are expressed as percentage of control plateau values (% inhibition). B, n = 13; C: 0 pm, n = 26; 10 pm, n = 8; 100 pm, n = 5; 1 nm, n = 8; D: Aβ1-42, n = 73; Aβ12-28, n = 15; ′Aβ′40-1, n = 6.
Figure 2.
Figure 2.
Sensitivity of nicotine-evoked presynaptic Ca2+ increases to various nicotinic antagonists and to Aβ for preparations from various regions of the brain. A, Average maximal Ca2+ responses to 500 nM nicotine in hippocampal synaptosomes in the absence (n = 34) or presence of 5 μm dihydro-β-erythroidine (DHβE;n=26), 500nmα-bungarotoxin (BgTx; n= 9), 10 μm mecamylamine (Mec; n = 18), or BgTx plus Mec (n = 11) during the second stimulation, using the successive stimulation protocol described in the legend to Figure 1. B, Inhibitory effects of 100 nm Aβ1-42 on nicotine-evoked Ca2+ responses in synaptosomes from hippocampus (Hippo; n = 17), cortex (CTX; n = 13), or striatum (Str; n = 10). Qualitatively similar results were obtained using 100 pm Aβ1-42. *p < 0.05; t test with paired control.
Figure 5.
Figure 5.
Sensitivity of Aβ-evoked Ca2+ increases in isolated hippocampal nerve endings to nicotinic receptor antagonists. Average maximal responses Ca2+ responses to Aβ1-42 in the absence or presence of 500 nm α-bungarotoxin (BgTx; A) (100 pm, n = 14; 100 nm, n = 22), 10 μm mecamylamine (Mec; B) (100 pm, n = 14; 100 nm, n = 14), 500 nm α-bungarotoxin plus 10 μm mecamylamine (Bg/Mec; C) (100 pm, n = 12; 100 nm, n = 6), or 5 μm dihydro-β-erythroidine (DHβE; D) (100 pm, n = 22; 100 nm, n = 16). *p < 0.05; t test with paired control.
Figure 3.
Figure 3.
Aβ-evoked Ca2+ increases in individual hippocampal nerve endings. Composites of Ca2+ responses in individual synaptosomes to Aβ1-42 (A) and Aβ12-28 (B) over an extended time course. Top, Representative initial phases of successive Ca2+ responses in an individual synaptosome to 100 nM Aβ1-42. Data sampling were 4-sec intervals. C, Concentration dependence: 1 pm, n = 26; 10 pm, n = 20; 100 pm, n = 116; 100 nm, n = 175. D, Insensitivity to prior treatment with 50 μm ZnCl2 (n = 15). E, Average maximal Ca2+ responses to 100 nM Aβ1-42 (n = 11) or 100 pM Aβ1-42 (n = 14) before or after filtration of the Aβ solutions through 0.2 μm filters.
Figure 4.
Figure 4.
Dependence of Aβ-evoked Ca2+ increases in isolated hippocampal nerve endings on Ca2+ entry and voltage-gated Ca2+ channels. A, Average maximal responses Ca2+ responses to 100 nm (n = 16) or 100 pm (n = 7) Aβ1-42 in the absence or presence of 1 mm external Ca2+. B, Average maximal Ca2+ responses to 100 nM Aβ1-42 (n = 22) or 100 pM Aβ1-42 (n = 17) in the absence or presence of a mixture of voltage-gated Ca2+ channel blockers (toxins: 200 nm agatoxin TK; 500 nm conotoxin GVIA; 500 nm conotoxin MVIIC), which have previously been shown to block completely K+ depolarization-induced synaptosomal Ca2+ responses (Rondé and Nichols, 1998). *p < 0.05; t test with paired control.
Figure 6.
Figure 6.
Ca2+ responses in isolated nerve endings to Aβ, followed by various concentrations of nicotine (A, B) compared with Ca2+ responses to nicotine, followed by various concentrations of Aβ (C,D). Averaged responses in A (n = 6) and C (n = 13) are means ± SEM. Averaged maximal responses in B (500 nm, n = 2; 5 μm, n = 4; 50 μm, n = 3; 200 μm, n = 5) and D (10 pm, n = 14;1nm, n = 9; 10 nm, n = 9; 100 nm, n = 8) denote the second agent as a percentage of the maximal response to the first agent. Insets, Representative responses.
Figure 7.
Figure 7.
Ca2+ responses in isolated hippocampal nerve endings to Aβ after prior depolarization by KCl compared with Ca2+ responses to depolarization by KCl after Aβ. The averaged responses in A (n = 26) and C (n = 18) are means ± SEM. Individual peak responses to the second agent in B (n = 71) and D (n = 16) are correlated to the initial peak responses of the first agent, with each response to the second agent normalized to the response to the first agent as a percentage.
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